and Drug Therapies
Amgen, Inc., Thousand Oaks, California, USA
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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]
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
Part One 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
2. Central Glucose Sensing and Control of Food Intake and Energy
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
6. Energy Metabolism in Skeletal Muscle and its Link to Insulin
Part Two Metabolic Diseases and Current Therapies
7. Mechanisms and Complications of Metabolic Syndrome 179
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
Part Three Drug Targets for Antidiabetic Therapies
11. GLP-1 Biology, Signaling Mechanisms, Physiology, and Clinical
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 391
16. 11b-Hydroxysteroid Dehydrogenase Type 1 as a Therapeutic
Target for Type 2 Diabetes 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
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 cardio-
vascular 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
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.
Remy Burcelin, Rangueil Institute of Molecular Medicine, INSERM U858,
Cendrine Cabou, Rangueil Institute of Molecular Medicine, INSERM U858,
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 Laborato-
ries, Lilly Corporate Center, Indianapolis, IN, USA
Shobha H. Ganji, Department of Veterans Affairs Healthcare System, Atheroscle-
rosis Research Center, Long Beach, CA, USA; Department of Medicine,
University of California, Irvine, CA, USA
Pierre Gourdy, Rangueil Institute of Molecular Medicine, INSERM U858, Tou-
Wei Gu, Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA,
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,
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
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, Atheroscle-
rosis 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,
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,
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 Labora-
tories, 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,
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,
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
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,
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,
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
The Physiology of
Under Normal and
Gut as an Endocrine Organ:
the Role of Nutrient Sensing
in Energy Metabolism
Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA
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 duode-
num (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 subse-
quently 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 Ó 2011 John Wiley Sons, Inc.
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
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 gluconeo-
genesis 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
FOOD INTAKE AND NUTRIENT-SENSING SYSTEMS
IN THE GI TRACT
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
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
to the release of GI peptides, which subsequently act on the stomach and the CNS to
4 Chapter 1 Gut as an Endocrine Organ
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 short-
term 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
Food (carbohydrates, fat, protein, etc.)
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.
Food Intake and Nutrient-Sensing Systems in the GI Tract 5
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.)infusionoflipids didnotaffect foodintake (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, admin-
istration 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 robustsecretion 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 administra-
tion (21). Peptones also induced CCK secretion and transcription in STC-1 cells, an
established L cell line (22, 23). Peptones made from both albumin egghydrolysate 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
6 Chapter 1 Gut as an Endocrine Organ
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-depen-
dent 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 sweet-
eners (35). The activity was inhibited by the sweet taste inhibitor lactisole (35).
These data indicatethat the T1R2/T1R3 complex mediates sweetsensation alongwith
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
Molecular Mechanisms of Nutrient Sensing 7
Closure of KATPchannels
Opening of voltage-
Secretion of GI peptides
CaR, GPR93, and others
FFAs Amino acids and peptides
G proteins (Gs or Gq)
Secretion of GI peptides
Secretion of GI peptides
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.
the T1R class of GPCRsare 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 compo-
nents 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 secre-
tion (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 cotran-
sporter 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
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
Molecular Mechanisms of Nutrient Sensing 9
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 glucose-
dependent insulin secretion (46). This machinery includes components such as ATP-
sensitive potassium (KATP) channels and glucokinase (Figure 1.2). In this pathway,
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
accumulation of intracellular Ca2 þ
, which triggers insulin release. Both the KATP
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 glu-
cose-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 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
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 SGLTinhibitor
compound (48). Moreover, the EC50 value of glucose for glucose-induced GLP-1
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 (Fig-
ure 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
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).
10 Chapter 1 Gut as an Endocrine Organ
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
, aromatic amino acids had no effect on CaR-mediated signaling (52), suggest-
ing 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
lization assay (52). In addition, L-phenylalanine can activate CCK secretion, pre-
sumably 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-depen-
dent 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 long-
chain FFAs (60). Activation of GPR120 both in vitro and in vivo led to increased GLP-
1 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 oleoylethanola-
mide (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 long-
chain 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
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
Molecular Mechanisms of Nutrient Sensing 11
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 cardiopro-
tective. 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
GIP is a 42-amino acid secreted peptide initially isolated from intestinal mucosa.
It was named gastric inhibitory peptide but later renamed glucose-dependent insu-
linotropic 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
12 Chapter 1 Gut as an Endocrine Organ
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 neuropep-
tides (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 proglu-
cagon 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. Somato-
statin-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,
Regulation of Incretin Secretion 13
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 GLP-
1 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 concentra-
tions of infused GIP was completely abrogated with selective hepatic branch
14 Chapter 1 Gut as an Endocrine Organ
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 beenvalidated 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 contributeto the disease pathogenesis. If this is one of
the major causes of the defective glucose homeostasis in type 2 diabetes, adminis-
tration 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.
Other GI Peptide Hormones or Neurotransmitters 15
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 PYYare 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).
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). More-
over, 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 CCK-
58 (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
16 Chapter 1 Gut as an Endocrine Organ
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, oxyntomo-
dulin 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 dem-
onstrated that dual activation of both GLP-1 and glucagon receptors with oxynto-
modulin- 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 ErbB-
dependent 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
The Physiological Importance of the Gut: Lessons Learned from Gastric Bypass 17
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. Late-
onset 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 PYYare 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 sur-
gery (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
Bypassed section transports bile and
pancreatic fluid into the gut
The upper portion is
separated from the
Figure 1.3 Illustration of Roux-en-Y gastric bypass.
18 Chapter 1 Gut as an Endocrine Organ
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
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 Goto-
Kakizaki (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 gluco-
neogenesis in the distal intestine post DJB but not gastric banding (160), and the
increased local glucose concentration is detected by a GLUT2-dependent hepato-
portal 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
The Physiological Importance of the Gut: Lessons Learned from Gastric Bypass 19
play important roles via distinct mechanisms to achieve beneﬁcial metabolic effects
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 assim-
ilation. 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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28 Chapter 1 Gut as an Endocrine Organ
Central Glucose Sensing
and Control of Food Intake
and Energy Homeostasis
AND BERNARD THORENS
Department of Physiology, University of Lausanne, Lausanne, Switzerland
Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland
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
/glucose cotransporter SGLT1 and the translocation of the glucose transporter
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 Ó 2011 John Wiley Sons, Inc.
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 counter-
regulation; termination of food intake; and stimulation of ﬁrst-phase insulin secre-
tion (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: glucose-
excited (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 glucose-
inhibited 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 Bernardﬁrstimplicatedthebrainsteminglucosehomeostasiswhen 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 cellularglucoprivation (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 nu-
clei (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), havealsobeen identiﬁedin the Arc (35, 42).
30 Chapter 2 Central Glucose Sensing and Control of Food Intake
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
channels. The inﬂux of calcium then triggers insulin secre-
tion. 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
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 charac-
terized (Figure 2.1b). However, the effect of glucose on GI neurons may be controlled
by changes in Naþ
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
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 offeeding behavior and energy expenditure by CNS is a complex
process that involves hypothalamic and brainstem neuronal circuits. In the hypo-
thalamus, 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
Physiological Functions Modulated by Central Glucodetection 31
Classical model Alternative models
Classical model Alternative models
Figure 2.1 Models for the control of the electrical activity of GE and GI neurons by glucose. (a)
Glucose activation ofﬁring 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
, 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
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
channels and/or a reduction in the activity of the Naþ
ATPase, plasma depolarization, and the
entry of Ca2þ
that triggers the secretion of neurotransmitters. Alternatively, a new pathway involving
metabolism, possibly through interaction with a putative speciﬁc receptor. KC: Kþ
voltage-dependent calcium channel.
32 Chapter 2 Central Glucose Sensing and Control of Food Intake
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 NPYin the
Arc (73, 74).
When blood glucose concentrations fall below the euglycemic level, a rapid counter-
regulatory 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
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 decere-
brated 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.
Multiplicity of Sensing Mechanisms 33
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
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 neu-
rons (89). Homozygous Glut3 knockout mice die during embryogenesis whereas
heterozygous knockout mice have normal glucose homeostasis and feeding behav-
ior (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 homeosta-
sis (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
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 local-
ization 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 Glut2À/À
express a transgenic glucose transporter in their b-cells to restore normal glucose-
stimulated insulin secretion (ripglut1;glut2À/À
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
34 Chapter 2 Central Glucose Sensing and Control of Food Intake
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 non-
metabolizable analogues 3-O-methyl-D-glucose (3-O-MDG) and a-methyl-D-
glucopyranoside (a-MDG) and is inhibited by phlorizin (107, 109). SGLT1 is also
expressed in the hypothalamus and ependymal cells of the third and fourth ven-
tricles (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 phlorizin-
sensitive 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
Following its uptake, glucose is phosphorylated by hexokinases. In pancreatic b-cells,
glucokinase controls the ﬂux of glucose metabolism and the dose response of glucose-
stimulated 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
Multiplicity of Sensing Mechanisms 35
GK decreases the activity of GE neurons and increases that of GI neuronsshowingthat
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
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
/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
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).
36 Chapter 2 Central Glucose Sensing and Control of Food Intake
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
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).
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 abol-
ished 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
Multiplicity of Sensing Mechanisms 37
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
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 produc-
tion (159–161). Indeed, initial studies of UCP2À/À
mice showed that their macro-
phages generated 80% more ROS than those from control mice (159). Importantly,
ROS are also intracellular signaling molecules (162) that can regulate the activity of
channels (163, 164) or Ca2þ
inﬂux (165–167). In b-cells, ROS may
participate in the coupling between glucose metabolism and insulin secre-
tion (168–174). In the hypothalamus, there is evidence that glucose sensing may
involveROS 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).
KATP Channel-Dependent Mechanisms
The KATP channel plays a fundamental role in coupling changes in glucose metab-
olism 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
38 Chapter 2 Central Glucose Sensing and Control of Food Intake
of KATP in brain and especially in hypothalamus and brainstem (34, 46, 118, 156, 176,
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 glucose-
sensing neurons is also controlled by mechanisms not involving the KATP channel.
Table 2.1 lists these channels and their distribution in the hypothalamus and
Electrophysiological recording of neurons from Kir6.2À/À
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
The ﬁring activity of GI neurons in response to decreased extracellular glucose
may involve reduced activation of the Naþ
ATPase (30, 191), blockade of a ClÀ
conductance (37, 192), or inhibition of acid-sensitive two-pore domain Kþ
In the LHA, local application of glucose hyperpolarizes the GI orexin neurons,
an effect that is prevented by ouabain (a blocker of the Naþ
ATPase) and azide (an
inhibitor of energy production), suggesting that glucose exerts its inhibitory effect
ATPase (30). The response of the orexin neurons may involve a
conductance (55), which based on the sensitivity to pH and
halothane may be the K2p Twik1-related acid-sensitive Kþ
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).
Multiplicity of Sensing Mechanisms 39
Table 2.1 Distribution in the Hypothalamus and Brainstem of the Proteins Involved in Central Glucose Sensing
Glut2 ? þ þ þ þ þ ? þ þ þ þ (96–101)
Glut1/3 þ þ þ þ þ þ þ þ þ þ þ (51, 97, 99)
SGLT1/3 SGLT1/3 is detected in hypothalamic cultured neurons and in adult hypothalamus ND ND þ ND þ (52, 110)
Glucokinase þ þ À À þ þ À þ þ þ þ (99, 117, 118)
AMPK þ þ þ þ þ ND þ þ þ þ þ (130, 135)
UCP2/ROS þ þ À þ þ þ þ þ þ ND þ (148, 156, 158, 175)
TRP þ À ND þ þ ND TRP1 is detected in brainstem tissue extract (35)
þ ND þ þ þ ND À À þ ND ND (191, 192)
þ þ þ þ þ þ þ þ þ þ þ (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,
þ , Positive; À, negative; ND, undetermined; ?, conﬂictual data.
In Arc GI neurons, a role for ClÀ
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 Naþ
ATPase by strophanthidin or
ouabain suppressed the inward currents of GI neurons and a role for ClÀ
be excluded (191).
Changes in glycemia are monitored by several systems located at different ana-
tomical 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 Naþ
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
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 nonme-
tabolizable 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
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Abnormalities in Insulin
Secretion in Type 2
AND PIERRE-JEAN GUILLAUSSEAU
APHP, Department of Internal Medicine B, Hoˆpital Lariboisiere, APHP Paris
Universite Paris 7, Paris, France
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 quanti-
tative 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 Ó 2011 John Wiley Sons, Inc.
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 physiolog-
ical processes: insulin secretion, tissue glucose uptake, and hepatic glucose produc-
tion (HGP). In the fasting state, plasma glucose is almost exclusively provided by
hepatic production via glycogenolysis and gluconeogenesis. Hepatic glucose appear-
ance 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 stim-
ulation 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
absorptivestate duringor after a meal, ﬁrst-phaseinsulin secretion exertsan 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-sensitiveglucose transport system.Insulinpromotes conversionof glucose
54 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
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 thegold 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 Â
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
Measuring Insulin Secretion and b-Cell Function 55
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 Â ﬁrst-phase insulin response to glucose stimu-
lation) 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 abol-
ished 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
56 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
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 exo-
cytosis 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
First-Phase Insulin Secretion in Initial Stages of T2DM or First-
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
insulinsecretion takes place early in thecourseof the disease,as ithas been reportedin
subjects with impaired glucose tolerance (IGT) and IFG (31) as well as normogly-
cemic 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 ofﬁrst-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 Organiza-
tion (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
QUANTITATIVE AND QUALITATIVE ALTERATIONS IN INSULIN
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
Quantitative and Qualitative Alterations in Insulin Secretion 57
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 sensi-
tivity 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
up, were compared to subjects who remained NGT. Progression to IGT was accom-
progression from IGT to diabetes. Increases in body weight and a 31% decrease 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
normoglycemic relatives of patients with T2DM (44). b-Cell function, evaluated by
but by only 20% in subjects who remained NGT.
Progressionof Abnormalities of Insulin SecretioninOvertT2DM
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.
58 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
MECHANISMS OF b-CELL FAILURE
Glucose-Stimulated Insulin Secretion and Glucotoxicity
In pancreatic b-cells,glucose is transportedacross cytoplasmic membranevia speciﬁc
transporters, glucose transporter 1 (GLUT1) and 2 (GLUT2), and is rapidly phos-
phorylated 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
(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 supraphy-
siological glucose concentrations along with the characteristic decreases in insulin
synthesis and secretion caused by decreased insulin gene expression (56). Gluco-
toxicity, 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 sensitiveto small changesin 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 hyper-
glycemia 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
Mechanisms of b-Cell Failure 59
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.
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
hexosamines. Del Guerra et al., using islets isolated from the pancreas of patients with
T2DM and matched nondiabetic controls, demonstrated that several functional and
glucose transporters and glucokinase, reduced activation of AMP-activated protein
kinase (AMPK) and alterations in some transcription factors regulating b-cell differ-
-deoxyguanosine, were signiﬁcantly higher in T2DM than in control islets,
and correlated with the degree of impairment in glucose-stimulated insulin release. The
suggested by diminished levels of nitrotyrosine), improved glucose-stimulated insulin
that the functional impairment of T2DM islets could be, at least in part, reversible by
reducingisletcell oxidative stress (62). Itisimportant to emphasize thatin this study the
percentage of b-cells was only slightly ($10%), although signiﬁcantly, reduced in
diabetic islets compared withcontrol islets (62). As proposed byRobertson et al. (56), if
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 path-
ways 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).
T2DM is associated with dyslipidemia characterized by an increase in circulating
FFAs and changes in lipoprotein proﬁle. Acute elevation of FFAs in healthy humans
60 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
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 concen-
trations 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
Mechanisms of b-Cell Failure 61
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
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 resistancewas 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. Nonethe-
less, 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),
62 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
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 con-
trols, 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 meth-
ods) 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
Reduction in b-Cell Mass in T2DM 63
LINKAGE OF REDUCED b-CELL MASS AND DYSFUNCTION
The role of reduced b-cell mass in the alterations of insulin secretion that char-
acterizes 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 interrelation-
ship between the two parameters. A large proportion of liver-related pancreatic
donors who underwent a 50% pancreatectomy developed diabetes (84). Pharmaco-
logical 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 nondi-
abetic 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 IGTand 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
64 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
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 ﬁrst-
degree 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 (ATP-
channel), NOTCH2 [Notch homolog 2 (Drosophila, WFS1 for Wolfram
syndrome 1; wolframin)], CDKAL1 (CDK5 regulatory subunit associated protein 1-
like 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 suscep-
tibility 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 IGTand 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
Origin of b-Cell Dysfunction 65
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 func-
tions 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).
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. Identi-
ﬁcation 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
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100. GAROFANO, A., P. CZERNICHOW, and B. BREANT. 1999. Effect of ageing on beta-cell mass and function in
rats malnourished during the perinatal period. Diabetologia 42:711–718.
101. BLONDEAU, B., A. GAROFANO, P. CZERNICHOW, and B. BREANT. 1999. Age-dependent inability of the
endocrine pancreas to adapt to pregnancy: a long-term consequence of perinatal malnutrition in the
rat. Endocrinology 140:4208–4213.
102. PHILLIPS, D.I., D.J. BARKER, C.N. HALES, S. HIRST, and C. OSMOND. 1994. Thinness at birth and insulin
resistance in adult life. Diabetologia 37:150–154.
103. FLANAGAN, D.E., V.M. MOORE, I.F. GODSLAND, R.A. COCKINGTON, J.S. ROBINSON, and D.I. PHILLIPS.
2000. Fetal growth and the physiological control of glucose tolerance in adults: a minimal model
analysis. Am J Physiol Endocrinol Metab 278:E700–E706.
104. JAQUET, D., D. CHEVENNE, P. CZERNICHOW, and C. LEVY-MARCHAL. 2000. No evidence for a major beta-
cell dysfunction in young adults born with intra-uterine growth retardation. Pediatr Diabetes
and C. JOGLEKAR. 1999. Insulin resistance syndrome in 8-year-old Indian children:small at birth, big at
8 years, or both? Diabetes 48:2422–2429.
72 Chapter 3 Abnormalities in Insulin Secretion in Type 2 Diabetes Mellitus
Adipokine Production by
Adipose Tissue: A Novel
Target for Treating Metabolic
Syndrome and its Sequelae
, DANIELLE STRINGER
, RYAN HUNT
CARLA G. TAYLOR
, AND 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
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
Metabolic Syndrome: Underlying Mechanisms and Drug Therapies Edited by Minghan Wang
Copyright Ó 2011 John Wiley Sons, Inc.
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
MetS AND OBESITY
MetS refers to acluster 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 organiza-
tions as central characteristics of MetS, the diagnostic criteria arevariable (3). Several
health organizations, including the World Health Organization, the National Cho-
lesterol 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,
74 Chapter 4 Adipokine Production by Adipose Tissue
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 consid-
erably during adolescence (16). In adults that maintain energy balance, cell prolif-
eration 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 mani-
fested 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 macro-
phage inﬁltration of the adipose tissue, leading to twice as many macrophages in
visceral adipose tissue compared to subcutaneous adipose tissue (21). The accumu-
lation 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
MetS and Adipose Tissue 75
and macrophages is one of the factors that leads to the hypersecretion of proathero-
genic, proinﬂammatory, and prodiabetic adipokines, and the reduced secretion of
protective, anti-inﬂammatory adipokines from adipose tissue that are characteristic of
MetS (20, 24, 25).
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). Subcu-
taneous 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 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 is expressed in adipose tissue. In the circulation, it exists in two forms
full-length (primarily as trimers, hexamers, and multimeric complexes) and globular
76 Chapter 4 Adipokine Production by Adipose Tissue
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
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 subcu-
taneous adipose tissue (39). In humans, similar results have been observed, with
abdominal adipose expressing and secreting more resistin than subcutaneous adipose
tissue (74, 75).
Proinﬂammatory molecules such as TNF-a, C-reactive protein (CRP), and interleu-
kin-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).
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
MetS and Adipose Tissue 77
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
arelower in patients withCHD (92, 93).In humans, thethickness ofepicardial 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
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 adipo-
cytes 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
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
78 Chapter 4 Adipokine Production by Adipose Tissue
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 progres-
sing to this stage (97). Fifteen percent of patients with ﬁbrosis advance to cirrho-
sis (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. Conse-
quently, there is an increased ﬂux of FFAs from the visceral adipose tissue into the
portal vein, resulting in direct delivery offatty 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-inﬂamma-
tory 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,
MetS and the Liver: Nonalcoholic Fatty Liver Disease 79
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 TNF-
a 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, he-
patic lipid content, serum alanine transaminase (ALT), TAG, and FFA after only
2 weeks of treatment (121). In humans (both adults and children), plasma concentra-
tions 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 liverbecomes 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 hyperlepti-
nemia 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
80 Chapter 4 Adipokine Production by Adipose Tissue
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
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 proin-
ﬂammatory 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
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 hyper-
secretion of insulin and no detrimental effects on functional characteristics of b-cells
in the presence of pancreatic fat deposition (138). In humans, histological exami-
nation 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).
MetS and the Pancreas 81
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,
energyexpenditure, or adiposity when comparedto impairedcentralleptinsignaling 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, pre-
pro-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. Endocannabi-
noids 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 mar-
ijuana 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
82 Chapter 4 Adipokine Production by Adipose Tissue
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 expres-
sion 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 intracerebroven-
tricular 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 promot-
ing 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
areexacerbated byobesity,underliesthepathophysiological changesthat 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
thisapproach 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
MetS and the Cardiovascular System 83
common cardiovascular disorder associated with MetS is atherosclerosis, a progres-
sive 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 hemo-
dynamic 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
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 adipo-
kines 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 indivi-
duals (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 contribu-
tion 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.
84 Chapter 4 Adipokine Production by Adipose Tissue
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
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 pro-
duction 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
(b) Vascular Tone. Blood pressure is maintained by a balance of vasoconstric-
tory 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.
MetS and the Cardiovascular System 85
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 endo-
thelial 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 never-
theless 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
(c) Vascular Injury. The onset and progression of vascular disease is closely
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 con-
tractile 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,
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
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
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
86 Chapter 4 Adipokine Production by Adipose Tissue
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
LDL receptor, ApoE and leptin. This study indicates that a lack of leptin is
insufﬁcient topreventatherosclerosis under conditions where extremehyper-
lipidemic conditions exist. An alternate view has been proposed by Bohlen
A similar ﬁnding was made by Nair et al. (214) with airway smooth muscle.
Procopioet al.(199)recently reportedthat leptin stimulates eNOS expression
by endothelial cells via AMPK, which could explain how leptin inhibits cell
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 leukocytesas 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 endo-
thelial function (216). Additionally, the altered surface properties of dys-
functional 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
MetS and the Cardiovascular System 87
results in release ofintracellular 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 resis-
tance 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 capillar-
ies (225). Thus, leptin supports the progression of MetS by assisting in the
formation of vessels to carry nutrients during the expansion phase of
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, produc-
tion 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 impor-
tance in vascular disease onset has been established by Zhang et al. (233),
who showed that increased expression of the receptors increases the anti-
inﬂammatory actions of adiponectin.
88 Chapter 4 Adipokine Production by Adipose Tissue
(b) Vascular Tone. An inverse correlation between hypertension severity and
adiponectin levels has been identiﬁed(190), butacausal 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 hyperten-
sion (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 ani-
mal (238). These data suggest adiponectin can inﬂuence vascular tone,
however, this may be mediated through the central nervous system rather
(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 vascu-
lature 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 sup-
pressed 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 adecrease in the response
of vessels to factors that trigger vasodilation (246), by activating the AMPK-
NOS 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,
MetS and the Cardiovascular System 89
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 adipo-
nectin 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 adi-
ponectin 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
Resistin is an adipokine that is primarily produced by adipocytes in rodents, but
macrophagesare theprimary source of theresistinexpressed byhuman adipose tissue.
The inﬁltration of macrophages into adipose tissue likely explains the increase in
circulatingresistinseen in MetS (265).Onthe other hand, ithas also beenreportedthat
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
vasodilatorysubstances (270).Resistin is associatedwith inﬂammation (271),and can
promote the release of proinﬂammatory cytokines from endothelial cells (272).
90 Chapter 4 Adipokine Production by Adipose Tissue
Interestingly, resistin secretion is elevated in hyperhomocysteinemia and stimulation
of SMC migration may lead to neointimal hyperplasia under these conditions (273).
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).
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 subse-
quently 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
MetS and the Cardiovascular System 91
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 progres-
sion 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.
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 is synthesized by cardiomyocytes (301). Furthermore, Skurk et al. (302)
have suggested adiponectin production by cardiac tissue is controlled via a mech-
anism 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 levelsof 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
92 Chapter 4 Adipokine Production by Adipose Tissue
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).
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 failurewill 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 long-
term 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
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 negativeconsequences for recovery after aheart attack and likely promote
the development of heart failure in persons with MetS.
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
Therapeutic Interventions 93
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
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.
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
94 Chapter 4 Adipokine Production by Adipose Tissue
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).
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. Addi-
tionally, 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
Therapeutic Interventions 95
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
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 improve-
ments in the function of various organs, speciﬁcally the liver, kidneys, and pancre-
as (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 diet-
induced 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 omega-
3 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 differ-
entiation 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
96 Chapter 4 Adipokine Production by Adipose Tissue
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 food-
based 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.
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
Therapeutic Interventions 97
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 charac-
terized as a marked loss of adipose tissue (379).
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
to restore leptin responsiveness in diet-induced obese rats (382). Increased respon-
siveness 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 Æ 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 metre-
leptin yielded weight loss of 8.4 Æ 0.9% and 8.2 Æ 1.3%, respectively (146).
Like leptin, adiponectin has been shown to have a wide range of physiological
effects. In particular, increasing serum adiponectin levelsis 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 endo-
thelial 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 atheroscle-
rosis. HIV protease inhibitors reduce adiponectin secretion from 3T3-L1 adipocytes,
98 Chapter 4 Adipokine Production by Adipose Tissue
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).
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 over-
come 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, adenovirus-
delivered adiponectin lowered elevated blood pressure in KO mice (56) and signi-
ﬁcantly 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.
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
Therapeutic Interventions 99
improve parameters of MetS (392, 393). In general, it is assumed that weight
reduction through pharmacological management will result in the same improve-
ments 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 expres-
sion (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 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 prolifer-
ation 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 hyperglyce-
mia (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
100 Chapter 4 Adipokine Production by Adipose Tissue
NASH show increased plasma adiponectin and reduced TNF-a levels after 24 months
of atorvastatin treatment (405). However, 6 months of treatment with atorvasta-
tin (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 LDL-
cholesterol 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
Both ﬁbrates and thiazolidinediones (TZDs), like statins, were developed as agents for
cholesterol lowering and increasing insulin sensitivity, respectively. These com-
pounds 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 3T3-
L1 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
Therapeutic Interventions 101
adiponectin while decreasing FFA levels suggests this effect is mediated by adipo-
cytes. 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. Adiponectin-
stimulated 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 ifit 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 compo-
nents 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 com-
pounds 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
102 Chapter 4 Adipokine Production by Adipose Tissue
implementing these therapies. This statement is based on the fact that some treatments
designed to reduce blood pressure may negatively inﬂuence adiponectin. For in-
stance, 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
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 arolein 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 endog-
enous 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 melli-
tus (458). Rimonabant is recommended for patients with a BMI greater than 30 kg/
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,
Therapeutic Interventions 103
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 antag-
onizing 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 6.5% (463) suggests this class of drugs
may not provide the panacea from obesity as it ﬁrst appeared.
Cox-2 inhibition has been recently examined as a mechanism of reducing inﬂam-
matory disease. Obesity is considered an inﬂammatory disease due to the high
numbers of macrophages that inﬁltrate the adipose tissue (21). Lijnen et al. (464)
recently examined the effect of the Cox-2 inhibitor rofecoxib (Vioxx) in an animal
model of diet-induced obesity. These investigators found that the treatment attenuated
weight gain and reduced adipocyte size. Furthermore, there were fewer macrophages
present in the adipose tissue and 30% less leptin was produced. No differences in
adiponectin or TNF-a were detected. Similar results were obtained with wild-type
mice (465). Although some Cox-2 inhibitors have been withdrawn from the market,
these compounds might represent an alternative route for modulating adipose
function. The efﬁcacy of these drugs may be due to the ability of adipocyte-derived
prostaglandin E2 (PGE2) to stimulate the secretion of leptin by adipocytes (466). In
retrospect, these results indicate PGE2 could also be classed as an adipokine.
Inhibition of Monocyte Inﬁltration
Weisberg et al. (467) showed that antagonists of the CC chemokine receptor-2, which
is present on monocytes and binds MCP-1, increased adiponectin expression in
adipose tissue of obese mice without altering weight or adipose mass. Although gross
changes in body composition did not occur, there was a reduction in hepatic steatosis
and improved insulin sensitivity. It is presumed that a reduction in monocyte
inﬁltration into the adipose tissue is the primary cause of these positive changes.
Interestingly, deletion of the MCP-1 gene did not produce similar effects (468), but
this may be due to the fact that CC chemokine receptor-2 binds other members of the
MCP family and thus there is sufﬁcient redundancy to ensure that macrophages will
still be drawn to adipose tissue as it enlarges due to excess caloric intake. This novel
approach warrants further investigation given the signiﬁcant contribution of macro-
phage inﬁltration to inﬂammation-mediated adipose dysfunction.
104 Chapter 4 Adipokine Production by Adipose Tissue
All-trans-retinoic acid (RA) is the active metabolite of vitamin A and is a ligand of the
retinoid X receptor, a cofactor of numerous nuclear hormone receptors including the
PPARs. Several studies have investigated the contribution of RA to the actions of
nuclear hormone receptors, and as a matter of course have identiﬁed RA-speciﬁc
changes in cellular activity. One relevant example is a decrease in leptin expression in
3T3-L1 adipocytes and human adipose tissue explants with RA treatment in
vitro (469). In agreement with this observation, both RA and vitamin A have been
shown to lower plasma leptin levels in rats when given in pharmacological doses, but
there were no changes in body weight or adipose mass (470). Interestingly, there was
no effect of RA on adipokine levels in a human population except for a lowering of
resistin (471). Of note, 13-cis retinoic acid, a compound considered to have anti-
inﬂammatory properties and is used for the treatment of acne, was shown to increase
insulin resistance in adults. Paradoxically, it also increases plasma adiponectin
levels (472, 473). Effects on TNF-a have also been reported (474).
Ciliary Neurotrophic Factor
Ciliary neurotrophic factor (CNTF) is a neurocytokine that suppresses AMPK in the
hypothalamus (475) and thus exhibits similarities to leptin (476). At the same time, it
can act peripherally to modulate metabolism by stimulating AMPK in skeletal
muscle (477), and has been shown to induce weight loss in both animals and
humans (478, 479). This weight reduction has been linked to changes in adiponectin
production by the adipose tissue (480). It appears that remodeling of adipocytes by
CNTF may represent the mechanism by which it achieves these results (481). These
impressiveresults indicate there is strong potential for future development in this area.
The identiﬁcation of leptin in 1994 indicated for the ﬁrst time that adipose tissue plays
a signiﬁcant role in metabolic homeostasis, and its importance in this process is now
well established. Adipokine production by adipose tissue has a regulatory role in the
fasting/feeding cycle, with leptin contributing to satiation. Adipokines also link
adipose tissue to various organs (liver, skeletal muscle) that are important for lipid and
carbohydrate metabolism, but what has now become more accepted is the role of
adipokines in modulating the function of other tissues, such as the pancreas, brain,
heart, and blood vessels. At this point in time, exploration of the therapeutic potential
of adipokines has revealed that it is possible to manipulate their production. The
underlying mechanisms that inﬂuence adipokine levels in pathological conditions,
however, have not been delineated to the point that speciﬁc drug targeting is possible.
Until this transpires, careful investigation of the physiological effects of any inter-
vention will require close attention, particularly from the standpoint that their
manipulation has the potential to negatively affect a variety of metabolic pathways.
But on a positive note, this approach appears to have great potential for treating
metabolic diseases, even if weight loss is not achieved, by improving the function of
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