Metabolic Syndrome: Underlying Mechanisms and Drug Therapies

Metabolic Syndrome

Metabolic Syndrome Underlying Mechanisms and Drug Therapies Edited by

Minghan Wang Amgen, Inc., Thousand Oaks, California, USA

Copyright Ó 2011 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/ permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data: Metabolic syndrome : underlying mechanisms and drug therapies / edited by Minghan Wang. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-34342-5 (cloth) 1. Metabolic syndrome–Pathophysiology. 2. Metabolic syndrome–Chemotherapy. I. Wang, Minghan, 1966[DNLM: 1. Metabolic Syndrome X–drug therapy. 2. Metabolic Syndrome X–physiopathology. WK 820 M58695 2011] RC662.4.M53 2011 616.3’99–dc22 2010019505 Printed in the United States of America 10 9 8

7 6 5 4

3 2 1

Contents

Introduction

ix

Minghan Wang

Contributors

Part One

xi

The Physiology of Metabolic Tissues Under Normal and Disease States

1. Gut as an Endocrine Organ: the Role of Nutrient Sensing in Energy Metabolism

3

Minghan Wang

2. Central Glucose Sensing and Control of Food Intake and Energy Homeostasis

29

Lourdes Mounien and Bernard Thorens

3. Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus

53

Taly Meas and Pierre-Jean Guillausseau

4. Adipokine Production by Adipose Tissue: A Novel Target for Treating Metabolic Syndrome and its Sequelae

73

Vanessa DeClercq, Danielle Stringer, Ryan Hunt, Carla G. Taylor, and Peter Zahradka

5. Hepatic Metabolic Dysfunctions in Type 2 Diabetes: Insulin Resistance and Impaired Glucose Production and Lipid Synthesis

133

Ruojing Yang

v

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Contents

6. Energy Metabolism in Skeletal Muscle and its Link to Insulin Resistance

157

Minghan Wang

Part Two

Metabolic Diseases and Current Therapies

7. Mechanisms and Complications of Metabolic Syndrome

179

Minghan Wang

8. Emerging Therapeutic Approaches for Dyslipidemias Associated with High LDL and Low HDL

199

Margrit Schwarz and Jae B. Kim

9. Mechanism of Action of Niacin: Implications for Atherosclerosis and Drug Discovery

235

Devan Marar, Shobha H. Ganji, Vaijinath S. Kamanna, and Moti L. Kashyap

10. Current Antidiabetic Therapies and Mechanisms

253

Minghan Wang

Part Three

Drug Targets for Antidiabetic Therapies

11. GLP-1 Biology, Signaling Mechanisms, Physiology, and Clinical Studies

281

Remy Burcelin, Cendrine Cabou, Christophe Magnan, and Pierre Gourdy

12. Dipeptidyl Peptidase IV Inhibitors for Treatment of Diabetes

327

C.H.S. McIntosh, S.-J. Kim, R.A. Pederson, U. Heiser, and H.-U. Demuth

13. Sodium Glucose Cotransporter 2 Inhibitors

359

Margaret Ryan and Serge A. Jabbour

14. Fibroblast Growth Factor 21 as a Novel Metabolic Regulator

377

Radmila Micanovic, James D. Dunbar, and Alexei Kharitonenkov

15. Sirtuins as Potential Drug Targets for Metabolic Diseases Qiang Tong

391

Contents

16. 11b-Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic Target for Type 2 Diabetes

vii

423

Clarence Hale and David J. St. Jean, Jr.

17. Monoclonal Antibodies for the Treatment of Type 2 Diabetes: A Case Study with Glucagon Receptor Blockade

459

Hai Yan, Wei Gu, and Murielle Veniant-Ellison

Part Four

Lessons Learned and Future Outlook

18. Drug Development for Metabolic Diseases: Past, Present and Future

471

Minghan Wang

Index

489

Introduction

It has been more than 20 years since Reaven first introduced the concept of syndrome X or insulin resistance syndrome to describe the clustering of several cardiovascular risk factors. The concept has evolved over the years and is now commonly referred to as metabolic syndrome, which covers the individual metabolic abnormalities of obesity, insulin resistance, hyperglycemia, dyslipidemia (high triglycerides and low HDL), and hypertension. Patients with metabolic syndrome have increased risk of developing cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). Despite the debates surrounding the existence and definition of metabolic syndrome, the concept has been useful in understanding the interconnections of the various risk factors that are common in a large population of patients and thereby managing the overall disease risk. From the drug discovery standpoint, all the components of metabolic syndrome are therapeutic targets for the treatment of CVD and T2DM to reduce comorbidities and overall mortality. While there is a wealth of information concerning the clinical features and mechanisms of metabolic syndrome, putting them in the physiological context relevant to the development of therapeutics is essential for drug discovery. The goal of this book is to provide comprehensive understanding of the molecular and physiological abnormalities associated with metabolic syndrome and the therapeutic strategies for drug development. Part One is devoted to gaining an integrated understanding of the metabolic abnormalities at the tissue and pathway levels that are associated with disease states. In Part Two, metabolic syndrome is discussed at the physiological level and current therapies are summarized. These sections help lay the foundation to identify pathways and molecular targets for the development of antidiabetic therapies in Part Three. Since more than 80% type 2 diabetic patients have metabolic syndrome, a large portion of this book is devoted to antidiabetic therapies. Finally, the successes and failures in developing antidiabetic and cardiovascular drugs and lessons learned are discussed in Part Four. Although the chapters are contributed by different authors, the organization and the content of the book have been carefully designed so that the information is presented systematically. In the meantime, each chapter independently covers a subarea of metabolic or drug discovery topics, the reader has the flexibility to gain information on a specific tissue, pathway, or target in a time-efficient manner. Despite the exciting advances that have been made in developing antidiabetic and CVD therapies in the past several

ix

x

Introduction

decades, drug discovery in these areas continues to be a challenge. I hope this book will help the reader better understand the exciting science behind metabolic drug discovery and development and develop a greater appreciation of the complexity of metabolic syndrome as well as the treatment strategies.

MINGHAN WANG

Contributors

Remy Burcelin, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Cendrine Cabou, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Vanessa DeClercq, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada H.-U. Demuth, Probiodrug AG, Biocenter, Halle (Saale), Germany James D. Dunbar, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Shobha H. Ganji, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Pierre Gourdy, Rangueil Institute of Molecular Medicine, INSERM U858, Toulouse, France Wei Gu, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA Pierre-Jean Guillausseau, APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, Paris, France; Universite Paris 7, Paris, France Clarence Hale, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA U. Heiser, Probiodrug AG, Biocenter, Halle (Saale), Germany Ryan Hunt, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Serge A. Jabbour, Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

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xii

Contributors

Vaijinath S. Kamanna, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Moti L. Kashyap, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Alexei Kharitonenkov, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Jae B. Kim, Global Development, Amgen, Inc., Thousand Oaks, CA, USA S.-J. Kim, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Christophe Magnan, INSERM U858, Toulouse, France; University Paris Diderot, CNRS, Paris, France C.H.S. McIntosh, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Devan Marar, Department of Veterans Affairs Healthcare System, Atherosclerosis Research Center, Long Beach, CA, USA; Department of Medicine, University of California, Irvine, CA, USA Radmila Micanovic, BioTechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA Taly Meas, APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, Paris, France; Universite Paris 7, Paris, France Lourdes Mounien, Department of Physiology, University of Lausanne, Lausanne, Switzerland; Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland R.A. Pederson, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, Canada; Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, Canada Margaret Ryan, Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA Margrit Schwarz, Department of Metabolic Disorders, Amgen, Inc., South San Francisco, CA, USA

Contributors

xiii

David J. St. Jean, Jr., Department of Medicinal Chemistry, Amgen, Inc., Thousand Oaks, CA, USA Danielle Stringer, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Carla G. Taylor, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada Bernard Thorens, Department of Physiology, University of Lausanne, Lausanne, Switzerland; Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Qiang Tong, USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Department of Medicine, Baylor College of Medicine, Houston, TX, USA; Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA Murielle Veniant-Ellison, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA Minghan Wang, Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA Hai Yan, Department of Protein Science, Amgen, Inc., Thousand Oaks, CA, USA Ruojing Yang, Department of Metabolic Disorders – Diabetes, Merck Research Laboratories, Rahway, NJ, USA Peter Zahradka, Department of Physiology, University of Manitoba, Winnipeg, Canada; Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada; Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada

Part One

The Physiology of Metabolic Tissues Under Normal and Disease States

Chapter

1

Gut as an Endocrine Organ: the Role of Nutrient Sensing in Energy Metabolism MINGHAN WANG Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Energy homeostasis is balanced by food intake and energy expenditure. Both events are controlled by complex sets of neuronal and hormonal actions. Food intake is driven by a central feeding drive, namely, the appetite, which is induced under the fasting state after energy consumption through physical activities. Following food digestion, the passage of nutrients through the gastrointestinal (GI) tract generates signals that produce sensations of fullness and satiation. In particular, nutrients interact with receptors in the small intestine and stimulate the release of peptide hormones, the actions of which mediate physiological adaptations in response to energy intake. The commonly known GI peptides include the incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide or gastric inhibitory peptide (GIP), as well as peptide tyrosine tyrosine (PYY), cholecystokinin (CCK), and oxyntomodulin. These peptide hormones are secreted from different regions of the small intestine. GLP-1, oxyntomodulin, and PYY are secreted from endocrine L cells that are mainly distributed in the distal small intestine (1, 2), whereas GIP is secreted from endocrine K cells primarily localized in the duodenum (3, 4). CCK is secreted from I cells in the duodenum (5). Nutrients released through the digestive tract induce secretion of GI peptide hormones, which subsequently bind to their respective receptors and trigger a cascade of physiological events. These receptors are expressed in tissues such as the central nervous system (CNS), the GI tract, and pancreas, and upon activation lead to suppression of appetite, Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright
2011 John Wiley & Sons, Inc.

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Chapter 1

Gut as an Endocrine Organ

reduced gastric emptying, and assimilation of nutrients. Nutrients can also suppress the secretion of GI peptides. For example, ghrelin, a peptide hormone released by the stomach under the fasting state that stimulates food intake (6), is suppressed after food ingestion (6). GI peptides mediate two principal physiological events: (i) the feedback response on the CNS and the stomach to reduce food intake and slow gastric emptying, and (ii) the feedforward response, mediated particularly by the incretins, to prepare tissues for nutrient integration. In this regard, the small intestine is not only an organ for nutrient absorption but also a major site for providing hormonal regulation of energy intake and storage. GLP-1 and GIP are called incretins because they act on the pancreatic b-cells to increase insulin secretion at normal or elevated glucose levels. They also regulate glucagon secretion by pancreatic a-cells. These actions represent a critical step in preparing the body to switch from the fasting state to postprandial activities. By suppressing glucagon secretion, GLP-1 shut down hepatic gluconeogenesis and adipose lipolysis, two key biological pathways in maintaining energy homeostasis under the fasting state. In addition, GLP-1 can act directly on liver and muscle to regulate glucose metabolism independent of its incretin action (7). In the meantime, induction of insulin secretion by the incretins facilitates glucose uptake by the peripheral tissues. GLP-1 is also involved in the feedback response by acting on the CNS to suppress food intake. PYY and CCK exhibit a similar effect in the CNS underscoring the complexity of appetite regulation. The magnitude and potency of the feedback and feedforward responses depend on both the nutrient content and the length of small intestine exposed. Although both glucose and free fatty acids (FFAs) modulate the secretion of GI peptides, their actions are mediated by distinct mechanisms because they have different residence times in the small intestine and interact with different nutrient-sensing receptors. In fact, even the activity of FFAs varies with their chain length. Moreover, the intestinal length exposed to nutrients and the nutrient contact sites are important determinants in GI peptide secretion.

FOOD INTAKE AND NUTRIENT-SENSING SYSTEMS IN THE GI TRACT After ingestion, food chime is mixed with digestive juices in the stomach and propelled into the small intestine. The three segments of the small intestine, the duodenum, the jejunum, and the ileum, perform different digestive functions (Figure 1.1). Nutrients are generated from the digestion of carbohydrates, fat, protein, and other food components. The passage of nutrients through the small intestine not only facilitates absorption but also plays a role in regulating gastric emptying and satiety. The interaction of nutrients with the small intestine segments generates signals that regulate the rate of gastric emptying and food intake. The nutrient-sensing system consists of receptors, channels, and transporters in the open-type cells on the small intestine luminal surface. It responds to macronutrients and activates signaling pathwaysleading to the release of GI peptides, which subsequently act on the stomach and the CNS to

Food Intake and Nutrient-Sensing Systems in the GI Tract

5

Food (carbohydrates, fat, protein, etc.)

Stomach Target tissues

Ileum Duodenum

Neurons Circulation in bloodstream Colon

GI peptide

Jejunum Enterocyte Enteroendocrine cell

Figure 1.1 Localization of enteroendocrine cells in the GI tract. Enteroendocrine cells (exemplified by an L cell) are on the surface of the GI tract where their luminal sides detect nutrients passing in the lumen, leading to intracellular signals that stimulate the secretion of GI peptides. GI peptides exert biological effects by acting on their receptors in nearby neurons that transduce signals to target tissues. The peptides are carried to target tissues through the circulation and act locally on sites such as the CNS, the pancreas, and the stomach.

slow gastric emptying and suppress appetite, respectively (Figure 1.1). In addition, some peptides such as the incretins stimulate insulin secretion and regulate glucagon secretion to help integrate nutrients into tissues post absorption (Figure 1.1). Studies in pigs demonstrated that rapid injection of glucose into the duodenum during or immediately prior to feeding suppressed food intake (8, 9). The reduction in food intake far exceeded the energy content of the infused glucose (8, 9), suggesting that the effect of glucose on food intake is likely to be mediated by signaling events. In the meantime, hepatic portal or jugular infusion of glucose in pigs did not alter shortterm food intake (10). These data suggest that the regulatory effect of glucose on food intake is a preabsorptive event and the sites of regulation are in the GI tract. To further understand the mechanisms by which dietary carbohydrates regulate energy intake, glucose was infused into the stomach or different segments of the small intestine in pigs. The infusion started 30 min prior to the meal and continued until the pigs stopped eating (11). It was found that infusion of glucose into the stomach, duodenum, jejunum, or ileum each suppressed food intake (11). But comparatively, jejunal infusion caused more reduction in food intake than elsewhere (11). These data suggest that glucose may interact with receptors or other sensing components expressed in various parts of the small intestine to control short-term energy intake. In addition to glucose, FFAs released from fat digestion also play important albeit more complex roles in controlling energy intake. Healthy human volunteers receiving ileal infusion

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Chapter 1

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of lipids consumed a smaller amount of food and energy and had delayed gastric emptying (12). Ileal lipid infusion also accelerated the sensation of fullness during a meal (12). However, intravenous (i.v.) infusion of lipids did not affect food intake (12), suggesting that lipids may interact with ileal receptors to induce satiety and reduce food consumption. Further studies suggest that digestion is a prerequisite for the inhibitory effect of fat on gastric emptying and energy intake. For example, administration of a lipase inhibitor increased food intake in healthy subjects or type 2 diabetic patients receiving a high-fat meal (13, 14), suggesting that FFAs, the breakdown products of fat after ingestion, rather than triglycerides, are the active nutrients that exert the regulatory effects. Likewise, sugars from carbohydrate digestion, rather than carbohydrates themselves, are the active nutrients that induce intestinal signals. Although both glucose and FFAs can stimulate a set of GI peptides that regulate appetite, gastric emptying, and insulin and glucagon release, they have differential effects. For example, glucose stimulates robust secretion of both GLP-1 and GIP, whereas FFAs from a fat meal elicit only modest GLP-1 secretion despite equally robust GIP secretion (15). Further, not all FFAs are equally active since the stimulatory effect depends on their chain length. Although FFAs with a chain length of greater than C12 stimulate CCK release, further increase in chain length has no additional effect, and C11 or shorter FFAs are not active (16, 17). Like carbohydrate and fat meals, protein meals also activate the nutrient-sensing system but in different ways. In healthy human subjects, plasma GIP levels were elevated after both carbohydrate and fat meals but not a protein meal (15). However, intraduodenal amino acid perfusion in human subjects stimulated both GIP and insulin secretion (18, 19). Oral ingestion of mixed amino acids by healthy volunteers also increased plasma GLP-1 levels (20). These findings suggest that amino acids can function as nutrient-sensing agents, and a protein meal is likely to contribute to nutrient sensing in the GI tract. However, since mixed amino acids are not equivalent to a digested protein meal, GLP-1 secretion was studied in humans following a protein meal (15). A transient peak was observed at 30 min followed by a steady-state rise throughout the rest of the 3 h study period (15). The nutrients from the protein meal that stimulated GLP-1 secretion were a mixture of protein hydrolysates but not amino acids per se. It is important to carry out studies with protein hydrolysates that mimic the digested products in the GI tract. A protein hydrolysate (peptone) containing 31% free amino acids and 69% peptides induced the secretion of PYY and GLP-1 in the portal effluent of isolated vascularly perfused rat ileum after luminal administration (21). Peptones also induced CCK secretion and transcription in STC-1 cells, an established L cell line (22, 23). Peptones made from both albumin egg hydrolysate and meat hydrolysate stimulated the transcriptional expression of the proglucagon gene encoding GLP-1 in two L cell lines but not pancreatic glucagon-producing cell lines (24), suggesting that the signaling pathways mediating this effect are L cell/ small intestine specific. In STC-1 cells, the proglucagon promoter contains elements responsive to peptones (25). In contrast, the mixture of free amino acids is at best a weak stimulant (21, 24). These data suggest that free amino acids may have a limited role in protein meal-stimulated GLP-1 or PYY secretion. However, amino acids are indeed involved in nutrient sensing in the GI tract. Aromatic amino acids may play a

Molecular Mechanisms of Nutrient Sensing

7

role in gastrin secretion because they activate the calcium-sensing receptor (CaR) on gastrin-secreting antral cells (26, 27). In addition, amino acids also stimulate CCK release (28, 29) and gastric acid secretion (30). In addition to glucose, FFAs, amino acids, and digested peptides from proteins, other nutrients are also involved in the regulation of GI peptide secretion (21). At physiological concentrations, bile acids stimulate the secretion of PYY, GLP-1, and neurotensin (NT) (21). Interestingly, the threshold concentration of taurocholate for PYY and GLP-1 stimulation is about twofold that required for stimulating NT release (21), suggesting that there is a slight difference in the sensitivity of L cells and N cells to bile acids (21). In addition to the small intestine, the stomach plays an important role in terminating a meal. When rats were implanted with an extra stomach to which a liquid diet was infused, food intake was reduced regardless of whether food was allowed to empty into the small intestine or retained in the stomach (31). This effect is not likely to be mediated by neuronal mechanisms because the implanted stomach was completely denervated (31). This result suggests that the implanted stomach may have generated hormonal signals that affect food intake, and these hormonal signals may mediate the ability of the stomach to sense nutrient quality and quantity to alter the rate of gastric emptying and amount of food ingested (32, 33).

MOLECULAR MECHANISMS OF NUTRIENT SENSING It has been recognized that it is the monomeric nutrients that interact with luminal small intestinal receptors or other nutrient-sensing components and regulate the feedback and feedforward responses to food intake. What do we know about these receptors and their downstream pathways? The analogy between the intestinal nutrient sensing and taste reception by the tongue can shed new light on this question. Glucose sensing in taste buds is mediated by taste receptors expressed in the lingual epithelium (34). These receptors are G protein-coupled receptors (GPCRs) in the apical membranes of taste receptor cells (34). All the three members of the taste receptor family 1 (T1R) class of GPCRs are involved in this function by acting in combination to sense different tastes. The T1R2/T1R3 heterodimer senses sweet taste whereas the T1R1/T1R3 heterodimer senses amino acids and umami taste (35). These receptors activate a phospholipase C (PLC) b2-dependent pathway to increase intracellular Ca2 þ concentrations by coupling to the G proteins gustducin and/or transducin (34). The activated taste receptors may also stimulate the cAMP-dependent pathway (34). In an in vitro assay where T1R2/T1R3 were coupled to Ga15, a promiscuous G protein linked to PLC, T1R2/T1R3 responded to sweet taste stimuli, including glucose, fructose, lactose, and galactose, as well as synthetic sweeteners (35). The activity was inhibited by the sweet taste inhibitor lactisole (35). These data indicate that the T1R2/T1R3 complex mediates sweet sensation along with other components such as G proteins and PLC. Interestingly, the key components of the sweet taste transduction pathways are also expressed in the gut enteroendocrine cells (36), with the signaling events leading to GI peptide secretion by these cells (Figure 1.2). For example, the three members of

Carbohydrates

Fat

Protein

8 Glucose

FFAs

Amino acids and peptides

Glucokinase Taste receptors (i.e., T1R2/T1R3)

SGLT2

GPR40, GPR119, and GPR120

CaR, GPR93, and others

G proteins (Gs or Gq)

G proteins

ATP/ADP ratio Electrogenic activity G proteins (i.e., gustducin)

PLCβ2

Closure of KATP channels

Membrane depolarization

Intracellular [Ca2+ ]

Secondary messengers

or Intracellular cAMP Opening of voltagedependent Ca2+ channels

Secretion of GI peptides Secretion of GI peptides

Intracellular [Ca2+ ]

Secretion of GI peptides (i.e., GLP-1)

Figure 1.2

Potential signaling cascades that mediate GI nutrient sensing in response to main nutrients. Macronutrients, including sugars, FFAs, and amino acids/peptides, are derived through digestion from carbohydrates, fat, and protein. There are three potential pathways that can sense glucose: taste receptors, KATP channels, and SGLT1. FFAs and amino acids/peptides can activate GPCRs expressed in enteroendocrine cells. Activation of downstream signaling by these mechanisms triggers secretion of GI peptides.

Molecular Mechanisms of Nutrient Sensing

9

the T1R class of GPCRs are detected in brush cells, one form of solitary chemosensory cells (SCCs), in the apical membranes of rat jejunum (37). Also found in these cells are a-gustducin, transducin, and PLCb2 (37). In addition, a-gustducin is also expressed in brush cells of the stomach, the duodenum, and pancreatic ducts in rats (38, 39). Brush cells have a structure similar to lingual taste cells (39), suggesting that they may use similar nutrient-sensing pathways. Consistent with the findings in rats, T1R2, T1R3, and a-gustducin are expressed in mouse small intestine (40). Taste signaling elements, including the three subunits of gustducin (a-gustducin, Gb3, and Gg13), PLCb2, and taste receptors, were also found in human L cells (41). Taken together, these data suggest that the taste receptors and associated signaling components are present in gut cells and may be involved in nutrient sensing in a fashion similar to that by the lingual epithelium of the tongue. There are two functional consequences upon the activation of the taste receptor systems in the gut. The first is the release of GI peptides such as GLP-1, which mediates both feedback and feedforward responses to food intake as described above. Glucose induces GLP-1 secretion from enteroendocrine L cells by stimulating the taste receptors, the signal of which is mediated by the taste G protein gustducin. The role of gustducin in sugar sensing and glucose homeostasis was exemplified in a-gustducin null mice (41). In wild-type mice, ingestion of glucose induced a marked increase of GLP-1 secretion (41); in contrast, a-gustducin null mice exhibited defective GLP-1 secretion in response to glucose ingestion (41), suggesting that L cells of the gut sense glucose through similar mechanisms used by taste cells of the tongue. Thus, the gut cells can “taste” sugars and release mediators, such as the incretins, that in turn regulate food intake and nutrient assimilation. The second consequence of the taste receptor activation in the GI tract is elevated glucose transporter 2 (GLUT2) insertion on the apical membrane of the gut lumen to increase glucose absorption (37). The basal level of glucose absorption in the gut is mediated by sodium–glucose cotransporter 1 (SGLT1) and GLUT2 when glucose level is around 20 mM (37). At higher local glucose concentrations (30–100 mM), increased insertion of GLUT2 in the apical membrane occurs to facilitate additional glucose absorption (37). GLUT2 provides three to five times more capacity for glucose absorption than the SGLT1 pathway (37). Despite the above evidence that supports the role of the taste receptor system in mediating nutrient-sensing effects in the GI tract, several research groups have reported findings that dispute this notion. Although the artificial sweetener sucralose was shown to stimulate GLP-1 secretion from human L cells in vitro (41), it did not stimulate GLP-1 secretion in primary L cells (42). In addition, it did not stimulate GLP-1 or GIP release in healthy humans when delivered by intragastric infusion (43). This is in agreement with an earlier study in type 2 diabetic patients where the sweetener stevioside had no effect on GLP-1 or GIP release (44). Further, several sweeteners, including sucralose, were tested in Zucker diabetic fatty rats for their nutrient-sensing activity (45). Consistent with the previous reports, none of these sweeteners increased incretin secretion (45). Taken together, these data indicate that the role of the taste receptor system in GI nutrient sensing remains to be further clarified.

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Two additional signaling pathways in the GI tract have been proposed that could mediate GLP-1 secretion in response to glucose exposure. The first one is the classical glucose-sensing machinery employed by pancreatic b-cells for eliciting glucosedependent insulin secretion (46). This machinery includes components such as ATPsensitive potassium (KATP) channels and glucokinase (Figure 1.2). In this pathway, glucokinase serves as the rate-limiting step in glucose metabolism and therefore is also termed “glucose sensor.” Glucose metabolism increases the ATP/ADP ratio, which causes the closure of KATP channels and depolarization of the b-cell membrane. Next, membrane depolarization leads to opening of voltage-dependent Ca2 þ channels and accumulation of intracellular Ca2 þ , which triggers insulin release. Both the KATP channel subunits Kir6.2 and SUR1 and glucokinase were detected in GLUTag cells, an L cell line (46). In these cells, glucose concentrations between 0 and 20 mM decreased membrane conductance, caused membrane depolarization, and triggered action potentials (46). Tolbutamide also triggered action potentials in GLUTag cells (46), presumably by blocking the KATP channels. These data suggest that the classical glucose-sensing machinery involving glucokinase and KATP channels mediates glucose-induced GLP-1 release from L cells. However, if this notion is true, GLP-1 and GIP secretion following an oral glucose challenge should be lower in individuals with heterozygous glucokinase mutations that confer reduced activity. Unfortunately, when heterozygous glucokinase mutation carriers were subjected to oral glucose tolerance test (OGTT), they did not have altered GLP-1 or GIP secretion post oral glucose challenge compared to normal controls (47). This observation suggests that the glucokinase and KATP channel pathway does not mediate incretin secretion in the gut, or it is involved but there are other redundant pathways that can compensate for it. SGLT1 represents another novel glucose-sensing mechanism that triggers GLP-1 secretion (Figure 1.2). Both SGLT1 and SGLT3 are expressed in GLUTag cells (48), and GLP-1 secretion in response to glucose is inhibited by phlorizin, a SGLT inhibitor compound (48). Moreover, the EC50 value of glucose for glucose-induced GLP-1 secretion matches the Km of SGLT1 (49). These data suggest that SGLT1 could directly mediate glucose-induced GLP-1 release. This effect could be attributed to the electrogenic activity of SGLT1 because low glucose concentrations were shown to trigger small inward currents as they enter cells (48). This current could cause membrane depolarization, which could induce GLP-1 release (Figure 1.2). Like sugars, amino acids and FFAs also regulate endocrine response to food intake through activation of their respective GPCRs in enteroendocrine cells (Figure 1.2). L-Amino acids activate the T1R1/T1R3 heterodimer, which mediates umami taste in taste buds (35). These GPCRs are also expressed in the apical membranes of the gut (37) and couple to the G protein transducin to activate PLCb2 and stimulate Ca2 þ mobilization. Through this signaling system, amino acids may mediate GI peptide release and regulate food intake. In addition, the extracellular CaR may also act to sense amino acids released from protein digestion. CaR is abundantly expressed in epithelial cells and neurons of the stomach, the small intestine, and the large intestine (50). In the stomach, CaR is expressed on gastrin-releasing G cells and its activation stimulates intracellular Ca2 þ mobilization via the activation of PLC (51).

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CaR can be activated by aromatic amino acids (52), suggesting that it may act as a nutrient-sensing receptor in response to a protein diet. However, in the absence of Ca2 þ , aromatic amino acids had no effect on CaR-mediated signaling (52), suggesting that aromatic amino acids are not CaR agonists; rather, they may act as allosteric modulators to enhance the sensitivity of CaR to its agonist Ca2 þ . The proposed role of CaR in amino acid sensing has physiological support. Analysis of human jejunal content before and 3 h after ingestion of a protein-rich meal revealed that aromatic amino acids were more preferentially released than acidic, polar, and aliphatic amino acids (53). For example, the phenylalanine concentration in jejunum could reach 2 mM (53), a level similar to the EC50 value of phenylalanine in a Ca2 þ mobilization assay (52). In addition, L-phenylalanine can activate CCK secretion, presumably through CaR (54, 55). Further, protein hydrolysates directly activate GPR93 in enterocytes, suggesting that multiple GPCRs are involved in sensing of protein nutrients (23). The G protein species to which CaR and GPR93 are coupled are diverse; they depend on specific conditions in different cell types (56) and ligand species (23). As a result, these receptors stimulate the accumulation of a number of secondary messengers. Like glucose and amino acids, longer FFAs appear to interact directly with GPCRs in enteroendocrine cells. The FFA receptor GPR40 is a GPCR highly expressed in pancreatic b-cells mediating the FFA-stimulated glucose-dependent insulin secretion (57). GPR40 is activated by medium- and long-chain FFAs (57, 58). Interestingly, it is also expressed in endocrine L and K cells of the GI tract and mediates GLP-1 and GIP secretion (59). GPR120 is another GPCR expressed in the intestine especially in GLP-1 positive cells and acts as a receptor for unsaturated longchain FFAs (60). Activation of GPR120 both in vitro and in vivo led to increased GLP1 secretion (60), suggesting that GPR120 is a major intestinal FFA sensing receptor that mediates incretin release. Further, a recent study indicates that GPR120 also mediates the stimulation of CCK release by FFAs (61). GPR119, a receptor for endogenous ligands oleoyl-lysophosphatidylcholine (OLPC) and oleoylethanolamide (OEA) (62, 63), is expressed in pancreatic b-cells and upon activation enhances glucose-dependent insulin secretion (63). GPR119 is also localized in L cells and oral administration of a GPR119 agonist increased the release of both GLP-1 and GIP in normal but not GPR119 knockout mice (64), suggesting that GPR119 mediates longchain FFA-induced incretin release. The three GPCRs trigger different intracellular signaling pathways. GPR40 is coupled to the Gq-PLC pathway and upon activation increases the intracellular Ca2 þ accumulation (65), which leads to incretin secretion. Similarly, GPR120 also induces incretin release by triggering the accumulation of intracellular Ca2 þ (60). GPR119 is coupled to Gs and stimulates intracellular cAMP accumulation (66). In addition to enteroendocrine cells, the intestinal mucosa has two other types of sensory systems, neurons and immune cells (67). The sensory neurons are involved in the control of GI motility and signaling to the CNS that controls feeding behavior (67). The immune cells protect against harmful substances that may enter the GI tract. All the three sensing systems work in concert through direct contact with the intestinal contents.

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REGULATION OF INCRETIN SECRETION In 1902, Bayliss and Starling discovered that acid extracts of intestinal mucosa contained a hormone that could be carried to distal tissues via blood circulation and stimulate the exocrine secretion of the pancreas, and named this factor secretin (68). To test if this factor could be used to treat diabetes, Moore et al. administered duodenal mucosa extracts orally to several type 1 diabetics but did not see clear effects (69). The term “incretin” was first proposed by La Barre in 1932 to describe a hormone extracted from the upper gut mucosa with hypoglycemic effect (70). However, the existence of incretin was not proven until 1964, when two independent research groups discovered that an oral glucose load is associated with a significantly greater insulin response than intravenous administration of the same amount of glucose in human subjects (71, 72). The incretin activity was further evaluated by conducting i.v. glucose infusion isoglycemic to the profile generated from an oral glucose challenge. Despite the identical plasma glucose profiles generated by both the oral and the i.v. routes, the oral glucose challenge stimulated greater levels of insulin and C-peptide (73, 74), suggesting that intestinal factors may be released and involved in the stimulation of insulin secretion after oral glucose ingestion. This so-called “incretin effect” describes the important communication through enteroendocrine factors from the GI tract to pancreas in response to food ingestion. This response is a key part of the feedforward mechanism that increases insulin secretion in anticipation of rising blood glucose after food ingestion. There are two incretins, GLP-1 and GIP, both of which are rapidly released to the bloodstream after meal ingestion and stimulate glucose-dependent insulin secretion (GSIS) by pancreatic b-cells. In addition, GLP-1 also suppresses glucagon release by pancreatic a-cells, food intake, and gastric emptying, and is cardioprotective. In contrast, GIP does not exhibit these effects. GLP-1 is secreted from intestinal L cells, which are predominantly found in the distal jejunum, ileum, colon, and rectum (1). However, the distribution of L cells throughout the GI tract is somewhat species specific. The overall L cell density in rat or pig GI tract is greater than that in human gut (1), and higher levels are located in the distal jejunum, ileum, and rectum relative to other intestinal regions in humans (1). In dogs, L cells are predominantly concentrated in the jejunum and less so in the ileum (4). Recently, GLP-1 immunoreactive cells were detected in human duodenum (75), and GLP-1 and GIP were colocalized in a subset of endocrine cells in the small intestine (76). GIP is secreted from K cells located primarily in the duodenum (3), but they can be found in other parts of the small intestine (76). For instance, in dogs, GIP-secreting K cells are equally distributed in the duodenum and the jejunum (4). Both L and K cells are open-type endocrine cells that are in immediate contact with nutrients in the intestinal lumen, allowing nutrient-dependent regulation of incretin secretion. GIP is a 42-amino acid secreted peptide initially isolated from intestinal mucosa. It was named gastric inhibitory peptide but later renamed glucose-dependent insulinotropic peptide for its ability to stimulate insulin secretion (77). The secreted GIP from intestinal K cells is the active form GIP(1–42). GIP is rapidly cleaved at the

Regulation of Incretin Secretion

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N-terminus by dipeptidyl peptidase-4 (DPP-4) (also known as DPP IV, DP 4, CD26, and adenosine deaminase binding protein), an amino peptidase found in almost all organs and tissues (78), producing the inactive form GIP(3–42) (79). DPP-4 also processes other peptides such as GLP-1 (79), chemokines (80–82), and neuropeptides (83). GLP-1 is part of the proglucagon polypeptide that is expressed in both intestinal L cells and pancreatic a-cells. The proglucagon polypeptide is processed posttranslationally by prohormone convertases (PC) 1/3 and 2. PC1/3 is expressed in L cells whereas PC2 is expressed in a-cells. The tissue-specific expression of the convertase isoforms dictates which mature peptides are generated from the proglucagon polypeptides. In the small intestine, the posttranslational processing by PC1/3 produces GLP-1, GLP-2, glicentin, and oxyntomodulin (84, 85). In contrast, in pancreatic a-cells, PC2-mediated posttranslational processing generates glucagon, glicentin-related pancreatic peptide (GRPP), and the major proglucagon fragment (MPGF) that contains the GLP-1 and GLP-2 segments within its sequence (85). There are two equipotent active forms of GLP-1, GLP-1(7–36)amide and GLP-1(7–37). Both forms are prone to proteolytic cleavage by DPP-4 generating inactive GLP-1(9–36)amide and GLP-1(9–37), respectively (79). Carbohydrate, fat, and protein meals all stimulate GLP-1 secretion in human subjects with glucose being the strongest stimulant (15, 20). Unlike carbohydrates and fat that are also strong stimulants of GIP secretion, protein meals have no effect (15). The plasma concentrations of both hormones increase rapidly within 5–15 min after food ingestion (15, 20) but their actions are short lasting due to rapid proteolytic degradation by DPP-4 and other proteases. The plasma half-lives for intact GLP-1 and GIP are 1–2 and 7 min, respectively (86–88). DPP-4 is the main enzyme for incretin clearance as targeted disruption of the DDP-4 gene in mice led to improved stability of endogenous GLP-1 (89). The tissue distribution of DPP-4 plays an important role in GLP-1 degradation. There is a high level of DPP-4 in the endothelium of the capillaries surrounding L cells, and over 50% of newly secreted intact GLP-1 loses the N-terminal dipeptide and as a result is inactivated before entering the systemic circulation (90). The rapid rise of GLP-1 and GIP in the circulation ensures elevated GSIS in response to a meal, which is essential for the normalization of postprandial glucose. The disappearance of the incretins is in sync with the normalization of postprandial glucose. The first contributor of such a precise regulation is proteolytic degradation. In addition to DPP-4, the neutral endopeptidase 24.11 (NEP-24.11) is also involved in incretin degradation (91, 92). But DPP-4 is the main incretin degradation protease. Like DPP-4, NEP-24.11 is not selective against the incretins; it also processes other hormonal peptides (91). Its catalytic rates on vasoactive intestinal peptides (VIP) and glucagon are much faster than those on the incretins (91). The other factor that contributes to the rapid decline of plasma GLP-1 and GIP levels is a negative feedback mechanism, under which both hormones limit their own secretion by stimulating the somatostatin-mediated paracrine regulation. Somatostatin-positive D cells are located throughout the small intestine in close proximity to both L and K cells (4). In vitro, somatostatin inhibits GLP-1 secretion by L cells (93). In perfused porcine intestine, blocking somatostatin activity with a neutralizing monoclonal antibody increased GLP-1 secretion by 8–9-fold (94). Further,

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intravascular infusion of somatostain-28 strongly inhibited GLP-1 release in pigs (94). This finding is consistent with other somatostatin infusion studies in rats (95), sheep (96), and human subjects (97), where somatostatin inhibited GLP-1 or GIP secretion in vivo. These data suggest that GLP-1 secretion is tonically inhibited by the local release of somatostatin-28 from epithelial paracrine D cells. Compared to somatostatin-28, the enteric neuron-derived somatostatin-14 is much weaker in influencing GLP-1 secretion (94). The suppressive effect of somatostatin on GLP1 secretion is mediated by somatostatin receptor subtype 5 expressed in L cells (98). Since both GLP-1 and GIP stimulate somatostatin release (99, 100), somatostatin is believed to be a key player in a negative feedback loop that controls incretin release in the gut. The existence of the negative feedback loop on GLP-1 secretion is supported by further evidence in dogs and humans. Conscious dogs were orally given a DPP-4 inhibitor, which increased meal-induced active GLP-1 levels (101). However, the total GLP-1 levels in these dogs were reduced (101), presumably due to the inhibitory effect of elevated active GLP-1 on endogenous GLP-1 secretion. A similar result was observed in healthy human volunteers who received an oral dose of a different DPP-4 inhibitor (102). These data support the notion that GLP-1 can inhibit its own secretion in vivo as part of a negative feedback loop. In addition to direct stimulation by nutrients, GLP-1 secretion is also indirectly regulated by GIP released in the proximal intestine in rodents. After a meal, nutrients are expected to reach the distal L cells and stimulate GLP-1 release via direct contact. However, this does not explain the biphasic pattern of GLP-1 secretion after a meal, including a 15–30 min rapid rise after oral ingestion followed by a second minor peak at 90–120 min (15, 103). Since all the initial findings indicate that L cells are located in the distal intestine (ileum, colon, rectum), the rapid early rise of GLP-1 after food ingestion within 5–15 min is faster than the time required for unabsorbed nutrients to reach the L cells in the distal intestine. A proposed mechanism is the existence of a neuroendocrine loop that regulates GLP-1 secretion distally once the ingested nutrients reach the proximal intestine (duodenum). This regulatory mechanism is referred to as proximal–distal neuroendocrine loop or duodeno-ileal endocrine loop. Since high GIP levels can stimulate GLP-1 secretion (104, 105), it is possible that nutrient entry into the duodenum stimulates GIP release, which in turn stimulates GLP-1 secretion in the distal intestine even before the nutrients arrive. This notion is supported by several studies. First, intraarterial infusion of GIP into perfused rat colon strongly stimulated GLP-1 secretion (106). In another study, the flow of nutrients to the distal intestine was restrained in rats to prevent direct interaction of the luminal content with the distal L cells (107). Next, when fat or glucose was placed in the duodenal lumen of these animals, GLP-1 release was induced at a level comparable to that by directly placing nutrients into the ileum (107). In the meantime, a rapid rise in GIP was also observed (107). This finding suggests that GIP released from the proximal intestine may mediate the early secretion of GLP-1 in the distal intestine. The vagus nerve appears to play an important role in this regulation because bilateral subdiaphragmatic vagotomy abolished the GLP-1 secretion by fat placed into the duodenum (108). Further, GLP-1 secretion stimulated by physiological concentrations of infused GIP was completely abrogated with selective hepatic branch

Other GI Peptide Hormones or Neurotransmitters

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vagotomy (108). These data suggest that the vagus nerve mediates the GIP-stimulated GLP-1 response in the distal intestine in rats. The GIP-mediated regulation of GLP-1 release has not been validated in humans. Although there is an early rise of GLP-1 after oral ingestion in humans (15, 103), GIP does not play a role in mediating this response. Intraduodenal infusion of a small amount of glucose produced a rapid and short-lasting GLP-1 response but the GIP level did not change (20), suggesting that the GLP-1 response to the duodenal glucose infusion is not mediated by GIP. In a separate study, synthetic GIP was infused into both type 2 diabetic patients and normal subjects. The exogenously administered GIP increased insulin secretion but had no effect on circulating GLP-1 level in normal subjects (109, 110). A further study was carried out in patients with upper and lower gut resections (jejunal or ileal small intestinal resections and colectomy), and it was found that a clear and early (peak at 15–30 min) GLP-1 response after food ingestion was observed in the patients with gut resection as well as controls (111). These studies demonstrate that the early GLP-1 response to food ingestion is not mediated by GIP in humans. One proposed explanation is that the early rise in GLP-1 is also a direct effect of nutrients on L cells because in contrast to previous reports that L cells are primarily located in the distally lower jejunum, ileum, colon, and rectum (1), GLP-1 positive cells were also found in human duodenum in recent studies (75, 76). These data suggest that the early rise in GLP-1 in humans after a meal could be ascribed to GLP-1 secretion from L cells in the upper gut.

DISORDERS IN INCRETIN RESPONSE IN TYPE 2 DIABETES Meal- or oral glucose-induced GLP-1 response is decreased in type 2 diabetic patients as well as subjects with impaired glucose tolerance (IGT) (112, 113), but the GLP-1 response in IGT subjects trends higher than that in type 2 diabetics (112). This impairment could at least in part contribute to the disease pathogenesis. If this is one of the major causes of the defective glucose homeostasis in type 2 diabetes, administration of exogenous GLP-1 is expected to help normalize glucose control. It is encouraging that despite reduced GLP-1 secretion, type 2 diabetics still respond to GLP-1 infusion with augmented insulin release and improved glucose tolerance (109, 114). However, there are individual variations in response to exogenous GLP-1 administration among type 2 diabetics; glucose elimination is faster and lower glycemia was achieved in patients with lower baseline fasting plasma glucose (114). This finding suggests that GLP-1 treatment becomes less effective as the disease progresses. In contrast to GLP-1, GIP has diminished incretin effect in type 2 diabetic patients, suggesting that the GIP response is largely lost in the disease state (109). The underlying mechanism behind this observation is not clear. Based on these data, only GLP-1 is expected to have potential therapeutic value in treating type 2 diabetes.

OTHER GI PEPTIDE HORMONES OR NEUROTRANSMITTERS In addition to the incretins, other peptide hormones and neurotransmitters are also involved in the regulation of gastric emptying, food intake, and energy metabolism.

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There are many such peptides and some are yet to be assigned exact functional roles. They are secreted by different types of enteroendocrine cells distributed in different segments across the GI luminal surface. These cells sense luminal contents through direct interaction and secrete peptide hormones with regulatory effects. PYY and CCK are both involved in the regulation of food intake and gastric emptying. PYY-immunoreactive L cells are found in the distal small intestine, the colon, and the rectum (115). There are two forms of PYY, PYY(1–36) and PYY(3–36), in human blood (116), with PYY(3–36) derived from PYY(1–36) through DPP-4 proteolytic cleavage. Unlike GLP-1, both forms of PYY are bioactive. PYY(3–36) is the major form in human colonic mucosa. The plasma PYY level increases several fold after meal ingestion in humans. Compared to equivalent calories of protein and carbohydrate diets, fat is a more potent stimulus of PYY secretion (117). PYY can inhibit gastric acid and pepsin secretion and delay intestinal transit time (117), suggesting that PYY is a negative regulator of energy intake in response to food ingestion. PYY can interact with a family of Gi-coupled GPCRs, including Y1R, Y2R, Y4R, Y5R, and Y6R. Peripheral injection of PYY(3–36) was shown to inhibit food intake and reduce body weight in rats (118). PYY(3–36) also inhibited food intake in mice but not in Y2R-null mice (118), suggesting that the anorectic effects are mediated by Y2R. Consistent with the findings in animals, PYY(3–36) infusion significantly reduced appetite and food intake in human subjects of normal weight (118). Further, the circulating levels of PYY were significantly lower in obese subjects compared to lean controls, and like its effect in lean subjects, PYY infusion reduced food intake in obese individuals (119). These findings demonstrate that PYY is an anorectic agent and could be used to treat obesity. However, in contrast to peripheral administration, central administration of PYY increased food intake (120). Moreover, the anorectic effects of peripheral PYY(3–36) administration could not be reproduced by some research groups (121), although others have been successful in replicating the original findings (122, 123). These discrepancies remain to be resolved with further studies. Similarly, CCK is another gut peptide involved in the regulation of food intake and related physiological activities. CCK is expressed as a 115-amino acid peptide in cells and undergoes posttranslational proteolytic processing to generate CCK58 (124, 125), the main circulating form. CCK is secreted by both I cells in the proximal intestine and L cells in the distal intestine (5). CCK is also found in the brain (5). Further proteolytic cleavage of CCK-58 generates smaller but still biologically active CCKs, including CCK-39, CCK-33, CCK-22, CCK-12, and CCK-8 (126). CCK is secreted and released into the blood circulation upon food ingestion and induces satiety. Two CCK receptors mediate the CCK function: CCK-1 receptor, primarily expressed in the GI tract, and CCK-2 receptor, mainly expressed in the brain. CCK-1 receptor is also expressed in the hindbrain and hypothalamus. Part of the CCK action in the brain is mediated by suppressing the expression of orexins A and B, two peptides produced in the lateral hypothalamic areas that stimulate food intake (127). The suppression of food intake by CCK was demonstrated in animal models as well as humans. Rats deficient in CCK-1 receptor had increased meal size and developed obesity (128), suggesting that the satiation signal is mediated by CCK-1 receptor. CCK administration also decreased food intake in humans by

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shortening meals (129). The anorectic effects of CCK are weak because rats deficient in CCK-1 receptor developed only mild obesity (128), and CCK-1 receptor-null mice did not develop obesity (130). In addition, the anorectic effects were rapidly lost during repeated CCK administration (131), suggesting that behavioral tolerance may have developed under such a condition. These data question the suitability of CCK as an anti-obesity therapy. Other important GI peptides include oxyntomodulin, GLP-2, and ghrelin. Like GLP-1, oxyntomodulin and GLP-2 are proglucagon-derived peptides secreted from L cells (84, 85). Oxyntomodulin has been demonstrated to reduce food intake and body weight gain in rodents (132–134) and humans (135, 136). Interestingly, oxyntomodulin also increases energy expenditure in both animals and humans (133, 137). These effects are presumably mediated by GLP-1 receptor, although oxyntomodulin binds to it less avidly than GLP-1 (132). Oxyntomodulin also binds to glucagon receptor as its N-terminus contains the full glucagon sequence (138). However, it has a lower affinity than glucagon itself (138). The dual activation of both GLP-1 and glucagon receptors by oxyntomodulin might be a better explanation for the effects on food intake, body weight gain, and energy expenditure. Two independent studies demonstrated that dual activation of both GLP-1 and glucagon receptors with oxyntomodulin- or glucagon-derived peptides reduced food intake, body weight gain, body fat, hepatic steatosis, and blood glucose, and improved insulin sensitivity and lipid metabolism (138, 139). Although also derived from the proglucagon polypeptide and secreted from L cells, GLP-2 has no incretin effect. Rather, it is an intestinal growth factor. GLP-2 stimulates crypt cell proliferation and bowel growth in an ErbBdependent manner (140, 141). GLP-2 also increases intestinal lipid absorption through activation of CD36 (142), thereby mediating a key function in response to food intake. Ghrelin is secreted from the stomach (6, 143) and is the endogenous ligand of the growth hormone (GH) secretagogues receptor (143). There are acylated and unacylated forms of ghrelin and the acylation is essential for the activity (143). Unlike the incretins or PYY, it increases food intake and is involved in meal initiation marked by a pre-meal surge (144). Ghrelin is likely involved in the long-term regulation of body weight (145). Interestingly, ghrelin improved cardiac functions in rats with heart failure (146), suggesting that there may be a role of ghrelin in regulating cardiovascular function.

THE PHYSIOLOGICAL IMPORTANCE OF THE GUT: LESSONS LEARNED FROM GASTRIC BYPASS The metabolic role of the gut is further implicated in the fascinating findings from bariatric surgery, which produces dramatic and durable weight loss (147). Among many different types of bariatric surgical operations employed to treat severe obesity (147), the most commonly performed are laparoscopic adjustable gastric banding (LAGB), gastric bypass, and biliopancreatic bypass (147). Gastric bypass (or Roux-en-Y gastric bypass, RYGB) involves surgical reduction of the size of stomach and bypassing a portion of the proximal small intestine (Figure 1.3). The portion

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Food Duodenum The upper portion is separated from the main stomach

RYGB

Bypass

Bypassed section transports bile and pancreatic fluid into the gut

Figure 1.3

Illustration of Roux-en-Y gastric bypass.

bypassed is connected to the distal small intestine to allow the passage of pancreatic fluids and bile into the gut (Figure 1.3). This procedure causes dramatic weight loss and has been the most effective treatment of severe obesity. In a series of 608 patients with 95% follow-up for at least 16 years, the mean weight loss was 106 lb (148). Surprisingly, more than 80% of the patients with type 2 diabetes developed complete remission of the disease after the surgery (148, 149). Weight loss does not fully explain the remission of type 2 diabetes after gastric bypass because within days after surgery the hyperglycemia and hyperinsulinemia were totally normalized (148). Although the mechanisms behind the antidiabetic effect are not entirely clear, increased insulin secretion and improved b-cell function are likely involved. Lateonset hyperinsulinemic hypoglycemia has been observed in patients after the surgery (150–152), and some may even require partial or total pancreatectomy to prevent recurrent hypoglycemia (150, 152). This phenomenon underscores the robust improvement of pancreatic function achieved by RYGB. GLP-1 and PYY are two important gut hormones that are believed to mediate the more robust beneficial effects of RYGB compared to LAGB, a procedure that restricts food intake by banding the stomach but does not involve the bypass of the proximal intestine. The metabolic effects of LAGB are therefore results of reduced food intake and weight loss. The average reduction in body weight after LAGB is 28% compared to 40% after RYGB, and the remission of type 2 diabetes occurs in 48% relative to 84% in RYGB (153, 154). One of the key differences between these two different operations is the greater GLP-1 and PYY response post meal after RYGB surgery (155), suggesting that these peptide hormones may play an important role in promoting weight loss and improved insulin sensitivity. As mentioned above, RYGB results in improved insulin sensitivity before weight loss in the short term. This effect

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seems to persist even in the long run in a weight loss independent manner, although weight loss itself can lead to improved insulin sensitivity. When compared with a weight-matched group, the patients who underwent RYGB had lower fasting insulin and better insulin sensitivity (156), suggesting that in addition to weight loss something else leads to further improved insulin sensitivity in RYGB patients. In addition to suppressing appetite and weight loss after RYGB (157), the increased postprandial GLP-1 response could further improve insulin sensitivity by increasing b-cell mass and improving b-cell function. In fact, there is sustained elevation of GLP-1 secretion post meal in RYGB patients compared to normal controls (158). This may be counterintuitive because L cells are also found in human duodenum (75) and nutrient bypass of the proximal intestine is expected to cause reduction in GLP-1 release. It could be that this is a small loss relative to the robust increase in GLP-1 secretion by the distal intestine so that the total GLP-1 secretion is still elevated after RYGB. Two hypotheses have been proposed to explain the weight loss independent effect in RYGB based on the roles of the foregut and the hindgut. The hindgut hypothesis proposes that the beneficial effects result from the expedited delivery of nutrients to the distal small intestine and enhancement of physiologic signals that improve glucose homeostasis (159); the foregut hypothesis holds that the weight loss independent effect depends on the exclusion of the duodenum and proximal jejunum from nutrient passage, therefore preventing the secretion of a physiologic signal that promotes insulin resistance (159). Using nonobese diabetic GotoKakizaki (GK) rats, Rubino et al. demonstrated that duodenal–jejunal bypass (DJB), a stomach-preserving RYGB, improved oral glucose tolerance compared to a pair-fed sham-operated group (159). However, restoration of duodenal nutrient passage in the DJB rats reestablished impaired glucose tolerance (159), suggesting that the weight loss independent metabolic benefits in the DJB rats were likely to be driven by the nutrient bypass of the foregut. Why does the duodenal nutrient passage have a negative effect? These researchers proposed that a physiologic signal induced by duodenal nutrient passage might play a role. This negative signal could be an anti-incretin factor, which might be secreted from the proximal intestine in response to nutrient passage and stimulate insulin resistance (159). The anti-incretin factor may interfere with the incretin secretion and/or actions and ultimately inhibit insulin action (159). One of the possibilities is that the anti-incretin inhibits GLP-1 secretion and after nutrient bypass of the proximal intestine the suppression is relieved leading to elevated GLP-1 secretion. Although this hypothesis is consistent with the improved b-cell function in RYGB patients, it remains to be validated by identification of a factor with anti-incretin effect. While the anti-incretin concept helps explain the weight loss independent effects in RYGB patients, Rubino’s data do not exclude the involvement of the hindgut in the improvement of metabolic effects. In fact, a study in mouse models indicates that there is increased gluconeogenesis in the distal intestine post DJB but not gastric banding (160), and the increased local glucose concentration is detected by a GLUT2-dependent hepatoportal sensor, which leads to reduced food intake and body weight and improved insulin sensitivity (160). Thus, it seems that different sections of the small intestine

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play important roles via distinct mechanisms to achieve beneficial metabolic effects in RYGB.

SUMMARY In addition to its role in food intake and nutrient absorption, gut is also an endocrine organ for secreted GI peptides. The release of these peptides in response to food intake is mediated by the direct contact of macronutrients with enteroendocrine cells on the luminal side distributed throughout the GI tract. These GI peptides regulate a variety of physiological actions in response to food intake, including the feedback response to suppress food intake and the feedforward response for nutrient assimilation. The incretin GLP-1 plays important roles in both regulatory pathways. Different sets of GI peptides are stimulated in response to specific types of macronutrients. There are several potential nutrient-sensing mechanisms mediated by taste receptors, KATP channels, glucose transporters, and GPCRs. Further studies are required to clarify the relative contributions of these pathways. The robust metabolic benefits associated with RYGB suggest that changes in the secretion profiles of GI peptides may be beneficial, although the exact mechanism is still elusive. Further studies in gut biology will likely shed new light on the metabolic functions of GI peptides.

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Central Glucose Sensing and Control of Food Intake and Energy Homeostasis LOURDES MOUNIEN1,2 1 2

AND

BERNARD THORENS1,2

Department of Physiology, University of Lausanne, Lausanne, Switzerland Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland

INTRODUCTION Glucose plays an essential role in energy homeostasis by regulating the secretion of various hormones and the activation of neuronal circuits controlling feeding and energy expenditure (1, 2). Glucose-sensing systems are present at many anatomical sites including the mouth, the gastrointestinal tract, the hepatoportal vein, the liver, the endocrine pancreas, and the central nervous system (CNS). In the mouth, glucose activates taste receptors, which stimulate afferent fibers projecting to the brainstem and trigger the cephalic phase of insulin secretion (3–5). In the intestine, glucose stimulates the secretion of the gluco-incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), by a mechanism that may involve the sodium-dependent glucose transporter SGLT1 and the KATP channel (6) and possibly also the activation of sweet taste receptors (7–9). A local action of GLP-1 hormones is to increase the expression of the Naþ /glucose cotransporter SGLT1 and the translocation of the glucose transporter GLUT2 at the brush border of enterocytes, leading to increased glucose absorption (9). Glucose also activates autonomic and enteric neurons located in the gut mucosa (10, 11). Activation of the cholinergic neurons of the submucosal and myenteric plexus may be mediated by glucose binding to SGLT3 (12).

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright
2011 John Wiley & Sons, Inc.

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Glucose entering the portal vein activates vagal afferents that project to the CNS (13, 14) to control several adaptive responses such as stimulation of glucose storage by liver, soleus, heart, and brown adipose tissue; inhibition of counterregulation; termination of food intake; and stimulation of first-phase insulin secretion (10, 15, 16). In the liver, glucose stimulates glycogen synthesis (17) as well as the expression of genes involved in glycolysis and lipogenesis through transcriptional mechanisms mediated in large part by the glucose-sensitive transcription factor ChreBP (18, 19). In the pancreas, increase in plasma glucose triggers insulin secretion by b-cells (1, 20–22). In contrast, glucagon secretion by a-cells is triggered when glycemia falls below the euglycemic level (20, 23, 24). In the CNS, glucose modulates the activity of glucose-sensitive neurons located in the hypothalamus and brainstem. These belong to at least two classes: glucoseexcited (GE) neurons, whose firing activity is increased by rises in extracellular glucose concentrations, and glucose-inhibited (GI) neurons, which are activated when glucose concentrations decrease. These glucose-sensitive neurons control glucose homeostasis, feeding behavior, and energy homeostasis. The molecular basis for glucose monitoring and regulation of firing activity is being actively investigated. Present evidence indicates that there is a large diversity in the mechanisms of glucose sensing, which may define subpopulation of either glucose-excited or glucoseinhibited neurons. Recent experimental evidence also supports a role for glial cells in glucose sensing (25, 26). The complexity of these glucose-sensing mechanisms needs to be eventually completely understood to better manage pathologies caused by deregulated glucose and energy homeostasis. Here, we mainly focus on the mechanisms of glucose sensing by neurons.

BRAIN GLUCOSE SENSING Sites of Glucodetection Claude Bernard first implicated the brainstem in glucose homeostasis when he showed that puncturing the floor of the fourth cerebral ventricle of dogs rapidly induced diabetes (27). In 1953, Jean Mayer proposed that cells located in hypothalamus monitor plasma glucose levels by translating variations in glycemia into electrical or chemical signals to control feeding behavior (28). Several groups then identified GE neurons, whose electrical activity is increased by high glucose, and GI neurons activated by cellular glucoprivation (29–32). GE and GI neurons are mainly expressed in the hypothalamus, in particular in the arcuate (Arc), lateral (LHA), dorsomedial (DMH), ventromedial (VMH), and paraventricular (PVN) hypothalamic nuclei (32–38), and in the brainstem, in the area postrema (AP), the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMNX), and the basolateral medulla (BLM) (14, 33, 34, 36, 39–41). High glucose excited (HGE) and high glucose inhibited (HGI) neurons, whose firing activity is regulated over glucose concentration ranges (5–20 mM), have also been identified in the Arc (35, 42).

Physiological Functions Modulated by Central Glucodetection

31

Mechanisms of Glucodetection Glucose controls the electrical activity of GE neurons by a mechanism that shares similarities to glucose-induced insulin secretion by pancreatic b-cells (43, 44) (Figure 2.1a). Glucose signaling in these cells is initiated by the uptake of glucose by GLUT2 followed by its phosphorylation by glucokinase (hexokinase IV, Km for glucose 6 mM). Subsequent activation of mitochondrial metabolism and oxidative phosphorylation increases the ATP/ADP ratio. This leads to closure of ATP-sensitive potassium (KATP) channels, plasma membrane depolarization, and opening of voltage-sensitive Ca2þ channels. The influx of calcium then triggers insulin secretion. In GE neurons, rise in extracellular glucose also increases the cytosolic ATP/ADP ratio and induces closure of KATP channels (39, 45, 46), plasma membrane depolarization, Ca2þ entry, and neurotransmitter release (47, 48). However, recent studies suggest that some GE neurons may be activated by glucose in a KATP channel-independent manner (35, 49). Evidence also suggests that GK (50) and GLUT2 (51, 52) may not be required for activation of these neurons. The mechanisms through which GI neurons sense glucose are not well characterized (Figure 2.1b). However, the effect of glucose on GI neurons may be controlled by changes in Naþ /Kþ ATPase activity (30, 37) or by the opening of ATP-regulated chloride channels that leads to hyperpolarization (31, 50). In the LHA, inhibition by high glucose of the GI orexin neurons may depend on tandem-pore Kþ (TASK) or related channels (53–55).

PHYSIOLOGICAL FUNCTIONS MODULATED BY CENTRAL GLUCODETECTION Food Intake and Energy Expenditure The role of glucose in the control of food intake was demonstrated in many studies (2, 28). Particularly, it has been shown that initiation of feeding is preceded by a small drop in glycemia, and preventing it by infusing glucose suppresses initiation of feeding (56). In addition, peripheral or central administration of 2-deoxyglucose (2-DG), which induces neuroglycopenia, stimulates food intake (57–59). The modulation of feeding behavior and energy expenditure by CNS is a complex process that involves hypothalamic and brainstem neuronal circuits. In the hypothalamus, neurons integrate nutrient (lipid and glucose), hormonal (ghrelin, insulin, PYY3–36, leptin, CCK, GLP-1, and adiponectin), and nervous signals that convey information about food absorption and the levels of stored energy (10, 60–64). These signals are detected in large part by Arc neurons expressing the anorexigenic peptides POMC/CART or the orexigenic peptides NPY/AgRP. These neurons project to melanocortin 3 and 4 receptor-expressing neurons of the PVN and LHA (20, 65). Neurons in the PVN produce the anorexigenic neuropeptides TRH and CRF whereas neurons in the LH produce the orexigenic peptides MCH and orexin (62). Together these neurons form the melanocortin pathway and regulate peripheral metabolism

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(a) GLUCOSE GE neurons

Classical model

Alternative models GLUCOSE GLUT2 1,3

SGLT1,3

?

GK glucose

↑G6P VDCC

↑Ca2+

↑Ca2+

ATPase

↑ATP

↑ROS UCP2

Classical model

↑ROS ?

Alternative models GLUCOSE

ClC

C VD

GLUT2 1,3

Receptor

C

GK

↑Ca2+

TASK

?

↓pyruvate ↓ATP ATPase

↓ATP/ADP

Na+/K+ pump

Figure 2.1

?

↑H+

↓G6P

VDCC

VDCC

KC

GLUCOSE GI neurons

KC

UCP2 Ox. Ph.

KATP

(b)

↑Na+

↑pyruvate

↑ATP/ADP

TRP

GLUT2 1,3

KC

?

Ox. Ph. ↓H+

Models for the control of the electrical activity of GE and GI neurons by glucose. (a) Glucose activation of firing rate in GE neurons. In the b-cell model, glucose uptake through GLUT2, but in neurons possibly also by GLUT1 or GLUT3, is followed by its phosphorylation by GK. Pyruvate is then channeled into the mitochondria to eventually increase ATP production and induce a rise in the ATP/ADP ratio. This leads to the closure of KATP channel, membrane depolarization, and the entry of Ca2þ , which triggers the release of neurotransmitters. In an alternative mechanism, the activity of GE neurons is controlled by the electrogenic cotransport of Naþ and glucose by SGLT1, by activation of TRP channels, or by production of ROS following the activation of the oxidative phosphorylation. ROS could directly regulate the activity of Kþ channels or intracellular Ca2þ availability. UCP2 may serve as a regulator of ATP production through its decoupling activity or by reducing the production of ROS; both mechanisms lead to reduced glucose-induced neuronal activity. (b) Activation of GI neurons by a decrease in extracellular glucose concentration. A reduction in the ATP/ADP ratio leads to closure of Cl channels and/or a reduction in the activity of the Naþ /Kþ ATPase, plasma depolarization, and the entry of Ca2þ that triggers the secretion of neurotransmitters. Alternatively, a new pathway involving TASK channel may control the activity of GI neurons through a mechanism that does not involve glucose metabolism, possibly through interaction with a putative specific receptor. KC: K þ channel; VDCC: voltage-dependent calcium channel.

Multiplicity of Sensing Mechanisms

33

through activation of the autonomic nervous system and higher brain structures to control not only feeding behavior, but also arousal and reward (66–68). The hindbrain is also a site of glucodetection and may have a major role in regulating feeding in physiological conditions. Indeed, intracerebroventricular (i.c.v.) injection of 2-DG stimulates feeding only if it can have access to the brainstem (57, 69) and food uptake can be activated by injection of 5-thioglucose (5-TG) into NTS, DMNX, and BLM but not in hypothalamic nuclei (39–41, 70, 71). Glucose-sensitive neurons from the BLM are catecholaminergic and send projection to Arc and PVN (72). Destruction of these projections by immunotoxins suppresses the effect of 2-DG on food intake and on regulated expression of AgRP and NPY in the Arc (73, 74).

Counterregulation When blood glucose concentrations fall below the euglycemic level, a rapid counterregulatory response is activated to restore normoglycemia. This involves activation of the autonomic nervous system (75) that triggers glucagon secretion and the release of catecholamines from adrenal glands (76–78). The central sites of glucose detection that activate the autonomic nervous system are located in the hypothalamus and brainstem. In the hypothalamus, lesion studies as well as pharmacological and genetic approaches have provided evidence for an important role of the VMH in the control of glucagon secretion (79). For instance, glucagon release can be induced by direct injection of 2-DG in the VMH (80). In contrast, hypoglycemia-induced glucagon secretion can be suppressed by direct injection of glucose into this structure (81). Brainstem nuclei also play an important role in activating counterregulation. For instance, when the cerebral aqueduct is obstructed, 5-TG induces glucagon secretion when injected in the fourth but not in the third ventricle (69). In addition, injection of 5-TG directly in the NTS or the BLM nuclei containing the A1/C1 catecholaminergic neurons strongly stimulates glucagon secretion (70). In decerebrated rats, the hyperglycemic response to an i.p. injection of 2-DG is preserved (82) and c-fos staining revealed that the activated cells are present in the NTS, DMNX, and the catecholaminergic neurons of the BLM (72).

MULTIPLICITY OF SENSING MECHANISMS One important goal of current research is to identify each type of glucose-sensing neurons and to determine which physiological functions they control. One path to reach this goal is to identify the critical proteins that allow these neurons to respond to glucose and use these proteins as markers to identify the glucose-sensing neuron subpopulations, their topographical distribution, and the neuronal circuits they form. In recent years, many ion channels, transporters, or enzymes have been described to participate in central glucose sensing. Here, we review the list of these gene products and their role in glucose sensing.

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Glucose Transporters In most instances, glucose uptake and metabolism are required for glucose signaling in neurons. Glucose uptake may be catalyzed by either facilitated diffusion glucose transporters (Gluts) or Naþ -linked glucose transporters (SGLTs) (83–87), and both types of transporters have been associated with glucose sensing by hypothalamic and brainstem neurons. Glut1, Glut3, and Glut8 Glut1 is highly expressed in endothelial cells forming the blood–brain barrier and is required for glucose entry in the brain. Inactivating mutations of Glut1 reduce the concentration of glucose in the cerebrospinal fluid, which causes seizure and delayed development (88). Glut3 is a high-affinity glucose transporter expressed in neurons (89). Homozygous Glut3 knockout mice die during embryogenesis whereas heterozygous knockout mice have normal glucose homeostasis and feeding behavior (90), although they show defects in spatial learning and memory processing (91). Glut8 is a high-affinity glucose transporter expressed in neurons in different brain regions, including the hippocampus, the hypothalamus, and the brainstem (92, 93) but homozygous Glut8 knockout mice show no defect in glucose or energy homeostasis (94). Thus, Glut1, Glut3, and Glut8 play specific role in brain glucose metabolism unrelated to the glucose sensing and control of whole-body glucose and energy homeostasis. Glut2 Glut2 is a low-affinity glucose transporter, required for normal glucose sensing by pancreatic b-cells. In the brain, it is expressed in neurons, astrocytes, tanycytes, and epithelial cells lining the cerebral ventricles (95–99). The low level of expression of Glut2 makes its immunohistochemical detection challenging and its precise localization is still partly uncertain, although it is present in hypothalamic and brainstem nuclei (51, 95, 96, 100, 101). A role for central GLUT2 in glucose sensing has been suggested by i.c.v. or direct injection in the Arc of antisense oligonucleotides to reduce its expression (98, 102). This decreased feeding and body weight gain and suppressed 2-DG-induced feeding as well as the insulin response normally triggered by intracarotid glucose injection (98, 102). Studies with Glut2/ mice, which express a transgenic glucose transporter in their b-cells to restore normal glucosestimulated insulin secretion (ripglut1;glut2/ mice) (103), demonstrated a role for central Glut2 in the control of glucagon secretion in response to insulin-induced hypoglycemia or 2-DG-induced neuroglucopenia (26). Transgenic complementation studies revealed that Glut2 reexpression in astrocytes but not in neurons restored the counterregulatory response to hypoglycemia (26). The same mouse model was used to study the role of Glut2 in central glucose sensing and the control of feeding. It was demonstrated that in the absence of Glut2, the mouse presented defects in both feeding initiation and termination. This was

Multiplicity of Sensing Mechanisms

35

correlated with suppressed regulation of the hypothalamic anorexigenic (POMC, CART) and orexigenic (NPY, AGRP) neuropeptides during the fast to refed transition or following intracerebroventricular injection of glucose (104). In contrast to the impaired counterregulatory response, the abnormal feeding behavior of GLUT2-null mice did not rely on GLUT2 expression in astrocytes (105), suggesting that regulation of counterregulation and feeding behavior depends on Glut2 expression in different cell types, astrocytes and neurons, respectively. SGLT1 and SGLT3 SGLT1 is a Naþ –glucose symporter present in the brush border of intestinal epithelial cells, GLP-1 secreting L cells, and in the proximal straight tubule of the kidney nephrons (106–108). SGLT1 transports glucose and galactose as well as the nonmetabolizable analogues 3-O-methyl-D-glucose (3-O-MDG) and a-methyl-Dglucopyranoside (a-MDG) and is inhibited by phlorizin (107, 109). SGLT1 is also expressed in the hypothalamus and ependymal cells of the third and fourth ventricles (52, 110). A role for SGLT1 in L cell glucose sensing has been described (108, 111). As glucose uptake is electrogenic, it leads to membrane depolarization and GLP-1 secretion in a KATP-independent manner (112); GLP-1 secretion can also be triggered by the nonmetabolized substrates 3-O-MDG and a-MDG (113, 114). That SGLT1 may play a role in central glucose sensing is suggested by the finding that i.c.v. injection of phlorizin enhances food intake in rats (115) and inhibits activation of GE neurons in the VMH (44). On the other hand, hypothalamic GE neurons can be excited by a-MDG and 3-OMG (52). SGLT3 is expressed mainly in intestine, liver, kidney, and muscle (116). Pig SGLT3 transports glucose and a-MDG with relatively low affinity and, in contrast to SGLT1, does not transport galactose and 3-O-MDG (109). Human SGLT3 does not transport glucose when expressed in Xenopus laevis oocytes but may play a role as a glucose sensor in cholinergic neurons of the small intestine and at the neuromuscular junctions (12). In Xenopus oocytes expressing SGLT3, glucose produced a phlorizinsensitive inward current that depolarizes the membrane potential by up to 50 mV (12). SGLT3 mRNA is expressed in both cultured hypothalamic neurons and adult hypothalamus, suggesting that this transporter may also be involved in central glucose sensing (52).

Intracellular Mediators Glucokinase Following its uptake, glucose is phosphorylated by hexokinases. In pancreatic b-cells, glucokinase controls the flux of glucose metabolism and the dose response of glucosestimulated insulin secretion. In the brain, glucokinase is expressed in the Arc, LHA, DMH, VMH, and PVN, as well as in the brainstem and ependymocytes of the third and fourth ventricles (99, 101, 117, 118). Pharmacological inhibition of hypothalamic

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GK decreases the activity of GE neurons and increases that of GI neurons showing that GK is involved in both high glucose and glucopenia detection (32, 38, 44, 51, 119). Recently, GK has also been shown to be required for glucose sensing by GI and GE neurons in the NTS and DMNX (118). A role for central GK in glucagon secretion and feeding is evidenced by i.c.v. administration of the GK inhibitor alloxan that stimulates feeding (120). Reduction of GK activity in the VMH by injection of alloxan or by adenoviral-mediated transduction of a GK-specific shRNA showed that this enzyme is essential for the counterregulatory response to insulin-induced hypoglycemia (121) and for feeding (122). AMPK AMPK is a ubiquitous enzyme formed by a catalytic and b and g regulatory subunits (123, 124). AMPK is an intracellular fuel gauge activated by increased intracellular AMP/ATP ratio and by phosphorylation by the LKB1 and Ca2þ /Calmodulin-activated kinases (124, 125). AMPK turns on catabolic pathways such as fatty acid oxidation and turns off gluconeogenesis and lipogenesis (125). In pancreatic b-cells, activation of AMPK by low glucose suppresses glucose-induced glycolysis, mitochondrial oxidative metabolism, Ca2þ influx, and insulin secretion (126). A role for hypothalamic AMPK in metabolic regulation has been initially proposed based on studies indicating that its activity is inhibited by leptin, insulin, glucose, and refeeding. AMPK activity is regulated in these conditions only in the Arc and PVN but not in the VMH, DMH, and LHA nuclei (123). Adenoviral delivery of constitutively active or dominant negative forms of AMPK in medial hypothalamic nuclei activates or inhibits feeding, respectively (123), and i.c.v. administration or direct injection in the PVN of 5-amino-4-imidazolecarboxamide riboside (AICAR) stimulates feeding (127). Similarly, neuroglucopenia induced by i.c.v. injection of 2-DG increases hypothalamic AMPK activity and feeding, an effect that can be blocked by the AMPK inhibitor compound C (128). How AMPK activity in hypothalamic neurons controls feeding is not fully understood. In neuronal cell lines and on ex vivo hypothalamic explants, low glucose concentrations and AICAR increase AMPK activity and AgRP expression (129). In accordance with these observations, the specific deletion of the a-subunit of AMPK in POMC and AgRP neurons suppressed glucose sensing by these cells but preserved normal leptin or insulin action (130). AMPK may also be involved in the counterregulatory response to hypoglycemia or to neuroglucopenia (131–134). Stimulation of AMPK by microinjection of AICAR in the VMH increases endogenous glucose production whereas compound C or expression of a dominant negative form of AMPK in ARC and VMH impaired early counterregulation as evidenced by reduced glucagon and catecholamine responses to hypoglycemia (134). In contrast, overexpression of the dominant negative AMPK in the PVN attenuated late counterregulation and corticosterone responses (134).

Multiplicity of Sensing Mechanisms

37

Recently, it has been shown that the counterregulatory hormone response impaired by 2-DG-induced recurrent neuroglucopenia was partially restored by i.c.v. injection of AICAR (131). At the brainstem level, AMPK activity also contributes to energy homeostasis. For instance, AMPK activity is significantly increased in the NTS of fasted compared to ad libitum fed rats (135) and injection of compound C directly in the NTS induces a decrease in food intake and body weight (135). Regulation of AMPK in the brainstem may thus mediate the anorectic action of leptin, which is reversed by AICAR injections (135). mTOR The mammalian target of rapamycin (mTOR) is a conserved serine–threonine kinase that promotes anabolic pathways such as protein synthesis in response to growth factors, nutrients (amino acids and glucose), and stress (136). These mechanisms involve the regulatory proteins 70 kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein-1 (4EBP1), which are key regulators of protein synthesis (136, 137). mTOR exists in two distinct complexes. Target of rapamycin complex 1 (TORC1) is a functional association of mTOR with the scaffolding protein raptor, whereas TORC2 is the combination of mTOR with the protein rictor. mTORC1 functions as a nutrient/energy/redox sensor controlling protein synthesis and can be inhibited by rapamycin (138, 139). mTORC2 is an important regulator of the cytoskeleton and phosphorylates the serine/threonine protein kinase Akt/PKB at a serine residue S473 (140). TSC1 and TSC2 are tumor suppressors, and their gene products form a stable complex that inhibits mTORC1 activity (141). Glucose deprivation inhibits mTOR activity, and inhibition is abolished in TSC mutant cells. Interestingly, AMPK inhibits mTORC1 activity by phosphorylation of TSC2 as well as raptor (136). The group of McDaniel showed that glucose elevation activates mTOR/S6K1/ 4EBP1 and protein synthesis in an amino acid-dependent manner in both rodent and human islets (142–144). In rat brain, mTOR has been located in numerous brain structures including hypothalamus, thalamus, and cortex (145). In hypothalamus, mTOR is located in PVN and Arc (145). More precisely, 90% of NPY/AgRP neurons while 45% of POMC neurons also expressed mTOR protein (145). After fasting, there is a decrease in mTOR-positive cells in Arc suggesting that mTOR activity is low after glucose privation (145). In addition, leptin also stimulates mTOR activity and inhibition of mTOR with rapamycin blunts the anorexigenic effect of leptin (145). Recently, specific ablation of TSC1 in POMC neurons induced hyperphagic obesity and alteration of the morphology of POMC neurons (146). These phenotypes are reversed by treatment with rapamycin (146) suggesting an important role of POMC neurons in the control of metabolism. Interestingly, the activation of AMPK-dependent mechanism leads to the inhibition of mTOR activity (147). Thus, AMPK and mTOR may have reciprocal functions and interact in order to control energy homeostasis.

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UCP2/ROS Activation of the mitochondrial respiratory chain leads to Hþ extrusion from the mitochondria and establishment of an electrochemical gradient across the inner mitochondrial membrane. The transport of Hþ back into the mitochondrial matrix through the F0F1ATPase generates ATP. In pancreatic b-cells, the rise in intracellular ATP/ADP ratio is critical to link glucose metabolism to insulin secretion. The uncoupling protein UCP2, located in the inner mitochondrial membrane, can dissipate the electrochemical Hþ gradient, thereby decreasing the capacity of the cells to produce ATP (148). Accordingly, overexpression of UCP2 in islets or insulinoma cells blunts glucose-induced insulin secretion (149–151), and islets from UCP2/ mice have increased ATP levels and higher secretory response to glucose (152, 153). In brain, UCP2 is widely distributed and expressed at high levels in the hypothalamus, in particular in the Arc, VMH, PVN, and LHA, as well as in the brainstem (154) where it has been proposed to play a role in glucose sensing (155). For instance, increased expression of UCP2 in POMC neurons of mice fed a high-fat diet is associated with a loss of their glucose sensitivity, which can be prevented by genepin, an inhibitor of UCP2, or by UCP2 gene inactivation (156). UCP2 was also found to be critical for activation of NPY/AgRP neurons during fasting and in response to ghrelin (157, 158). Oxidative phosphorylation is also associated with the production of reactive oxygen species (ROS) and UCP2 may act as a negative regulator of ROS production (159–161). Indeed, initial studies of UCP2/ mice showed that their macrophages generated 80% more ROS than those from control mice (159). Importantly, ROS are also intracellular signaling molecules (162) that can regulate the activity of voltage-gated Kþ channels (163, 164) or Ca2þ influx (165–167). In b-cells, ROS may participate in the coupling between glucose metabolism and insulin secretion (168–174). In the hypothalamus, there is evidence that glucose sensing may involve ROS production (175). Exposure of hypothalamic slices to increase in glucose concentrations (from 5 to 20 mmol/L) stimulates ROS generation, which is reversed by addition of antioxidants. Intracarotid administration of antimycin or rotenone, which induces ROS formation, mimics the effect of glucose on Arc neurons’ activity and subsequent nervous-mediated insulin release (175).

Channels KATP Channel-Dependent Mechanisms The KATP channel plays a fundamental role in coupling changes in glucose metabolism to plasma membrane electrical activity (176). This channel is an octameric protein consisting of four copies of the pore-forming Kir6.2 channel (in pancreatic b-cells and in neurons) and four copies of the sulfonylurea receptor SUR1 (in pancreatic b-cells and brain) or SUR2B (in brain) (34, 177–180). The expression of these different subunits (Kir6.2, SUR1, and SUR2B) suggests a molecular diversity

Multiplicity of Sensing Mechanisms

39

of KATP in brain and especially in hypothalamus and brainstem (34, 46, 118, 156, 176, 177, 181–184). This channel is involved in central glucose sensing to control glucagon secretion and feeding. For instance, i.c.v. or intrahypothalamic injection of glibenclamide, a KATP channel inhibitor, blocks the counterregulatory response to a hypoglycemic clamp or induced by central administration of 5-TG (185). Genetic inactivation of Kir6.2 leads to impaired glucagon secretion in response to 2-DG administration and this is correlated with suppressed glucose-regulated firing of VMH neurons (46). In contrast, activation of this channel in the VMH amplifies counterregulatory hormone responses to hypoglycemia in normal and recurrently hypoglycemic rats (186). Kir6.2-null mice also have a smaller but significant feeding response than control mice to 2-DG injection (46). In addition, the acute regulation of membrane potential and firing of a subset of hypothalamic neurons by leptin and insulin is due to an action on the KATP channel that causes cell hyperpolarization (187–189). In a recent study, the role of the KATP channel in POMC neurons was addressed by transgenic expression in these neurons of a mutant Kir6.2 subunit that prevents ATP-mediated closure of the channel. This suppresses the response of these neurons to glucose without affecting feeding and induces mild glucose intolerance (156). KATP Channel-Independent Mechanisms Numerous studies suggest that the glucose-dependent firing activity of glucosesensing neurons is also controlled by mechanisms not involving the KATP channel. Table 2.1 lists these channels and their distribution in the hypothalamus and brainstem. Electrophysiological recording of neurons from Kir6.2/ mice showed firing activity triggered by increase in glucose concentrations from 5 to 20 mmol/L with a decrease in input resistance (35), suggesting that these HGE neurons used a KATP channel-independent mechanism to sense glucose. This response appears to depend on the opening of transient response potential (TRP) channels (35), which may play a role in maintaining intracellular Ca2þ concentration (190). The firing activity of GI neurons in response to decreased extracellular glucose may involve reduced activation of the Na þ /Kþ ATPase (30, 191), blockade of a Cl conductance (37, 192), or inhibition of acid-sensitive two-pore domain Kþ channels (TASK) (55). In the LHA, local application of glucose hyperpolarizes the GI orexin neurons, an effect that is prevented by ouabain (a blocker of the Na þ /K þ ATPase) and azide (an inhibitor of energy production), suggesting that glucose exerts its inhibitory effect through Naþ /Kþ ATPase (30). The response of the orexin neurons may involve a glucose-activated Kþ conductance (55), which based on the sensitivity to pH and halothane may be the K2p Twik1-related acid-sensitive Kþ channel subunit TASK3 (55). However, glucose-induced hyperpolarization of orexin neurons is unaffected not only in TASK3 knockout mice but also in TASK1 and TASK3/ TASK1-null mice suggesting that the exact mechanisms of activation of neurons by low glucose are still incompletely understood (53).

40

Table 2.1

Distribution in the Hypothalamus and Brainstem of the Proteins Involved in Central Glucose Sensing Hypothalamus

Brainstem

Arcuate Dorsomedial Lateral Paraventricular Ventromedial Ependymal Area Dorsal Nucleus Basolateral Ependymal nucleus nucleus hypothalamic nucleus nucleus layers postrema motor of the medulla layers of area of third nucleus of solitary (A1/C1) fourth ventricle the vagus tract ventricle Transporter Glut2 Glut1/3 SGLT1/3 Enzyme Glucokinase AMPK UCP UCP2/ROS Channels TRP CFTR/Cl Tandempore K þ Na/K ATPase KATP channels

? þ þ þ þ þ ? þ þ þ þ þ þ þ SGLT1/3 is detected in hypothalamic cultured neurons and in adult hypothalamus ND

þ þ ND

þ þ þ

þ þ ND

þ þ þ

(96–101) (51, 97, 99) (52, 110)

þ þ

þ þ

 þ

 þ

þ þ

þ ND

 þ

þ þ

þ þ

þ þ

þ þ

(99, 117, 118) (130, 135)

þ

þ



þ

þ

þ

þ

þ

þ

ND

þ

(148, 156, 158, 175)

þ þ þ

 ND þ

ND þ þ

þ þ þ

þ þ þ

ND ND þ

TRP1 is detected in brainstem tissue extract   þ ND ND þ þ þ þ þ

(35) (191, 192) (55, 191)

þ

þ

þ

þ

þ

ND

þ

þ

þ

þ

ND

(30, 191)

þ

þ

þ

þ

þ

ND

þ

þ

þ

þ

ND

(34, 46, 118, 156, 177, 181)

þ , Positive; , negative; ND, undetermined; ?, conflictual data.

References

41

In Arc GI neurons, a role for Cl conductance has been evidenced for the response to low glucose concentrations (37, 192). As gemfibrozil, a cystic fibrosis transmembrane regulator (CFTR) blocker, prevents activation of GI neurons in both the Arc and VMH, the CFTR may be involved in this response (192). In the dorsal vagal complex, inhibition of Na þ /K þ ATPase by strophanthidin or ouabain suppressed the inward currents of GI neurons and a role for Cl channels can be excluded (191).

CONCLUSION Changes in glycemia are monitored by several systems located at different anatomical sites. GE and GI neurons, located in hypothalamus and brainstem, control many physiological responses such as counterregulation, feeding behavior, and energy expenditure. At the molecular level, present evidence indicates that a large number of mechanisms have evolved to tightly monitor increases or falls in glycemia. SGLT1, GLUT2, GK, and KATP channel appear to be associated with the response of GE neurons to increase in glucose concentrations. However, not all GE neurons do express GLUT2 or GK, suggesting a diversity in the function of the GE neurons. GI neurons increase their firing activity in response to fall in glucose concentrations by mechanisms that are still unclear but that do not involve the KATP channel but rather Na þ /Kþ ATPase pump, chloride channels, or TRP or TASK channels. Again, available evidence indicates that these pump and channels may be differently required by GI neurons present in different locations. AMPK, mTOR, UCP2, and the production of ROS are also involved in the response of neurons to changes in glycemia and it is so far not known whether they all contribute to the response of all GE or GI neurons or only to subpopulations of glucose-sensitive neurons. Although not fully discussed in this chapter, it has been recently suggested that some GE and GI neurons may sense glucose independently of glucose metabolism. For instance, hypothalamic GE neurons in culture are also excited by the nonmetabolizable glucose analogue a-MDG, which is a substrate of SGLT. In addition, in GI neurons such as orexin cells, glucose-induced hyperpolarization and inhibition are unaffected by GK inhibitors and mimicked by 2-DG. Therefore, the CNS, which critically depends on glucose for its function, has evolved many glucose-sensing mechanisms to monitor all aspects of energy supply and need and modulate glucose and energy homeostasis. Deciphering the complexity of glucose sensing by the CNS and the structure of the glucose-sensing neuronal circuits that control glucose and energy homeostasis still represents a formidable challenge.

REFERENCES 1. MARTY, N., M. DALLAPORTA, and B. THORENS. 2007. Brain glucose sensing, counterregulation, and energy homeostasis. Physiology (Bethesda) 22:241–251.

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Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus TALY MEAS1,2 1 2

AND

PIERRE-JEAN GUILLAUSSEAU1,2

APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, APHP Paris Universite Paris 7, Paris, France

INTRODUCTION According to the World Health Organization (WHO) and the American Diabetes Association (ADA), type 2 diabetes mellitus (T2DM) is defined as resulting from defects in both insulin secretion and insulin sensitivity. Since the discovery of plasma insulin radioimmunoassay by Salomon Berson and Rosalyn Yalow (1), evidence has been obtained that insulin secretion is severely impaired in T2DM. Numerous functional defects such as b-cell dysfunction and other pathological abnormalities have been described in T2DM patients. Functional alterations that lead to b-cell dysfunction, including abnormalities in the kinetics of insulin secretion and quantitative and qualitative defects, all progress with time. Pathological abnormalities then can at least in part explain functional alterations, which include b-cell loss and its progression and reduced b-cell mass.

NORMAL GLUCOSE HOMEOSTASIS Normal glucose homeostasis represents the balance between glucose appearance in systemic circulation (endogenous or meal-derived) and tissue glucose uptake and

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright  2011 John Wiley & Sons, Inc.

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utilization. This balance is tightly regulated and plasma glucose concentrations are maintained within a narrow range. Normal fasting and 2 h post-glucose load plasma glucose levels are defined as 60–110 mg/dL and
140 mg/dL, respectively. Glucose homeostasis is maintained by the highly coordinated interaction of three physiological processes: insulin secretion, tissue glucose uptake, and hepatic glucose production (HGP). In the fasting state, plasma glucose is almost exclusively provided by hepatic production via glycogenolysis and gluconeogenesis. Hepatic glucose appearance rate in the circulation is matched with tissue glucose uptake rate, which occurs mostly in tissues that require glucose such as the central nervous system. Fasting plasma glucose (FPG) concentration is maintained at constant levels by the matched regulation of glucose production and glucose uptake. Following a meal, rising plasma glucose levels promote hepatic glucose uptake and insulin-independent glucose disposal and stimulate the release and the production of insulin by pancreatic b-cells. Increased plasma insulin concentrations suppress HGP primarily by decreasing glycogenolysis and gluconeogenesis and increasing glucose disposal through stimulation of peripheral glucose uptake (mostly in the muscle). These responses minimize hyperglycemia and ensure the return of mealtime glycemic levels to pre-meal values (2–4).

INSULIN SECRETION AND EFFECTS ON TARGET TISSUES Glucose is the primary regulator of pancreatic b-cells by direct stimulation of insulin secretion and by modulating the insulin response to gut hormones and neural factors released during nutrient consumption. Insulin, like many hormones, displays rapid variations in plasma concentrations with frequent secretory peaks (periodicity 5–10 min), and less frequent larger oscillations (periodicity 60–120 min) (5). Normal insulin secretion in response to intravenous glucose follows a two-phase pattern. The first phase of insulin secretion (early or acute secretion) is rapid and sharp, reaching a maximum at 3–5 min and lasting for approximately 10 min. It represents mainly the release of stored granules. Second phase (late secretion) is gradual and persists for as long as glucose levels remain elevated. It stems from both stored secretory granules and de novo insulin synthesis. Early phase of insulin secretion is pivotal in the transition from the fasting state to the fed state with several different functions: to suppress HGP (6, 7), to suppress lipolysis (7), and to cross the endothelial barrier to prepare target cells for insulin action (8). Liver is a pivotal site in glucose metabolism regulation, and is responsive to minute changes in portal plasma insulin and glucagon concentrations. Early in the absorptive state during or after a meal, first-phase insulin secretion exerts an inhibitory effect on HGP by suppressing glycogenolysis and gluconeogenesis rate. In the fasting state, liver is almost the exclusive source of plasma glucose and therefore the most important site of insulin-mediated basal glucose release. Insulin’s first effect on skeletal muscle occurs during the absorptive state. Muscle is the major site of insulin-mediated dietary glucose uptake through stimulation of the insulin-sensitive glucose transport system. Insulin promotes conversion of glucose

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into glycogen by enhancing glycogen synthase activity through regulation of a cyclic adenosine monophosphate (cAMP)-mediated cascade. Free fatty acid (FFA) metabolism plays an important role in maintaining glucose homeostasis both in the postabsorptive and absorptive states. Adipose tissue is highly sensitive to insulin. The meal-stimulated increase in plasma insulin inhibits lipolysis and FFA release from adipose tissue. Physiologic elevation of FFAs enhances hepatic gluconeogenesis and inhibits glucose uptake and utilization in insulin-sensitive tissues. Therefore, insulin action on adipose tissue affects glucose metabolism in liver and muscle, and in the mealtime suppression of FFA release contributes to the increase in peripheral glucose uptake and utilization (9–11).

MEASURING INSULIN SECRETION AND b-CELL FUNCTION Measurement of Insulin Secretion Insulin secretion is markedly influenced by the route of glucose administration. When glucose is administered via the gastrointestinal tract, a much greater stimulation of insulin secretion is observed compared with similar hyperglycemia created with intravenous glucose. The difference in insulin secretion between intravenous versus oral glucose administration is referred to as the incretin effect and is mediated by glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) (12). Insulin secretion in response to intravenous glucose also differs from oral glucose in its temporal pattern. Following glucose ingestion, there is a gradual rise in plasma glucose concentration reflecting the slow rate of glucose absorption, and this is accompanied by a gradual increase in plasma insulin. The abrupt rise in plasma glucose following intravenous administration causes a rapid and transient increase in plasma insulin concentration (first-phase insulin secretion), which lasts for 10 min. This is followed by a slower, sustained rise in plasma insulin (second-phase insulin secretion), which persists as long as plasma glucose remains elevated (13). The hyperglycemic clamp is considered the gold standard for measuring first- and second-phase insulin secretion. Intravenous glucose tolerance test (IVGTT) has been widely used to assess insulin secretion. The acute insulin response (AIR) (0–10 min) correlates with first-phase insulin response during the hyperglycemic clamp (14). A disadvantage of IVGTT is that the plasma glucose concentration declines rapidly following glucose injection, precluding any second-phase insulin secretory response measurement. Indexes of insulin secretion derived from oral glucose tolerance test (OGTT) provide an estimate of insulin secretion during the more physiological route of glucose administration. The insulinogenic index (increment in plasma insulin  increment in plasma glucose) during the first 30 min of the OGTT has been used widely in epidemiological studies as a surrogate measure of first-phase insulin secretion, although not extensively validated (15). In clinical studies, insulin secretion is evaluated by measuring plasma insulin or C-peptide response to oral or intravenous glucose. The amount of insulin secreted must be related to the increment in plasma glucose concentration, which provides the

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stimulus to b-cells. In normogluco-tolerant subjects, the amount of insulin secreted in response to glucose correlates inversely with peripheral insulin sensitivity (16). Reduced insulin sensitivity, through as-yet unidentified mechanisms, enhances plasma insulin response to any given glucose stimulus. Therefore, if one aims at comparing b-cell function between subjects with different insulin sensitivity, an insulin secretion/insulin resistance index (disposition index) should be used (17).

The Hyperbolic Sensitivity–Secretion Relationship To understand the role of b-cells, it has been useful to elucidate the quantitative relationship between insulin sensitivity and insulin action as it exists in nondiabetic individuals. Some years ago, R. Bergman postulated that, if b-cell function was normal, the sensitivity-secretion relationship could be expressed more efficiently as a rectangular hyperbola (18). The product of insulin sensitivity and insulin secretory response (insulin sensitivity index  first-phase insulin response to glucose stimulation) would equal a constant, which was named the “disposition index (DI).” Based on a limited data set obtained in human volunteers, this author postulated that shifts in insulin sensitivity would be accompanied by compensatory alterations in b-cell sensitivity to glucose. The single parameter, DI, can thus be envisioned to predict the normal b-cell response adequate for any degree of insulin resistance. The DI is thus a measure of the ability of b-cells to compensate for insulin resistance. It can be considered a measure of pancreatic functionality in nondiabetic individuals (17). S. Kahn et al. were the first to confirm the hyperbolic relationship in a cohort of 96 nondiabetic subjects. They assessed the relationship between insulin response to intravenous stimuli and insulin sensitivity by quantifying these two variables in a large cohort of healthy subjects of less than 45 years of age (16). The nature of this relationship implies that the product of insulin sensitivity and insulin response is a constant for a given degree of glucose tolerance. This hyperbolic relationship exists whether the insulin response is examined following intravenous administration of glucose or nonglucose insulin secretagogues. Based on these analyses, it is apparent that the variations in insulin release in response to differences in insulin sensitivity are due to changes in the secretory capacity of b-cells rather than their sensitivity to glucose (19). Additional confirmations have emerged from studies with large cohorts (20). It appears that the proposed relationship provides a quantitative and convenient approach to expressing normal metabolic functionality in vivo.

ALTERATIONS IN INSULIN SECRETION KINETICS IN T2DM Alterations in Pulsatile Insulin Release In nondiabetic subjects, when endogenous insulin secretion is experimentally abolished by somatostatin infusion, pulsatile insulin administration is more effective in controlling glycemia than its continuous administration (21). Moreover, in T1DM patients, pulsatile insulin administration compared to continuous subcutaneous

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administration is associated with a 40% reduction in insulin doses needed to maintain normal glycemic control (22). The lower efficacy of continuous administration is related to the downregulation of insulin membrane receptors. Pulsatile insulin release is related to oscillations in intracellular Ca2 þ concentrations, which regulate exocytosis of insulin granules (5). Lack of oscillatory secretion can be caused by excessive intracellular Ca2 þ concentrations and may alter islet pattern (23). Prolonged exposure of islets to high Ca2 þ concentrations has been shown to be associated with apoptotic signals in b-cells (5). b-Cell “pace-maker” is severely altered in T2DM patients where reduction or absence of rapid secretory peaks is observed, and these abnormalities are present in the early phases of the disease (24–27).

First-Phase Insulin Secretion in Initial Stages of T2DM or FirstDegree Relatives At the time of diagnosis of T2DM, first-phase insulin secretion is abolished (9, 28–30), and the late phase is reduced and delayed. Reduction in first-phase insulin secretion takes place early in the course of the disease, as it has been reported in subjects with impaired glucose tolerance (IGT) and IFG (31) as well as normoglycemic first-degree relatives of patients with T2DM (32). The abolition of first-phase insulin secretion has been found not only in patients with overt T2DM but also at the initial stage of the disease such as IGT and impaired fasting glucose (IFG). The abolition of first-phase insulin secretion predicts further conversion of IGT or IFG to overt diabetes. Therefore, use of first-phase insulin secretion as a marker of T2DM has been proposed by some researchers. The decrease in first-phase insulin secretion after intravenous glucose in patients with mild abnormalities of glucose tolerance has been reported well before IGT received its definition by the World Health Organization (33). Long-term follow-up studies of patients with IGT have demonstrated conversion from IGT to T2DM in more than 50% of the cases. Thus, IGT should be considered as an at-high-risk state for development of T2DM. Most of the studies performed in patients with IGT revealed abolition or decrease in first-phase insulin secretion (32–35).

QUANTITATIVE AND QUALITATIVE ALTERATIONS IN INSULIN SECRETION In T2DM, a marked decrease in basal and glucose-stimulated plasma insulin concentration has been reported (36, 37) regardless of whether body mass index (BMI) is increased. Specific measurement of insulin and prohormones in T2DM patients by an immunoradiometric assay according to Hales coworkers (38) revealed true insulin deficiency. This defect is masked in T2DM patients by elevated circulating insulin propeptides equally represented by proinsulin 32–33 and 64–65. These peptides account for more than 40% of the circulating peptides compared to 5% in nondiabetic subjects (38, 39). Excess in proinsulin in T2DM is not a consequence of hyperstimulation of b-cells as it is devoid of the states of

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secondary hyperinsulinemia such as obesity (40) and liver diseases (41). It seems to indicate a diseased b-cell state rather than an altered functional state.

PROGRESSION OF ABNORMALITIES OF INSULIN SECRETION Progression of Abnormalities of Insulin Secretion When Progressing from Initial Steps to Overt T2DM Longitudinal studies evaluating both early phase insulin secretion and insulin sensitivity have shown that defects in both functions can predict the development of overt diabetes (42, 43). Longitudinal studies have shown that the transition from normal glucose tolerance (NGT) to diabetes is associated with a progressive deterioration in early insulin secretion. Pima Indians, progressing to IGT during a mean 5.1 year followup, were compared to subjects who remained NGT. Progression to IGT was accompanied by a 27% reduction in AIR. A further 51% reduction in AIR was observed during progression from IGT to diabetes. Increases in body weight and a 31% decrease in insulin sensitivity were also observed in patients progressing to diabetes. In contrast, in patients who remained NGT, while a similar decrease in insulin sensitivity was observed, AIR increased by 30%. This compensatory effect of insulin resistance by the b-cells explains the absence of progression of these subjects to diabetes. Similar conclusions can be drawn from a long-term (7–9 years) longitudinal study performed in normoglycemic relatives of patients with T2DM (44). b-Cell function, evaluated by determining DI values, decreased by 38% in subjects who progressed from NGT to IGT but by only 20% in subjects who remained NGT.

Progression of Abnormalities of Insulin Secretion in Overt T2DM Over Time Worsening of insulin secretion deficiency with time is a characteristic of overt T2DM. This gradual reduction is evidenced by longitudinal studies of large cohorts (45, 46). Studies in the control group of the UKPDS indicated that residual insulin secretory capacity was decreased by 50% at the time of diagnosis of diabetes with a further decrease of 15% 6 years later (45). This decrease was linear at least during the 6 year follow-up period. If one extends the line toward the left as a way to hypothesize disease progression in the past, the actual beginning of the disease may have happened 10 years ago. This extrapolation is consistent with the results drawn from the retinal status at the time of T2DM diagnosis according to Harris et al. (47). If one extends the line toward the right as a way to predict potential progression in the future, the line crosses the abscissas axis 10–12 years after the date of T2DM diagnosis. Thus, these data suggest that the natural history of progressive b-cell death has a length of 20–25 years. Different mechanisms have been proposed to explain the progressive reduction in insulin secretion, including glucotoxicity (48), lipotoxicity (49), and the effects of advanced glycation end products (AGEs) (50, 51). The deposition of an islet amyloid substance, also known as amylin (52), may also play a role.

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MECHANISMS OF b-CELL FAILURE Glucose-Stimulated Insulin Secretion and Glucotoxicity In pancreatic b-cells, glucose is transported across cytoplasmic membrane via specific transporters, glucose transporter 1 (GLUT1) and 2 (GLUT2), and is rapidly phosphorylated by a specific glucokinase with a high Km for glucose. The combination of transport and phosphorylation determines metabolic flux through glycolysis in b-cells. Increased glycolytic flux in b-cells results in a rapid increase in the production of reducing equivalents and increased electron transfer to the mitochondrial matrix, leading to increased ATP production in mitochondria and increased ATP/ADP ratio in the cytoplasm. This in turn results in several sequential events: the closure of the ATP-sensitive K þ (KATP) channels, depolarization of the cytoplasmic membrane, influx of extracellular Ca2 þ , a rapid increase in intracellular Ca2 þ , and activation of protein kinases, which then mediate exocytosis of insulin (53, 54). As glucose is the key physiological regulator of insulin secretion, it appears possible that it also regulates the long-term adaptation of insulin production by regulating b-cell turnover. However, it is important to stress that in human b-cells in vitro, graded increase in glucose from a physiological concentration of 5.6 to 11.2 mmol/L and above induces apoptosis. However, in rat islets, the same graded glucose increment decreases apoptosis, indicating that glucose affects the survival of islets differently in these species. This difference has created some confusion in the field (55). It also highlights the importance of genetic backgrounds in glucose sensitivity of the islets. Glucotoxicity of the islets can be defined as nonphysiological and potentially irreversible b-cell damage caused by chronic exposure to supraphysiological glucose concentrations along with the characteristic decreases in insulin synthesis and secretion caused by decreased insulin gene expression (56). Glucotoxicity, on the other hand, implies the gradual, time-dependent establishment of irreversible damage to cellular components of insulin production and consequently to insulin content and secretion (56). Glucose-induced apoptosis in b-cells is probably linked to the relative specificity of this toxicity toward b-cells but not to other islet or most nonislet cell types. The b-cell is extremely sensitive to small changes in ambient glucose. When these changes are of short duration and lie within the physiological range, such as after a meal, they lead to insulin secretion. When changes are of longer duration and more pronounced in magnitude, they could be translated by the b-cell glucose-sensing pathways into proapoptotic signals (57, 58).

Reactive Oxygen Species The toxic role of oxygen species, which are produced in excess in uncontrolled diabetes, is a pertinent explanation of the b-cell apoptosis (59). Long-term hyperglycemia also induces the generation of reactive oxygen species (ROS), leading to chronic oxidative stress because the islets express very low levels of antioxidant enzymes and activity. In b-cells, hyperglycemia induces mitochondrial production of

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superoxides that activates uncoupling protein 2 (UCP2), resulting in a decrease in intracellular ATP/ADP ratio and reduced glucose-stimulated insulin secretion (60). Diabetic islets are characterized by reduction in glucose-evoked insulin secretion, decreased cytosolic ATP and ATP/ADP ratio, abnormal hyperpolarization of the mitochondrial membrane, hyperexpression of UCP2 of complexes I and V of the respiratory chain, and high levels of a marker of oxidative stress, nitrotyrosine (61). These observations support the role of ROS in reduced b-cell function in T2DM. ROS, particularly hydroxyl radicals, interfere with normal processing of the mRNA of pancreas duodenum homeobox-1 (PDX-1), a transcription factor required for insulin gene expression and glucose-induced insulin secretion as well a critical regulator of b-cell survival (55, 56). The generation of ROS and reactive nitrogen species ultimately activates stress-induced pathways such as nuclear factor kB (NF-kB), stress kinases, and hexosamines. Del Guerra et al., using islets isolated from the pancreas of patients with T2DM and matched nondiabetic controls, demonstrated that several functional and molecular defects are present inT2DMislets(62). HeconfirmedthatT2DM isletsrelease less insulin than control islets. This perturbation is accompanied by altered expression of glucose transporters and glucokinase, reduced activation of AMP-activated protein kinase (AMPK) and alterations in some transcription factors regulating b-cell differentiation and function. The levels of oxidative stress markers,such as nitrotyrosine and 8hydroxy-20 -deoxyguanosine, were significantly higher in T2DM than in control islets, and correlated with the degree of impairment in glucose-stimulated insulin release. The addition of glutathione in the incubation medium caused reduction of oxidative stress (as suggested by diminished levels of nitrotyrosine), improved glucose-stimulated insulin secretion and increasedinsulin mRNA expression(62). Thus,Del Guerraetal. concluded that the functional impairment of T2DM islets could be, at least in part, reversible by reducing islet cell oxidative stress (62). It is important to emphasize that in this study the percentage of b-cells was only slightly (10%), although significantly, reduced in diabetic islets compared with control islets (62). As proposed by Robertson et al. (56), if the steady decline in b-cell function in T2DM is attributable in any significant manner to chronic oxidative stress-induced apoptosis but not deterioration in b-cell replication, interference with apoptosis by antioxidants or any other therapy might provide a much needed new treatment approach to stabilize b-cell. Excessive ROS not only damage cells directly by oxidizing DNA, protein, and lipids, but also indirectly by activating stress-sensitive intracellular signaling pathways such as NF-kB, p38 MAPK, JNK/SAPK, hexosamine, and others. Activation of these pathways results in the increased expression of numerous gene products that may cause cellular damage and play a major role in the etiology of the late complications of diabetes. In addition, recent in vitro and in vivo data suggest that activation of the same or similar stress pathways results in insulin resistance and impaired insulin secretion (63).

Lipotoxicity T2DM is associated with dyslipidemia characterized by an increase in circulating FFAs and changes in lipoprotein profile. Acute elevation of FFAs in healthy humans

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induced hyperinsulinemia; there is also an increase in glucose-stimulated insulin secretion after prolonged FFA infusion (48 and 96 h) (64, 65), but not in nondiabetic individuals genetically predisposed to developing T2DM (65). In healthy control subjects, the FFA-induced insulin resistance was compensated by the enhanced insulin secretion, whereas persistently elevated FFAs may contribute to progressive b-cell failure (b-cell lipotoxicity) in individuals genetically predisposed to T2DM. Santomauro et al. (66) demonstrated that overnight administration of the nicotinic acid analogue acipimox lowered plasma FFAs as well as fasting insulin and glucose levels, reduced insulin resistance, and improved OGTT in lean and obese nondiabetic subjects and in subjects with IGTand T2DM. The significant decrease in insulin levels suggested that plasma FFAs support between 30 and 50% of basal insulin levels. A sustained (7 day) reduction in plasma FFA concentrations in T2DM with acipimox was also associated with enhanced insulin-stimulated glucose disposal (reduced insulin resistance), decreased content of intramyocellular long-chain fatty acyl metabolites, improvement in OGTT with a slight decrease in mean plasma insulin levels (67). These data suggest that physiological increases in plasma FFA concentrations in humans enhance glucose-stimulated insulin secretion and are unlikely to be “lipotoxic” to b-cells (11) but may contribute to progressive b-cell failure in at least some individuals who are genetically predisposed to developing T2DM (65). For all studies reported in relation to the FFA–b-cell interaction, it is important to emphasize that the stimulatory effects on glucose-stimulated insulin secretion are physiological in nature, particularly during the fasted-to-fed transition. Circulating FFAs help maintain a basal rate of insulin secretion, keeping adipose tissue lipolysis in check. In rodent islets, increased FFAs have been shown to be proapoptotic in b-cells (68). Exposure of cultured human islets to saturated FFAs such as palmitate is highly toxic to b-cells, inducing b-cell apoptosis, decreased b-cell proliferation, and impaired b-cell function. In contrast, monounsaturated FFAs such as oleate are protective against both palmitate and glucose-induced apoptosis and induce b-cell proliferation. The deleterious effect of palmitic acid is mediated by ceramide-mitochondrial apoptotic pathways, whereas induction of the mitochondrial protein Bcl-2 by oleic acid may contribute to the protective effect of monounsaturated FFAs such as palmitoleic or oleic acids (69). The physiological or pathological significance of the effects of fatty acids and glucose on pancreatic b-cell function is a matter of debate. Although one can reasonably assert that fatty acid-induced b-cell death is clearly a toxic manifestation, their effects on functional parameters such as insulin secretion or gene expression are more difficult to categorize as either beneficial or deleterious responses in a short time frame, although they are clearly deleterious in the long run.

Islet Cell Amyloid The relevance of amyloid deposition in the deterioration of b-cell function has been the subject of debate for many years. Deposits composed mainly of islet amyloid polypeptide (IAPP), also known as amylin, have been reported in up to 90% of T2DM

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individuals compared with 10–13% of nondiabetic counterparts (70). IAPP is a 37-amino acid b-cell peptide that is costored and coreleased with insulin from b-cells in response to insulin secretagogues. Transgenic mice expressing human IAPP (hIAPP) in b-cells were obese and spontaneously developed diabetes characterized by islet amyloid deposition and decreased b-cell mass (71). Prospective studies in these mice support the hypothesis that the mechanism of the decreased b-cell mass is increased apoptosis (70). Alternatively, it is possible that IAPP formation is secondary to the onset of hyperglycemia and not of primary importance in the pathophysiology of T2DM (72). In a recently published review of islet amyloid (73), the authors concluded that in human T2DM, islet amyloidosis largely results from diabetes-related pathologies such as diabetes-associated abnormal proinsulin processing, which could contribute to the destabilization of granular IAPP, and therefore, it is not an etiological factor for hyperglycemia.

REDUCTION IN b-CELL MASS IN T2DM The normal pancreas contains approximately 1 million islets of Langerhans, and each islet includes b-cells (60–80%), a-cells (20–30%), somatostatin secreting d-cells (5–15%), and pancreatic polypeptide secreting cells (PP-cells). As mentioned above, b-cell mass is regulated by apoptosis, hypo- and hyperplasia, replication and neogenesis (74, 75). In other words, regulation of b-cell mass is a dynamic process where the actual mass represents the net balance between replication, growth, and neogenesis on one side and necrosis/apoptosis on the other. The phenomenon is also known as b-cell plasticity and allows adaptation to changes in demand of b-cell function (76). Such process is disrupted in T2DM where functional defects and decreased b-cell mass coexist. Both impaired proliferation and increased apoptosis may contribute to the loss of b-cell mass. Increased apoptosis has been observed in Zucker diabetic fatty (ZDF) rats, an animal model of T2DM (77). In these animals, expansion of b-cell mass in response to insulin resistance was shown to be inadequate. However, no defects in proliferation or neogenesis could be identified, suggesting that excessive rate of cell death by apoptosis could play a major role. It is difficult to distinguish the two mechanisms, cell formation and cell death, from each other in human tissue sections because dead cells are rapidly removed from the islet by macrophages and neighboring cells, making it hard to quantify cell death. Nonetheless, apoptosis is currently believed to represent the main cause for loss of b-cell mass in T2DM. This view is supported by necropsy data where pancreatic tissues from T2DM patients were compared to those from nondiabetic subjects (72). Moreover, elevated activities of apoptotic mediators caspase-3 and -8 have been found in b-cells from islets of T2DM patients (78). Most studies addressing the issue of b-cell loss have concluded that there is a marked reduction in the number of b-cells in postmortem specimens of pancreas obtained at necropsy of T2DM patients. In contrast to the adaptive increases in b-cell mass observed in rodent models of obesity (79) and obese human subjects (80),

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a marked reduction in b-cell mass in patients with T2DM has been reported by numerous groups. Recent data have provided new insights into islet pathology of T2DM and the mechanisms responsible for decreased b-cell mass (72). Pancreatic tissue samples from 124 autopsies have been examined with analysis stratified according to BMI (less or above 27 kg/m2). In this study, 91 obese (41 patients with T2DM, 15 subjects with IGT, and 35 nondiabetic subjects) and 33 lean subjects (16 patients with T2DM and 15 nondiabetic subjects) were included. Relative b-cell volume, frequency of b-cell apoptosis, b-cell replication, and neogenesis (new islet formation from exocrine ducts) were assessed. Compared to weight-matched controls, pancreas from overweight and lean T2DM patients presented with 63 and 41% deficits in relative b-cell volume, respectively. A similar decrease (41%) was observed in subjects with IGT. No difference was seen regardless of what previous T2DM treatments the patients received (diet, sulfonylureas, or insulin). Relative b-cell volume was increased in overweight patients compared to lean ones due to increased neogenesis. b-Cell replication was found to be low in all groups. Neogenesis, while increased in overweight patients, was not different between overweight T2DM patients and nondiabetic subjects, or between the lean T2DM patients and nondiabetic subjects. The most remarkable abnormality observed in islet samples from T2DM patients was increased b-cell apoptosis. Frequency of b-cell apoptosis was increased tenfold in normal weight patients and threefold in overweight patients compared to respective control groups. In this study, islet amyloid was present only in a minority of cases, around 10%, of patients with T2DM or IGT. There are two potential explanations for these results either small islet amyloid pancreatic peptide oligomers (nondetectable by light microscopy) are present and responsible for b-cell loss, or islet amyloid is not crucial in the pathogenesis of T2DM. The authors concluded that b-cell mass is decreased in T2DM due to increased b-cell apoptosis. A confirmation has been provided by recent in vitro data, indicating an increased rate of apoptosis in islets exposed to high glucose concentrations (81). Another recent study (82) quantified the b-cell mass in pancreas obtained at autopsy of 57 T2DM and 52 nondiabetic subjects of European origin. Sections from the body and tail of pancreas were immunostained for insulin. The b-cell mass was calculated from the volume density of b-cells (measured by point-counting methods) and the weight of the pancreas. The pancreatic insulin concentration was measured in some of the subjects. The main findings of this study were that b-cell mass slightly increases with BMI in both nondiabetic and T2DM subjects. On average, b-cell mass is 35–39% lower in T2DM than in nondiabetic subjects and so is the concentration of pancreatic insulin; but the variations of individual values are large. b-Cell mass does not correlate with the age of T2DM subjects at diagnosis of the disease, but decreases throughout the duration of clinical diabetes. Whether the loss of b-cell mass precedes and precipitates the clinical onset of the disease remains uncertain. It is also unclear if it accounts alone for the defects in insulin secretion. Prospective noninvasive studies measuring b-cell mass, insulin secretion, and insulin action in the same individuals are necessary to unequivocally address these questions (83).

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LINKAGE OF REDUCED b-CELL MASS AND DYSFUNCTION The role of reduced b-cell mass in the alterations of insulin secretion that characterizes T2DM has not yet fully elucidated. Assessing the possible contribution of a low b-cell mass to the development of T2DM is not easy. Does one become diabetic when the number of properly functioning b-cells has decreased, or when b-cell function has deteriorated beyond a threshold level needed to maintain normal glucose homeostasis, or both? Longitudinal in vivo measurements of b-cell mass, b-cell function, and insulin action in the same individuals would be needed to unequivocally address the issue. Still, clinical observations and experimental data support a close interrelationship between the two parameters. A large proportion of liver-related pancreatic donors who underwent a 50% pancreatectomy developed diabetes (84). Pharmacological or surgical reduction of b-cell mass in rodents results all in impaired insulin secretion (85). More recently, Matveyenko and Butler (86) carefully analyzed the effect of 50% pancreatectomy in normal dogs and showed that partial pancreatectomy resulted in IFG and IGT. Partial pancreas resection was associated with reduction of both basal and glucose-stimulated insulin secretion. Altogether, these data support a mechanistic role of reduced b-cell mass in the development of alterations in glucose homeostasis and progression toward T2DM. In conclusion, it seems that the major defect leading to decreased b-cell mass in T2DM is inappropriate apoptosis, while new islet formation and b-cell replication are normal. Therefore, therapeutic approaches designed to arrest apoptosis could have quite an impact on prevention and treatment of the disease.

THE COMPENSATION OF INSULIN RESISTANCE BY b-CELLS In nondiabetic controls, b-cell adapts its secretion rate to the level required by insulin sensitivity so that plasma glucose concentrations remain normal. A hyperbolic relation has thus been observed between insulin secretion and sensitivity in nondiabetic subjects (42). In uncomplicated obesity, insulin resistance is compensated by increased b-cell mass and insulin hypersecretion (19, 87). If compensation is absent or even incomplete, plasma glucose concentrations rise gradually defining the incipient stages IFG or IGT and then overt diabetes. Inability of the b-cell to adjust its secretion rate to increased insulin demand explains why glucose intolerance appears in the physiological setting of aging (88) and gestational diabetes.

ORIGIN OF b-CELL DYSFUNCTION As indicated above, b-cell dysfunction is present at the early stages of the disease, that is, IFG or IGT, and in normoglycemic first-degree relatives of patients with T2DM (32, 89, 90). These results rule out the hypothesis of a hyperinsulinemic state preceding T2DM, which was evoked from findings using nonspecific insulin assays (over-estimating “true” insulin concentrations), or from pseudo-longitudinal

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studies describing the “Starling curve of the pancreas.” But plasma glucose levels and insulin-sensitivity status were not taken into account in these studies.

Impacts of Genetics and/or Environment Studies of genetic susceptibility for T2DM in families revealed high concordance for T2DM in monozygotic twins compared with a lower occurrence in heterozygotic ones (80–90% compared to 40–50%). There is also a high frequency of T2DM in subjects with family history of diabetes (50% if both parents affected; 25–30% if only one firstdegree relative is affected). In monogenic subtypes of diabetes (MODY or MIDD), insulin deficiency is predominant. However, these subtypes represent only a minority of T2DM. In the recent years, multiple loci associated with T2DM have been discovered by gene candidate and/or genome scan strategies in large cohorts of patients and relatives (91). Presently, 18 susceptibility variants for T2DM have been described (92, 93), but their individual weight in the development of the disease is weak. Relative risk for developing T2DM in association with these variants ranges from 1.06 for ADAMTS9 to 1.37 for TCF7L2 (91). In a study aimed at evaluating the risk associated with the 18 loci in a large cohort of Scandinavian subjects with a mean follow-up period of 23.5 years, variants in 11 genes were associated with T2DM. These genes include TCF7L2 (Transcription factor 7-like 2), PPARG (peroxisome proliferator-activated receptor g), FTO (fat mass and obesity), KCNJ11 (ATPsensitive K þ channel), NOTCH2 [Notch homolog 2 (Drosophila, WFS1 for Wolfram syndrome 1; wolframin)], CDKAL1 (CDK5 regulatory subunit associated protein 1like 1), IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2), SLC30A8 [solute carrier family 30 (zinc transporter) member 8], JAZF1 (JAZF zinc finger 1), and HHEX (hematopoietically expressed homeobox) (94). Among these 11 variants, 8 were associated with alterations in insulin secretion. Recently, a major type 2 diabetes susceptibility gene, TCF7L2, which accounts for 20% of all cases, was identified by Grant et al. in Icelanders (95). Studies conducted in European Caucasian, Asian Indian, and Afro-Caribbean populations of both sexes have confirmed the ubiquitous distribution of the association (96). TCF7L2 is associated with alterations in insulin secretion. Genotype–phenotype relationship studies disclosed severely impaired insulin secretion in carriers of T2DM susceptibility variants (97). Nongenetic factors, particularly insufficient supply of nutrients during fetal development and the first years of life, may also be involved in a defective development of the islets. This defect may result in a reduced b-cell mass, and/or a reduction in the ability to compensate when insulin resistance is present in cases of pregnancy, overweight or obesity, low physical exercise levels, and aging. In this respect, Hales et al. have shown that subjects with birth weight in the lowest quintiles are more prone to IGT and T2DM in adulthood (98). Barker coworkers proposed that T2DM associated with a low birth weight could be the consequence of impaired b-cell function. This may result from in utero undernutrition during a critical period of fetal life and lead to abnormal development of the endocrine pancreas. This hypothesis has

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been supported by studies using animal models (99). If rodents are subjected to an overall reduction in maternal food intake (50% of the normal daily ration) during the last week of pregnancy and throughout the lactation, the offspring showed intra utero growth restriction. They were born with a reduced b-cell proliferation rate. Moreover, these alterations have consequences during adulthood. Inadequate pancreatic functions can develop in situations of increased insulin demand such as ageing or pregnancy. Further, fetal (or in utero) programming is associated with deterioration in glucose tolerance, insulinopenia, and b-cell mass reduction (100, 101). In humans, Barker reported that low birth weight was associated with defective insulin secretion in 21 year old adults during OGTT (102). But these data are not confirmed by other groups. A pathological study has shown that small gestational age does not alter fetal pancreas development and morphology in comparison to appropriate growth for gestational age (103). In a case study comparing young adults born SGA or appropriate for gestational age, subjects born SGA did not demonstrate any evidence of impairment of either the first- or the second-phase insulin secretion (104). Using another model, Flanagan et al. reached the same conclusion in a different adult cohort (103). In 8 year old Indian children, low birth weight is associated with insulin resistance without abnormality of insulin secretion (105).

CONCLUSIONS Substantial evidence supports the view that T2DM is a heterogeneous disorder. There is a progressive deterioration in b-cell function over time in T2DM. The UKPDS indicated that pancreatic islet function has been found to be at about 50% of normal capacity at the time of T2DM diagnosis regardless of the degree of insulin resistance. The decline of b-cell function is the limited capacity to compensate for insulin resistance. Both insulin resistance and b-cell dysfunction are usually present in classical T2DM as well as most individuals with IGT. The defect of insulin secretion in T2DM is related to two confounding components, insulin deficiency and b-cell secretory defect. On the other hand, there is an impaired glucose sensing in the b-cells. The reduction of b-cell mass is attributable to accelerated b-cell apoptosis. Identification of the factors conferring susceptibility (mainly genetic) and those that may accelerate such process (mainly environmental and metabolic) represents a major imperative, which could lead to new therapeutic strategies of slowing if not arresting b-cell loss, thus contributing to more robust glycemic control and reduction of the risk of developing long-term diabetic complications. Although difficult, with the new understanding of b-cell biology, it seems that we may be a little bit closer to a solution.

REFERENCES 1. YALOW, R.S., and S.A. BERSON. 1960. Immunoassay of endogenous plasma insulin in man. J Clin Invest 39:1157–1175. 2. DINNEEN, S., J. GERICH, and R. RIZZA. 1992. Carbohydrate metabolism in non-insulin-dependent diabetes mellitus. N Engl J Med 327:707–713.

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Adipokine Production by Adipose Tissue: A Novel Target for Treating Metabolic Syndrome and its Sequelae VANESSA DECLERCQ2,3, DANIELLE STRINGER2,3, RYAN HUNT2,3, CARLA G. TAYLOR2,3, AND PETER ZAHRADKA1,2,3 1

Department of Physiology, University of Manitoba, Winnipeg, Canada Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada 3 Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, Canada 2

INTRODUCTION The topic of this chapter is adipokines, hormones produced and secreted by adipose tissue that have multiple effects on metabolism. In normal healthy adults, adipokines regulate the utilization and storage of lipids and help to coordinate their distribution throughout the body. Glucose metabolism is also modulated by various adipokines. Additionally, certain adipokines can influence the functions of specific target tissues, of which the heart, vasculature, brain, pancreas, and liver are included in this chapter. For instance, adipokines have a major role in maintaining the normal function of vascular tissues, independent of its metabolic state. Thus, when adipokine production is altered by obesity, a variety of changes can ensue, involving one or more of these tissues. For this reason, therapeutic strategies to correct adipokine imbalance will not only be useful for treating metabolic disorders, they will also help to minimize the end organ damage.

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright
2011 John Wiley & Sons, Inc.

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We have attempted to provide an overview of the role of adipokines and described the changes that occur in obesity as they relate to the metabolic syndrome (MetS). As well, we have outlined the state-of-the-art surgical, lifestyle, dietary, genetic, and pharmacological strategies available for manipulating adipokine levels. This provides a platform for understanding the impact that may be achieved by interventions designed to influence adipokine production, such as lifestyle, diet, surgery, and pharmaceuticals.

MetS AND OBESITY MetS refers to a cluster of metabolic abnormalities characterized by the coexistence of abdominal obesity, dyslipidemia, hypertension, insulin resistance, and glucose intolerance (1). It has been recently proposed that inflammation and nonalcoholic fatty liver disease (NAFLD) also be considered as elements of MetS (2). Although obesity and insulin resistance are generally recognized by different health organizations as central characteristics of MetS, the diagnostic criteria are variable (3). Several health organizations, including the World Health Organization, the National Cholesterol Education Program, the European Group for the Study of Insulin Resistance, and the American Association of Clinical Endocrinology have proposed their own diagnostic criteria for MetS; consequently, it has been difficult to estimate its true prevalence. Based on the criteria of the National Cholesterol Education Program and the results of the Third National Health and Nutrition Examination Survey, it was estimated that 47 million people in the United States have characteristics of MetS (4). In Canada, estimates reach as high as 26% of the population, or roughly 8 million people; however, the prevalence among ethnic subsets of the population range from 11% in the Inuit to as high as 45% in First Nations people (5). Furthermore, the prevalence of MetS among adolescents is increasing. Based on data collected from the Fourth National Health and Nutrition Examination Survey (1999–2000), the overall prevalence of MetS was 6.4%, or approximately 2 million adolescents, representing an increase of 2.2% from the period of 1988–1994. The rising prevalence of MetS is likely attributable to increasing obesity rates. Regardless of definition or diagnostic criteria, older age, reduced physical fitness, and higher percentage of body fat are associated with increased risk of MetS (5). In 1979, 13.8% of Canadian adults were obese. Results from the recent Canadian Community Health Survey reveal that, presently, 36.1% of adult Canadians have a body mass index (BMI) between 25 and 30, while 23.1% have a BMI greater than 30 (6). Globally, approximately 1.6 billion people are overweight, and 400 million people are obese (World Health Organization, 2005), and these individuals are at increased risk of developing MetS. In addition to obesity, MetS is considered a risk factor for diabetes, insulin resistance, and cardiovascular disease. Although all three conditions are interrelated, they are linked to distinct organ systems. The liver, pancreas, heart, and blood vessels, in particular, are affected by MetS and their failure is ultimately responsible for death (7). While it has long been recognized that obesity has a significant role in MetS,

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only in 1994 with the discovery of leptin, a hormone that is produced by adipose tissue (8), was it possible to begin to understand how adipose tissue exerts its effects on the metabolic state of other tissues.

MetS AND ADIPOSE TISSUE Adipose tissue contributes to both the onset and progression of MetS. Among the factors that influence the emergence of MetS are the total amount of adipose tissue and adipose tissue distribution, both of which affect the endocrine, inflammatory, and metabolic functions of adipose tissue. These changes in adipose properties coincide with adipocyte enlargement, which implicates cellular hypertrophy as the underlying cause. This state has been termed adipocyte dysfunction, and several excellent reviews on this topic have been written recently (9–12). White adipose tissue is the main type of adipose associated with obesity and related pathologies such as insulin resistance, hyperlipidemia, hypertension, coronary heart disease (CHD), and type 2 diabetes (13). Traditionally, adipose tissue was thought to provide cushioning, heat insulation, and energy storage. We now recognize that adipose tissue is an important endocrine organ, secreting adipokines that operate as hormones and paracrine factors that have key roles in modulating glucose and lipid metabolism (14, 15). White adipose tissue consists of adipocytes and adipocyte precursors, vascular tissue, and immune cells, primarily macrophages (13). Adipocytes, which represent the specialized cells of adipose tissue that store lipid, only account for approximately 50% of the cellular content of adipose tissue (9). Mature adipocytes are spherical in shape and vary greatly in size. During the first year of life, proliferation and differentiation of adipocytes is the highest, and these processes slow down considerably during adolescence (16). In adults that maintain energy balance, cell proliferation remains rather stable. However, when energy intake exceeds expenditure, expansion of adipose mass occurs (17, 18), initially by adipocyte hypertrophy and subsequently by hyperplasia (17, 18). Alterations in adipokine production in MetS occur primarily as a result of adipocyte hypertrophy. The metabolic effects of adipocyte dysfunction are manifested primarily through increased leptin secretion and decreased adiponectin secretion, however, secretion of adipokines such as monocyte chemoattractant protein-1 (MCP-1), which attracts macrophages to the adipose tissue (19, 20), is also elevated. Consequently, the increase in adipose tissue mass promotes macrophage infiltration of the adipose tissue, leading to twice as many macrophages in visceral adipose tissue compared to subcutaneous adipose tissue (21). The accumulation of macrophages in white adipose tissue in response to MCP-1 occurs through the CC chemokine receptor-2 (22), however, the extracellular matrix may contribute to macrophage-independent adipose inflammation due to the stress it places on adipocytes as they begin to enlarge (23). The increase in macrophages leads to chronic inflammation of the adipose tissue, since they are the source of many proinflammatory cytokines (20, 24). In fact, it now appears that the interaction between adipocytes

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and macrophages is one of the factors that leads to the hypersecretion of proatherogenic, proinflammatory, and prodiabetic adipokines, and the reduced secretion of protective, anti-inflammatory adipokines from adipose tissue that are characteristic of MetS (20, 24, 25). The blood vessels that permeate adipose tissue also secrete a number of cytokines, but their contribution to MetS has received limited attention. It is well established that vascular function can be altered in response to proinflammatory cytokines that originate from macrophages (26). Thus, adipose inflammation in MetS may be a factor in vascular dysfunction. However, at least one molecule released from vascular tissue, nitric oxide (NO), may also contribute to adipose tissue dysfunction in MetS.

Adipose Tissue: Visceral Versus Subcutaneous Dysregulation of adipokine production and secretion from site-specific adipose depots (subcutaneous and visceral) plays an important role in mediating the insulin resistance, diabetes and cardiovascular disease that accompany MetS (27). Subcutaneous adipose tissue mainly accumulates around the gluteal and femoral regions, whereas visceral adipose tissue is composed of omental and mesenteric adipose (28). However, intra-abdominal adipose appears to have the strongest association with MetS (29–31). The association between visceral adipose and MetS may be linked to an increase in free fatty acids (FFAs) entering the portal circulation (32, 33), and the resultant overabundance of circulating FFAs can in turn contribute to the development of insulin resistance (34, 35). Alternatively, the fact that subcutaneous and visceral adipose tissues demonstrate differences in the production of adipokines (36–40) suggests that they have different roles in modulating metabolism. For example, while leptin mRNA levels are higher in subcutaneous adipose, angiotensinogen mRNA levels are higher in visceral adipose, whereas tumor necrosis factor (TNF)-a mRNA levels are similar in both depots (38). These differences underscore how preferential expression of certain adipokines by different adipose depots can affect metabolism. Leptin Leptin regulates body weight and energy expenditure (41) as well as glucose and lipid metabolism, angiogenesis, immunity, and blood pressure homeostasis (27). Leptin has also been shown to cause vasodilatation in coronary arteries (42), but in obese individuals leptin-induced NO production is impaired due to leptin resistance (42, 43). Circulating levels of leptin are directly related to obesity, with increasing adipose mass associated with increases in serum leptin (44). Subcutaneous adipose produces approximately 80% of the circulating leptin (45), however, expression and secretion of leptin are correlated with cell size in both depots (46, 47). Adiponectin Adiponectin is expressed in adipose tissue. In the circulation, it exists in two forms full-length (primarily as trimers, hexamers, and multimeric complexes) and globular

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adiponectin (48, 49). Adiponectin has been shown to have antiatherogenic (50, 51) and anti-inflammatory (52) properties and is significantly reduced in patients with cardiovascular disease (53) and CHD (54, 55). Hypoadiponectinemia is also associated with the development of obesity-related hypertension (56) and type 2 diabetes mellitus (57). Unlike most other adipokines, circulating adiponectin concentrations are reduced in obesity (especially with increased visceral adipose tissue), type 2 diabetes mellitus, CHD, and MetS (58– 61). Likewise, secretion of adiponectin from adipose tissue is decreased with increased adipose mass (62), with omental adipose secreting more than subcutaneous adipose (63–65). Resistin Resistin is expressed by human adipocytes, but the majority is expressed by the macrophages embedded in adipose tissue (66). Data on levels of circulating resistin in obese humans are very inconsistent (67–69), however, there is good evidence that expression of resistin in adipose tissue is differentially regulated depending on the disease model (obesity, diabetes, and insulin resistance) (70–72). The visceral depot in mice appears to have the highest resistin expression (73). Likewise, Zucker diabetic fatty (ZDF) rats have higher resistin mRNA levels in visceral compared to subcutaneous adipose tissue (39). In humans, similar results have been observed, with abdominal adipose expressing and secreting more resistin than subcutaneous adipose tissue (74, 75). Inflammatory Cytokines Proinflammatory molecules such as TNF-a, C-reactive protein (CRP), and interleukin-6 (IL-6) are increased in the plasma of obese individuals (76–80). Adipose TNF-a levels are increased in obesity (81–83) and TNF-a appears to be produced equally by both subcutaneous and visceral adipose depots in humans (84). In contrast, about 30% of circulating IL-6 in obese individuals originates from adipose tissue, primarily from the visceral adipose (27, 37, 84). Renin-Angiotensin System Angiotensinogen is a major component of the renin-angiotensin system (RAS), and a fundamental regulator of systemic blood pressure. Angiotensinogen is the precursor to the vasoconstrictor angiotensin II (AngII), and thus plays an important role in hypertension (85) and vascular inflammation (86). Angiotensinogen is expressed in adipocytes (14), with higher levels in visceral adipose compared to subcutaneous adipose (38). Adipose tissue also has the ability to produce AngII due to the presence of a tissue-localized RAS (87). Interestingly, an increase in the activity of adipose tissue RAS is observed in individuals with obesity-related hypertension (88).

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Perivascular Adipose Perivascular adipose is the term that is applied to the adipose tissue located around vascular structures, including those present on the heart. A correlation has been shown between epicardial adipose tissue and some of the components of MetS such as waist circumference, diastolic blood pressure, and fasting insulin, but not circulating triglycerides or HDL (89). Rat aortic rings surrounded by perivascular adipose tissue display a lower contractile response compared to aortic rings without perivascular adipose (90). Similarly, vessels that have perivascular adipose such as the mesenteric arteries also show a reduced contractile response (91). Perivascular adipose has recently been shown to produce a variety of adipokines and be involved in vascular inflammation. For example, in epicardial adipose (includes surface of the heart especially around the coronary arteries), adiponectin mRNA levels are lower in patients with CHD (92, 93). In humans, the thickness of epicardial adipose tissue correlates with abdominal visceral adipose tissue and fasting insulin, and it is thought to behave like visceral adipose tissue (89). IL-6 and plasminogen activator inhibitor-1 (PAI-1) are higher in abdominal omental adipose compared to epicardial adipose, whereas leptin levels in subcutaneous abdominal adipose are higher than in epicardial adipose (93). More recently, Cheng et al. (94) found that adiponectin levels were lower in abdominal adipose compared to epicardial adipose, whereas TNF-a, IL-6, leptin, and visfatin were higher in abdominal compared to epicardial fat. In a recent study of subcutaneous, visceral, and perivascular adipose, Chatterjee et al. (95) reported that mice had higher levels of adiponectin and leptin in abdominal (epididymal) adipose compared to subcutaneous adipose, whereas levels of adiponectin and leptin were lower in perivascular adipose compared to subcutaneous adipose. When mice were fed a high fat (42% energy) diet for 2 weeks, leptin levels increased in all tissue depots but adiponectin levels were significantly reduced only in the perivascular adipose compared to chow fed animals (95). In primary human adipocytes, release of inflammatory mediators such as IL-8, IL-6, and MCP-1 was highest, and leptin and adiponectin secretion was lowest in cells from perivascular adipose relative to subcutaneous and visceral (peri-renal) adipose (95). Interestingly, perivascular adipocytes were smaller in size than adipocytes from subcutaneous or visceral adipose, a finding that correlated with a reduction in lipid droplet accumulation (95). While adipose tissue has a significant role in MetS, the morbidity associated with MetS is the result of changes in the properties of tissues that are targets for the actions of adipokines. Thus, while alterations in adipokine production may be a prime factor in the onset of MetS, the response of these target tissues is likely responsible for MetS progression.

MetS AND THE LIVER: NONALCOHOLIC FATTY LIVER DISEASE The term nonalcoholic fatty liver disease encompasses a spectrum of hepatic disorders, beginning with simple hepatic steatosis characterized by intracytoplasmic

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lipid droplets within hepatocytes (96). Inflammation and necrosis of hepatocytes marks the progression to the second stage, nonalcoholic steatohepatitis (NASH). Further inflammatory damage leads to fibrosis, with half of NASH patients progressing to this stage (97). Fifteen percent of patients with fibrosis advance to cirrhosis (97). If not detected and treated, cirrhosis can cause portal hypertension, hepatocellular carcinoma, and even liver failure (98, 99). It is estimated that 3% of patients with NAFLD develop liver failure or require liver transplantation (97). Insulin resistance is a precursor for the development of NAFLD. It is estimated that up to 75% of patients with type 2 diabetes mellitus have some form of NAFLD, and past history of type 2 diabetes mellitus is associated with a 26-fold increase in the risk of steatohepatitis (100, 101). Ninety-eight percent of patients with NASH are insulin resistant, and 87% exhibit attributes of MetS (102). Persons with type 2 diabetes mellitus and fatty liver have substantially higher insulin resistance than those with diabetes but without fatty liver (103). Studies have also shown that insulin resistance, elevated serum triacylglycerol (TAG) levels, and hyperinsulinemia are associated with NAFLD, regardless of body weight and BMI (104). Although there is strong evidence for an association among obesity, insulin resistance and NAFLD, nondiabetic and/or normal weight patients with NASH can also exhibit markers of insulin resistance (105). Obesity is also closely correlated with NAFLD, as the risk and severity of hepatic steatosis and steatohepatitis in obese patients is proportional to the degree of obesity (101). Although BMI and hepatic fat content are positively correlated, the relationship between waist circumference and hepatic fat content is stronger, highlighting a role for visceral adiposity in the development of hepatic steatosis (106– 108). It has been suggested that 30–40% of the variation in hepatic fat content can be explained by the variability in visceral adipose tissue (103). Hypertrophy of visceral adipose tissue and the resulting inflammatory response is a potential explanation for the strong relationship between visceral adipose tissue and fatty liver (109). The inflammatory response initiated by expanding visceral adipose tissue recruits macrophages which then secrete various proinflammatory cytokines. Prolonged exposure of adipocytes to these proinflammatory cytokines induces insulin resistance and leads to impaired insulin-mediated suppression of lipolysis. Consequently, there is an increased flux of FFAs from the visceral adipose tissue into the portal vein, resulting in direct delivery of fatty acids to the liver (110). Once they reach the liver, these FFAs can then be taken up by hepatocytes and bound to coenzyme A (CoA). The fatty acyl-CoAs can form hepatic TAG, but they can also interfere with insulin signaling and cause hepatocyte insulin resistance (111). In addition to increased release of FFAs, altered production and release of adipokines by hypertrophic adipose tissue represents another possible link between obesity and hepatic steatosis (112). In particular, increased production of IL-6 and TNF-a (20, 24, 113) can suppress the production of adiponectin, an anti-inflammatory adipokine that appears to have an important role in the development of hepatic steatosis (114). The relationship between adiponectin and hepatic steatosis is highlighted by the strong inverse relationship between circulating adiponectin and hepatic fat content (115, 116) as well as hepatic insulin resistance (116). In addition,

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genetic variability in the adiponectin receptor gene affects hepatic fat accumulation, supporting the important role of adiponectin signaling in the pathophysiology of hepatic steatosis in humans (117). However, the mechanism has yet to be elucidated.

Adipokines and NAFLD Much of our knowledge of the contribution of adipokines to the development of hepatic steatosis is derived from studies in obese and/or insulin resistant animal models, where it is evident that certain adipokines are prosteatotic, while others are antisteatotic. Expression of hepatic TNF-a is elevated in the ob/ob mouse, a model which develops obesity and insulin resistance due to a mutation in the leptin gene (118). Fatty liver, which is characteristic of this mouse model, is reversed upon treatment with a neutralizing TNF-a antibody (119), suggesting a likely role for TNFa in the development of hepatic steatosis. However, the actual therapeutic potential for modulating TNF-a expression may be limited, as circulating levels of this adipokine do not reflect expression in tissue (120). Conversely, ob/ob mice given adiponectin display reduced hepatomegaly, hepatic lipid content, serum alanine transaminase (ALT), TAG, and FFA after only 2 weeks of treatment (121). In humans (both adults and children), plasma concentrations of adiponectin are significantly lower in patients with NAFLD compared to both obese and healthy people (122–125). Furthermore, plasma adiponectin concentration is inversely associated with hepatic insulin sensitivity and hepatic lipid content (116). The amelioration of hepatic steatosis by adiponectin is hypothesized to occur via three possible mechanisms:stimulation of lipid oxidation via activation of AMP-activated protein kinase (AMPK), suppression of lipogenesis by decreasing activity of key lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and activation of peroxisome proliferator activated receptor-a (PPARa), a transcription factor involved in lipolytic gene expression (114, 121, 126). Leptin is considered another important regulator of hepatic fat, although the mechanisms responsible for the protective effect of this adipokine are not fully understood. The observation that leptin deficiency leads to hepatic steatosis, which is reversible upon leptin treatment, suggests a protective role for this adipokine against hepatic fat accumulation (127). Even more convincing evidence for a direct effect of leptin signaling in the process of hepatic lipid deposition comes from studies in the ZDF rat (128), which lacks a functional leptin receptor. When infused with a recombinant adenovirus containing the gene for a functional leptin receptor, hepatic steatosis is markedly decreased and, since almost all of the infused functional leptin receptor construct is taken up by the liver, the liver becomes the only leptin-responsive tissue (128). Therefore, any reduction in hepatic lipid content resulting from infection with the normal leptin receptor is due to the direct action of endogenous hyperleptinemia on the now leptin-responsive liver. Potential mechanisms responsible for this protective effect include stimulation of lipid oxidation by activating AMPK (129), reduced lipid synthesis, and increased lipid export (130). Development of leptin resistance due to chronic exposure of hepatocytes to high levels of circulating leptin is

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the likely explanation for hepatic steatosis despite elevated leptin in obesity. An interaction between resistin and leptin could also be responsible for the development of hepatic steatosis, since a cross between resistin-null and ob/ob mice does not exhibit fatty liver even though the degree of obesity is equivalent to that of ob/ob mice (131). Over time, hepatic steatosis will progress to steatohepatitis and finally fibrosis. The latter represents the point at which cirrhosis becomes evident. Hepatic stellate cells are normally found in a quiescent state in the healthy liver and become activated in response to liver injury. When this occurs, they begin to secrete collagen, which, if not stopped, is ultimately the cause of fibrosis. Evidence that adipokines influence fibrosis is now accumulating (132). Adiponectin, in particular, may have a protective role because it can suppress stellate cell activation by platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF) (133). In contrast, the proinflammatory effects of resistin may promote disease progression (134).

MetS AND THE PANCREAS The metabolic consequences of MetS on the pancreas are not as well defined as those of other organs such as liver and skeletal muscle. In one study of 104 adults with MetS, fatty pancreas, as detected by sonography, was present in 77% of the participants (135). Interestingly, coexistence of fatty pancreas and fatty liver were observed in 68% of the participants. After adjustment for age, BMI and lipid profile, fatty pancreas was independently related to insulin resistance (HOMA-IR), visceral adipose and ALT; furthermore, the number of MetS characteristics was significantly higher in the fatty pancreas group compared to the nonfatty pancreas group. Although increased pancreatic fat content has been negatively correlated with b-cell function (136), the relationship between pancreatic fat content and reduced b-cell function remains controversial (137), as other research has observed hypersecretion of insulin and no detrimental effects on functional characteristics of b-cells in the presence of pancreatic fat deposition (138). In humans, histological examination of fatty pancreas has revealed that the majority of fat is present in adipocytes within the exocrine tissue or in adipose tissue within the interlobular space, not within the islets themselves (139). This is contrary to what is observed in animal models such as the ZDF rat and high fat/high sucrose-fed swine, where higher pancreatic fat is associated with fibrotic, irregular, atrophied, and vacuolar islets containing lipid droplets and reduced insulin content (140). Adipokines likely influence pancreatic function, but few studies have examined this link. For instance, it was recently shown that the adiponectin type 1 receptor (AdipoR1) is reduced in the pancreas of obese mice (141). Leptin can inhibit glucagon release by a-cells in culture (142). Circulating leptin, but not adiponectin, levels also appear to be altered in acute pancreatitis, but a causal relationship has not been identified (143). Proinflammatory adipokines such as IL-6 and MCP-1 have also been reported to participate in the pathogenesis of pancreatitis (144).

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MetS AND THE BRAIN The effects of MetS on the brain result in changes in appetite, both in the fed and fasted states. The brain responds to short-term updates on food intake from gut-derived hormones such as cholecystokinin, peptide YY, and ghrelin while signals of long-term adiposity come from adipose tissue via the adipokine leptin (145, 146). Abdominal obesity, elevated serum leptin levels, and hypothalamic leptin resistance are common features of the MetS (147). Instead of the normal reduction in food intake and increase in energy expenditure that should accompany increased leptin secretion from adipose tissue, a leptin resistant state inhibits the amplified satiety message from reaching the hypothalamus. The leptin signal is only as strong as what is able to cross the blood–brain barrier, a process mediated by the short-form leptin receptor (ObRa). The leptin resistant state results in hypothalamic leptin insufficiency despite elevated blood levels (148). A decrease in central leptin levels affects pancreatic insulin secretion, energy expenditure and glucose metabolism as a result of impaired hypothalamic signaling (148, 149). These changes, in turn, can lead to obesity, hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. This phenotype is mirrored in the db/db mouse model of type 2 diabetes mellitus, which lacks a functional leptin receptor. Impaired peripheral leptin signalling does not significantly alter insulin levels, energy expenditure, or adiposity when compared to impaired central leptin signaling or resistance (150). Hypothalamic neurons in the arcuate nucleus produce the appetite stimulating peptides neuropeptide Y (NPY) and agouti-regulated peptide (AGRP), both of which are inhibited by leptin. A third peptide that is stimulated by leptin, prepro-opionmelanocortin (POMC), is a precursor to a-melanocyte stimulating hormone (a-MHS), which suppresses appetite and leads to increased energy expenditure (151, 152). The disruption in signaling between leptin and NPY may be central to leptin resistance induced by energy rich diets (148). The interaction of the signal transduction and activator of transcription-3 (STAT-3) and the leptin receptor is important in the regulation of energy homeostasis in NPYand AGRP expressing neurons in the arcuate nucleus (153). Restoring the leptin-induced restraint on NPY release may be an important therapeutic target in future treatment of obesity and MetS. Dysregulation of the endocannabinoid system (ECS) along with leptin resistance is linked to abdominal obesity and may exacerbate key risk factors that lead to the development of cardiovascular disease and type 2 diabetes mellitus. Inflammation, an altered blood lipid profile, hepatic steatosis, and insulin and leptin resistance have all been associated with chronic endocannabinoid receptor stimulation. Endocannabinoids are lipid mediators, produced endogenously from membrane phospholipid precursors and triglycerides in response to elevated intracellular calcium (154). The majority of research has focused on two main endocannabinoids, arachidonoyl ethanolamide (or anandamide, AEA), and 2-arachidonoylglycerol (2-AG), which are derived from arachidonic acid (155). AEA and 2-AG are able to mimic the pharmacological effects of D9-tetrahydrocannabinol, the active compound of marijuana that stimulates appetite (156). Both AEA and 2-AG concentrations are elevated in the plasma and adipose tissue of obese humans (156). Diabetes also plays a role, as

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obese type 2 diabetic patients have greater 2-AG levels than nondiabetic weight matched controls (157). Two G protein-coupled ECS receptors are currently known. Cannabinoid receptor 1 (CB1) is highly abundant in the central nervous system, in particular the hypothalamus. CB1 receptors are also found peripherally in liver, gut, skeletal muscle, pancreas, and white adipose tissues. This receptor is linked to increased fat mass, decreased adipocyte proliferation, accelerated adipogenesis, elevated expression of hepatic sterol regulatory element binding protein-1 (SREBP-1), ACC and FAS, and decreased glucose uptake by various peripheral tissues (158). The CB2 receptor is expressed by immune and hematopoietic cells as well as in the pancreas and white adipose tissue. The effect of the CB1 receptor on metabolism, however, is not limited to appetite. Activation of CB1 can affect glucose, insulin, cholesterol, TAG, and leptin levels, all independent of food intake. Upregulation of PPARg mRNA mediated by CB1 activation leads to TAG accumulation in adipocytes (159). Cross talk exists between the leptin and ECS signaling systems, however, it seems to occur downstream of the leptin receptor. Both ob/ob (leptin deficient) and db/db mice, which have elevated levels of AEA and 2-AG in their hypothalamus, show reduced appetite following blockade of CB1 (160, 161). CB1-null mice remain sensitive to intracerebroventricular leptin injection while having relatively low fasting leptin levels when challenged with a high fat diet (162). CB1-null mice are also resistant to diet-induced obesity and weigh approximately 30% less than regular adult mice due to decreased food intake (163).

MetS AND THE CARDIOVASCULAR SYSTEM Numerous epidemiological studies have established a relationship between MetS and cardiovascular disease risk (164). Although the mechanisms responsible for promoting the progression of cardiovascular disease in MetS remain to be identified, a strong case can be made that the onset of insulin resistant and hyperglycemic states, which are exacerbated by obesity, underlies the pathophysiological changes that ensue (165). This concept is supported by the fact that accelerated atherosclerosis and restenosis are highly prevalent in diabetes, even in the absence of obesity (166). On the other hand, evidence is mounting that adipose tissue can influence vascular and cardiac tissues directly via adipokines (10). A clear understanding of the positive and negative actions of these hormones may assist in the development of interventional strategies that can be directed at the adipose tissue and/or the relevant target organs. Although this approach may seem paradoxical, altering adipokine production is now recognized as a plausible means of combating cardiovascular disease.

Diseases of the Heart and Vasculature The majority of cardiovascular disorders stem from either a reduction in the pumping efficiency of the heart or a diminution of blood flow through the vessels. The most

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common cardiovascular disorder associated with MetS is atherosclerosis, a progressive decrease in the diameter of the vessel lumen due to presence of plaque (167). Although the hyperlipidemia component of MetS may feature prominently in these circumstances, there is also evidence that MetS-induced alterations in the levels of various circulating factors may independently influence atherosclerosis. Regardless of the underlying cause, the resultant inability to provide adequate oxygen and nutrients eventually leads to organ failure. Whether this produces an acute (heart attack, stroke) or a chronic (heart failure, renal failure, neuropathy) response ultimately determines the degree of morbidity associated with the condition. Arterial narrowing and stiffening due to atherosclerosis can also affect cardiovascular hemodynamics, which in turn can cause hypertension and an increase in cardiac workload. These conditions promote cardiac hypertrophy, which in time develops into heart failure even in the absence of a heart attack. Certain features of hemodynamic disorders may also be attributed to MetS independent of atherosclerotic disease (167). On the other hand, genetic abnormalities and alterations in electrical conductance are forms of cardiovascular disease that typically are not a consequence of MetS (168), although dietary intake of long-chain omega-3 fatty acids has been shown to reduce the mortality attributable to arrhythmia (169).

Vascular Actions of Adipokines The vascular system serves as a conduit for blood, and thus provides the oxygen and nutrients needed for cells to function. A constant flow rate must therefore be maintained, and this requires tight control of blood pressure within a certain range. Likewise, vessels must be able to repair themselves if they are injured. The vascular response to these stimuli is mediated by various hormones, including adipokines. The incidence of cardiovascular disease in MetS, which is typically associated with higher circulating leptin and lower levels of circulating adiponectin, begs the question whether adipokines are causal factors. Certain studies have linked adipokines to endothelial dysfunction (170), whereas recent prospective studies by Sattar et al. (171, 172) have revealed that neither leptin nor adiponectin are strongly correlated with CHD risk. In fact, the association between leptin and CHD may be a consequence of the close correlation of leptin with BMI (172). These results are supported by an independent assessment of adipokine levels in elderly individuals (173). Indeed, leptin may more accurately predict diabetes than cardiovascular disease (174). The latter observation may clarify the association between leptin and cardiovascular disease, since accelerated atherosclerosis is a hallmark of the diabetic state. Regardless of these results with CHD, it remains plausible that adipokines contribute to other forms of cardiovascular disease. Adipokines have been reported to have multiple vascular effects. The contribution of specific adipokines is described in relation to normal and pathological blood vessel function. Various reviews have been written on this topic (12, 175–177) and we have therefore tried to emphasize the more recent publications and concepts.

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Leptin This hormone was originally identified by its ability to regulate food intake (178). However, evidence that leptin stimulated the sympathetic nervous system led to experiments that showed it was capable of increasing blood pressure (179). More recently, it was shown that leptin receptors are present on cells of the vasculature (180), which suggests that leptin may be able to directly influence their function. This has become an area of intense research, given that leptin levels are elevated in MetS. (a) Vascular Expression. Leptin is primarily secreted by adipose tissue. Other cells that have been shown to express leptin, albeit in small amounts, include fibroblasts and osteoblasts (181, 182). There is no evidence of leptin production by cells of the healthy vasculature, however, Reyes et al. (183) have shown it is produced by sinusoidal endothelial cells. The importance of this localized production has not been determined. Nevertheless, its presence may be sufficient to promote vascular disease progression. The leptin receptor (ObR) has been detected on coronary endothelial cells in culture (184) and on the endothelial and smooth muscle cells (SMCs) of normal vascular tissue (180). These data are consistent with leptin’s ability to directly affect these cells in culture (185, 186). Changes in ObR expression apparently occur during atherogenesis. Schroeter et al. (180) detected a decrease in ObR staining of SMCs of atherosclerotic lesions, whereas strong staining was associated with macrophages. There was no apparent change in endothelial ObR levels. A subsequent study by the same group confirmed that ObR levels in atherosclerotic plaque were associated with macrophage infiltration (187). (b) Vascular Tone. Blood pressure is maintained by a balance of vasoconstrictory and vasodilatory factors. Blood pressure is affected by MetS, as indicated by the prevalence of hypertension in this condition (188). A close association with obesity is evidenced by reports of improvements in blood pressure in conjunction with weight loss (189). The latter may be linked to leptin, which also declines with weight (190), since it has been shown that leptin can act both directly and indirectly to modulate vascular tone. The sympathetic nervous system is a key regulatory system for vascular tone. Leptin is known to act centrally via the hypothalamus to suppress appetite (191). As part of this process, leptin activates sympathetic neurons, and their activation may increase blood pressure (192, 193). The most direct experiment to demonstrate this relationship involved injection of leptin into the ventromedial hypothalamus of healthy rats, which resulted in an increase in blood pressure (194). At the same time, it has been difficult to clearly establish the link between these systems in the hypertensive state (195), and therefore it is not clear whether leptin contributes to hypertension via this mechanism. It is quite plausible that sympathetic activation of the kidney may be the mechanism by which leptin operates. Alternatively, leptin may function by affecting the vascular wall directly.

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Hypertension is closely associated with stiffening of the arterial wall and hypertrophy/hyperplasia of the medial smooth muscle layer. Leptin has been shown to stimulate SMC hypertrophy (196) and proliferation (197), and activation of either process is a prerequisite for medial enlargement. On the other hand, leptin has also been reported to block SMC proliferation (198). Inhibition of cell proliferation may be the result of a leptin-dependent increase in the production of the vasodilator NO via stimulation of endothelial nitric oxide synthase (eNOS) (199). As well, leptin has been shown to interfere with AngII-dependent vasoconstriction (200), a major factor in hypertension. Based on these data, it may therefore be presumed that leptin resistance could result in hypertension due to the lack of NO and loss of vasoconstrictor inhibition, although both processes may be connected (201). Alternatively, leptin may alter the balance between NO and peroxynitrite production (202), thus inducing endothelial dysfunction by increasing the levels of molecules associated with oxidative stress (203). Although more direct links between hypertension and leptin are lacking, there are nevertheless a number of studies that show a correlative relationship between circulating leptin levels and hypertension (190, 204, 205). It is also possible that these actions of leptin are indirect, and are the result of sympathetic activation by leptin (206, 207), which is a feature of MetS (208). Evidence that the vagal afferent nerves are targeted by leptin, thus interfering with baroreflex function and increasing blood pressure, has recently been reported (209). (c) Vascular Injury. The onset and progression of vascular disease is closely linked to the development of endothelial dysfunction, a consequence of injury to the vessel wall. Hyperleptinemia is associated with endothelial dysfunction and arterial stiffening (210). Leptin resistance, however, may serve as an adaptive mechanism to prevent this outcome (210). A major consequence of endothelial dysfunction is activation of the underlying SMCs. This process requires the modulation of SMC phenotype, which switches from the contractile state present in the healthy vessel to the synthetic state that is characterized by migration, proliferation, and secretion of extracellular matrix proteins (211). Similar events occur when the vascular wall is injured, either mechanically (e.g., bypass graft surgery) or by chemical mediators (e.g., hyperlipidemia). The resultant attempt to repair the vessel wall often leads to the formation of a lesion that can interfere with blood flow. Although both atherosclerotic (chronic injury) and restenotic (acute injury) lesions may have different constituents, it is multiplication of SMCs in the intimal space (neointimal hyperplasia) that underlies lesion formation. Leptin promotes neointimal hyperplasia (212), a fact which may explain the higher atherosclerosis rates in diabetes with hyperleptinemia. Leptin likely operates by stimulating SMC proliferation (197). Leptin could promote proliferation by increasing the responsiveness of cells to mitogenic agents. Juan et al. (71) reported that leptin increased endothelin-1 type A receptor

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expression, leading to enhanced proliferation in response to endothelin-1. Although indirect stimulation of SMC proliferation by leptin is supported by the lack of atherosclerosis in mice and rats that do not express leptin or lack a functional leptin receptor, a recent study by Lloyd et al. (213) has shown that atherosclerosis still occurs in a triple knockout mouse that does not express the LDL receptor, ApoE and leptin. This study indicates that a lack of leptin is insufficient to prevent atherosclerosis under conditions where extreme hyperlipidemic conditions exist. An alternate view has been proposed by Bohlen et al. (198) who found that leptin prevents vascular SMC proliferation in vitro. A similar finding was made by Nair et al. (214) with airway smooth muscle. Procopio et al. (199) recently reported that leptin stimulates eNOS expression by endothelial cells via AMPK, which could explain how leptin inhibits cell proliferation. These latter observations do not agree with the findings of Bodary et al. (215). These researchers compared neointimal formation in wild type, leptin deficient (ob/ob), and leptin receptor defective (db/db) mice and found that their inability to respond to leptin protection against formation of a neointimal lesion. Interestingly, lesion formation was equivalent to wild type in mice with a leptin receptor deficient in STAT-3 signalling (leptrs/s), although these animals were as obese as the db/db mice (215). These data suggest that exacerbation of vascular lesion formation by leptin is not a function of STAT-3-dependent signaling, but STAT-3 does mediate the effects of leptin on obesity. (d) Inflammation and Thrombosis. Vascular injury triggers an inflammatory response that results in attachment and infiltration of leukocytes as well as the differentiation of monocytes into macrophages. This process is driven by the release of chemoattractants from the injured endothelial cells and SMCs, and results in the release of inflammatory cytokines that further disturb endothelial function (216). Additionally, the altered surface properties of dysfunctional endothelial cells lead to greater adhesion of leukocytes. This in turn can precipitate formation of a thrombus or blood clot. Leptin can indirectly influence inflammation and thrombosis through an increase in the production of CRP (217). CRP is an acute phase protein that originates primarily from the liver, but sites of extrahepatic production include vascular SMCs and macrophages (218). Elevated levels of CRP in the circulation have been linked to increased thrombosis, possibly as a result of its ability to stimulate the expression of adhesion molecules by endothelial cells. Thus, the resultant increase in leukocyte attachment promotes both progression of atherosclerotic lesion formation and elevation of the risk of thrombosis. In parallel, innate production of CRP by the vascular and inflammatory cells may exacerbate the inflammatory state of adipose tissue and thus intensify the resultant dysfunction caused by inflammation. Leptin also enhances thrombosis by increasing platelet aggregation (219). The leptin receptor (ObRb) is present on platelets (220), and leptin binding

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results in release of intracellular calcium (221). Additionally, leptin enhances aggregation in response to ADP (221). Although leptin has no additional effect on platelet characteristics, and obesity does not trigger leptin resistance in platelets, the increased circulating levels of leptin may be sufficient to explain the increased platelet aggregation observed in obesity (222). (e) Angiogenesis. Tissue hypoxia results in the release of paracrine factors that promote the formation of new blood vessels to perfuse the region that is oxygen deficient. This process can thus enhance recovery after a heart attack by providing new blood vessels to the damaged region of the heart. At the same time, angiogenesis allows the enlargement of atherosclerotic plaques by providing oxygen to the cells that form the core of the lesion. Leptin appears to be a potent proangiogenic factor (223, 224) that operates by promoting endothelial dysfunction and cell proliferation (225). Leptin enhances the rate of angiogenic tube formation through the release of matrix metalloproteinases, enzymes that degrade the extracellular matrix and thus provide channels for elongation of the nascent capillaries (225). Thus, leptin supports the progression of MetS by assisting in the formation of vessels to carry nutrients during the expansion phase of obesity (226). Adiponectin Adiponectin is currently regarded as a potent vasoprotective hormone based on its ability to prevent atherosclerosis (227). Adiponectin likely operates through the endothelial cells since an inverse association exists between circulating adiponectin levels and endothelial dysfunction (228). As such, it is expected that adiponectin will affect a variety of vascular functions. But whether adiponectin functions directly on the vascular tissues or indirectly through induction of other cytokines remains unclear in many circumstances. (a) Vascular Expression. The abundant production of adiponectin by normal adipose tissue greatly exceeds that of other tissues. For this reason, production by other cell types has only recently been recognized. It was shown by Wolf et al. (229) that endothelial cells are capable of secreting adiponectin, at least in certain vascular beds. Interestingly, adiponectin is highly expressed in fetal SMCs (230), but is apparently not found in SMCs of the adult vasculature. Regardless, adiponectin secreted from periadventitial adipose tissue, the adipocytes found around blood vessels, may be more pertinent to its role in vascular function than production by cells of the vasculature itself, and possibly even with respect to circulating adiponectin (231). On the other hand, the primary adiponectin receptors, AdipoR1 and AdipoR2, are expressed by both vascular SMCs and endothelial cells (232). Their importance in vascular disease onset has been established by Zhang et al. (233), who showed that increased expression of the receptors increases the antiinflammatory actions of adiponectin.

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(b) Vascular Tone. An inverse correlation between hypertension severity and adiponectin levels has been identified (190), but a causal relationship remains to be proven. Cao et al. (234) have reported that blood pressure decreases when adiponectin levels increase. Fesus et al. (235) suggest that adiponectin functions as a vasodilator. This effect can certainly be linked to the fact that vascular tone is controlled in part by periadventitial adipose, a major source of local adiponectin (236), and the fact that changes in the properties of periadventitial adipose tissue have been linked to the onset of hypertension (237). The most compelling evidence of a link to blood pressure regulation is the fact that adiponectin induces expression of eNOS and can stimulate production of NO (56). Only one study, however, has examined directly the effect of introducing adiponectin into a hypertensive animal (238). These data suggest adiponectin can influence vascular tone, however, this may be mediated through the central nervous system rather than systemically. (c) Vascular Injury. The primary cause of most vascular disease is failure of the endothelial cell barrier, and this is especially true when blood vessels are injured. Several recent reviews of the effects of adiponectin on the vasculature as it relates to the development of atherosclerotic disease have been published (239–242). However, an interesting question has recently emerged: is a decrease in circulating adiponectin levels a cause of endothelial dysfunction? If this is the case, as suggested by Cao et al. (228), then changes in adiponectin production by adipose tissue may be the causal factor for the onset of cardiovascular disease in obesity. The consequence of the loss of a protective agent such as adiponectin may thus be disease progression. For instance, glucose-induced formation of reactive oxygen species is suppressed by adiponectin in endothelial cells (243). Likewise, secretion of adiponectin is linked to paraoxonase-1 (PON1) (244), a peroxidase that protects LDL from oxidation and is associated both with a reduction in atherosclerotic disease and increased longevity (245). Adiponectin also improves endothelial dysfunction, characterized as a decrease in the response of vessels to factors that trigger vasodilation (246), by activating the AMPKNOS pathway (247), and NO is a potent anti-proliferative agent (248). Although adiponectin may affect vascular remodeling in response to injury via this mechanism, there is also evidence that adiponectin can influence SMCs directly. Both SMC proliferation and migration are restricted in the presence of adiponectin (249, 250). These actions would likewise explain the inhibition of restenosis observed with adiponectin (251), which is supported by the negative association of adiponectin and restenosis (252). Interestingly, adiponectin also protects against arterial calcification (253). (d) Inflammation and Thrombosis. Production of adiponectin by macrophages may provide some positive benefits (229), particularly if accompanied by the release of anti-inflammatory cytokines. Increased NO release in response to adiponectin would also reduce inflammation (239). Additionally,

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adiponectin may suppress reactive oxygen species formation in endothelial cells, thereby reducing both oxidative and nitrative stress (254). The prevention of endothelial dysfunction by adiponectin reduces the expression of adhesion molecules, which reduces the risk of thrombosis by decreasing leukocyte adherence (61). The anti-thrombotic properties of adiponectin may also result from a decrease in platelet aggregation (255), although adiponectin was unable to block propylgallate-induced platelet aggregation in vitro when added to the blood of healthy humans (256). Interestingly, the CD40 ligand (CD40L) is elevated in MetS and has been shown to exacerbate inflammation (257). Since CD40L is a target of adiponectin, it has been proposed that the anti-inflammatory actions of adiponectin result from its ability to lower circulating levels of CD40L (257). The relationship between adiponectin and its truncated globular adiponectin version is not a topic of this review, but it has been shown that globular adiponectin can cause platelet activation through an interaction with the collagen receptor (258). (e) Angiogenesis. Low levels of circulating adiponectin are correlated with a decrease in collateral vessel formation in persons with occluded coronary arteries (259), while the converse is true when high levels are present (260). Although other factors affected by MetS may also be responsible, and addressing this point will only be possible by intervention studies, there is other experimental evidence that supports an inhibitory role for adiponectin in angiogenesis. Adiponectin blocks endothelial cell migration in response to vascular endothelial growth factor (261). Cyclooxygenase-2 (Cox-2) may mediate this process, since angiogenesis in response to adiponectin does not occur in Cox-2 deficient mice (262). Caloric restriction also promotes revascularization, and involves an adiponectin-dependent mechanism that relies on AMPK and eNOS (263). Interestingly, adiponectin promotes migration of endothelial progenitor cells (264), which may provide an additional explanation for its ability to block both atherosclerotic disease and restenosis.

Resistin Resistin is an adipokine that is primarily produced by adipocytes in rodents, but macrophages are the primary source of the resistin expressed by human adipose tissue. The infiltration of macrophages into adipose tissue likely explains the increase in circulating resistin seen in MetS (265). On the other hand, it has also been reported that circulating resistin levels are not correlated with MetS in humans (266). Interestingly, SMCs subjected to cyclical stretch or hypoxic conditions also produce resistin (267, 268), although the physiological relevance of this process has not been investigated. Resistin induces fatty acid binding protein in endothelial cells, possibly promoting hypertension via this mechanism (269). Alternatively, resistin blocks the effect of vasodilatory substances (270). Resistin is associated with inflammation (271), and can promote the release of proinflammatory cytokines from endothelial cells (272).

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Interestingly, resistin secretion is elevated in hyperhomocysteinemia and stimulation of SMC migration may lead to neointimal hyperplasia under these conditions (273). Visfatin Visfatin, also known as pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is specifically released by adipocytes (274). Visfatin is the secreted form of Nampt and likely has a different function than the intracellular protein, as indicated by the fact that these forms have different molecular masses (274). Visfatin is elevated in the proinflammatory state, and circulating levels increase in parallel with waist circumference (275). However, the available evidence suggests visfatin does not correlate with the presence of MetS (276). Rather, visfatin levels appear indicative solely of visceral fat accumulation (277). The lack of an association with MetS may be puzzling given that intracellular Nampt regulates Sirt1 activity, and this protein is closely linked with cell metabolic state and the positive actions of caloric restriction (278). Other Adipokines Vaspin (visceral adipose tissue-derived serine proteinase inhibitor) is secreted primarily by visceral adipose tissue and circulating levels vary with nutritional state (279). Furthermore, vaspin is associated both with endothelial dysfunction (280) and proatherogenic inflammation of smooth muscle (281). At this time, however, there is no evidence to link vaspin with atherosclerosis (282). Although apelin is secreted by white adipose tissue, it is also produced by many other cell types. Nevertheless, circulating apelin levels are increased in obesity, suggesting it may function as a hormone to influence other tissues (283). Apelin correlates with CHD, but not diabetes (284). Although it is claimed that there is a link between apelin and vascular injury, this view is not supported by the fact injury is also prevalent in diabetes (285). Apelin expression is induced by hypoxia, and subsequently promotes endothelial proliferation (286). A consequence of this interaction is the upregulation of angiogenesis (287), which would enable an increase in adipose mass and therefore obesity through the formation of blood vessels to oxygenate the new tissue (287). The localized production of apelin by other tissues may have a similar function and thereby ensure tissue perfusion under conditions when blood flow is reduced (288). This would be beneficial in the case of cardiac ischemia (289). However, whether secretion of apelin by adipose can influence these other tissues has not been determined.

Adipokines and the Heart The heart serves primarily as a pump to move nutrients, oxygen, and waste products to and from our tissues via the bloodstream. To accomplish this task, the heart has an efficient system for deriving energy from fatty acids. For this reason, alterations in

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metabolic state such as those that occur in MetS can significantly affect cardiac function. This altered metabolic state can have multiple effects on the cardiovascular system, from increasing the resting heart rate (290) to increasing the mortality of patients with heart failure (291, 292). While most heart disease can be attributed to changes in vascular function, MetS can also affect the heart muscle directly. Heart failure is the most serious consequence of reduced blood flow as well as increased workload due to hypertension or a valve disorder. Each of these conditions is associated with ischemia, a reduction in oxygen and nutrient access. As a result, the heart modifies its metabolism as a means of adapting to the lower oxygen levels. In parallel, the heart may begin a physical transformation depending upon the degree of damage inflicted on the muscle, especially if there is an acute loss of blood flow such as occurs during a heart attack. Ischemia also results in the secretion of cytokines from the heart that can promote the growth of new blood vessels and thus improve oxygen transport. What has become clear recently is that adipokines can influence progression of heart failure independent of their effects on blood vessels (293). And, as was seen with vascular disease, leptin and adiponectin have received the most attention with respect to heart failure. Leptin The LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) study recently released results that indicate circulating leptin levels are predictive of recurrent cardiovascular events such as death, stroke, and heart attack in males who have experienced a heart attack or been hospitalized for angina (294). Interestingly, adiponectin was not associated with recurrences, which suggests leptin secretion is only elevated when the heart muscle itself is compromised. Currently, the source of this leptin is unknown, but it can be speculated that it is secreted from the pericardial adipose tissue. The leptin receptor is expressed by cardiomyocytes, which explains their ability to respond to leptin. Three distinct responses to leptin have been identified: (i) stimulation of fatty acid oxidation (295), (ii) decreased cardiac contractility (296), and (iii) cardiac hypertrophy (297). It is the latter response that likely explains the positive correlation between circulating leptin levels and poor prognosis for those with heart failure (298–300). Alternatively, leptin levels become elevated as a means of suppressing cardiac hypertrophy, and leptin resistance in MetS counters the benefits expected from this response (173, 293). Adiponectin Adiponectin is synthesized by cardiomyocytes (301). Furthermore, Skurk et al. (302) have suggested adiponectin production by cardiac tissue is controlled via a mechanism that is distinct from the adipose tissue. Their study also revealed that cardiac adiponectin production is suppressed in heart failure, while AdipoR1 and AdipoR2 remain unchanged. Interestingly, high levels of adiponectin are associated with poorer prognosis for persons with heart failure (303), although this is converse to the findings of Soderberg et al. (294). In part, these different conclusions may reflect different

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responses between healthy and obese individuals (304). As well, adiponectin might induce the expression of proinflammatory cytokines that negatively affect the heart tissue (305). Adiponectin may also directly stimulate enlargement of the heart and thus promote heart failure (306). Natriuretic peptides released by the heart stimulate secretion of adiponectin from adipocytes, even in patients with congestive heart failure (307), but whether this has positive or negative consequences on heart failure has yet to be determined. In contrast to its actions on heart failure, adiponectin provides protection against ischemia-reperfusion injury. Endothelial cells appear to have a significant role in this process. Reduction of oxidative and nitrative stress may represent the mechanism of action (254, 308). The AMPK-NOS pathway also contributes to the protective effects of adiponectin on this process (309, 310). Cardiac-specific production of adiponectin in response to ischemia is elevated through activation of hypoxia-inducible factor-1 (hif-1) (311). Adiponectin also is activated in the ischemic brain, and likely represents the underlying mechanism for its cerebroprotective actions (312). Other Adipokines There is evidence that high serum resistin levels are associated with a risk for heart failure (313, 314), possibly due to its relationship with cardiac injury (315). Both ischemic injury and heart failure will affect the heart’s pumping action, so the increase in resistin levels may be explained by the fact that mechanical stretch induces resistin expression by cardiomyocytes (316). A recent report indicates that resistin protects against myocardial infarction (317). It is therefore possible that resistin has a cardioprotective role in acute injury, but that it is ineffective with respect to longterm cardiac dysfunction such as heart failure. Apelin may protect against cardiac ischemia-reperfusion injury (289, 318), but whether adipose tissue, in particular epicardial adipose, plays a role in this process is uncertain. Visfatin also appears to have cardioprotective actions. In a recent study by Lim et al. (319), it was shown that administration of visfatin was capable of reducing cell death during an episode of ischemia-reperfusion. In contrast, fatty acid binding protein-4 (FABP4), an adipokine released in higher levels in MetS, has been shown to suppress cardiac contractility (320). This activity would have negative consequences for recovery after a heart attack and likely promote the development of heart failure in persons with MetS.

THERAPEUTIC INTERVENTIONS The ability to alter adipokine levels is expected to provide a means of improving health given the strong links between adipokines and certain disease states. On the other hand, implementing approaches that can successfully manipulate adipokine levels is fraught with difficulty, since little is yet known about the mechanisms that regulate adipokine synthesis and secretion. On the other hand, it is possible to propose

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that small molecule agonists or antagonists of adipokine receptors may be equally effective. At the same time, will the expected positive effects be realized? It is generally accepted that raising adiponectin levels will have numerous health benefits. Even so, there is the chance that negative outcomes may result, as was suggested by Wannamethee (321) who reported that elevated levels of adiponectin may increase the incidence of CHD. The following section will describe research studies designed to address these issues, and provide an overview of behavioral, nutritional, and pharmacological intervention strategies that have been tested in either animal models or humans.

Surgery Altered adipokine expression is tightly linked to adipocyte dysfunction, which is primarily due to adipocyte hypertrophy. Both animal and human studies have shown that weight loss restores the adipokine balance to one with fewer proinflammatory cytokines and more adiponectin (322, 323). These improvements are clearly linked to a reduction in adipocyte size (324). Bariatric surgery likewise improves adipocyte function according to the observed drop in circulating leptin levels and increase in circulating adiponectin (325). However, short- and long-term changes in glucose metabolism and insulin resistance due to caloric restriction and fat mass reduction that transiently affect adipokine production could influence interpretation of the results (326). Also noteworthy is the fact that removal of subcutaneous adipose exacerbates the inflammatory response, but this is followed by a reduction in proinflammatory adipokines (327, 328). Thus, surgical removal of adipose tissue appears generally beneficial to the subject, but whether this is the best treatment approach remains to be determined.

Lifestyle Greater caloric intake than utilization has long been recognized as the major cause of obesity. Consequently, increased exercise or decreased food consumption have been touted as the best means for managing this disease. This approach alone will not be successful for those individuals whose obesity is caused by genetic or endocrine abnormalities. Behavioral strategies (hypocaloric diets and exercise programs) that target weight loss have resulted in increased plasma adiponectin levels in adults with MetS (329, 330), diabetes (60, 323), and obesity (331), however, this does not seem to be the case in obese adolescent girls (332). Although few studies reported to date have addressed the question of adipocyte dysfunction directly, weight reduction has been confirmed to alter biomarkers of obesity. Choi et al. (333) have shown that levels of circulating adipocyte fatty acid binding protein (A-FABP) correlate with BMI. Furthermore, it was shown that A-FABP, which is higher in obese individuals, decreases when weight is lost. In dogs, weight loss is also associated with a reduction in proinflammatory adipokines such as TNF-a (322). Interestingly, some adipokines such as leptin and TNF-a are

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consistently improved with weight loss regimens, while others such as adiponectin are not (323). At the same time, adiponectin has been the focus of considerable study due to its association with metabolic and cardiovascular disease (334), as well as its status as a surrogate for adipocyte dysfunction (335). The concept that weight loss leads to improved adipose tissue function is strongly supported by the results of Pasarica et al. (336). In their study, the authors showed correlations among adipocyte size, adipokine production, and weight loss induced by lifestyle modification. Specifically, a 13% reduction in weight led to a decline in the number of large adipocytes, and this resulted in a 36% increase in circulating adiponectin levels. Interestingly, major proinflammatory adipokines (IL-6, TNF-a) did not change. Specific cardiovascular risk factors were not examined in this cohort, but significant improvements in glucose utilization were obtained. Similar data have been reported by Varady et al. (324), who found the level of improvement was dependent upon the amount of weight loss. Thus, it is reasonably clear that weight loss regimens can have a positive effect on the production of adipokines. Lifestyle interventions such as diet or physical activity that induce weight loss improve all factors of the MetS (337–339).

Dietary Components In relation to changes in dietary patterns, Bradley et al. (340) have recently shown that no significant changes in circulating adipokine levels occur when successful weight loss programs emphasize the reduction of specific macronutrients (e.g., carbohydrate or fat) from the diet. However, there may be other dietary constituents in our food that are capable of influencing adipokine levels. Some specific dietary components have been shown to increase plasma adiponectin levels. For instance, Decorde et al. (341) have shown that a melon extract provided to obese hamsters increased adiponectin levels by 61%. With the same animal model, Decorde et al. (341) showed that a diet enriched in grape phenolics could also positively modify adipokine levels. Additionally, resveratrol, a compound found in the skin of red grapes, has shown beneficial effects for reducing epididymal adipocyte size in mice (342) as well as increasing the circulating concentration of adiponectin, reducing TNF-a production and enhancing eNOS expression in visceral adipose tissue of obese Zucker rats (343). This study in rats also showed that resveratrol improved several parameters of MetS including dyslipidemia, hypertension, hyperinsulinemia, and inflammatory markers (343). Likewise, millet can increase adiponectin levels in mice with type 2 diabetes mellitus (344). Recently, Jobgen et al. (345) showed that dietary supplementation with L-arginine for 12 weeks reduces adipocyte size in diet-induced obese rats, and lowers serum concentrations of glucose, TAG, and leptin while increasing levels of NO metabolites. On the other hand, serum insulin and adiponectin levels were not affected by L-arginine supplementation (345). Oolong tea consumption for 1 month increased plasma adiponectin levels in patients with previous myocardial infarction and stable angina pectoris (346). Interestingly, omega-3 fatty acids from fish, specifically docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), also elevate circulating adiponectin in mice, and this occurs regardless of food intake and

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adiposity (347). Similar results have been reported with Rhizoma Dioscoreae Tokoronis extracts, Pollack (fish) protein, and mushroom-derived chitosan (348– 350), but none of these studies examined the relationship with adipocyte dysfunction. A definitive link between adipocyte size and the secretion of cardioprotective and proinflammatory cytokines has been identified (351, 352). While this association is being increasingly recognized, few studies have utilized this parameter to determine whether specific dietary components can improve adipocyte function. Pilvi et al. (353) showed recently that incorporation of lactalbumin into low-fat rodent chow resulted in smaller adipocytes after weight loss than the low-fat chow alone. Likewise, both a dietary herb and fiber combination (354) and persimmon leaf (355) have been shown to reduce adipocyte size in conjunction with improving adipocyte functional parameters. While the increase in smaller adipocytes and improved function are typically a product of the weight loss properties of these supplements, it has also been shown that improved function can be induced without a corresponding loss of weight. Noto et al. (356) conducted a study in which a mixture of conjugated linoleic acid (CLA) isomers was fed to obese (fa/fa) Zucker rats for 8 weeks. CLA has been proposed as a weight loss agent, but evidence of efficacy remains equivocal (357–360). In this case, the CLA regimen failed to reduce adipose mass, but there were significant improvements in the function of various organs, specifically the liver, kidneys, and pancreas (356, 361, 362). These changes in physiological parameters were associated with a decrease in adipocyte size, with a concomitant increase in cell number, and elevated circulating levels of adiponectin (363). As well, there were decreases in a number of proinflammatory mediators (361). These data and similar results from Nagao et al. (364) suggest it is possible to dissociate obesity from the end organ damage that typically occurs with increased weight. Interestingly, Lasa et al. (365) examined the effects of a single CLA isomer in the Syrian Golden hamster, a model of dietinduced obesity. Although many reports suggest trans-10,cis-12 CLA is the most biologically active isomer, no changes in adipose mass or adipocyte size were obtained. No other organs were examined, so it is not possible to ascertain whether this treatment had additional effects on the physiology of these animals. Likewise, there have been no beneficial effects observed with CLA treatment in most human studies on obesity management (366). However, the combination of CLAwith omega3 fatty acids over a 12-week period increased plasma adiponectin levels in young obese (BMI 30–36 kg/m2) men (367). Additional studies have been directed solely at adipocytes in culture based on the rationale that compounds that inhibit the differentiation of preadipocytes would interfere with weight gain (368). Whether these strategies would be effective in vivo has not been stringently tested. An argument can be made that maintaining cells in the preadipocyte state would not promote the production of protective adipokines such as adiponectin, although a reduction in proinflammatory cytokines might be expected. A number of phytochemicals have been tested for their ability to interfere with adipogenesis. These include xanthohumol, a prenylflavonoid present in hops and therefore found in small quantities in beer (369); genistein, quercetin, and resveratrol, polyphenols found in many plant species (370); epigallocatechin-3-gallate from

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tea (371); and guggulsterone, a compound present in tree gum that has been reported to exhibit cholesterol lowering properties (372). Of this brief list, resveratrol has also been reported to inhibit leptin release from adipocytes (373). An interesting alternative to utilizing a food product directly is to modify the item prior to ingestion. An example that has shown some success is biotransformation with bacteria. Vuong et al. (374) used this approach to modify blueberry juice. The resultant material was not only able to prevent adipocyte differentiation in vitro, its incorporation into the diet of obese mice also raised adiponectin levels, reduced weight by decreasing appetite, and was able to prevent onset of diabetes and obesity when provided to young animals. In all cases, however, none of these positive results have been translated to human studies, and concerns still exist regarding their implementation in humans (375). The data that have accumulated suggest there are a number of strategies that can be used to restore, at least in part, the normal functioning of adipose tissue in the absence of pharmacological intervention. However, it has to be noted that most studies have utilized animal models, and for this reason it is difficult to extrapolate to humans. Furthermore, few studies have examined the pharmacological aspects of using foodbased therapies for modulating the morbidities associated with obesity. In particular, the issue of concentration has received little consideration. While isolated compounds may be effective when used in vitro, delivering a similar dose to an animal or human may be very difficult, especially if it is at low concentrations in a product intended for oral consumption. As well, the form of the compound may be distinct from the pharmacological version. For instance, quercetin is typically found in a glucosidic form (rutin) in plants, and the activity of the conjugate may be different than the aglycoside. Within this context, passage through the gastrointestinal system or subsequently through the liver might also affect activity through the metabolic actions of these organs. Nevertheless, the indication that it is possible to improve overall health without a requirement to lose adipose mass, as was suggested by Noto et al. (363), does present a novel concept for the development of new therapeutic agents that can operate successfully without the need to induce weight loss.

Adipokine Therapy Pharmacological approaches to alter serum adipokine levels represent the state of the art in this field (242). However, before drugs with these adipokine modulatory properties were identified, direct delivery of adipokines was used in the first attempts to modulate obesity. The discovery of leptin was hailed as a breakthrough because it provided the first indication that obesity and appetite were controlled by biological factors. Numerous attempts were subsequently made to test the assumption that elevating leptin levels by direct infusion would lead to reduced weight as a consequence of a decrease in food intake. Leptin infusion into the mediobasal hypothalamus of rats both suppressed white adipose tissue lipogenesis and decreased vascular tone (376). Inhibition of lipogenesis occurs by reducing the expression of SREBP-1c and PPARg and their target genes FAS and ACC (163). Sympathetic

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innervations stimulate lipolysis through the activation of hormone-sensitive lipase along with increased expression of phosphoenol-pyruvate carboxykinase (PEPCK) and uncoupling protein-2 (UCP2). Independently of feeding behavior, leptin is able to simultaneously reduce de novo lipogenesis and FFA uptake in white adipose while preserving lean body mass through promotion of protein synthesis (376–378). While these data were encouraging, leptin infusion yielded few positive results in humans, and the best results were obtained in cases of leptin deficiency. The consequence of leptin replacement, therefore, is the amelioration of lipodystrophy, which is characterized as a marked loss of adipose tissue (379). The use of exogenous leptin as a method for treating MetS or obesity is not practical unless it is injected directly in to the hypothalamic circulation because most MetS patients already have highly elevated endogenous leptin production as well as leptin resistance. Treatment with recombinant leptin has minimal effect on adiposity in obese patients, even at supraphysiological doses (380). However, infusion of recombinant rat leptin into Fisher Brown Norway and Sprague-Dawley rats for seven days significantly reduced total intra-abdominal fat and caused a NO-dependent decrease in mean arterial pressure (381). Likewise, infusion of leptin and amylin in combination has been shown to restore leptin responsiveness in diet-induced obese rats (382). Increased responsiveness to leptin was determined by the increased phosphorylation of STAT-3 in the ventromedial hypothalamus (146). The improved sensitivity to leptin resulted in decreased caloric consumption and weight loss. The increased leptin sensitivity was not explained by the reduction in caloric intake alone (146). The same effect was demonstrated in humans, with combination therapy of pramlintide (amylin-analogue) and metreleptin (recombinant human leptin) resulting in a mean weight loss of 12.7
0.9% after 24 weeks, with the weight loss being significantly different from the control group after 4 weeks. In contrast, monotherapy of pramlintide and metreleptin yielded weight loss of 8.4
0.9% and 8.2
1.3%, respectively (146). Like leptin, adiponectin has been shown to have a wide range of physiological effects. In particular, increasing serum adiponectin levels is expected to influence both insulin sensitivity and vascular function. At this point, however, no direct infusion studies have been performed in humans, although numerous animal experiments have shown the validity of the basic premise (383). In contrast to employing the full-length protein, Lyzogubov et al. (384) describe a novel approach that uses a peptide containing sequence present in the globular domain of adiponectin that is responsible for AdipoR1 binding. Interperitoneal injection of this peptide prevented ocular neovascularization in an animal model of macular degeneration by blocking endothelial cell proliferation. Infusion of recombinant resistin in Sprague-Dawley rats worsened glucose homeostasis (385) and, in a similar way, intraperitoneal injections of resistin into mice impaired glucose homeostasis and insulin action (66). These results suggest that resistin may contribute to insulin resistance. Even so, longer studies using different animal models are needed. Clinical therapy with HIV protease inhibitors reduces plasma adiponectin concentrations and causes metabolic disorders such hyperlipidemia and atherosclerosis. HIV protease inhibitors reduce adiponectin secretion from 3T3-L1 adipocytes,

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however, infusion of adiponectin in mice ameliorates protease inhibitor-induced elevations of TAG and FFAs (386). Infusion of recombinant adiponectin significantly reduces insulin resistance by decreasing TAG content in muscle and liver in obese mice, thus ameliorating hyperglycemia and hyperinsulinemia (387). Infusion of peptides can also affect pancreatic function. Kapica et al. (388) have shown that leptin, apelin, and obestatin promote output of fluid and protein from the pancreas. As well, administration of exogenous resistin improved blood flow to the pancreas (389). Given these roles in pancreatic function, it is not surprising that circulating adipokine levels may also be linked to pancreatitis (143).

Gene Therapy Treatment of MetS may also be possible through the application of gene therapy. This field of research is in the early stages; however, some promising progress has been made. One type of gene therapy involves the use of adenoviruses, double-stranded DNA molecules carrying the genetic code for a particular gene. Some studies have used adenoviruses to transfer adipokine genes and study disease progression or treatment. For example, adenoviral transfer of the leptin gene into nonobese rats reverses streptozotocin-induced diabetes (390). Leptin insufficiency may be overcome by an intravenous injection of recombinant adeno-associated viral vectors that encode the leptin gene (rAAV-lep). A single injection leads to increased circulating leptin levels and normalizes body weight in obese rodents such as the ob/ob mouse. Experiments in genetically obese, diet-induced obese, or wild-type rodents given leptin either intracerebroventricularly or in specific hypothalamic sites results in leptin-induced downregulation of NPY signaling for the rodent’s lifetime (149). This restraint on NPY signaling leads to decreased levels of circulating triglycerides and FFAs and prevents insulin hypersecretion, a process that would normally precede weight gain (149). A single rAAV-lep intracerebroventricular injection normalizes blood glucose levels and prolongs life span of streptozotocin-induced diabetic mice and rats as well NOD (nonobese diabetic) mice by inhibiting the normal catabolic effects of a total lack of insulin (390). Apolipoprotein E-deficient mice treated with recombinant adenovirus expressing full-length adiponectin have a 30% reduction in aortic lesions (50). Furthermore, adiponectin-knockout (KO) mice developed hypertension; however, adenovirusdelivered adiponectin lowered elevated blood pressure in KO mice (56) and significantly decreased plasma TAG levels in normal mice (391). Based on these results, it would appear that viral delivery can successfully be used to elevate adipokine levels. Whether this approach will be feasible in the long term will depend upon development of vectors capable of constitutive, tissue-specific expression of these proteins.

Pharmacological Interventions The identification of drugs for weight control has received considerable interest. Drugs such as orlistat or sibutramine have been shown to reduce visceral obesity and

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improve parameters of MetS (392, 393). In general, it is assumed that weight reduction through pharmacological management will result in the same improvements in health as would be expected from lifestyle changes (394, 395). In particular, the changes would involve reductions in leptin and proinflammatory cytokines (e.g., IL-6, TNF-a) in conjunction with elevated adiponectin levels. As well, it is becoming evident that some of these pharmacological agents operate by modifying adipocyte dysfunction. The sections below describe the effect of both established and recently introduced pharmacological agents on adipokine production. It should be noted that compounds not currently employed may be more effective than those already available, but their utility as drugs has never been examined. For instance, it has been shown that induction of hypoxia inducible factor-1 (hif-1) or heme oxidase-1 (HO-1) under conditions of low oxygen levels will increase adiponectin expression (311). While activation of HO-1 can also be achieved with SnCl2 (234), it is unlikely that this compound will ever be used clinically since it is a strong irritant of mucosal membranes. However, the knowledge that HO-1 is a potential therapeutic target may lead to interventions that are feasible (396). Statins Statins are drugs developed to inhibit a key regulatory enzyme in the cholesterol synthesis pathway, HMG-CoA reductase. By reducing the ability to synthesize cholesterol in the liver, circulating cholesterol levels decrease. While this relationship is well recognized, statins also have pleiotropic effects that are independent of their cholesterol lowering actions. Targets of statins that have received considerable attention include RhoA and Rac1 (397). RhoA and Rac1 are key intracellular signaling proteins that regulate numerous cellular functions, among them proliferation and differentiation. It has recently been established that statins can prevent the differentiation of 3T3-L1 adipocytes in culture (398). This would explain their ability to modulate expression of leptin, resistin, and adiponectin in animals and humans (399–401). These data would also explain the weight loss attained with statin treatment in obese individuals with type 2 diabetes (402), although weight loss has not been reported in other statin trials. Given the relative safety profile of statins, there appears to be considerable potential for these drugs as weight-loss agents. On the other hand, if weight loss is not a major characteristic of statins, or it is limited to specific conditions such as type 2 diabetes mellitus, there is still substantial evidence that statins improve adipose function. In the latter case, increases in adiponectin levels could explain why statins ameliorate the vascular effects of obesity, and this improvement could explain the well-known cardioprotective actions of statins. For instance, in patients with CHD, 6 months of pravastatin treatment significantly increased plasma adiponectin levels along with improving other factors of MetS such as lowering CRP levels, total cholesterol, LDL-cholesterol, and improving hyperinsulinemia and hyperglycemia (403). Similarly, a recent study by Nomura et al. (404) demonstrated significant increases in serum adiponectin levels and reductions in total and LDL-cholesterol after 6 months of treatment with pravastatin. Likewise, hyperlipidemic patients with

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NASH show increased plasma adiponectin and reduced TNF-a levels after 24 months of atorvastatin treatment (405). However, 6 months of treatment with atorvastatin (406) or 3 months of rosuvastatin (407) in patients with type 2 diabetes mellitus did not alter plasma adiponectin levels but did improve, as expected, total and LDLcholesterol levels. The conflicting data may be due to the fact that different statins were used in these studies, as was indicated by Koh et al. (408). These investigators showed that a 2-month treatment with simvastatin significantly decreased plasma adiponectin levels and reduced insulin sensitivity while pravastatin significantly increased adiponectin levels and improved insulin sensitivity. Atorvastatin in patients with steatohepatitis and hyperlipidemia improved steatosis and steatohepatitis, and this was paralleled with a 25% increase in serum adiponectin; however, it is unknown if the improvement in steatohepatitis was related to the increase in circulating adiponectin (405). PPAR Agonists Both fibrates and thiazolidinediones (TZDs), like statins, were developed as agents for cholesterol lowering and increasing insulin sensitivity, respectively. These compounds operate by activating PPARs, key regulators of body metabolism. Fibrates are PPARa ligands, and therefore stimulate b-oxidation of fatty acids (409). Pathological weight loss is a side effect noted for the most commonly prescribed fibrate, fenofibrate, and these results are supported by evidence that this compound also prevents weight gain in animals (410). The increase in b-oxidation induced by fenofibrate may also explain its ability to reduce adipocyte size, and concomitantly decrease circulating levels of proinflammatory adipokines (411). In agreement with these data, it has been shown that fibrates are capable of increasing adiponectin levels in humans with hypertriglyceridemia (412). These results therefore support the concept that fibrates positively affect adipocyte function, and likely provide benefits that extend beyond their ability to lower serum lipids. The TZDs, in contrast to the fibrates, are ligands for PPARg and work as insulin sensitizers (413). PPARg is also an essential mediator of adipogenesis, and therefore agents such as rosiglitazone and pioglitazone are capable of affecting adipocyte function (414, 415). The latter likely explains why rosiglitazone increases plasma adiponectin levels and decreases resistin in obese persons with type 2 diabetes mellitus (416). However, the data showing effects of TZDs on resistin in these studies is not consistent (417–419). Even in animal studies, the data on TZDs and resistin are not as clear as that for adiponectin. Some studies have shown that TZDs upregulate resistin mRNA levels in adipose tissue of ob/ob mice and ZDF rats (420) whereas others report downregulated mRNA levels in adipose tissue of db/db mice and in 3T3L1 adipocytes (72, 421, 422). Both human and animal studies have shown that PPARg agonists increase plasma adiponectin levels (423–425). Many studies have shown the benefits of TZDs in increasing adiponectin levels and reducing proinflammatory mediators such as IL-6 and CRP in patients with MetS (328, 417–419). Work by Krzyzanowska et al. (426) suggests there is a relationship between adiponectin and FFA levels. The fact that rosiglitazone treatment promotes an increase in plasma

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adiponectin while decreasing FFA levels suggests this effect is mediated by adipocytes. The effect on adiponectin would also explain why rosiglitazone protects against endothelial dysfunction induced by FFAs (427). Randomized, placebo-controlled trials of pioglitazone treatment in patients with nonalcoholic steatohepatitis have shown that as little as 6 months of treatment can reduce hepatic lipid content, normalize liver function, and improve hepatic insulin sensitivity (428, 429). The reduction in liver lipid content was inversely associated with an increase in plasma adiponectin. Paradoxically, although patients given pioglitazone experienced an increase in percentage of body fat, plasma adiponectin increased. Adiponectinstimulated activation of AMPK is thought to be an important factor in mediating the metabolic effects of TZDs in the liver (429). Renin-Angiotensin System Inhibition AngII is a vasoactive molecule that is not only essential for normal cardiovascular function, but can also have detrimental effects if it is present at chronically high levels. The pathological effects of AngII include atherosclerosis and hypertension. Typically, angiotensinogen is secreted by the liver. It is then cleaved to angiotensin I by renin, which is produced by the kidney. AngII is then produced by cleavage with angiotensin converting enzyme (ACE), which is present on the luminal surface of endothelial cells. An important discovery was the identification of angiotensinogen, a precursor to AngII, as an adipokine (27). Furthermore, adipose tissue contains all of the components of the renin-angiotensin system necessary to convert angiotensinogen to AngII. Local production of AngII therefore does not require the circulating enzymes, but adipose-derived AngII can influence systemic levels. In this way, AngII production by adipocytes can promote the development of hypertension. Another way that AngII can impact health is through suppression of adiponectin production (430), which in turn can exacerbate endothelial dysfunction. The negative actions of AngII suggest that inhibitors of AngII production will have beneficial health effects. This has been seen with ACE inhibitors, a popular class of antihypertensive agents. These compounds function by lowering systemic AngII levels, and thus reduces blood pressure. In addition, they have been shown to lower body weight and increase plasma adiponectin levels in rats (431), presumably by decreasing adipocyte size (432). In humans, AngII receptor blockers (ARBs), as well as ACE inhibitors, increase plasma adiponectin concentrations (433–435). Specifically, ACE inhibitors such as ramipril and valsartan have been shown to increase adiponectin concentrations in patients with MetS (436). These results may explain the broad protective effects against renal, cardiovascular, and neural disease ascribed to ACE inhibitors (437). Interestingly, ARBs are not as effective as ACE inhibitors in raising adiponectin. The sole exception is telmisartan, an ARB that stimulates PPARg as well as blocks the AngII AT1 receptor (target of ARBs) (438, 439). The fact that these observations were made in human studies suggests this approach may have a great value in the treatment of various diseases linked to vascular dysfunction. While it is clear that elevating adiponectin may be a useful therapeutic approach for treating cardiovascular disease (440), some caution may be advised before

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implementing these therapies. This statement is based on the fact that some treatments designed to reduce blood pressure may negatively influence adiponectin. For instance, ACE inhibitors have been shown to increase circulating adiponectin, but in combination with a diuretic, adiponectin levels decrease (441). This may be due to the fact that diuretic monotherapy is also associated with a reduction of adiponectin (442). AMPK Activation AMPK plays an important role in regulating glucose and lipid metabolism. Activation of AMPK results in reduced deposition of lipids and enhances oxidation of stored fat, thus it may be a possible target for treatment of MetS (443). In humans, metformin, a member of the biguanide family of antidiabetic drugs that activate AMPK, has been shown to reduce serum leptin, insulin and glucose concentrations despite having no affect on body weight or adipose tissue mass (444). Interestingly, adiponectin also activates AMPK resulting in free fatty acid oxidation and glucose uptake by skeletal muscle (126, 445) and suppression of glucose production (126). Metformin also increases resistin protein levels in abdominal (epididymal) adipose tissue in db/db mice (446). Interestingly, metformin can also affect the expression of resistin in hepatic tissues and downregulation of resistin levels by metformin may lead to improved insulin sensitivity (447). Endocannabinoid Receptor Antagonists The ECS has been identified as an important modulator of metabolism (448), and it is now clear that the ECS plays a role in food intake and adipose accumulation in humans and animals (448–450). The discovery of two G protein-coupled receptors (CB1 and CB2) for D9-tetrahydrocannabinol has led to the identification of numerous endogenous cannabinoid receptor ligands and the development of various receptor agonists and antagonists. Activation of CB1 increases food intake, while blocking the CB1 receptor suppresses food intake. The CB2 receptor, in contrast, appears to modulate insulin secretion by the pancreas (451) and has been recently reported to influence hepatic steatosis, inflammation, and insulin resistance in obesity (452). Treatment with a CB1 receptor antagonist (rimonabant) increases plasma adiponectin in both obese humans and rats (453–455). Recently, Despres et al. (456) have shown that one year treatment with rimonabant, significantly reduces the ratio of intra-abdominal (visceral) adipose to subcutaneous adipose tissue while increasing serum adiponectin. While these studies show rimonabant is linked to an elevation of adiponectin levels, it appears this effect may be a consequence of weight loss (457). Five placebo-controlled randomized clinical trials have been completed using rimonabant, two of which included participants with type 2 diabetes mellitus (458). Rimonabant is recommended for patients with a BMI greater than 30 kg/ m2 and/or abnormal blood lipids. Pooled, one year data from the Rimonabant in Obesity (RIO) program showed that 20 mg/day of rimonabant resulted in significant weight loss as well as improvements in other end points such as HDL cholesterol,

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TAG, fasting glucose levels, as well as a 0.6% reduction in hemoglobin A1c (HbA1c) levels in diabetes (458, 459). With respect to other adipokines, a CB1 knockout mouse shows increased leptin resistance in association with hepatic steatosis, but little is known about the relationship between the ECS and leptin. It is possible that they operate as antagonizing systems in the hypothalamus in terms of satiety (460, 461), which agrees with evidence that leptin may regulate CB1 expression in the hypothalamus (160). Interestingly, resveratrol has been recently shown to bind to the CB1 receptor (462), which may explain its ability to affect leptin release. Further investigation will be necessary to fully understand the effects of the ECS on other adipokines. At the same time, the withdrawal of rimonabant from the market due to psychiatric side effects associated with depression (155) and an increase in death rate as a result of intensive treatment intended to lower HbA1c levels to 70 years of age. Am J Cardiol 104:602–605. 174. WELSH, P., H.M. MURRAY, B.M. BUCKLEY, A.J. DE CRAEN, I. FORD, J.W. JUKEMA, P.W. MACFARLANE, C.J. PACKARD, D.J. STOTT, R.G. WESTENDORP, J. SHEPHERD, and N. SATTAR. 2009. Leptin predicts diabetes but not cardiovascular disease: results from a large prospective study in an elderly population. Diabetes Care 32:308–310. 175. FANTUZZI, G., and T. MAZZONE. 2007. Adipose tissue and atherosclerosis: exploring the connection. Arterioscler Thromb Vasc Biol 27:996–1003.

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472. HELIOVAARA, M.K., A. REMITZ, S. REITAMO, A.M. TEPPO, S.L. KARONEN, and P. EBELING. 2007. 13-cisRetinoic acid therapy induces insulin resistance, regulates inflammatory parameters, and paradoxically increases serum adiponectin concentration. Metabolism 56:786–791. 473. KOISTINEN, H.A., A. REMITZ, V.A. KOIVISTO, and P. EBELING. 2006. Paradoxical rise in serum adiponectin concentration in the face of acid-induced insulin resistance 13-cis-retinoic. Diabetologia 49:383–386. 474. KRSKOVA-TYBITANCLOVA, K., D. MACEJOVA, J. BRTKO, M. BACULIKOVA, O. KRIZANOVA, and S. ZORAD. 2008. Short term 13-cis-retinoic acid treatment at therapeutic doses elevates expression of leptin, GLUT4, PPARgamma and aP2 in rat adipose tissue. J Physiol Pharmacol 59:731–743. 475. STEINBERG, G.R., M.J. WATT, B.C. FAM, J. PROIETTO, S. ANDRIKOPOULOS, A.M. ALLEN, M.A. FEBBRAIO, and B.E. KEMP. 2006. Ciliary neurotrophic factor suppresses hypothalamic AMP-kinase signaling in leptin-resistant obese mice. Endocrinology 147:3906–3914. 476. MATTHEWS, V.B., and M.A. FEBBRAIO. 2008. CNTF: a target therapeutic for obesity-related metabolic disease? J Mol Med 86:353–361. 477. WATT, M.J., N. DZAMKO, W.G. THOMAS, S. ROSE-JOHN, M. ERNST, D. CARLING, B.E. KEMP, M.A. FEBBRAIO, and G.R. STEINBERG. 2006. CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK. Nat Med 12:541–548. 478. BLUHER, S., S. MOSCHOS, J. BULLEN, JR., E. KOKKOTOU, E. MARATOS-FLIER, S.J. WIEGAND, M.W. SLEEMAN, AND C.S. MANTZOROS. 2004. CILIARY NEUROTROPHIC FACTORAX15 ALTERS ENERGY HOMEOSTASIS, DECREASES BODY WEIGHT, AND IMPROVES METABOLIC CONTROL IN DIET-INDUCED OBESE AND UCP1-DTA MICE. DIABETES 53:2787–2796. 479. CEDARBAUM, J.M. for the ALS CNTF Treatment Study Group. 1996. A double-blind placebocontrolled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis. ALS CNTF Treatment Study Group. Neurology 46:1244–1249. 480. BLUHER, S., J. BULLEN, and C.S. MANTZOROS. 2008. Altered levels of adiponectin and adiponectin receptors may underlie the effect of ciliary neurotrophic factor (CNTF) to enhance insulin sensitivity in diet-induced obese mice. Horm Metab Res 40:225–227. 481. CROWE, S., S.M. TURPIN, F. KE, B.E. KEMP, and M.J. WATT. 2008. Metabolic remodeling in adipocytes promotes ciliary neurotrophic factor-mediated fat loss in obesity. Endocrinology 149:2546–2556.

Chapter

5

Hepatic Metabolic Dysfunctions in Type 2 Diabetes: Insulin Resistance and Impaired Glucose Production and Lipid Synthesis RUOJING YANG Department of Metabolic Disorders – Diabetes, Merck Research Laboratories, Rahway, NJ, USA

INTRODUCTION Insulin resistance is a condition in which liver, muscle, and adipose cells fail to respond to normal amount of circulating insulin to promote the storage of carbohydrates, lipids, and proteins. Insulin resistance is closely associated with type 2 diabetes mellitus (T2DM), central obesity, dyslipidemia, atherosclerosis, hypertension, and inflammation (1). The International Diabetes Federation estimates that more than 285 million people worldwide have diabetes and 438 million people will have this disease within 20 years. The American Diabetes Association estimated that the total annual cost of diabetes in the United States was $174 billion in 2007. There are two types of diabetes mellitus resulting in hyperglycemia. Type 1 diabetes mellitus (T1DM) is characterized by loss of the insulin-producing b-cells in the pancreas leading to insulin deficiency. T2DM accounts for more than 90% of all diabetes and is

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characterized by insulin resistance combined with relatively reduced insulin secretion. Increased obesity, aging population, and sedentary lifestyles are the leading causes to the rising prevalence of T2DM (1). The pathogenesis of T2DM remains poorly understood. Current therapies for T2DM focus on strict glycemic control, which lowers the risk of diabetic complications including those in heart, kidney, eye, and nerves. T2DM develops from insulin resistance and is a progressive disease in the loss of insulin action (2, 3). In early stages, pancreatic b-cells can compensate for insulin resistance by secreting more insulin leading to increased circulating insulin levels to maintain normal glycemia (Figure 5.1). When b-cells can no longer secrete enough insulin to compensate for insulin resistance, glucose intolerance occurs leading to postprandial hyperglycemia. Insulin resistance occurs in liver with increased glucose production and impaired glycogen metabolism, in muscle with decreased glucose uptake, and in adipose tissue with increased lipolysis to increase circulating free fatty

Obesity Aging Lifestyle

Liver Increased glucose production Impaired glycogen metabolism

Skeletal muscle Decreased glucose uptake

Adipose tissue Increased lipolysis

Insulin resistance Increased free fatty acid

Pancreatic beta cell compensation

Hyperinsulinemia

Pancreatic beta cell decompensation

Glucose intolerance

Lipotoxicity Glucotoxicity

Diabetes

Pancreatic beta cell destruction

Figure 5.1 Progressive development of type 2 diabetes from insulin resistance. Obesity, aging, and sedentary lifestyles lead to insulin resistance in different tissues: (i) increased glucose production and impaired glycogen metabolism in liver; (ii) decreased glucose uptake in muscle and increased lipolysis in adipose tissue leading to increased plasma free fatty acid (FFA) levels. At early stage, pancreatic b-cells produce more insulin to compensate for insulin resistance and to maintain normal glycemia leading to hyperinsulinemia. When b-cells can no longer compensate for insulin resistance, glucose intolerance ensues leading to postprandial hyperglycemia. High plasma glucose and FFAs result in glucotoxicity and lipotoxicity to the cells, leading to more severe insulin resistance in muscle and liver and damaging b-cells to develop more severe diabetes (permission from The International Journal of Biochemistry & Cell Biology, 2008, Vol 40, Iss 12, page 5).

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acid (FFA) levels. Increased circulating glucose and FFAs cause lipotoxicity and glucotoxicity to cells, which further increase insulin resistance in muscle and liver and decrease insulin secretion by damaging b-cells, leading to more severe diabetes. Thus, T2DM occurs as a consequence of the failure of several regulatory systems: (i) reduced capacity of insulin to induce glucose uptake and to inhibit lipolysis in muscle and fat; (ii) dysregulated insulin release from b-cells; (iii) abnormally increased glucose production in liver. In T2DM, defects in suppression of hepatic glucose production and liver glycogen storage are observed and account for approximately one-third of the defect in total body glucose homeostasis (4, 5). This chapter focuses on the regulatory role of insulin in regulation of hepatic glucose and lipid metabolism and contribution of hepatic insulin resistance to the development of T2DM.

BALANCING HEPATIC GLUCOSE DISPOSAL AND PRODUCTION BY GLUCOSE-6-PHOSPHATE SYSTEM Liver senses changes in circulating glucose concentration and plays a major role to maintain glucose homeostasis: (i) it increases glucose disposal and storage at high glucose concentrations; (ii) it produces glucose via gluconeogenesis and glycogenolysis at low glucose concentrations. In T2DM, an imbalance between hepatic glucose production and disposal makes a major contribution to the development of hyperglycemia and other perturbations in fuel homeostasis (6). A membrane-bound glucose transporter, GLUT2, plays a major role to facilitate glucose diffusion in and out of the hepatocytes (7, 8). Hepatocytes with genetic disruption of GLUT2 had more than 95% reduction in glucose uptake (8). However, GLUT2 knockout mice had normal glucose production suggesting the existence of an alternative membrane traffic mechanism for glucose in this extreme condition (8). The balance between hepatic glucose production and glucose disposal and storage is ultimately determined by the relative rates of glucose phosphorylation and glucose-6-phosphate (G6P) hydrolysis (Figure 5.2). The hydrolysis of G6P to free glucose is catalyzed by the glucose-6-phosphatase (G6Pase) enzyme complex. The complex is comprised of a catalytic subunit sequestered within the endoplasmic reticulum (ER), a G6P translocase known as T1, and putative ER glucose and inorganic phosphate transporters (T2, T3) that move the reaction products back into the cytosol (6, 9, 10) (Figure 5.2). Overexpression of the G6Pase catalytic subunit or the T1 translocase in primary hepatocytes increased G6P hydrolysis and lowered intracellular G6P levels, which led to substantial decreases in glycolytic flux and glycogen deposition, and a parallel increase in gluconeogenesis (11, 12). Hepatic overexpression of G6Pase in rats exhibited several of the abnormalities associated with early-stage T2DM, including glucose intolerance, hyperinsulinemia, a marked decrease in hepatic glycogen content, and increased peripheral (muscle) triglyceride stores (13). These findings are consistent with the notion that increased activity of the G6Pase complex in liver can make a significant contribution to the development of T2DM, and clearly establish the importance of the tight control on the balance between glucose phosphorylation and G6P hydrolysis in the regulation of hepatic glucose metabolism.

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Chapter 5 Hepatic Metabolic Dysfunctions in Type 2 Diabetes Glucose GLUT2 GKRP

Phosphate

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Figure 5.2

Glucose-6-phophate (G6P) system balances hepatic glucose disposal and production. Glucose enters cells via GLUT2 transporter and is phosphorylated by glucokinase to form G6P. Dissociation of glucokinase from the glucokinase regulatory protein (GKRP) in the nucleus releases the glucokinase into cytoplasm and activates its enzymatic activity. G6P can be stored into glycogen via activationofglycogen synthase (GS) and metabolizedthrough glycolysis promotinglipogenesis. When the plasma glucose levels are low, liver produces G6P through gluconeogenesis or glycogen breakdown via activation of glycogen phosphorylase (GPH) and its kinase (GPHK). G6P can be translocated into endoplasmic reticulum (ER) via glucose-6-phosphatase (G6Pase) translocase T1, where G6P is dephosphorylatedbyG6Paseandtheglucoseandphosphate istransportedout oftheERviaT2andT3translocase, respectively. Glycogen-targetingsubunit(GTS) ofproteinphosphatase1(PP1)isa scaffold proteinbinding to GS, GPH, GPHK, and glycogen to activate glycogen synthesis and suppress glycogen breakdown.

Glucose enters liver and is primarily phosphorylated by glucokinase (hexokinase IV) to form G6P (6) (Figure 5.2). This enzyme has a lower affinity for glucose and a higher catalytic capacity than other members of its gene family, and is limited in terms of its tissue distribution to liver, the islets of Langerhans, and certain specialized neuroendocrine cells in the pituitary and gastrointestinal tract (14, 15). In liver, glucokinase is sequestered in the nucleus bound to glucokinase regulatory protein (GKRP) at low glucose concentrations (16). Increased glucose concentrations trigger the translocation of glucokinase to the cytoplasm to generate G6P (Figure 5.2). Fructose-1-phosphate synergistically potentiates glucose-induced glucokinase translocation (17). Overexpression of glucokinase in primary hepatocytes led to profound increases in glycogen deposition and glycolytic rate, while overexpression of hexokinase I had a very limited impact on both variables (18). This difference in efficacy is likely explained by the fact that hexokinase I activity is strongly inhibited by the product of the reaction, G6P, while glucokinase is not subject to such regulation (19). Hepatic overexpression of glucokinase by sixfold increased liver glycogen content and robustly decreased blood glucose levels and insulin levels accompanied by a robust increase in circulating triglycerides and FFAs (20). Chronic increase in hepatic glucokinase activity by twofold decreased blood glucose levels

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without increases in circulating triglycerides and FFAs (21–23). In humans, loss-offunction mutations on glucokinase result in maturity onset diabetes of the young type 2 (MODY-2), while gain-of-function mutations lead to persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Heterozygous glucokinase knockout in mice resulted in hyperglycemia and defective glucose-induced insulin secretion, while homozygous knockout was lethal (24–26). Pancreatic b-cell specific glucokinase knockout mice had profound hyperglycemia and died within three days of birth (27, 28). Liver-specific glucokinase knockout mice were mildly hyperglycemic when fasted with impaired glucose-induced insulin secretion (28). Thus, MODY-2 results from loss of glucokinase function in liver and b-cells, but the defect in b-cells plays a dominant role leading to hyperglycemia. Significant progress has been made to develop allosteric small molecule activators of glucokinase (29). Several glucokinase activators have been reported to lower blood glucose in insulin resistance animals or in T2DM patients (29–31). The activators stimulated glucokinase activity both in liver and pancreatic islets to enhance glucose disposal and increase insulin secretion.

REGULATION OF HEPATIC GLUCOSE METABOLISM BY INSULIN Insulin promotes glucose uptake in muscle and fat cells to increase circulating glucose disposal by stimulating the translocation of GLUT4 transporter to the plasma membrane (32). Insulin does not promote glucose uptake in liver since GLUT2 is the major glucose transporter and is located in the plasma membrane. Insulin increases circulating glucose disposal in liver by stimulating glucose utilization and storage as glycogen and lipids. Skeletal muscle is the major tissue for insulindependent glucose disposal (33). In the fasted state, liver is the main organ to produce glucose to maintain circulating glucose homeostasis. Liver produces glucose through two processes: (i) glycogenolysis that breaks down glycogen stores into glucose and (ii) gluconeogenesis that generates glucose using 3-carbon substrates. Nuclear magnetic resonance (NMR) studies in humans demonstrated that hepatic glycogenolysis and gluconeogenesis each contributed 50% to endogenous glucose production after 6–12 h fasting, but they account for 4 and 96% of glucose production, respectively, during prolonged fasting for 46–64 h (34). Insulin inhibits hepatic glucose production and promotes glucose disposal via several different mechanisms (32, 35), which will be described in more details below.

INSULIN-SIGNALING PATHWAY Insulin receptor belongs to a subfamily of receptor tyrosine kinases and consists of two a-subunits and two b-subunits (32). Insulin binds to the a-subunit to increase the kinase activity of the b-subunit, which autophosphorylates to further increase its tyrosine kinase activity. Activated insulin receptor phosphorylates tyrosine residues of its intracellular substrates, including four insulin receptor substrate (IRS-1 to -4) proteins (Figure 5.3). Genetic studies suggest that different IRS proteins play distinct

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I P -Y

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P- Y S -P

AKT

P- Y

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mTOR p70

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Serine kinase

Stress inflammatory signals

GTS

Glycogen synthesis

Glycogenolysis

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Figure 5.3 Insulin regulates hepatic glucose and lipid metabolism. Insulin acts on its receptor to increase tyrosine phosphorylation of the insulin receptor and insulin receptor substrates (IRSs). Tyrosine phosphorylated IRS proteins lead to activation of the PI3 kinase pathway to increase Akt phosphorylation. Increased Akt phosphorylation regulates different metabolic pathways in liver. (i) Akt activates glycogen synthase to promote glycogen synthesis via inhibiting glycogen synthase kinase 3 (GSK3) and activation of glycogen-targeting subunit (GTS) of protein phosphatase 1 (PP1). Activation of GTS–PP1 complex also contributes to the suppression of hepatic glycogenolysis. (ii) Akt enhances hepatic glycolysis and lipogenesis via upregulation of genes in these pathways via sterol regulatory element binding protein-1 (SREBP-1). (iii) Akt inhibits gluconeogenesis via inhibition of several transcription factors, including forkhead-related proteins (FKHR), PPARg coactivator 1 (PGC-1), and hepatocytes nuclear factor (HNF). (iii) Akt also promotes protein synthesis via activation of mTOR/p70 (S6kinase). Activation of mTOR (mammalian target of rapamycin) signaling triggers IRS protein degradation, a feedback inhibition of insulin signaling, through kinases that increase serine/threonine phosphorylation of IRS. Protein tyrosine phosphatase 1B (PTP-1B) also inhibits insulin signaling through tyrosine dephosphorylation of insulin receptor and IRS proteins. Inflammatory signals and stress lead to insulin resistance via activation of those serine/threonine kinases to inhibit insulin signaling.

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roles in different tissues. Mice with genetic disruption of IRS-1 developed insulin resistance in peripheral tissues and impaired glucose tolerance (36, 37). IRS-2 knockout mice developed insulin resistance in both peripheral tissues and liver and impaired pancreatic b-cell function leading to the development of T2DM (38). IRS-3 or IRS-4 knockout mice displayed mild metabolic phenotypes (39). Several groups have proposed the model that IRS-1 regulates lipid metabolism in liver while IRS-2 regulates hepatic glucose production (40). However, recent studies demonstrated that double knockout of both IRS-1 and IRS-2 resulted in much more severe diabetes compared to either single knockout, suggesting that the two proteins are effectively interchangeable in relaying insulin action (41, 42). Tyrosine phosphorylated IRS proteins activate phosphatidylinositol 3-kinase (PI3K) pathway to increase Akt phosphorylation. Increased Akt phosphorylation regulates different metabolic pathways: (i) it promotes GLUT4 translocation to the membrane to increase glucose uptake in muscle and adipose tissue; (ii) it downregulates gluconeogenesis through forkhead-related protein (FKHR) in liver, such as FoxO1; (iii) it upregulates glycogen synthesis through phosphorylation of glycogen synthase kinase 3 (GSK3); (iv) it also increases protein synthesis through activation of mTOR/p70 (S6 kinase) (Figure 5.3). Activation of mTOR (mammalian target of rapamycin) signaling has been shown to be capable of triggering IRS-1 degradation, a feedback inhibition of insulin signaling, through kinases that increase phosphorylation of IRS-1 on serine-307 (43–46) (Figure 5.3). Several groups demonstrated that c-Jun N-terminal kinase (JNK), IkB kinase b (IKKb), and protein kinase C u (PKCu) inhibit insulin signaling via serine/ threonine phosphorylation of IRS-1 (47–49). These kinases can be activated by stress and inflammatory signals leading to the development of insulin resistance under these conditions (3, 50, 51). Insulin action can also be attenuated by tyrosine phosphatases. Cytoplasmic protein tyrosine phosphatase-1B (PTP1B) has been shown to play a crucial role in the negative regulation of insulin action via tyrosine dephosphorylation of insulin receptor or IRS proteins. Mice with genetic disruption of PTP1B were resistant to diet-induced obesity and had increased tyrosine phosphorylation of insulin receptor and IRS proteins and improved insulin sensitivity (52). Inhibition of PTP1B with antisense oligonucleotides (ASO) improved insulin sensitivity in insulin-resistant mice and in monkeys (53).

INSULIN REGULATES GLYCOGEN SYNTHESIS AND BREAKDOWN Insulin increases glycogen storage in cells through activation of glycogen synthesis and inhibition of glycogen degradation. In muscle and adipose tissue, insulin promotes GLUT4 translocation to increase glucose uptake, which further increases glycogen synthesis by increasing substrate availability and allosteric activation of glycogen synthesis enzymes (2, 6). In liver, elevated blood glucose promotes glucose uptake through GLUT2 to increase glycogen accumulation, which does not require insulin action. Although glucose plays a major role in glycogen synthesis in liver, insulin activates glycogen synthesis enzymes to further facilitate this process. Insulin

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promotes phosphorylation of GSK3 via Akt to inactivate its activity, which decreases the phosphorylation rate of glycogen synthase and increases its activity (54) (Figure 5.3). Glycogen synthase is the rate-limiting enzyme in glycogen synthesis and is activated when dephosphorylated by protein phosphatase 1 (PP1), which is dependent on insulin action (55, 56). Insulin does not promote activation of PP1 globally, but rather specifically activates PP1 localized in glycogen particles via physical association to glycogen-targeting subunits (GTS) (2, 32, 57) (Figure 5.3). Glycogen-targeting subunits of PP1 are scaffolding proteins that organize and regulate key enzymes of glycogen metabolism (57). Five members of GTS have been described with varying tissue distributions: the major skeletal muscle isoform, GM (also known as RGl or PPP1R3); a liver enriched form, GL (also known as PPP1R4); ubiquitously expressed protein targeting to glycogen (PTG or PPP1R5) and PPP1R6; and recently identified PPP1R3E with different tissue distributions in humans and rats (57, 58). All of these proteins share PP1 and glycogen-binding motifs and also, to varying degrees, bind to the glycogen-metabolizing enzymes glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. Insulin administration restored glycogen-associated phosphatase activity in diabetic rats (59, 60). In insulin-deficient diabetic rats, hepatic GL and PTG expressions are reduced compared to wild-type rats and can be restored by insulin administration (61, 62). In liver, insulin acutely activates glycogen-bound PP1 through modulation of cAMP levels and reduction of phosphorylase a, which binds to the Cterminus of GL to inhibit PP1 activity (63–68). Inhibitors of the insulin-downstream target, PI3K, blocked insulin-induced activation of glycogen-associated PP1 (32). Glycogen breakdown requires glycogen phosphorylase and debranching enzymes. Glycogen phosphorylase controls the rate-limiting step of glycogenolysis to remove glucose from glycogen (69). Glycogen phosphorylase is regulated by phosphorylation as well as allosteric factors, including AMP, ATP, G6P, glucose, and caffeine (70). Catabolic hormones, such as epinephrine and glucagon, convert the inactive dephosphorylated glycogen phosphorylase b form to the active glycogen phosphorylase a form through cAMP-dependent protein kinase and phosphorylase kinase (68). In liver, glycogen phosphorylase is mainly activated by phosphorylation, whereas the allosteric factor AMP increases glycogen phosphorylase b activity by 10–20% and fails to activate glycogen phosphorylase a (71). Insulin indirectly inhibits activation of glycogen phosphorylase by suppressing the release of those catabolic hormones. Several groups demonstrated that insulin directly inhibited glycogen phosphorylase activity by promoting phosphorylase a to phosphorylase b, while other reports disputed their results (72–76). It has been shown that insulin activates GTS of PP1 to dephosphorylate glycogen phosphorylase and thereby inhibits glycogenolysis (55) (Figure 5.3).

EFFECT OF INSULIN ON GLUCONEOGENESIS One of the characteristics of insulin resistance is elevated hepatic glucose production leading to fasting hyperglycemia. Reduced suppression of hepatic glucose production by insulin also contributes to impaired glucose tolerance and postprandial

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hyperglycemia (77). Insulin has a direct role in the suppression of hepatic glycogenolysis and gluconeogenesis (78). Liver-specific insulin receptor knockout (LIRKO) mice completely blocked the insulin-signaling pathway in liver (79). In LIRKO mice, insulin failed to suppress hepatic glucose production, suggesting that an intact insulin-signaling system is required for insulin-mediated suppression of hepatic glucose production. Insulin suppresses the expression of several key genes in gluconeogenesis, including phosphoenolpyruvate carboxylase (PEPCK), glucose6-phosphatase, and fructose-1,6-bisphosphatase (35, 80). The factors involved in the insulin-mediated transcriptional regulation of gluconeogenesis genes have remained elusive. Intensive studies have indicated that the FKHR family of transcription factors, such as FoxO1, play a crucial role in insulin-mediated inhibition of gluconeogenic gene expression (35, 80). Several lines of evidence have shown that FKHR binds to the promoter region of several gluconeogenic genes to activate their transcription, and this effect can be blocked by insulin treatment (81–83). Insulin triggers phosphorylation of FKHR proteins via a PI3K-dependent pathway. Several groups demonstrated that phosphorylation of FKHR by activated Akt resulted in nuclear exclusion of FKHR proteins and consequently decreased the transcription of their target genes (84–86). However, there are conflicting reports indicating that Akt and FKHR are not required for insulin-mediated inhibition of gluconeogenic gene expression (87–90). The peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1a) functions as a master regulator of gluconeogenic gene expression in liver (91). Glucagon and glucocorticoids increase hepatic PGC-1a expression via cAMP response element binding protein (CREB) to induce gluconeogenic gene expression (92). PGC-1a binds to and activates FoxO1, glucocorticoid receptor, and HNF-4a, and fully activates the transcription of gluconeogenic genes (91, 93). PGC1a is strongly induced in several mouse models deficient in insulin action, whereas insulin does not have a direct effect on PGC-1a expression (91, 93). Recent studies demonstrated that insulin directly inhibited PGC-1a activity through Aktmediated phosphorylation of PGC-1a (94). Insulin also blocked PGC-1a activity on the expression of gluconeogenic genes by disrupting PGC-1a and FoxO1 interaction (93). Besides the direct effect of insulin on hepatic gluconeogenesis, several lines of evidence have demonstrated that insulin indirectly inhibits hepatic glucose production by limiting substrate availability for gluconeogenesis (95–100). Insulin acts on muscle and fat tissue to inhibit the release of gluconeogenic substrates and FFAs resulting in the suppression of hepatic glucose production (101–104). Insulin also indirectly inhibits hepatic glucose production by suppressing glucagon release since glucagon controls majority of basal glucose production (96, 97, 105–107). The flux of FFAs into liver plays a key role in controlling glucose production as supported by the following evidence: (i) a strong correlation between FFAs and hepatic glucose production; (ii) lack of insulin-mediated suppression of glucose production at basal FFA concentrations; (iii) reduction of FFAs leads to reduced glucose production regardless of insulin administration (108, 109). The flux of FFAs into liver provides energy and induction signals for gluconeogenesis through b-oxidation. During clamp study at well-controlled hormonal and metabolic

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conditions, increased plasma FFA levels within the physiological range robustly induced G6Pase expression in liver (110). Glycogenolysis and gluconeogenesis have similar contributions to the net hepatic glucose production except under the condition of prolonged fasting (more than 16 h) (111). In T2DM, the relative contribution of gluconeogenesis to hepatic glucose production is significantly increased after prolonged fasting based on NMR studies, which has not been confirmed by some studies (112–116). Either augmenting gluconeogenesis via infusion of gluconeogenic substrates or inhibition of gluconeogenesis did not alter circulating glucose levels in humans (117, 118). Inhibition of glycogenolysis by pharmacologic inhibitors of glycogen phosphorylase lowered blood glucose levels in diabetic animals (119–121). However, these inhibitors also significantly reduced gluconeogenic rate in diabetic animals through unknown mechanisms (121). These observations indicate that both gluconeogenesis and glycogenolysis have to be inhibited to reduce hepatic glucose production (68).

HEPATIC LIPID METABOLISM AND INSULIN RESISTANCE Insulin plays an essential role in the regulation of hepatic lipogenesis. Glucose enters liver cells via GLUT2 transporter and then is phosphorylated by glucokinase to produce G6P. G6P can be stored as glycogen or further metabolized through glycolysis and pentose pathways to generate substrates and NADPH for de novo FFA synthesis (35). The newly synthesized FFAs can then be esterified into triglycerides and packed and secreted in very low density lipoproteins (VLDL), which carry lipids to adipose tissue for storage. Insulin directly induces FFA and triglyceride synthesis through upregulation of genes in the lipogenic pathway, including glucokinase, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), S14, liver pyruvate kinase (L-PK), and stearoyl-CoA desaturase (SCD) (35). The induction of lipogenic gene expression by insulin is mediated by the transcription factor sterol regulatory element binding protein-1C (SREBP-1C) (35) (Figure 5.3). Insulin not only induces SREBP-1C expression but also activates its activity by converting the inactive membrane-bound precursor to active nuclear form (122–125). The mechanism by which insulin regulates SREBP-1C expression is not clear. Recent studies have indicated a direct role of the liver X receptors (LXRs) in insulin-induced activation of SREBP-1C promoter activity (126). Insulin might directly activate LXRs, promote the production of LXR activating ligands such as oxygenated derivatives of cholesterol (127, 128), or activate LXR coactivators such as PGC-1 and ASC-2 (129–131). The transcription coactivator PGC-1b has recently been identified as a key activator of not only hepatic lipogenesis through coactivation of SREBP-1C but also lipoprotein secretion through coactivation of LXRa and Foxa2 (132, 133). Hepatic overexpression of PGC-1b significantly increased circulating triglyceride levels in rodents (132). Recent studies demonstrate an important role of PGC-1b in the development of fructose-induced insulin resistance (134, 135). Knockdown of PGC-1b by ASO normalized fructose-induced plasma triglyceride levels and reduced hepatic

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SREBP-1 and lipogenic gene expression (134, 135). Knockdown of PGC-1b prevented fructose-induced insulin resistance in liver and adipose tissue (134, 135). Insulin exerts effects on both hepatic glucose production and lipid metabolism: (i) suppressing glucose production through the FoxO1 pathway, (ii) promoting lipogenesis through SREBP-1C (35, 136). In T2DM, the FoxO1 pathway is resistant to insulin so that insulin fails to suppress hepatic glucose production, but the lipogenic pathway remains sensitive to insulin. This alteration in hepatic insulin action is named “selective” insulin resistance in T2DM (136). Hyperinsulinemia in T2DM contributes to the increased hepatic lipogenesis, leading to elevated circulating triglyceride levels and increased flux of triglycerides from liver to peripheral tissues (137, 138). Elevated circulating triglycerides increase lipid storage in various tissues and exert detrimental effects, namely “lipotoxicity,” to exacerbate insulin resistance in liver, muscle, and adipose tissue and damage pancreatic b-cells. Liver-specific insulin receptor knockout mice result in “total” insulin resistance in liver: hyperinsulinemia fails to suppress glucose production and also fails to activate SREBP-1C (78, 139). LIRKO mice were insulin resistant and developed hyperglycemia and hyperinsulinemia (78). However, LIRKO mice had low circulating triglycerides and liver triglyceride content (139). The hyperglycemia of LIRKO mice decreased with age, while hyperglycemia in “selective” insulin resistance mice progressed with age (78, 136). The less severe diabetes in LIRKO mice supports a long-term detrimental effect of hypertriglyceridemia on the progression of T2DM. This raises concerns about insulin therapy or insulin secretion-stimulating antidiabetic drugs over their potential of causing longterm detrimental effects via activation of hepatic lipogenesis (136). Given the unmet medical needs for T2DM patients, new drugs are preferred to improve whole body insulin sensitivity to normalize overall metabolic profiles. Hepatic steatosis, also known as fatty liver, is the most common cause of liver dysfunction and is associated with insulin resistance, T2DM, and other metabolic diseases (140, 141). Hepatic steatosis occurs as a result of increased de novo lipogenesis and excessive delivery of FFAs from adipose tissue, while lipid oxidation and export plays a minor role (142). In patients with nonalcoholic fatty liver disease (NAFLD), liver triglyceride content arises from different sources: 60% from FFAs, 30% from de novo lipogenesis, and 10% from the diet (143). Genetic disruption of hormone sensitive lipase (HSL) in mice inhibited lipolysis, reduced plasma FFAs, and prevented steatosis in liver (144, 145). These mice also had increased hepatic insulin sensitivity, suggesting that plasma FFAs play an important role in the development of hepatic steatosis and insulin resistance. Genetic studies to investigate the role of de novo lipogenesis in the development of liver steatosis and insulin resistance have been complicated by the effects on FFA oxidation. Inhibition of both ACC1 and ACC2 by ASO-inhibited lipogenesis, activated FFA oxidation, reduced liver triglyceride contents, and improved insulin sensitivity (146). Genetic disruption of SCD1 or inhibition of SCD1 by ASO prevented diet-induced hepatic steatosis and insulin resistance due to combined effects of reduced lipogenesis and increased FFA oxidation (147–150). Liver-specific knockout of FAS (FASKOL) significantly reduced circulating insulin levels with no effect on liver triglycerides in normal chowfed mice (151). When fed with zero-fat diet, FASKOL mice unexpectedly increased

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liver triglyceride content, similar to PPARa knockout mice, and can be corrected with a PPARa agonist. These led to the hypothesis that newly synthesized fat via FAS reaction serves as PPARa activators to suppress the fatty liver phenotype (151). Shulman’s group has demonstrated that metabolites from de novo lipogenesis, such as acyl-CoA, lysophosphatidic acid (LPA), and diacylglycerol (DAG), contribute to the development of insulin resistance (152). Despite substantial evidence for the association of hepatic steatosis and insulin resistance, several studies suggested an opposite role for hepatic steatosis with respect to insulin resistance (153–157). These investigators suggested that the accumulated triglycerides in liver are not toxic but rather protect cells from lipotoxicity presumably by alleviating FFA-triggered cytotoxicity (153, 154).

TARGETING HEPATIC INSULIN RESISTANCE FOR THE TREATMENT OF T2DM T2DM is a complex disorder of impaired metabolism, insulin resistance, and b-cell dysfunction. Substantial evidence indicates that strict glycemic control lowers the risk for diabetic complications, including cardiovascular disease, renal failure, and visual impairment. Current therapies achieve glycemic control in T2DM via different mechanisms. First, insulin secretagogues, such as sulfonylureas and meglitinides, increase circulating insulin levels by increasing insulin secretion from b-cells. Exogenous insulin therapy plays a major role in T2DM despite the side effects of hypoglycemia and weight gain. A high proportion of T2DM patients fail to respond to oral drugs (secondary failure) and ultimately require exogenous insulin to control hyperglycemia (158, 159). Although many physicians avoid exogenous insulin therapy in T2DM patients, some suggest that earlier use of insulin in carefully selected patients has long-term benefits (159–162). Second, thiazolidinediones (TZDs) and metformin enhance insulin action in insulin responsive tissues, such as liver, muscle, and adipose tissue. TZDs, such as rosiglitazone and pioglitazone, are agonists for the nuclear peroxisome proliferator-activated receptor gamma (PPARg) and mainly increase insulin sensitivity in peripheral tissues to promote glucose utilization. Consistent with previous reports, recent studies indicate that TZDs improve hepatic insulin sensitivity and suppress glucose production (163, 164). TZDs have become second-line therapies for T2DM despite several side effects, such as weight gain, edema, and increased risk of bone fractures. Recent studies have indicated that rosiglitazone therapy increases cardiovascular mortality in T2DM (165, 166). Others claim that the studies are not conclusive and more investigation is needed before considering rosiglitazone for removal from the market (167). Metformin is the only biguanide available for the treatment of T2DM. The principal action of metformin is to reduce hepatic glucose production and the mechanism is not fully understood (168–170). Metformin also improves insulin sensitivity, increases glucose uptake in skeletal muscle, and suppresses inflammation, which contributes to the glucose lowering effect in T2DM (158, 163). Recent studies have shown that metformin may act on adenosine-monophosphate-activated protein kinase (AMPK)

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to regulate cellular glucose and lipid metabolism (171). Third, a-glucosidase inhibitors, such as acarbose, decrease the rate of intestinal carbohydrate digestion and delay carbohydrate absorption to reduce postprandial hyperglycemia (158). Last, dipeptidyl peptidase-4 (DPP-4) inhibitors, such as Januvia, and glucagon-like peptide-1 (GLP-1) analogues, such as exenatide, increase the incretin effect of GLP-1 to reduce hyperglycemia. These drugs not only potentiate glucose-stimulated insulin secretion from b-cells but also suppress hepatic glucose production through direct action in liver and indirect effects by suppression of the glucagon release (172–175). Hepatic insulin resistance is characterized by excessive hepatic glucose production, which plays a major role in the development of fasting hyperglycemia in T2DM. As described above, insulin reduces hepatic glucose production via both direct and indirect means. A modest rise in plasma insulin leads to robust reduction in hepatic glucose production (98). Metformin reduces hyperglycemia in T2DM mainly through suppression of hepatic glucose production and this effect requires the presence of adequate insulin (159). This indicates that enhancing hepatic insulin sensitivity to suppress glucose production can reduce hyperglycemia in T2DM. Glucagon is the counter regulatory hormone of insulin, which binds to the glucagon receptor (GCGR) to promote hepatic gluconeogenesis and glycogenolysis leading to increased glucose production (176). Inhibition of glucagon action by glucagonneutralizing antibodies, antagonistic glucagon peptide analogues, or GCGR ASO inhibits hepatic glucose production and reduces blood glucose levels in diabetic animals (177–183). Substantial efforts have been made to identify small molecule inhibitors of GCGR. Several studies demonstrated that small molecule GCGR antagonists lower glucose in diabetic animals (184–192). An oral GCGR antagonist has been shown to inhibit glucagon-induced increase of blood glucose in humans (193). Suppression of hepatic gluconeogenesis or glycogenolysis enzymes leads to reduced hepatic glucose production and lowers glucose levels in animals. Inhibitors of G6Pase translocase or glycogen phosphorylase reduce blood glucose in animals (119, 194, 195). The major concerns about suppression of hepatic glucose production in T2DM are hypoglycemia and accumulation of hepatic glycogen, but neither side effect has been reported in T2DM patients treated with metformin or GCGR antagonists. However, mutations in G6Pase or G6Pase translocase in humans lead to glycogen storage diseases (196). It is important to understand the actions of future drug targets that are aimed at suppressing hepatic glucose production so that these potential side effects can be avoided.

SUMMARY This chapter describes our current understanding of how insulin regulates glucose and lipid metabolism in liver. Circulating glucose concentration plays a major role in activation of hepatic glycolysis, lipogenesis, and glycogen synthesis. Insulin further upregulates expression of genes in lipogenesis and glycolysis via SREBP-1 to potentiate these pathways. Insulin induces phosphorylation of Akt to activate glycogen synthase via inhibition of GSK3 and activation of GTS–PP1 complex. Insulin

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suppresses hepatic glucose production via inhibition of gluconeogenesis and glycogen breakdown. Insulin suppresses the release of catabolic hormones and inhibits lipolysis in adipose tissue, indirectly suppressing hepatic glucose production. Insulin signaling directly inhibits gluconeogenesis by downregulation of gluconeogenic gene expression via transcription factors, such as FoxO1, PGC-1a, and HNF-4. Insulin signaling also directly inhibits glycogenolysis via activation of GTS–PP1 complex. Elevated hepatic glucose production in T2DM is the major cause of fasting hyperglycemia and contributes to the development of postprandial hyperglycemia. T2DM develops from insulin resistance and normal levels of insulin fail to suppress hepatic glucose production in the disease state. However, the insulin-signaling pathway that promotes hepatic lipogenesis and glycolysis remains to be responsive to normal insulin levels. Elevated lipogenesis in the face of hyperinsulinemia leads to lipid accumulation in liver and muscle, which further increases insulin resistance in these tissues causing more severe diabetes. This suggests that targeting hepatic insulin resistance to achieve beneficial effects on both glucose and lipid metabolism will be more effective to control hyperglycemia in a long term in T2DM. Indeed, the most popular drug for T2DM, metformin, suppresses hepatic glucose production via activation of AMPK, which regulates both hepatic glucose and lipid metabolism. Recent progress in the development of glucagon receptor antagonists further supports that suppression of hepatic glucose production is clinically beneficial to control hyperglycemia in T2DM. The US Food and Drug Administration recently require evaluation of cardiovascular risks of new antidiabetic drugs. Impaired hepatic lipid metabolism contributes to multiple cardiovascular disease (CVD) risk factors, such as liver steatosis, elevated triglycerides, and decreased HDL. Thus, targeting genes that enhance hepatic insulin sensitivity to regulate both glucose and lipid metabolism will not only control hyperglycemia but also provide beneficial effects on the CVD risk factors in T2DM.

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Chapter 5 Hepatic Metabolic Dysfunctions in Type 2 Diabetes MONTEITH, N. PORKSEN, R.A. MCKAY, B.P. MONIA, S. BHANOT, L.M. WATTS, and M.D. MICHAEL. 2004. Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors. J Clin Invest 113:1571–1581. JOHNSON, D.G., C.U. GOEBEL, V.J. HRUBY, M.D. BREGMAN, and D. TRIVEDI. 1982. Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science 215:1115–1116. AHN, J.M., M. MEDEIROS, D. TRIVEDI, and V.J. HRUBY. 2001. Development of potent truncated glucagon antagonists. J Med Chem 44:1372–1379. KIM, R.M., J. CHANG, A.R. LINS, E. BRADY, M.R. CANDELORE, Q. DALLAS-YANG, V. DING, J. DRAGOVIC, S. ILIFF, G. JIANG, S. MOCK, S. QURESHI, R. SAPERSTEIN, D. SZALKOWSKI, C. TAMVAKOPOULOS, L. TOTA, M. WRIGHT, X. YANG, J.R. TATA, K. CHAPMAN, B.B. ZHANG, and E.R. PARMEE. 2008. Discovery of potent, orally active benzimidazole glucagon receptor antagonists. Bioorg Med Chem Lett 18:3701–3705. LAU, J., C. BEHRENS, U.G. SIDELMANN, L.B. KNUDSEN, B. LUNDT, C. SAMS, L. YNDDAL, C.L. BRAND, L. PRIDAL, A. LING, D. KIEL, M. PLEWE, S. SHI, and P. MADSEN. 2007. New beta-alanine derivatives are orally available glucagon receptor antagonists. J Med Chem 50:113–128. CHANG, L.L., K.L. SIDLER, M.A. CASCIERI, S. de LASZLO, G. KOCH, B. LI, M. MACCOSS, N. MANTLO, S. O’KEEFE, M. PANG, A. ROLANDO, and W.K. HAGMANN. 2001. Substituted imidazoles as glucagon receptor antagonists. Bioorg Med Chem Lett 11:2549–2553. de LASZLO, S.E., C. HACKER, B. LI, D. KIM, M. MACCOSS, N. MANTLO, J.V. PIVNICHNY, L. COLWELL, G.E. KOCH, M.A. CASCIERI, and W.K. HAGMANN. 1999. Potent, orally absorbed glucagon receptor antagonists. Bioorg Med Chem Lett 9:641–646. DUFFY, J.L., B.A. KIRK, Z. KONTEATIS, E.L. CAMPBELL, R. LIANG, E.J. BRADY, M.R. CANDELORE, V.D. DING, G. JIANG, F. LIU, S.A. QURESHI, R. SAPERSTEIN, D. SZALKOWSKI, S. TONG, L.M. TOTA, D. XIE, X. YANG, P. ZAFIAN, S. ZHENG, K.T. CHAPMAN, B.B. ZHANG, and J.R. TATA. 2005. Discovery and investigation of a novel class of thiophene-derived antagonists of the human glucagon receptor. Bioorg Med Chem Lett 15:1401–1405. QURESHI, S.A., M. RIOS CANDELORE, D. XIE, X. YANG, L.M. TOTA, V.D. DING, Z. LI, A. BANSAL, C. MILLER, S.M. COHEN, G. JIANG, E. BRADY, R. SAPERSTEIN, J.L. DUFFY, J.R. TATA, K.T. CHAPMAN, D.E. MOLLER, and B.B. ZHANG. 2004. A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects. Diabetes 53:3267–3273. SHEN, D.M., F. ZHANG, E.J. BRADY, M.R. CANDELORE, Q. DALLAS-YANG, V.D. DING, J. DRAGOVIC, W.P. FEENEY, G. JIANG, P.E. MCCANN, S. MOCK, S.A. QURESHI, R. SAPERSTEIN, X. SHEN, C. TAMVAKOPOULOS, X. TONG, L.M. TOTA, M.J. WRIGHT, X. YANG, S. ZHENG, K.T. CHAPMAN, B.B. ZHANG, J.R. TATA, and E.R. PARMEE. 2005. Discovery of novel, potent, and orally active spiro-urea human glucagon receptor antagonists. Bioorg Med Chem Lett 15:4564–4569. LIANG, R., L. ABRARDO, E.J. BRADY, M.R. CANDELORE, V. DING, R. SAPERSTEIN, L.M. TOTA, M. WRIGHT, S. MOCK, C. TAMVAKOPOLOUS, S. TONG, S. ZHENG, B.B. ZHANG, J.R. TATA, and E.R. PARMEE. 2007. Design and synthesis of conformationally constrained tri-substituted ureas as potent antagonists of the human glucagon receptor. Bioorg Med Chem Lett 17:587–592. SLOOP, K.W., M.D. MICHAEL, and J.S. MOYERS. 2005. Glucagon as a target for the treatment of type 2 diabetes. Expert Opin Ther Targets 9:593–600. PETERSEN, K.F., and J.T. SULLIVAN. 2001. Effects of a novel glucagon receptor antagonist (Bay 279955) on glucagon-stimulated glucose production in humans. Diabetologia 44:2018–2024. HERLING, A.W., D. SCHWAB, H.J. BURGER, J. MAAS, R. HAMMERL, D. SCHMIDT, S. STROHSCHEIN, H. HEMMERLE, G. SCHUBERT, S. PETRY, and W. KRAMER. 2002. Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(–)) by the glucose-6-phosphate translocase inhibitor S 3025. Biochim Biophys Acta 1569:105–110. PARKER, J.C., M.A. VANVOLKENBURG, C.B. LEVY, W.H. MARTIN, S.H. BURK, Y. KWON, C. GIRAGOSSIAN, T.G. GANT, P.A. CARPINO, R.K. MCPHERSON, P. VESTERGAARD, and J.L. TREADWAY. 1998. Plasma glucose levels are reduced in rats and mice treated with an inhibitor of glucose-6-phosphate translocase. Diabetes 47:1630–1636. SHIN, Y.S. 2006. Glycogen storage disease: clinical, biochemical, and molecular heterogeneity. Semin Pediatr Neurol 13:115–120.

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Energy Metabolism in Skeletal Muscle and its Link to Insulin Resistance MINGHAN WANG Metabolic Disorders Research, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Tissue insulin resistance is a main target for antidiabetic treatment. In patients with type 2 diabetes mellitus (T2DM), hepatic insulin resistance leads to increased glucose output. Further, insulin-stimulated glucose disposal in the peripheral tissues is impaired due to insulin resistance. Both abnormalities directly contribute to the hyperglycemic state in T2DM. The skeletal muscle accounts for 40% of body mass and about 75% of total insulin-stimulated glucose uptake and therefore, is a main tissue for insulin-dependent glucose utilization. In T2DM, insulin resistance in skeletal muscle is exemplified by the decreased ability of insulin to cause translocation of GLUT4, the main glucose transporter that mediates glucose uptake. Unlike in the insulin-sensitive state, GLUT4 in insulin-resistant cells is much less efficiently translocated to the plasma membrane, where it mediates glucose uptake into the cells. This is due to the impairment of intracellular insulin signaling in muscle cells of T2DM patients because signals downstream of insulin signaling trigger GLUT4 translocation under normal conditions. Several potential mechanisms are responsible for the development of insulin resistance in skeletal muscle in T2DM, including accumulation of intramyocellular fatty acid metabolites, mitochondrial dysfunction, and increased inflammatory state. Muscle insulin resistance also impacts the metabolism of other insulin-sensitive tissues, either by releasing secreted factors with metabolic functions

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or through altering whole-body energy homeostasis. The regulation of skeletal muscle growth is also critical in insulin sensitivity. Therefore, insulin resistance in skeletal muscle is a major pathologic factor in the development of hyperinsulinemia and hyperglycemia in metabolic syndrome and T2DM. Mitigation of skeletal muscle insulin resistance is critical in improving the systemic glycemic homeostasis.

GLUCOSE METABOLISM IN SKELETAL MUSCLE: THE IMPORTANCE OF GLUCOSE UPTAKE Glucose Transporters Glucose enters cells by facilitated diffusion and this process, namely glucose uptake, is mediated by glucose transporters (GLUTs). These transporters are encoded by genes that belong to the family of solute carriers 2A (SLC2A). The protein symbol for the family of the transporters is GLUT. To date, there are at least 14 members that have been described in this family (1–3). GLUT proteins share common structural characteristics, including 12-membrane helices, an N-linked glycosylation site, and intracellular N- and C-termini but exhibit significant differences in tissue distribution, substrate specificity, and kinetics of transport (4). They are located in the plasma membrane of cells and also found in intracellular vesicles. Based on sequence homology and other characteristics, the family members are divided into three classes. GLUT1–4 are categorized in class I (4). They are low-affinity and high-capacity glucose transporters and play important roles in glucose metabolism by mediating basal and insulinstimulated glucose uptake into tissues. GLUT1 is found in proliferating cells in the early developing embryo, the blood–brain barrier, human erythrocytes and astrocytes, and cardiac muscle (5). GLUT1 is also expressed in skeletal muscle and white adipose tissue (6, 7). GLUT1 expression is regulated at both the transcriptional and the posttranslational levels (8–11). GLUT2 mediates basal glucose transport in liver, small intestine, pancreas, kidney, and brain (12, 13) and its expression is subject to regulation by a variety of dietary and metabolic conditions (14–18). GLUT3 is primarily expressed in neuronal tissues (19–21). GLUT3 has higher affinity for glucose than the other class I glucose transporters and at least fivefold greater transport capacity than GLUT1 and GLUT4 (22). This is significant for its role in mediating glucose uptake by neurons since the glucose level surrounding neurons is only 1–2 mM compared with 5–6 mM in serum (22). GLUT4 is responsible for insulin-stimulated glucose uptake in skeletal muscle, adipose, and other insulin-sensitive tissues (23–28). The regulation of GLUT4 translocation by insulin signaling represents the major pathway for insulin-dependent glucose uptake in skeletal muscle.

The Important Role of GLUT4 in Glucose Uptake in Skeletal Muscle Glucose uptake by skeletal muscle cells is the rate-limiting step of glucose metabolism under normoglycemic conditions. Both GLUT1 and GLUT4 are expressed in skeletal

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muscle butGLUT4 is the main glucose transporter in this tissue. GLUT1 mediates basal glucose uptake, whereas GLUT4 mediates insulin-stimulated glucose uptake (29). Both GLUT4 protein level and glucose uptake are much higher in red oxidative muscle fibers than white glycolytic fibers in rat skeletal muscle (30, 31). This difference is smaller in human skeletal muscle (32, 33). GLUT4 is localized in intracellular membrane vesicles in the basal state. Upon insulin stimulation, GLUT4 is translocated from the intracellular pools to the plasma membrane, which was demonstrated in human skeletal muscle biopsies (34). In addition to insulin stimulation, exercise also induces glucose uptake in skeletal muscle (35, 36). Exercise and insulin appear to stimulate the translocation of two separate pools of intracellular glucose transporters to the plasma membrane (35), suggesting that exercise triggers GLUT4 translocation through a mechanism distinct from that by insulin (35). There are distinct intracellular GLUT4 pools that can be recruited by insulin and exercise, respectively (37). The muscle glucose uptake is also regulated by changes of GLUT4 expression. Muscle GLUT4 expression is induced by insulin, thyroid hormone, and exercise (38–41). One important link of insulin signaling to GLUT4 translocation is Akt substrate of 160 kDa (AS160), which is a Rab GTPase activating protein (GAP) (42–44). In fat cells, AS160 is phosphorylated by PKB/Akt upon insulin stimulation and inactivation of its GAP activity leads to GLUT4 vesicle translocation (42–44). Although the role of ASP160 in skeletal muscle GLUT4 translocation remains to be validated, it has been demonstrated in adipocytes that a dominant-inhibitory mutant of ASP160 blocked insulin-stimulated GLUT4 exocytosis (42). The exercise-stimulated GLUT4 translocation is believed to be mediated by adenosine 50 -monophosphate (AMP)activated protein kinase (AMPK), a key metabolic switch that regulates energy metabolism. It has been demonstrated that muscle contraction activates AMPK, which can stimulate glucose uptake in skeletal muscle in an insulin-independent manner (45). In T2DM patients, AMPK has the full capacity to stimulate glucose uptake in skeletal muscle (45). Therefore, exercise in T2DM induces AMPK activation in a way similar to normal insulin-sensitive individuals (46), although insulin-stimulated glucose uptake is impaired. The AMPK activation-stimulated glucose uptake is mediated by GLUT4 translocation to the plasma membrane (46), and muscle contraction stimulates GLUT4 translocation in an AMPK-dependent manner (5). Increased GLUT4 content in skeletal muscle after endurance training has been observed in both diabetic animals and humans (47, 48). In addition to induced translocation, transcription and translation of GLUT4 are increased in response to exercise, which is likely to be mediated by the activation of AMPK (49). Further, pharmacologic activation of AMPK by injection of adenosine analogue 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased GLUT4 content in muscle (50). The regulation of GLUT4 activity is not only by translocation but also through an unknown mechanism at the plasma membrane level. Overexpression of GLUT1 in mouse skeletal muscle increased basal glucose transport by several fold (51). In the GLUT1-overexpressing skeletal muscle of these animals, insulin-stimulated increase in cell surface GLUT4 content was identical to that in wild-type muscle (52, 53).

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However, there was no elevated GLUT4-mediated glucose uptake in the transgenic muscle upon stimulation with a number of stimuli, including insulin and those with glucose transport activation mechanisms distinct from insulin (51). This defect in GLUT4-mediated activation of glucose uptake does not involve the insulin receptor (51). These data demonstrate that the level of basal glucose uptake can impact insulin-stimulated GLUT4-mediated glucose transport, and this effect can be exerted without changing insulin-stimulated GLUT4 translocation to the plasma membrane. It is possible that the activity of the translocated GLUT4 in the plasma membrane is regulated by the basal rate of glucose transport. Under conditions of elevated basal glucose transport, a regulatory mechanism is activated so that the GLUT4 in the plasma membrane is not fully active. In GLUT4 knockout mice, there is decreased longevity associated with cardiac hypertrophy and severely reduced adipose tissue deposits (54). They have elevated postprandial hyperinsulinemia, suggesting insulin resistance in these animals. Compensation for this defect by increasing other GLUT members occurred in these animals, with GLUT2 elevated in liver and GLUT1 in heart, but not in skeletal muscle (54). These compensatory responses impaired the ability to conclusively determine the absolute role of GLUT4 in glucose homeostasis.

Mechanisms of GLUT4 Translocation GLUT4 recycles between the intracellular vesicular compartments and the plasma membrane; the process involves multiple steps such as endocytosis, sorting into vesicles, docking, and fusion with the plasma membrane. Insulin stimulates glucose uptake in skeletal muscle by enriching cell surface GLUT4 protein, which is associated with both elevated exocytosis and reduced endocytosis (55, 56). Insulin-stimulated GLUT4 translocation is rather a complex process and involves many proteins that mediate individual steps. It is believed that two separate and parallel pathways downstream of insulin signaling regulate this process. One pathway involves the activation of phosphoinositol 3-kinase (PI3K), which leads to Akt activation and phosphorylation of RabGAP AS160 (42). AS160 activity has been demonstrated to be required for GLUT4 translocation as a dominant negative mutant of AS160 impaired insulin-stimulated GLUT4 translocation (42). A second pathway that works in parallel to PI3K involves Cbl and TC10. Cbl is associated with Cbl-associated adapter protein (CAP) via interaction with the SH3 domain of CAP (57). Upon insulin receptor activation, Cbl is recruited along with CAP to the insulin receptor and becomes phosphorylated (57). After phosphorylation, the Cbl/CAP complex dissociates from insulin receptor and translocates to lipid rafts, where it triggers a cascade of signaling events eventually leading to the activation of small G proteins TC10a and TC10b (58, 59). Activated TC10 interacts with a number of potential effector proteins, including Exo70, a component of the exocyst complex (60). The exocyst complex plays an important role in docking of secretory vesicles containing GLUT4.

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INSULIN SIGNALING AND MECHANISMS OF INSULIN RESISTANCE IN SKELETAL MUSCLE Insulin receptor and other signaling components are expressed in skeletal muscle. The signaling cascade is similar to that in other tissue as described in other chapters in this book. Insulin binds to the a subunits of insulin receptor and induces phosphorylation of tyrosine residues on the b subunits, resulting in the increased receptor tyrosine kinase activity. The activated receptor then phosphorylates tyrosine residues on intracellular substrates including the family of insulin receptor substrates (IRSs). IRS-1 and -2 are both expressed in skeletal muscle and believed to mediate insulin signaling in this tissue. The tyrosine-phosphorylated IRS-1 and -2 help recruit PI3K to the plasma membrane. PI3K has two subunits, the 110 kDa regulatory subunit that binds to IRS and the 85 kDa kinase subunit that phosphorylates and activates downstream signaling molecules, including PDK-1 and -2. Subsequent phosphorylation and activation of Akt by PDKs leads to further activation of a signaling cascade that stimulates the translocation of GLUT4 to the plasma membrane. In type 2 diabetic patients, reduction in insulin-stimulated glucose uptake in the peripheral tissues is one of the main metabolic deficiencies. This is not caused by deficiency in GLUT4 expression in the cell because the total GLUT4 level does not change in insulin resistance cells in T2DM; rather, the trafficking and translocation of GLUT4 in response to insulin stimulation is impaired in the skeletal muscle of these patients (61–63). As a result, there is insufficient GLUT4 at the plasma membrane following insulin stimulation (61–63). These data suggest that defective insulin signaling impaired the ability of insulin to stimulate GLUT4 translocation from subcellular pools to the plasma membrane. Interestingly, physical training increases muscle GLUT4 translocation as well as expression (41), suggesting that AMPK activation can bypass the insulin-resistant state. Identification of the defective point in the insulin-signaling cascade in T2DM muscle is critical in understanding the mechanisms of insulin resistance. It has been reported that insulin receptor phosphorylation in skeletal muscle is reduced (64) or unaltered (62) in T2DM compared with normal subjects. However, IRS-1 tyrosine phosphorylation is reduced in skeletal muscle of T2DM patients while the IRS-1 protein level remains unchanged (62). The reduced IRS-1 phosphorylation upon insulin stimulation is expected to result in less PI3K activation and reduced downstream signaling. There are several mechanisms that may lead to reduced IRS-1 phosphorylation. Proinflammatory and stress signals are associated with macrophage infiltration into adipose tissue and skeletal muscle and can cause activation of JNK1 and IKKb, both of which can phosphorylate IRS-1 on its serine residues and impair its ability to be tyrosine-phosphorylated and activated. Therefore, macrophage-derived proinflammatory cytokines such as tumor necrosis factor a (TNFa), and interleukin (IL)-1b, -4, and -6 are likely involved in the development of skeletal insulin resistance. Alternatively, increased fatty acid uptake by muscle cells can lead to insulin resistance, causing intramyocellular accumulation of lipids (triglycerides) and fatty acyl metabolites. Two such metabolites, ceramide and diacylglycerol (DAG), play important

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roles in fatty acid-induced muscle insulin resistance. Ceramide inhibits insulindependent activation of Akt2/PKB (65), while DAG activates PKC isoforms, which promotes IRS-1 serine phosphorylation and inhibition of insulin signaling (66). Thus, hyperlipidemia is likely to contribute to the development of muscle insulin resistance. Since hepatic insulin resistance contributes to the development of hyperlipidemia with elevated free fatty acid (FFA) levels, insulin resistance in skeletal muscle could be at least in part a consequence of reduced insulin sensitivity in liver. On the other hand, skeletal muscle insulin resistance is likely to impact hepatic insulin sensitivity. In a study with young, lean, insulin-resistant individuals, it was found that decreased muscle glycogen synthesis due to insulin resistance increased carbohydrate flow to the liver and caused elevated lipogenesis (67), supporting the notion that insulin resistance in skeletal muscle may negatively impact hepatic insulin sensitivity. The intramyocellular lipid accumulation in insulin-resistant muscle results from an imbalance between FFA oxidation and uptake. Overwhelming evidence suggests that alterations in fatty acid oxidation in skeletal muscle play an important role in the development of muscle insulin resistance. There is reduced mitochondrial oxidation capacity in the muscle of insulin-resistant offspring of T2DM patients (68). Further, in obese, insulin-resistant individuals, there is reduced rate of fatty acid oxidation (69, 70). The overall activity of the mitochondrial respiratory chain is reduced in T2DM and obese individuals compared with lean controls and the mitochondria in skeletal muscle of T2DM and obese subjects is smaller than those from lean controls (71). While the fatty acid oxidation rate is reduced, fatty acid uptake by skeletal muscle is increased in obesity and insulin resistance. Fatty acid transporters in the plasma membrane of skeletal muscle cells from obese and diabetic humans are increased (72). Interestingly, high-fat diet caused a rapid increase of fatty acid transporter CD36 in the plasma membrane of skeletal muscle cells in rats with correlated increase in DAG and ceramide (73). This occurs at two weeks after on high-fat diet, which precedes the onset of insulin resistance (73), indicating that increased fatty acid uptake and intracellular accumulation of fatty acyl metabolites may be the cause of insulin resistance. Consistent with this, a hypothesis was proposed that incompletely oxidized products from fatty acid b-oxidation induce insulin resistance in cells. In summary, mitochondrial overload contributes to the development of insulin resistance (74).

KEY PATHWAYS THAT IMPACT MUSCLE METABOLISM PGC-1a and Biogenesis in Skeletal Muscle The peroxisome proliferator-activated receptor g (PPARg) coactivator 1a (PGC-1a) is a cold-inducible nuclear transcriptional activator that binds to PPARg and increases its transcriptional activity (75). PGC-1a is expressed in tissues with high metabolic activity, including brown adipose tissue, skeletal muscle, heart, liver, and brain (75–77). It stimulates mitochondrial biogenesis by increasing the expression of key enzymes of the respiratory chain and the cellular content of mitochondrial

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DNA (75, 78). In muscle cells, PGC-1a stimulates mitochondrial biogenesis and respiration via induction of uncoupling protein 2 (UCP-2) and the nuclear respiratory factors (NRFs), NRF-1 and NRF-2. (78). In addition, PGC-1a binds to and coactivates the transcriptional function of NRF-1 to increase mitochondrial DNA replication and transcription. PGC-1a also increases the expression of the genes involved in oxidative phosphorylation (OXPHOS) (79, 80), suggesting that it plays a key role in energy metabolism in tissues with high metabolic activity. Since increased intramyocellular accumulation of fatty acyl metabolites has been demonstrated as a cause of muscle insulin resistance, PGC-1a induced mitochondrial biogenesis is expected to increase b-oxidation and improve insulin sensitivity. Although it is not clear if the dysregulation of PGC-1a expression or activity is involved in the pathogenesis of type 2 diabetes, several lines of evidence suggest alterations in PGC-1a expression or function are associated with insulin resistance. A Gly482Ser polymorphism in PGC-1a increased the risk of type 2 diabetes in Danish and Japanese populations (81, 82). However, no association of this polymorphism with type 2 diabetes was noted in French or Australian populations or Pima Indians (83–85). The expression of PGC-1a and OXPHOS genes is reduced in muscle biopsies of type 2 diabetic patients, first-degree relatives of type 2 diabetics, and subjects with impaired glucose tolerance (79, 80) as well as animal models of insulin resistance and type 2 diabetes (86, 87), suggesting that the reduction in PGC1a expression could lead to decreased biogenesis and insulin resistance in the skeletal muscle of type 2 diabetic patients. Consistent with this notion, reduction in PGC-1a is associated with elevated fasting insulin levels and body mass index (BMI) in Pima Indians (88). However, caution should be taken when interpreting these results because according to a separate study, PGC-1a expression in the muscle of type 2 diabetic patents was unchanged despite reduced mitochondrial density and insulin signaling (89). The important role of PGC-1a in muscle metabolism is implicated in its ability to regulate glucose uptake and fiber-type switch. PGC-1a is likely to be one of the mediators of metabolic effects by muscle contraction during exercise. PGC-1a expression is induced by either a single bout of exercise (90, 91) or exercise training (87, 92, 93). AMPK is activated in skeletal muscle during contraction and stimulates PGC-1a expression (87, 94). In vitro, PGC-1a overexpression increased insulin-stimulated glucose uptake by stimulating GLUT4 expression in C2C12 cells (95). However, in vivo only when overexpressed modestly, PGC-1a induced GLUT4 expression and increased insulin-dependent glucose uptake (96). When PGC1a was overexpressed in very high levels, both GLUT4 expression and insulin sensitivity were reduced (97), probably due to indirect effect of increased fatty acid uptake. The physiological role of PGC-1a in determining muscle fiber type was revealed by genetic studies. In humans, PGC-1a expression is higher in red oxidative muscle fibers than white glycolytic muscle fibers (93). Transgenic overexpression of PGC-1a in the skeletal muscle of mice induced fiber-type conversion (98). In these animals, the glycolytic type II fibers were redder and had increased expression of genes of mitochondrial oxidative metabolism (98), which is characteristic of the oxidative type I fibers (98). Consistent with these findings, skeletal muscle-specific

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PGC-1a knockout mice had a shift from oxidative type I and type IIa fibers toward type IIx and IIb fibers (99). Further, these knockout animals had reduced endurance capacity (99), consistent with the observation of fiber-type switch. These findings suggest that PGC-1a is an important factor in regulating muscle fiber type determination. Paradoxically, despite the increased muscle mitochondrial density and correlated ATP synthesis in the animals with muscle-specific overexpression of PGC-1a, there was no change in whole-body energy expenditure (100). More surprisingly, the transgenic mice were more prone to fat-induced insulin resistance with decreased insulin-stimulated muscle glucose uptake (100). In these animals, PGC-1a overexpression was sixfold (100). Consistent with another study with 10- to 13-fold overexpression, both muscle GLUT4 expression and whole-body insulin sensitivity were reduced (97). These effects could be due to the elevated intramyocellular lipid accumulation caused by superphysiologic PGC-1a overexpression (100). With modest PGC-1a overexpression by 25%, there was increased muscle GLUT4 expression and insulin-stimulated glucose uptake (96). Taken together, these data support the role of PGC-1a as a metabolic regulator in skeletal muscle that mediates beneficial effects. Consistent with this notion, skeletal musclespecific PGC-1a knockout mice had reduced muscle GLUT4 expression and exhibited impaired glucose tolerance (101).

Myostatin Signaling Myostatin is a member of the TGFb superfamily of secreted factors. It exists in a protein complex including its propeptide and follistatin-like 3 where its activity is inhibited. Myostatin is a key secreted factor that regulates muscle mass (102). Loss of function in myostatin leads to strong muscle growth in mice and cattle (102, 103). Myostatin mutation is associated with muscle hypertrophy in a child (104). Inhibition of myostatin by neutralizing antibodies or antagonists resulted in increased muscle mass in adult normal mice or mice with muscular dystrophy (105–107). Both activin receptor type IIA (ActR-IIA) and IIB (ActR-IIB) mediates myostatin signaling, although myostatin has a higher affinity for ActR-IIB (108, 109). Further, both receptors bind multiple ligands (110). Activation of these receptors results in the activation of several Smad proteins, leading to their oligomerization and translocation of the complex to the nucleus, where it suppresses the transcription of genes important in muscle growth (111, 112). Myostatin-deficient mice have increased muscle growth and reduced fat mass, and corresponding increases in muscle strength (113). These changes are also associated with alterations in muscle fiber type distribution with a greater proportion of type IIb fibers (113), which explains short contraction and relaxation times in these animals. A separate study reported similar findings that there are more fast and glycolytic fibers in myostatin-deficient mice (114). Loss of myostatin function not only resulted in increased muscle mass but also improved insulin sensitivity (115). Myostatin knockout mice had increased carbohydrate utilization for energy but the overall lipid utilization per animal did not

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change (115). The knockout mice had improved glucose and insulin tolerance, and reduced blood glucose and insulin levels (115). Further, they had increased wholebody glucose utilization rate as revealed in a hyperinsulinemic-euglycemic clamp study (115), with correlated increases in glucose utilization by white and brown adipose tissues and a trend toward elevation in skeletal muscle (115). The mechanism of myostatin inhibition was investigated by overexpressing a dominant negative myostatin molecule in either muscle or fat to block myostatin signaling tissuespecifically. Muscle-specific blockade had profound effects, resulting in increased muscle mass and reduced fat mass; while fat-specific blockade had no effect on body composition, and glucose and insulin tolerance remained unchanged in these animals (115). These data suggest that the metabolic effects by myostatin inhibition are mainly mediated by blockade of signaling in skeletal muscle, and the decreased fat mass is an indirect result of metabolic changes in skeletal muscle (115). Further, these findings indicate that myostatin action in skeletal muscle is more relevant to insulin sensitivity. Other studies showed that genetic deficiency in myostatin led to reduced fat accumulation and increased muscle mass, and conferred resistance to diet-induced obesity (116–118), protected liver against obesity-induced insulin resistance (119). Consistent with these findings, transgenic overexpression of myostatin propeptide, which is expected to bind to myostatin and neutralize its activity, prevented dietinduced obesity and insulin resistance (120).

Adipokines and Myokines A number of adipokines from adipose tissue mediate metabolic effects in skeletal muscle and impacts its insulin sensitivity. Adiponectin and leptin are two important players. In addition, secreted factors from skeletal muscle with endocrine functions (commonly termed myokines) may also regulate skeletal muscle insulin sensitivity. Together, adipokines and myokines may be part of a network of secreted factors from tissues that work in concert to mediate tissue cross talk and ultimately whole-body metabolic homeostasis. Adiponectin improves muscle insulin sensitivity (121), at least in part by stimulating fatty acid oxidation in skeletal muscle and decreasing lipid accumulation (122, 123). This effect is mediated by the activation of AMPK (122, 123). There are two isoforms of adiponectin receptors, AdipoR1 and AdipoR2, although there is some controversy as to whether these receptors indeed mediate adiponectin signaling. AdipoR1 is the main isoform expressed in skeletal muscle. The skeletal muscle from obese and insulin-resistant humans confers adiponectin resistance, which is characterized with impaired AMPK activation and reduced stimulation of FA oxidation (124, 125). The onset of adiponectin resistance precedes lipid accumulation and the development of insulin resistance in skeletal muscle (73), suggesting that adiponectin response in skeletal muscle may be critical suppressor of lipid-derived signals that cause muscle insulin resistance. Like adiponectin, leptin also improves muscle insulin sensitivity (126, 127). Leptin stimulates fatty acid oxidation (128) and decreases fatty acid uptake (129). The leptin effect is at least partially mediated by AMPK activation (130). Under obese and insulin-resistant

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state, there is leptin resistance. The mechanism is not clear but at least impaired AMPK activation is involved. IL-6 is both an adipokine and a myokine in that it is secreted from both tissues under different metabolic states (131). In addition, IL-6 is also secreted by endothelial cells, fibroblasts, pancreatic b-cells, and osteoblasts as well as immune cells such as monocytes and macrophages at inflammatory sites (131). IL-6 level is increased in inflammatory state and serves as a key regulator of inflammation. In addition, IL-6 also has metabolic functions. IL-6 suppresses insulin action in both liver and adipose tissue (132); in contrast, it sensitizes insulin action in muscle (132). IL-6 release from muscle is elevated in response to exercise (133, 134), which may work in an autocrine fashion to stimulate glucose uptake and fatty acid oxidation in muscle (132). Given the complex metabolic functions of IL-6 in different tissues, its therapeutic value is likely to be limited. Fibroblast growth factor 21 (FGF-21) is metabolic regulator involved in the control of glucose and lipid metabolism (135, 136). FGF-21 is characterized as a myokine because in addition to other tissues, it is expressed in muscle and upon Akt activation its expression is elevated (137). In addition, FGF-21 expression is upregulated in cultured skeletal muscle cells by insulin stimulation (137). Interestingly, FGF-21 expression in human skeletal muscle is induced by hyperinsulinemia (138). These findings suggest that FGF-21 is regulated by insulin action in skeletal muscle and may play a role in mediating the cross talk between skeletal muscle and other metabolic tissues. The details of FGF-21 biology are covered separately in Chapter 14 of this book.

PPARb/d Peroxisome proliferator-activated receptor b or d (PPARb/d) is a nuclear receptor that controls the expression of genes involved in fatty acid oxidation. Transgenic overexpression of PPARd in skeletal muscle induced a muscle fiber type switch toward increased numbers of type I fibers (139, 140). These animals had increased exercise endurance and were resistant to high fat-induced obesity and insulin resistance (139, 140). These data suggest that PPARd activation in skeletal muscle may activate fatty acid metabolism and increase insulin sensitivity. In humans, the whole-body insulin sensitivity is positively correlated with the relative abundance of slow-twitch oxidative fibers and negatively with that of glycolytic type II fibers (141), suggesting that fatty acid metabolism in skeletal muscle is an important factor for insulin sensitivity. Exercise is a metabolic state where increased lipid oxidation occurs. Both short and endurance training increased PPARd expression in human and rodent skeletal muscle (142, 143). Since PPARd upregulates the expression of genes involved fatty acid uptake and oxidation (144, 145), this change is a key part of the regulatory mechanism to activate muscle lipid metabolism in response to exercise. In the meantime, PPARd either directly upregulates genes involved in mitochondrial biogenesis or increases PGC-1a expression (146, 147), which is a master regulator of mitochondrial function. These data demonstrate that PPARd is an important

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metabolic regulator of lipid oxidation and mitochondrial biogenesis in skeletal muscle and its activity is important in muscle insulin sensitivity.

MUSCLE TYPES AND ASSOCIATED METABOLIC DIFFERENCES The skeletal muscle consists of both oxidative and glycolytic fibers. These fibers have different preferences for metabolic substrates and play distinct roles in muscle energy metabolism. The red muscles (type I and type IIa fibers) are mitochondria-rich and oxidative using FFAs as substrates. They are involved in prolonged physical activities/ fasting state when FFA oxidation is the main supply of energy source. The major fibers in the red muscles are slow-twitch type I and fast-twitch type IIa. The white muscles are fast-twitch, glycolytic type IIx fibers in humans (IIx and IIb in rodents) (148). Fiber switch from fast-twitch to slow-twitch type I fibers has been observed in mice with skeletal muscle-specific transgenic overexpression of either PGC-1a (98) or PPARd (139), implicating the importance of these transcriptional regulators in muscle substrate utilization. Interestingly, insulin sensitivity is positively associated with the relative abundance of slow-twitch oxidative fibers and negatively with that of glycolytic type II fibers (141). Not only the mitochondrial density is higher (149), insulin-stimulated glucose uptake is also greater in slow-twitch muscle fibers than fast-twitch muscle fibers (32, 150). These observations support the notion that increased proportion of slow-twitch fibers may provide metabolic benefits such as increased energy expenditure and improved muscle insulin sensitivity. However, this is not to say that fast-twitch, glycolytic fibers are not metabolically beneficial. As a matter of fact, absolute increase in muscle mass, even in the form of glycolytic type IIb fibers, leads to improved insulin sensitivity. This has been exemplified in myostatindeficient mice (113, 115).

EFFECTS OF MUSCLE INSULIN RESISTANCE ON OTHER TISSUES The existence of adipokines and myokines suggest that there is metabolic cross talk between tissues. Muscle-specific transgenic overexpression of Akt induced the expression of myokines that mediate metabolic effects in distant tissues such as liver and adipose. Muscle-specific Akt transgenic mouse induced the expression of myokines that mediate metabolic effects in distant tissues such as liver and adipose (151). Further, improvement of insulin sensitivity in one tissue could indirectly leads to better insulin sensitivity in another tissue. This could be achieved through the beneficial effects on the improvement of the whole-body glucose homeostasis and insulin sensitivity. Overexpression of malonyl-CoA decarboxylase (MCD) in liver is expected to reduce malonyl-CoA level and activate FFA oxidation, which results in the improvement of hepatic insulin sensitivity. In the meantime, insulin sensitivity in skeletal muscle was also improved (152). This effect may be mediated directly by a secreted factor. Alternatively, it could be an indirect effect of improved overall

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metabolic homeostasis. Another example for tissue cross talk is that the impairment of metabolism in one tissue leads to the increased pressure for another tissue to take on more metabolic substrates. Insulin resistance in skeletal muscle is characterized with decreased muscle glycogen synthesis, which can change the ingested carbohydrate away from muscle glycogen synthesis to hepatic lipogenesis (67). In this case, skeletal muscle promotes the development of dyslipidemia, due to increased hepatic triglyceride synthesis and secretion (67). This notion is exemplified by increased hepatic lipogenesis following ingestion of high carbohydrate meals in young, lean, insulinresistant subjects compared with age–weight–BMI–activity-matched, insulinsensitive control subjects (67).

SUMMARY Skeletal muscle is one of the major carbohydrate and FFA utilizing tissues in the body, and its metabolic state plays an important role in glucose homeostasis. Insulin sensitivity and mitochondrial biogenesis in skeletal muscle are important factors in energy expenditure, glucose homeostasis, and the development of lipid disorders. Improvement of skeletal muscle insulin sensitivity by either increasing energy metabolism or directly improving insulin sensitivity will be major targets for the treatment of obesity and T2DM.

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Part Two

Metabolic Diseases and Current Therapies

Chapter

7

Mechanisms and Complications of Metabolic Syndrome MINGHAN WANG Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Metabolic syndrome is a cluster of metabolic abnormalities that are usually found in individuals with high risk of non-insulin-dependent diabetes mellitus (NIDDM) or type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). The abnormalities usually include obesity, insulin resistance (hyperinsulinemia), dyslipidemia (hypertriglyceridemia and low high-density lipoprotein (HDL)), hypertension, and hyperglycemia. Not every element of these disorders is found in the same patient. However, existence of three or more of these factors warrants diagnosis of the syndrome. Metabolic syndrome is a major risk factor to develop CVD and T2DM. For example, about 80% of T2DM patients have metabolic syndrome, underscoring the importance of disease awareness and the need of early treatment of high-risk populations. However, the concept of metabolic syndrome is debatable for a number of reasons. First, there is no single unifying mechanism for all the metabolic disorders involved, raising questions about the existence of the syndrome. Further, the criteria for the definition of the syndrome are different among various medical groups, making it difficult to have consistent diagnosis. Since the syndrome is not recognized as a disease by regulatory authorities, there is no clear clinical and regulatory path for drug development and approval. Despite these challenges, metabolic syndrome is still a useful concept to identify and manage risk factors in subjects with high risk of developing T2DM and CVD. It is also important to identify a high-risk T2DM and/or

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright
2011 John Wiley & Sons, Inc.

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CVD population for prevention. Since each of the individual risk factors needs to be managed, understanding the etiology and interactions of these elements is important. Thus, drug discovery efforts to treat and minimize these risk factors could benefit from the concept of metabolic syndrome.

THE DEFINITION OF METABOLIC SYNDROME In 1988, Reaven first discovered that several cardiovascular risk factors tend to cluster together, including resistance to insulin-stimulated glucose uptake, glucose intolerance, hyperinsulinemia, increased plasma very-low-density lipoprotein (VLDL) triglyceride concentration, decreased HDL cholesterol concentration, and hypertension (1). At that time he defined this series of metabolic abnormalities as “syndrome X” (1). The insulin-stimulated glucose uptake in his observation was glucose disposal rate (Rd), which was measured by glucose clamp in human subjects, and therefore represents whole-body insulin sensitivity. Glucose intolerance refers to the reduced ability for the body to normalize plasma glucose in response to a 75 g oral glucose challenge. Since glucose normalization under such as a condition is driven by the insulin sensitivity of the peripheral tissues and the ability of pancreatic b-cells to secrete sufficient insulin, glucose intolerance is the result of insulin resistance or reduced glucose-dependent insulin secretion or combination of both. Reaven did not include obesity in the definition of syndrome X, but he indicated that the degree of obesity and sedentary lifestyle are correlated with the extent of insulin resistance regardless of genetic influences (1). The concept of syndrome X has evolved over the past 20 years and is more widely referred to as metabolic syndrome. In the meantime, the individual metabolic abnormalities are more closely defined as obesity (or central obesity), insulin resistance (hyperinsulinemia), hyperglycemia, dyslipidemia (high triglycerides and low HDL), and hypertension. The exact definition of metabolic syndrome varies among several professional organizations. However, the common features are similar. The National Cholesterol Education Program’s Adult Treatment Panel III report (ATPIII) definition includes the following risk factors: abdominal obesity (given as waist circumference), hypertriglyceridemia, low HDL, hypertension, and fasting hyperglycemia (2). While the World Health Organization (WHO) definition has more detailed criteria, it includes similar risk factors (2, 3). In addition, the WHO criteria include microalbuminuria (2, 3). Other risk factors such as family history of T2DM and high-risk ethnic group are included in the American Association of Clinical Endocrinologists (AACE) definition (4). Not all the metabolic abnormalities are found in the same individual; the diagnosis can be made if three or more characteristics are found in the same patient. In addition to the factors included in the variety of definitions, there are risk factors that are not apparent or easily measured but are critical in the development of CVD, such as endothelial dysfunction, increased vascular smooth muscle proliferation, vascular inflammation, and atherosclerosis. In obese individuals, there is increased inflammatory state in the adipose tissue characterized by macrophage accumulation (5), which is believed to be a major contributor to the

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development of insulin resistance. The main clinical outcomes of metabolic syndrome are CVD and T2DM. In the Kuopio Ischemic Heart Disease Risk Factor Study, 1209 Finnish men aged 42–60 years at baseline (1984–1989) who were initially without T2DM, CVD, or cancer were followed up through December 1998 (6). The study found that men with metabolic syndrome were several times more likely to die of CVD (6). Metabolic syndrome as defined by the WHO was associated with several times higher CVD mortality and about two times higher all-cause mortality (6). In the Framingham study, metabolic syndrome alone predicted about 25% of newly onset CVD (2). In the absence of diabetes, metabolic syndrome did not raise the 10-year risk of CVD to more than 20% (2). Almost half of the population-attributable risk for T2DM could be explained by the presence of metabolic syndrome (2). In addition, individuals with the syndrome are also susceptible to other conditions such as polycystic ovary syndrome, fatty liver, cholesterol gallstones, asthma, sleep disturbances, and cancer (2). Using the ATPIII definition and survey data collected between 1988 and 1994, a study found that the age-adjusted prevalence of metabolic syndrome among the U.S. adult population is more than 20% (7). The prevalence increases with age from 6.7% among people at 20–29 years of age to 43.5% among those at 60–69 years of age (7). Both men and women have similar age-adjusted prevalence (7). Mexican Americans have the highest prevalence and among both African and Mexican Americans, women have higher prevalence than men (7). In a study involving subjects from the European population between 1978 and 1987, the prevalence of metabolic syndrome was much lower (slightly over 10%) (8). A recent report using data from 1999–2000 indicates that the age-adjusted prevalence increased by 23.5% among women and 2.2% (not statistically significant) among men (9). Much of the increase is accounted for by increases in high blood pressure, waist circumference, and hypertriglyceridemia, particularly among women (9).

OBESITY AND INSULIN RESISTANCE Although decades of research efforts have been devoted to understanding the etiology of metabolic syndrome, there is no single causal mechanism that can explain the constellation of the risk factors in metabolic syndrome. Obesity and insulin resistance are believed to be central components but there is no concrete mechanistic evidence to support this notion. Insulin resistance is a common attribute in metabolic syndrome and T2DM. Tissue insulin resistance leads to glucose intolerance, which is compensated by increased insulin secretion by pancreatic b-cells to maintain normoglycemia. As a result, the patient develops hyperinsulinemia. Insulin resistance and the resultant hyperinsulinemia are believed to be involved in the development of other disorders in metabolic syndrome, such as hypertension and dyslipidemia (10). In this regard, some researchers even suggest that insulin resistance is likely the initiating factor of T2DM and other related complications. However, insulin resistance alone does not cause diabetes; b-cell failure is an important causal factor as well. Many insulin-resistant individuals do not develop T2DM because their b-cells can secrete

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sufficient insulin to counter insulin resistance. Nonetheless, insulin resistance is an important metabolic alteration that may set the stage for incremental loss of b-cell mass leading to b-cell failure. Insulin resistance in tissues that are important in metabolic response and energy homeostasis such as liver, adipose, and skeletal muscle is exemplified by distinct biological alterations. In liver, the ability of insulin to suppress glucose production is attenuated; in adipose tissue and skeletal muscle, there is reduced insulin-stimulated glucose uptake. These metabolic alterations result in both fasting and postprandial hyperglycemia. Impaired free fatty acid (FFA) metabolism could also contribute to increased hepatic glucose production and hyperglycemia (11). One of the normal functions of insulin is to suppress lipolysis in adipose tissue. Visceral fat is hyperlipolytic compared to subcutaneous fat and is resistant to the antilipolytic effect of insulin (12). Visceral obesity is associated with increased lipolysis due to its reduced sensitivity to insulin. This leads to increased FFA flux to liver promoting lipogenesis and hepatic insulin resistance. These disturbances further promote hyperglycemia and dyslipidemia (11). One important aspect of insulin resistance is the concomitant hyperinsulinemia, which occurs as a result of increased insulin release by pancreas to overcome the reduced insulin potency. Insulin resistance is believed to act through hyperinsulinemia to promote the development of other CVD risk factors (10). Although genetic defects and ethnic susceptibility are important contributors, the widespread obesity epidemic in the industrialized nations is primarily due to excessive energy intake and sedentary lifestyles. Compelling evidence indicates that Pima Indians have very high prevalence of obesity and T2DM. A study found that Pima Indians in Arizona are more obese than Mexican Pimas (13), and their prevalence of T2DM is several times higher than that in Mexican Pimas (13). This finding suggests that despite the genetic predisposition to these conditions, traditional lifestyles play a role in the development of obesity and T2DM. The link between obesity and insulin resistance has been well established. It is known that obesity, especially visceral (central) obesity, is correlated with insulin resistance and other cardiovascular risk factors (1). The prevalence of diabetes in an urban Asian Indian population is several times higher than that in the population living in a rural area with lower degree of obesity (14). This observation underscores the importance of obesity in the development of insulin resistance and diabetes. Obese people have postprandial hyperinsulinemia, which is characteristic of insulin resistance (15). Insulin sensitivity declines as body weight increases (16). The hyperinsulinemia in obese individuals correlates with the degree of insulin resistance but fails to fully compensate for insulin resistance (16). Therefore, hyperglycemia is often associated with insulin resistance. One potential underlying mechanism for obesity-induced insulin resistance is fat accumulation in liver and skeletal muscle. Elevated plasma FFA levels are observed in obese subjects and are associated with insulin resistance (17). This results in increased delivery of FFA into cells causing accumulation of fatty acyl metabolites, which can induce insulin resistance in liver and muscle (18). Thus, obesity plays an important role in the development of insulin resistance. However, obesity is not the absolute cause of insulin resistance because not every obese individual is insulin resistant (19). Rather, there are large variations in insulin

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sensitivity within the obese population (19). Some individuals are relatively insulin sensitive in the obese population (19), suggesting that there is a component of insulin sensitivity independent of obesity. Consistent with this notion, insulin sensitivity also varies in lean subjects due to differences in body fat distribution (20). These observations suggest that simple obesity per se does not necessarily induce insulin resistance. Obese individuals with only high subcutaneous fat depot are less likely to develop insulin resistance as those with central obesity (more fat distributed in the abdominal area). So when it comes to the link between insulin resistance and obesity, body fat distribution is as important as obesity itself. However, it is not clear if visceral fat is a causal factor or simply a better marker for insulin resistance. Compelling evidence indicates that insulin resistance is more closely associated with visceral adiposity. For this reason, the NCEP-ATPIII definition of metabolic syndrome identifies waist circumference rather than body mass index (BMI) as a risk factor to recognize the importance of central obesity (2). Certain ethnic groups are more prone to central obesity, such as Asians (21, 22), which may explain higher prevalence of T2DM at low BMI values. Less subcutaneous fat and more visceral adiposity are usually associated with more fat accumulation in liver, leading to elevated intracellular fatty acyl metabolites as mentioned above and greater hepatic insulin resistance (18). As a matter of fact, fatty liver alone is associated with strong insulin resistance. For instance, hepatic insulin resistance is found in patients with nonalcoholic steatohepatitis (NASH) or fatty liver disease (NAFLD) (23). Central obesity and reduced subcutaneous fat are found in patients with Cushing’s syndrome due to glucocorticoid excess (24). These patients develop severe insulin resistance (24), which at least can be explained by altered fat distribution. Similar findings were also made in some human immunodeficiency virus (HIV)-infected patients who were on combined highly active antiretroviral therapy (HAART) and developed a lipodystrophic syndrome. The condition is characterized with loss of subcutaneous fat, accumulation of abdominal fat, hypertriglyceridemia, and insulin resistance (25). This condition is also referred to as pseudo-Cushing’s syndrome because the fat distribution in these patients is similar to that in Cushing’s syndrome. Reducing hepatic lipid content is believed to improve insulin sensitivity. One of the beneficial effects of the thiazolidinediones (TZDs) is the reduction of hepatic fat accumulation (26), probably by increasing subcutaneous fat deposition (27).

HEPATIC INSULIN RESISTANCE AND DYSLIPIDEMIA Hypertriglyceridemia and low HDL are observed in individuals with metabolic syndrome and T2DM. Elevated low-density lipoprotein (LDL) levels are often seen in these patients. These are the characteristics of dyslipdemia that may have developed as a result of hepatic insulin resistance. Under normal conditions, plasma insulin has two hepatic actions. First, insulin suppresses gluconeogenesis by downregulating the expression of key gluconeogenic genes such as phosphoenolpyruvate

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carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). This process is mainly mediated by insulin-stimulated phosphorylation of forkhead box O1 (FoxO1), a transcription factor that controls the expression of gluconeogenic genes (28). When phosphorylated, FoxO1 is retained in the cytosol and cannot enter the nucleus to induce gene expression (28). In addition, insulin also stimulates hepatic glycogen synthesis. As a result of these effects, hepatic glucose production is suppressed by insulin action. Second, insulin activates sterol regulatory element binding protein-1c (SREBP1c), a transcription factor that controls the expression of lipogenic genes (29, 30). Under the condition of hepatic insulin resistance, the FoxO1 pathway is not responsive to insulin suppression. Despite high insulin levels, the expression of hepatic gluconeogenic genes remains elevated. However, insulin is still capable of activating SREBP1c and inducing lipogenesis (30, 31). In the insulin-resistant state, there is compensatory hyperinsulinemia to overcome insulin resistance. The elevated plasma insulin is bound to increase lipogenesis at a level much higher than that under normal conditions, thereby causing elevated hepatic lipogenesis (31). The increased triglycerides are secreted in VLDL leading to hyperlipidemia. It is well known that HDL level is decreased in metabolic syndrome and T2DM patients. Although the precise mechanism remains to be elucidated, epidemiological studies have demonstrated an inverse relationship between plasma insulin and HDL levels (32–34). Consistent with these findings, HDL degradation rate is enhanced in T2DM patients (35). Thus, dyslipidemia is at least in part caused by insulin resistance and its resultant hyperinsulinemia. Dyslipidemia can further induce peripheral insulin resistance (36) since it leads to elevated lipid accumulation in muscle and adipose and impairment of insulin signaling (18). Although mounting evidence supports the notion that the gluconeogenic pathway is resistant to insulin action in T2DM and metabolic syndrome patients, a recent study indicates that the expression of hepatic PEPCK and G6Pase is not elevated in T2DM patients (37). This observation suggests that the failure of FoxO1 to respond to insulin action may not explain the increased gluconeogenesis under the insulin-resistant state. Rather, an alternative pathway may mediate hepatic insulin resistance. Recent work indicates that a transcription complex involving the cAMP response element binding protein (CREB), CREB binding protein (CBP), and transducer of regulated CREB activity 2 (TORC2) could play a role in connecting hepatic insulin resistance and increased gluconeogenesis (38). The CREB/TORC2/CBP complex controls the transcription of peroxisome proliferator-activated receptor g (PPARg) coactivator-1a (PGC-1a), which regulates the transcription of gluconeogenic genes. Since CREB is phosphorylated and constitutively occupies the PGC-1a promoter, the binding states of CBP and TORC2 determine the transcriptional activity of PGC-1a (38). Insulin negatively regulates the assembly of this complex. In the insulin-resistant state, the complex is more active leading to increased gluconeogenesis. This new finding helps further define the nature of hepatic insulin resistance and does not change the fact that augmented gluconeogenesis and dyslipdemia are the outcomes of hepatic insulin resistance. More details of hepatic insulin resistance are covered in Chapter 5.

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DEVELOPMENT OF HYPERTENSION Many studies have demonstrated the correlation of circulating insulin levels and blood pressure in obese individuals (39–41). In addition, healthy persons with hyperinsulinemia and normal glucose tolerance have increased blood pressure (42). Blood pressure elevation was not associated with body fat mass in obese women (43), and a decrease in blood pressure was not associated with a decrease in body fat (43). These findings suggest that insulin resistance is more likely related to hypertension than obesity, and hyperinsulinemia may be the link between hypertension and insulin resistance. After a period of chronic physical training, both systolic and diastolic blood pressure decreased in obese women (43), and these declines were associated with decreases in fasting insulin levels (43). Insulin resistance and glucose intolerance are not improved when hypertension is controlled using blood pressure lowering agents. However, improvement of insulin sensitivity and the resultant lowering of plasma insulin levels generally improve endothelial function and lower blood pressure. These observations suggest that hypertension may be a downstream disorder of insulin resistance, which may act through hyperinsulinemia and play a causal role in the development of hypertension. This, however, does not fully exclude the possibility that insulin resistance and hypertension can develop simultaneously under certain pathological conditions. And the progression of hypertension can be independent of insulin resistance and hyperinsulinemia. At the molecular level, insulin resistance can lead to hypertension through the regulatory effect on nitric oxide levels in endothelial cells. Nitric oxide is produced in vascular endothelial cells by endothelial nitric oxide synthase (eNOS) and regulates vasodilation. In insulin resistant state, there is reduced insulin-stimulated NO production in endothelial cells and increased NO destruction resulting in endothelial dysfunction and hypertension (44). A second mechanism for hypertension is also mediated by reduction of endothelial NO level. Mitochondrial dysfunction can occur in multiple tissues. In endothelial cells, it leads to elevated levels of reactive oxygen species (ROS), which decreases bioavailable NO resulting in endothelial dysfunction and hypertension (44). In the meantime, mitochondrial dysfunction in insulinsensitive tissues leads to insulin resistance (44, 45). In this case, hypertension develops in conjunction with insulin resistance but not through insulin resistance per se. Similarly, glucocorticoid excess can induce both insulin resistance and hypertension through separate mechanisms. Glucocorticoid excess induces insulin resistance by activating glucocorticoid receptor (GR) but it induces hypertension independently of insulin resistance by directly activating mineralocorticoid receptor (MR) (46).

MECHANISMS OF INSULIN RESISTANCE There are several potential mechanisms that may explain the development of insulin resistance. Although insulin resistance can occur in lean individuals, obesity, precisely central obesity, is closely correlated with insulin resistance in most subjects

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with metabolic syndrome. One hypothesis is that the elevated plasma FFAs in obese and T2DM patients are the primary cause of insulin resistance. First, increased FFAs can acutely induce insulin resistance (47). On the other hand, rapid reduction of FFAs by inhibiting lipolysis improves insulin sensitivity and glucose tolerance in lean and obese nondiabetic subjects and in obese patients with T2DM (48). These observations suggest that FFAs may contribute to the development of insulin resistance in subjects with metabolic syndrome. Compelling evidence suggests that increased circulating FFAs can induce insulin resistance in different ways. Intracellular accumulation of fatty acyl metabolites can induce insulin resistance by interfering with the insulin signaling pathway (18). Alternatively, the influx of FFAs from abdominal fat to liver can induce hepatic insulin resistance and augment glucose production (11). In a canine model with visceral adiposity promoted by high-fat diet, Bergman et al. demonstrated that hepatic insulin resistance occurred at a time when there was no peripheral insulin resistance (11, 49), suggesting that it is the portal FFAs from visceral fat that induced insulin resistance in liver before reaching the peripheral tissues. The peripheral insulin resistance is found mainly in adipose tissue and skeletal muscle. The common mechanism underlying insulin resistance in these tissues is the reduced ability of insulin to stimulate glucose uptake. Insulin-stimulated glucose uptake is mediated by glucose transporter 4 (GLUT4). In the insulin resistant state, there is a deficiency in insulin-stimulated GLUT4 translocation in the peripheral tissues. Shulman and colleagues demonstrated that this is caused by the impaired insulin signaling pathway in the cell (18). Elevated intracellular content of fatty acyl metabolites such as diacylglycerol (DAG) and fatty acyl-CoA due to hyperlipidemia and increased plasma FFAs activate a serine/threonine kinase pathway including protein kinase C (PKC) (18). These metabolites activate PKC leading to serine/ threonine phosphorylation of insulin receptor substrate 1 (IRS-1) and 2 (IRS-2), which inhibits insulin-stimulated tyrosine phosphorylation of these signaling molecules and reduces the ability of these molecules to activate phosphoinositide 3-kinase (PI3-kinase) (18). Insulin resistance can be induced by increased actions of proinflammatory cytokines and/or declined adipokine actions. It has been widely recognized that adipose tissue is not only a fat storage site but also an endocrine organ. Adipose tissue releases cytokines and adipokines that are involved in the regulation of insulin sensitivity. Recent research indicates that there is macrophage accumulation in adipose tissue as a result of increased fat cell apoptosis in obesity (5). In this regard, obesity is an inflammatory state where elevated cytokines such as TNFa and IL-6 promote insulin resistance. Activation of various inflammatory pathways leads to insulin resistance. This is supported by overwhelming evidence that inflammatory signals activate the c-Jun N-terminal protein kinase 1 (JNK1) and the inhibitor of NF-kB kinase-b (IKK-b), which interfere with the intracellular insulin signaling pathway (50, 51). In addition, increased adiposity is associated with reduced levels of adiponectin, an adipokine that increases insulin sensitivity by stimulating AMPK activation (52). Reduced adiponectin action in obesity may contribute to the development of insulin resistance. Other mechanisms that induce insulin resistance include

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glucocorticoid excess. In patients with Cushing’s syndrome, there is increased circulating cortisol level due to adrenal adenoma or elevated adrenocorticotropic hormone (ACTH) from pituitary tumors. Elevated circulating cortisol induces obesity and insulin resistance. As a result, patients with Cushing’s syndrome have central obesity, reduced subcutaneous fat, and hepatic steatosis. In addition, they exhibit severe insulin resistance along with hyperglycemia, hypertension, and dyslipidemia. These symptoms are typical of the constellation of the metabolic disturbances in metabolic syndrome. Given the remarkable resemblance of the set of disorders, these findings actually led to the hypothesis that metabolic syndrome could be caused by glucocorticoid excess. However, glucocorticoid excess is found only in a single percentage of T2DM patients, suggesting that glucocorticoid excess is not the primary cause of T2DM. Another mechanism that leads to insulin resistance is the age-related mitochondrial dysfunction. There is mounting evidence that as the body ages, the ability of mitochondria to oxidize substrates decreases. Increased lipid accumulation in cells leads to mitochondrial overload producing metabolites from incomplete FFA oxidation (53). As a result, partially oxidized products and reactive oxidative species are damaging to cells, which leads to insulin resistance (53).

DEVELOPMENT OF TYPE 2 DIABETES Although the development of diabetes is the result of incremental changes in glucose homeostasis, there are strict criteria for the definition of diabetes. Two tests can be used to diagnose diabetes. The blood glucose tests can be done under fasting condition. If an individual’s fasting glucose reaches 126 mg/dL or higher and confirmed thereafter, the individual is diagnosed to have diabetes. If the fasting glucose is between 100 and 125 mg/dL, it is considered impaired fasting glucose (IFG) or a prediabetes state. A second test is oral glucose tolerance test (OGTT). If the blood glucose level is between 140 and 199 mg/dL 2 h after taking 75 g oral glucose, the person has a form of prediabetes called impaired glucose tolerance (IGT). If the 2 h glucose level reaches 200 mg/dL or above and confirmed by repeating the test on another day, it means the person has diabetes. Both forms of prediabetic states implicate higher risk of developing diabetes. The manifestation of T2DM is marked by insulin resistance and b-cell failure. Before the onset of overt diabetes, individuals with obesity and insulin resistance have several risk factors for T2DM. The hepatic insulin resistance is characterized by increased gluconeogenesis and glucose output. In the meantime, the peripheral tissue insulin resistance impairs the ability of insulin to stimulate sufficient glucose uptake by these tissues, exemplified by decreased insulin-stimulated GLUT4 translocation. The insulin-resistant state is compensated by increased insulin secretion by the pancreatic b-cells. Increased insulin release can initially overcome the insulin resistance of peripheral tissues and sufficiently facilitate glucose transport. With the progression of insulin resistance, excessive insulin secretion cannot be maintained, at which point the insulin secretory capacity cannot keep up with the need to offset insulin resistance. Over time, b-cell exhaustion develops and further progresses into

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b-cell failure, at which stage hyperglycemia is further exacerbated to meet the criteria of diabetes. It is important to understand that the clinical course of the development of T2DM is progressive and the onset of the disease is a process of reduced b-cell mass. It is only at a point where b-cell failure leads to a certain degree of hyperglycemia that the diagnosis of T2DM can be made. At this point, the number of b-cells is reduced by about 50% (54), and the remaining cells operate at lower secretory capacity (55). IGT and IFG are two characteristics for the so-called prediabetic state, a condition in metabolic syndrome patients in transition to T2DM, depending on the state of b-cell health/failure. IGT and IFG are useful indicators/measures that may help predict the likelihood of T2DM manifestation. Prediabetes is an intermediate but alarming state prior to overt T2DM. It is associated with hyperglycemia but the glucose levels are not high enough to warrant diagnosis of diabetes. Compelling evidence suggests that increased cardiovascular risk factors at the prediabetic stage contribute to diabetic complications as the disease progresses into overt diabetes. Currently, there are 57 million people in the United States who have prediabetes. Most people with obesity and insulin resistance do not develop severe hyperglycemia or T2DM because the pancreatic b-cells increase insulin secretion sufficiently to overcome the reduced efficacy of insulin action. This observation underscores the importance of b-cell health in the pathogenesis of T2DM. Despite the fact that there are many proposed mechanisms for b-cell failure in T2DM patients, it is not clear what the major factor is. The decline in b-cell function is progressive just like the T2DM disease itself. Indeed, b-cell dysfunction exists in high-risk individuals even when their glucose levels are normal (56). It is widely accepted that in T2DM patients, glucotoxicity and lipotoxicity contribute significantly to b-cell dysfunction, loss of pancreatic b-cell mass, and eventually b-cell failure. Since insulin resistance is associated with hyperglycemia, elevated glucose levels could have toxic effects on b-cell function and these harmful effects can be mitigated by glucose lowering therapy (57). Under normal FFA levels, insulin secretion is not affected. However, chronic exposure of elevated FFAs to b-cells is associated with impaired insulin secretion and biosynthesis (58, 59). The lipotoxic effect is relevant because both hyperlipidemia and elevated FFA levels are observed in T2DM patients. Further, mitochondrial dysfunction in pancreatic b-cells may impair the capacity of b-cells to secrete insulin because of the importance of ATP/ADP ratio in insulin secretion (45).

MECHANISMS OF INCREASED CARDIOVASCULAR RISK IN T2DM Both T2DM patients and nondiabetic subjects with metabolic syndrome have increased risk of developing macrovascular complications, including coronary heart disease, stroke, and peripheral vascular disease, which may share the common pathogenic features of atherosclerosis, inflammation, and the prothrombotic state. In addition, microvascular complications, including retinopathy, nephropathy, and neuropathy, are commonly found in patients with late-stage T2DM. Hyperglycemia,

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hyperinsulinemia, and other metabolic disorders are major contributors to these metabolic consequences. There are several underlying mechanisms that are associated with the increased cardiovascular risk in individuals with T2DM and metabolic syndrome. These mechanisms are not only promoted directly by several elements of metabolic syndrome, such as hypertriglyceridemia, high LDL, low HDL, and hypertension, but also promoted by common metabolic disturbances such as oxidative stress, inflammation, endothelial dysfunction, vascular smooth muscle proliferation, and increased prothrombotic state. Some of these disturbances form the basis for the development of atherosclerosis. Hyperglycemia promotes vascular complications by inducing the expression of adhesion molecules in endothelial cells (60). Through the adhesion molecules, monocytes are recruited to the vascular wall and are involved in the pathogenesis of atherosclerotic lesions. Advanced glycation end products (AGEs) formed in the hyperglycemic state generate reactive oxygen species that can damage the vascular wall (61). In the meantime, both elevated glucose and AGE inhibit nitric oxide production by endothelial cells (62, 63), leading to impaired endothelial relaxation and vascular injury. Endothelial damage promotes the entry of activated monocytes into the subendothelial space, which secretes proinflammatory cytokines and differentiates into macrophages. Increased lipid uptake by macrophages results in the formation of foams cells, the hallmark of atherosclerosis. The development of atherosclerosis also involves proliferation and migration of vascular smooth muscle cells in concert with inflammation and apoptosis. In particular, the proinflammatory cytokines within atheroma direct leukocyte migration into the intima and induce lipid uptake by macrophages (64). C-reactive protein (CRP), an important biomarker and predictor for inflammation, is strongly correlated with the risk of atherosclerotic complications (64). The prothrombotic state in the atherosclerotic process involves several important molecules. PAI-1, fibrinogen, and von Willebrand factor (vWF), which are important markers of hemostasis and fibrinolysis, are associated with abdominal obesity and can be used as potential predictors for cardiovascular risk (65). Plasma PAI-1 levels are elevated in individuals with metabolic syndrome and are more closely related to liver steatosis than adipose tissue accumulation, although it is highly expressed in both visceral and subcutaneous adipose tissue (66). PAI-1 is also regulated by inflammatory signals. CRP increases PAI-1 expression in endothelial cells (67), suggesting that increased inflammation could induce the risk of thrombosis. Given the role of hyperglycemia in endothelial dysfunction, antidiabetic treatments may help reduce cardiovascular risks. In the UKPDS, metformin treatment was associated with a 39% reduction in cardiovascular disease (68). Insulin and SU treatments, although achieved similar glycemic control, exhibited trends of improvement of cardiovascular outcomes but statistical significance was not reached (68). Therapies that correct dyslipidemia, including LDL lowering and HDL raising agents, are effective approaches to improve cardiovascular outcomes. Typically used drugs are statins for lowering LDL, fibrates for reducing triglycerides, and niacin for raising HDL. In addition, antihypertensives and aspirin, which is used to reduce vascular inflammation, are commonly used. Weight loss has been an effective approach to mitigate multiple risk factors and decrease cardiovascular risk.

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ETHNIC VARIATIONS IN THE DEVELOPMENT OF METABOLIC ABNORMALITIES Studies involving individuals with different ethnic backgrounds indicate that certain ethnic groups are more susceptible to metabolic disorders than others. It is well known that Pima Indians have much higher risks of developing obesity and T2DM than other ethnic groups. They have inheritable genetic traits that predispose individuals to obesity and insulin resistance. In a study involving 384 Pima Indians with IGT, it was found that the cumulative incidence of T2DM was 25% and 61% at 5 and 10 years, respectively (69). While the high prevalence of obesity in Pima Indians is a risk factor for insulin resistance, other metabolic disturbances may also contribute to insulin resistance because Pima Indians were 17% more insulin resistant than Caucasians after accounting for differences in the degree of obesity (70). Pima Indians also have exaggerated early insulin response even after accounting for differences in insulin action, suggesting that the altered insulin secretion cannot be explained by insulin resistance (70). The enhanced b-cell sensitivity to glucose may play a role in the predisposition of this ethnic group to T2DM (70). However, decreased early insulin response could be a good predictor for the development of T2DM. For example, Mexican Americans have higher degree of insulin resistance and fasting hyperinsulinemia than Caucasians. In contrast to Pima Indians, they have decreased early insulin response to glucose excursion (71). It is not clear why opposite early insulin response patterns contribute to the risk of diabetes in these two high-risk ethnic groups. Other ethnic groups such as African-Americans and Hispanics have several fold elevated risk of developing T2DM compared to non-Hispanic Whites. Both nondiabetic African-Americans and Hispanics have increased insulin resistance and higher acute insulin response than nondiabetic non-Hispanic Whites (72). These alterations may be in large part responsible for the higher prevalence of T2DM in these minority groups (72). In a study comparing South Asians settled overseas with a European group, the South Asian group had several times higher prevalence of diabetes (19% versus 4%) than the European group (73). Moreover, they had higher blood pressures, higher fasting and post-glucose serum insulin concentrations, higher plasma triglyceride levels, and lower HDL concentrations (73). Ethnic differences are also associated with increased cardiovascular risk. Asian Indians have at least double the risk of coronary artery disease compared to Whites. This increased risk appears to be due to dyslipidemia characterized by plasma apolipoprotein B, lipoprotein(a), and triglycerides and low HDL levels. In addition, there is dysfunction in the small, dense HDL particles found in this population (74). In England and Wales, high mortality rate from CVD was found in all the migrant groups from South Asia, including Hindus and Sikhs from India and Muslims from Pakistan and Bangladesh (75, 76). Although Pima Indians have higher prevalence of developing T2DM, they have unique lipoprotein profiles. The plasma triglyceride levels in Pimas are higher than those in Europeans but cholesterol is lower (77). This could be due to differences in lipoprotein metabolism in Pimas. Interestingly, after accounting for the differences in the prevalence of T2DM, the mortality rates from

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CVD in Pimas are lower than those in Europeans (78). This could be due to the lower LDL concentrations in Pimas (79).

GENETIC MUTATIONS AS A RISK FACTOR In addition to the general risk factors such as excessive energy intake and sedentary lifestyles, genetics also plays a role in the development of metabolic abnormalities. Although T2DM in the general population is a polygenic disease, loss or gain of function in a single metabolic gene caused by mutations can increase the risks of insulin resistance and T2DM. The most common examples in which genetic mutations cause metabolic diseases are maturity onset diabetes of the young (MODY). There are eight types of MODYs corresponding to mutations in eight different metabolic genes (80). In MODY1, a loss-of-function mutation in HNF4a, a transcription factor that is involved in the regulation of pancreatic development, causes b-cell dysfunction (80). In MODY2, gain-of-function mutations in glucokinase activate the glucose sensor activity and increase insulin secretion, leading to hyperinsulinemic hypoglycemia (80). There are six additional types of MODYs involving other genes that may be important in the metabolic functions (80). Many studies have demonstrated that single nucleotide polymorphisms (SNPs) in certain genes predispose individuals to higher risks of obesity, insulin resistance, and T2DM. For example, Pima Indians are an ethnic group with increased risks of developing obesity and T2DM, and research efforts have identified a number of SNPs that may help explain the high prevalence of T2DM. These include SNPs in genes such as the Cav2.3 subunit of voltage-activated Ca2þ channels (81), 11b-hydroxysteroid dehydrogenase (11b-HSD1) (82), uncoupling protein-2 (UCP2) (83), and the activating transcription factor 6 gene (ATF6) (84). Using the same approach, many SNPs in a variety of other genes have been identified to be linked to risks of obesity and T2DM. The Pro12Ala polymorphism in PPARg is associated with decreased risk of T2DM (85). Strong linkage of the transcription factor 7-like 2 gene (TCF7L2) to susceptibility of T2DM has been demonstrated through SNP analysis. TCF7L2 is a transcription factor involved in the WNT signaling pathway and acts as a nuclear receptor for b-catenin (80). The strong association of TCF7L2 with T2DM has been demonstrated in populations of both European and Chinese ancestry (86, 87). Recent successes in genome-wide association (GWA) studies allow precise typing of a large number of SNPs and help capture many more genetic variants present in the human genome (80).

SUMMARY Metabolic syndrome is a group of metabolic disorders that are commonly found in subjects with increased T2DM and CVD risks. These disorders are associated with each other but the pathogenesis is not clear. Specifically, there is no single underlying mechanism that can explain the development of these metabolic abnormalities despite the fact that they cluster in the same patients. Recent evidence suggests that insulin

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Excessive energy intake Sedentary lifestyles

Other unknown factors (important to β-cell health)

Glucotoxicity and lipotoxicity

Genetic predisposition and susceptibility

Metabolic syndrome • Central obesity • Insulin resistance • Dyslipidemia • Hypertension • Hyperglycemia Additional abnormalities: • Increased inflammatory state • Endothelial dysfunction • Prothrombotic state • Microalbuminuria • Atherosclerosis

β-cell failure

T2DM

Microvascular complications

Figure 7.1

CVD Stroke Peripheral vascular disease

Macrovascular complications

The development of metabolic syndrome, T2DM, and downstream complications. Among the variety of metabolic abnormalities discovered in metabolic syndrome, peripheral insulin resistance in combination with b-cell failure leads to T2DM. Both T2DM and metabolic syndrome lead to vascular diseases but T2DM is a more important and frequent cause than metabolic syndrome (highlighted by a thick arrow). Due to the lack of specific mechanisms, this figure represents only the general relationships of the disorders on the conceptual level.

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resistance might play a central and causal role in the development of dyslipidemia, hypertension, and hyperglycemia. Obesity is a major risk factor in the development of insulin resistance in general but insulin resistance can occur in lean individuals. Although some of the interrelationships between various metabolic dysfunctions have been proposed based on available evidence to date (Figure 7.1), mechanistic understanding of these links remains elusive. An important puzzle in understanding the development of T2DM is why some individuals with obesity and insulin resistance do not develop b-cell failure and hence do not have diabetes. Future studies should focus on dissecting molecular mechanisms that link the risk factors of metabolic syndrome and approaches to enhance the effectiveness in disease prevention, treatment, and management.

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8

Emerging Therapeutic Approaches for Dyslipidemias Associated with High LDL and Low HDL MARGRIT SCHWARZ1 1 2

AND JAE

B. KIM2

Department of Metabolic Disorders, Amgen, Inc., South San Francisco, CA, USA Global Development, Amgen, Inc., Thousand Oaks, CA, USA

INTRODUCTION Dyslipidemia is defined as a disorder of lipoprotein metabolism and is a major independent risk factor for the development of atherosclerosis and cardiovascular disease (CVD). Perturbations of lipoprotein metabolism, including lipoprotein overproduction or deficiency, are also associated with other morbid components of metabolic syndrome such as type 2 diabetes and obesity. With the preponderance of data supporting a causal link between elevated levels of low-density lipoprotein cholesterol (LDL-C) and increased incidence of CVD, contemporary therapeutic interventions are primarily focused on lowering LDL-C. Decreasing cholesterol synthesis via inhibition of HMG-CoA reductase (statins) is the therapy of choice for lowering LDL-C, but the need for safely attaining ever more aggressive lipid management goals recommended in recent clinical guidelines has engendered several novel therapeutic approaches based on new and discrete mechanisms of action. Many of these approaches, as described in this chapter, are now at various stages of clinical development. Albeit most dyslipidemias are characterized by elevations of lipids carried by lipoproteins in the blood, the spectrum of lipoprotein disorders is far more diverse and may be perturbed by diet and lifestyle in addition to mutations in certain

Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang Copyright
2011 John Wiley & Sons, Inc.

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genes involved in lipoprotein metabolism. Certain types of dyslipidemias are characterized by reduced levels of high-density lipoprotein cholesterol (HDL-C). An inverse correlation between CVD and the levels of cholesterol carried in HDL fractions has been observed in epidemiological studies; however, clinical proof of concept for a causal link has yet to be established. There is currently no marketed pharmacological intervention designed to specifically raise HDL-C, but the clinical research in this arena is highly active and provocative. This chapter attempts to briefly summarize derangements of lipoprotein metabolism and their metabolic consequences, as well as discuss emerging therapeutic strategies that target various components of lipoprotein production, transport, and clearance.

LIPOPROTEIN METABOLISM There have been numerous advances in the understanding of lipoprotein metabolism, function, and interactions over the past several decades. A large number of metabolic roles have been identified for apolipoproteins (apos), the principal protein moieties of lipoproteins, in addition to being cofactors that mediate clearance of lipids by their target tissues. A brief review of the major lipoprotein particles and their constituent molecules in this section is helpful in understanding the various lipid disorders, their relation to atherosclerotic disease, and potential therapeutic opportunities. Lipoproteins are biochemical entities carrying both lipids and proteins and have been classified by centrifugal separation characteristics (size and density), apo content, and the types of lipids they transport (1). A schematic overview of lipoprotein metabolism pathways is presented in Figure 8.1. Chylomicrons (density
0.95 g/mL) are very large particles that carry dietary (exogenous) cholesterol and triglycerides. They are formed in the enterocytes of the small intestine where they incorporate cholesterol and triglycerides absorbed from the gut with apoB48, and to a lesser extent apoC2 and apoE. The intestinal (apoB48) and hepatic isoforms (apoB100) of human apoB are encoded in a single gene and arise from differential splicing of the same primary APOB mRNA transcript, with apoB48 representing the amino-terminal 47% of apoB100 (2, 3). ApoB48 does not bind to the hepatic receptor for low-density lipoprotein (LDLR). Therefore, chylomicrons are not removed from the circulation where they are delivered via the lymphatic system; rather, they are acted upon by various lipoprotein lipases (LPLs) that hydrolyze the triglyceride core and release free fatty acids. The resulting chylomicron remnants are then cleared by the liver. Very-low-density lipoproteins (VLDLs; density 0.95–1.006 g/mL) are associated with apoB100, apoC2, and apoE and carry endogenous triglycerides and a small amount of cholesterol. VLDLs are synthesized and secreted by the liver, and their formation is controlled by the amount of lipids and apoB available in the hepatocyte, as well as by the enzymatic activities of acylCoA acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP). Once released into the plasma, VLDL can also serve as a substrate for LPLs, releasing free fatty acids and yielding intermediate-density lipoproteins (IDLs), which are either cleared by the liver or acted upon by hepatic lipase (HL) to form

B

B

B LPL

IDL HL

ox LDL

E

LPL

VLDL E

C2

C2

C2

LPL

LDL CD36 LOX1 SR-A

Remnant

B

B

E

Chylomicron CETP

LDL-R

Cholesterol pool

MTP ACAT

LRP

Liver

HDL LCAT ABCA1 ABCG1 SR-BI

Macrophage

HDL

A1 A1

Nascent HDL

Mature HDL

HDL

SR-BI

Small intestine

Fecal excretion

Figure 8.1 Overview of lipoprotein metabolism. Chylomicrons are formed in the enterocytes of the small intestine, incorporating cholesterol and triglycerides

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absorbed from the gut with apoB. LPLs hydrolyze their triglyceride core and release free fatty acids. The resulting remnants are cleared by the liver. VLDLs are associated with apoB, apoC2, and apoE and carry endogenous triglycerides and cholesterol. Synthesized and secreted by the liver, their formation is regulated by MTP and ACAT. LPLs release free fatty acids from VLDLs, yielding IDLs, which are converted to LDLs by the action of HL. LDLs primarily transport cholesteryl esters and some triglycerides and are being cleared by the hepatic LDLR. A portion of LDL is converted to oxLDL and taken up by macrophages via CD36, LOX-1, or SR-A, contributing to the formation of atherogenic foam cells. HDLs contain apoA1 and phospholipids, and are formed in the liver and small intestine. Lipid-poor apoA1 interacts with ABCA1, ABCG1, and SR-B1 and accepts free cholesterol from peripheral tissues to form nascent HDL. These are remodeled by LCAT to yield mature, cholesteryl ester-rich HDL particles. Mature HDLs are converted to IDL and VLDL by CETP-mediated exchange of cholesteryl esters for triglycerides and cleared via the hepatic LDLR pathway or via SR-B1. The HDL-mediated flux of cholesterol from peripheral tissues into the plasma and to the liver for clearance is known as RCT and is thought to form the mechanistic basis for the antiatherogenic properties of HDL.

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low-density lipoproteins (LDLs; density 1.006–1.019 g/mL). Small molecule inhibitors of ACAT and MTP as well as antisense oligonucleotides targeting APOB are being developed as pharmacological interventions for decreasing plasma levels of LDL via this pathway. LDL particles contain apoB100, the main ligand for the LDLR, and primarily transport cholesteryl esters along with some triglycerides. Because the liver is the principal organ responsible for the clearance of LDL, plasma LDL-C levels are directly determined by the activity of the hepatic LDLR in most species including humans. Upon binding to the hepatic LDLR, LDL is endocytosed and transported to endosomes where it dissociates from the receptor and is subsequently catabolized in lysosomes. The endosomal LDLR, however, recycles back to the cell surface. Regulating the synthesis, expression, or recycling rate of the hepatic LDLR has been the primary target of contemporary pharmacological interventions such as statins, and more recently, inhibitors of proprotein convertase subtilisin-like kexin type 9 (PCSK9). Finally, high-density lipoproteins (HDLs; density 1.063–1.21 g/mL) are small particles containing apoA1 and phospholipids and are formed in the liver and small intestine. Lipid-poor apoA1 interacts with cell surface cholesterol transporters expressed on macrophages and other tissues, specifically ATP-binding cassette (ABC) transporters A1 (ABCA1) and G1 (ABCG1) and scavenger receptor class B, type 1 (SRB-1). ApoA1 accepts free cholesterol to form nascent HDL particles, which are subsequently remodeled by lecithin–cholesterol acyltransferase (LCAT) to yield mature, cholesteryl ester-rich HDL particles. Mature HDL particles are converted to IDL and VLDL by cholesterol ester transfer protein (CETP)-mediated exchange of cholesteryl esters for triglycerides, and then cleared via the hepatic LDLR pathway. HDL can also be taken up directly by the liver via hepatic SRB-1; however, the quantitative contribution of this pathway varies substantially from species to species, and it is generally believed that the CETP-mediated pathway is the main route of HDL clearance in humans. The HDL-mediated flux of cholesterol from peripheral tissues into the plasma and to the liver for clearance is known as reverse cholesterol transport (RCT) and is thought to form the mechanistic basis for the antiatherogenic properties of HDL. Pharmacological approaches aimed at inhibiting CETP, activating LCAT, or mimicking the activity of apoA1 seek to exploit this hypothesized link between RCT and reversal of atherosclerosis.

THE ROLE OF LIPOPROTEINS IN ATHEROGENESIS The metabolism of plasma lipoproteins is highly interrelated and forms a complex network designed to maintain lipid homeostasis and protect against vascular inflammation. The “lipid hypothesis” asserts that an imbalance in any aspect of this network attributable to dysfunctional formation, transport, or clearance of cholesterol-carrying lipoprotein particles is highly causal for the formation of atherosclerosis in vascular walls. Specifically, retention of LDL in the subendothelial space of the vasculature is thought to be the major initiating factor for atheromas. This was first proposed by Virchow in 1856 (4) and supported by early studies in rabbits (5). Since then, numerous

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studies have shown that the atherosclerosis process can be recapitulated in animal models by modifying the concentrations of lipids carried in lipoproteins either genetically or by increasing dietary cholesterol, in the absence of any other risk factor. In addition, numerous epidemiological, genetic, and pharmacological studies have successfully demonstrated a direct link between plasma LDL cholesterol levels and the incidence of atherosclerotic CVD in humans. Taken together, this research has resulted in the general acceptance of the lipid hypothesis by much of the scientific community (6). LDL is the major cholesterol-carrying lipoprotein and is the only lipoprotein fraction markedly elevated in the genetic disorder known as familial hypercholesterolemia (FH), the most thoroughly investigated lipoprotein disorder (7). As such, LDLC lowering has emerged as the primary goal for therapeutic intervention in the treatment of hyperlipidemias and CVD. It is difficult to argue against the overwhelming evidence provided by millions of patients who respond to LDL-C lowering agents with a decreased incidence of CVD. However, the “cholesterol controversy” (8) is still fueled by the fact that despite the wide use of statins, CVD remains the underlying cause of >35% of deaths in the United States (9). Clearly, some patients are exposed to residual CVD risk that is independent of their blood cholesterol levels. A growing body of evidence suggests that molecular events downstream of subendothelial LDL retention are also major contributors to plaque formation and growth, a hypothesis first put forward by Ross and Glomset in 1973 (10). The theory of atherosclerosis as an inflammatory disease has since gained support, and this does not necessarily contradict the lipid hypothesis (11). Endothelial injury and deposition of LDL in the subendothelial space both promote local inflammation, leading to the release of signaling molecules and the expression of endothelial cell adhesion molecules, which in turn recruit monocytes to the arterial wall (12). These monocytes differentiate into macrophages—a process driven by cytokines, interaction with the extracellular matrix, and the upregulation of scavenger receptors capable of binding and internalizing oxidized forms of LDL (oxLDL). Because these scavenger receptors are not subject to the same feedback regulations as the hepatic LDLR, oxLDL accumulates in macrophages and leads to the formation of foam cells (13, 14). However, oxLDL has also been shown to independently promote proinflammatory cytokine release and platelet aggregation, both of which further contribute to atherosclerotic lesion formation. Together, these processes result in the growth and destabilization of plaques, ultimately leading to plaque rupture and thrombosis, which in turn are directly linked to acute cardiovascular events such as stroke and myocardial infarction. In the context of LDL atherogenicity, Lp(a) lipoprotein, a macromolecule consisting of an LDL-like particle covalently linked to a specific apolipoprotein (a), may play an important role. Lp(a) was discovered in 1963 (15), and the human gene (LPA) encoding Lp(a) was cloned in 1987, revealing homology to plasminogen (16). LPA polymorphisms associated with high Lp(a) levels confer an increased risk of coronary artery disease (17, 18), but the physiological function of Lp(a) is still not well understood. Clinical studies have identified a strong correlation between Lp (a) levels and oxLDL, suggesting that the atherogenicity of Lp(a) may be mediated in part by associated proinflammatory oxidized phospholipids (19). In addition, its homology to plasminogen suggest a function of Lp(a) within the coagulation

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system (20). Because the causality of the link between Lp(a) function and heart disease has not been clearly proven, Lp(a) is not yet accepted as a well-established risk factor for cardiovascular disease. In contrast to LDL, HDL is thought to play an atheroprotective role, which was first postulated based on the observation that differences in HDL levels between men and premenopausal women correlate with differences in age-adjusted risk of CVD (21). These initial observations have been firmly validated in large epidemiological studies (22). HDL is thought to exert its atheroprotective effect by mediating RCT from vessel wall macrophages back to the plasma, and ultimately to the liver for catabolism and secretion, a hypothesis developed decades ago by Glomset based on his studies of LCAT (23). Today, the RCT hypothesis is generally well accepted, although the precise mechanism of in vivo RCT is still being debated (24, 25). HDL possesses other biological activities that may contribute to its antiatherogenic properties, such as the ability to prevent LDL oxidation and to inhibit inflammation (26, 27). Much of the recent research in the field has focused on understanding the heterogeneity of HDL particles and the functional role of its protein components. Certain subspecies of HDL acquire and carry proteins that may either protect or harm the artery wall, and oxidative damage inflicted by enzymes such as myeloperoxidase may generate “dysfunctional” HDL characterized by the loss of its antiatherogenic properties (28). Analyzing the HDL proteome from patients with established CVD and comparing it to that of healthy controls will help elucidate RCT-independent protective properties of HDL (29). In the meantime, the focus of therapeutic strategies that target HDL is shifting from a definition of efficacy based upon simply raising circulating HDL-C to a more comprehensive definition encompassing fundamental properties of HDL heterogeneity and functionality.

ETIOLOGY AND CLASSIFICATION OF DYSLIPIDEMIAS Dyslipidemia is a broad term that refers to a range of lipoprotein disorders. The original classification of dyslipidemias by Fredrickson is based on the pattern of lipoproteins obtained with electrophoresis or ultracentrifugation (30). The Fredricksen classification has been adopted by the World Health Organization (WHO) and remains a popular system mostly because of its historical relevance, but its prognostic value and clinical utility are limited. Conceptually, dyslipidemias can be classified as primary (i.e., caused by a genetic defect) or secondary, when they arise as a consequence of diet, metabolic or endocrine disorders, or medication (31, 32). The most common conditions associated with secondary dyslipidemia are elevated blood pressure, impaired glucose tolerance, and increased visceral fat—a combination of metabolic abnormalities seen in patients with the metabolic syndrome (33, 34). Genetic lipoprotein disorders are summarized in Table 8.1. They can affect any of the lipoprotein classes; however, LDL-dominant lipid disorders such as FH are the most important from a clinical perspective. FHs are autosomal dominant disorders that affect approximately 1 in 500 individuals (7). They are characterized by significantly elevated LDL-C, usually to levels well above 190 and up to 1000 mg/dL, and require

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Etiology and Classification of Dyslipidemias Table 8.1

Genetic Lipoprotein Disorders

Disorder

Triglyceride-rich lipoproteins Lipoprotein lipase deficiency APOC2 deficiency Abetalipoproteinemia Remnant lipoproteins Dysbetalipoproteinemia III Hepatic lipase deficiency LDL particles Familial hypercholesterolemia Familial defective apoB100 Autosomal dominant hypercholesterolemia Autosomal recessive hypercholesterolemia Hypobetalipoproteinemia Familial sitosterolemia Familial Lp(a) hyperlipoproteinemia HDL particles ApoA1 deficiency Tangier disease Familial LCAT deficiency CETP deficiency

Gene

LPL APOC2 MTP

Effects on LDL-C

Effects on HDL-C

Effects on TG

#

## ## $

""" """ #

#

""

APOE HL LDLR APOB PCSK9

"" "" ""

ARH

"

APOB ABCG5/G8 APO(a)

# $ $

APOA1 ABCA1 LCAT CETP

## ## ## "

Adapted from Ref. 31.

aggressive treatment with LDL-C lowering drugs. FH can be caused by defects in the LDLR gene, a defective APOB gene, or missense mutations in PCSK9. In addition, a defect in the gene encoding the LDLR adapter protein 1 (ARH) has been linked to autosomal recessive forms of hypercholesterolemia (HC) (7). Triglyceride-dominant lipid disorders can arise as a consequence of defects in LPL, APOC2, APOA5, or MTP. These disorders are characterized by moderate to severe elevations of plasma triglycerides (up to 1000 mg/dL), and are usually associated with low levels of HDL-C. In addition to presenting an atherogenic risk, severe hypertriglyceridemias can lead to pancreatitis. Disorders characterized by low HDL (hypoalphalipoproteinemia) are caused by defects in APOA1, ABCA1 (Tangier disease), LCAT, or CETP. Affected individuals have HDL-C levels below 20 mg/dL and, in some cases, an increased risk of premature CVD. In addition, a large percentage of patients present with mixed dyslipidemia, characterized by elevated triglycerides, low HDL-C, and the presence of small, dense LDL particles. The causes can be primary or secondary, or both.

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EPIDEMIOLOGY The prevalence of dyslipidemia is very high and rising worldwide, with an estimated 50–70% of the U.S. adult population affected (9). Dyslipidemia is a major risk factor for developing CVD, and the WHO estimates that globally it is associated with more than half of all cases of ischemic heart disease, and more than 4 million deaths per year. Of the patients diagnosed with dyslipidemia, 26% are classified as having low risk for CVD, 46% as having moderate risk, and 28% as having high risk. Undertreatment for dyslipidemia is highly prevalent, due to factors that include insufficient detection, underprescribing, and low compliance. High LDL-C (>130 mg/dL) accounts for one-third of all specific dyslipidemia cases, and dyslipidemia associated with low HDL-C (