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Diabetes and Cancer. Epidemiological Evidence and Molecular Links
Frontiers in Diabetes
Vol. 19

Series Editors

M. Porta Turin
F.M. Matschinsky Philadelphia, Pa.
Diabetes and Cancer
Epidemiological Evidence and Molecular Links

Volume Editors

K. Masur Witten
F. Thévenod Witten
K.S. Zänker Witten

17 figures, 11 in color, and 2 tables, 2008

Basel · Freiburg · Paris · London · New York · Bangalore ·

Bangkok · Shanghai · Singapore · Tokyo · Sydney
Frontiers in Diabetes
Founded 1981 by F. Belfiore, Catania

Kai Masur, PhD Frank Thévenod, MD, PhD

Institute of Immunology and Experimental Department of Physiology and
Oncology Pathophysiology
University of Witten/Herdecke University of Witten/Herdecke
Witten, Germany Witten, Germany

Kurt S. Zänker, MD, DVM, PhD

Institute of Immunology and Experimental
Oncology
University of Witten/Herdecke
Witten, Germany

Library of Congress Cataloging-in-Publication Data

Diabetes and cancer : epidemiological evidence and molecular links /

volume editors, K. Masur, F. Thévenod, K.S. Zänker.
p. ; cm. – (Frontiers in diabetes, ISSN 0251–5342 ; v. 19)
Includes bibliographical references and index.
ISBN 978–3–8055–8640–5 (hard cover : alk. paper)
1. Diabetes–Epidemiology. 2. Diabetes–Molecular aspects.
3. Metabolic syndrome–Complications. 4. Cancer–Etiology.
I. Masur, K. (Kai) II. Thévenod, F. (Frank) III. Zänker, Kurt S. IV. Series.
[DNLM: 1. Diabetes Mellitus–epidemiology. 2. Diabetes Mellitus–metabolism.
3. Risk Factors. 4. Metabolic Syndrome X–complications.
5. Neoplasms–etiology. W1 FR945X v.19 2008 / WK 810 D53715 2008]
RC660.D443 2008
616.4⬘6207–dc22
2008025244

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and PubMed/MEDLINE.
Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and
contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty,
endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the
editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products
referred to in the content or advertisements.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this
text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research,
changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader
is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and
precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by
any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and
retrieval system, without permission in writing from the publisher.
© Copyright 2008 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)
www.karger.com
Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel
ISSN 0251–5342
ISBN 978–3–8055–8640–5
Contents

VII Preface
Masur, K.; Thévenod, F.; Zänker, K.S. (Witten)
1 Pathophysiology of Diabetes Mellitus Type 2: Roles of Obesity,
Insulin Resistance and ␤-Cell Dysfunction
Thévenod, F. (Witten)
19 In vivo ␤-Cell Imaging in Diabetes, ␤-Cell Hyperplasia, and Insulinoma
Gotthardt, M. (Nijmegen); Béhé, M. (Marburg); Lasser, T. (Lausanne)
30 Incretin-Based Therapies for the Treatment of Type 2
Diabetes – DPP-4 Inhibitors and Incretin Mimetics
Gallwitz, B. (Tübingen)
44 Janus Face of Glucose and Glucose-Regulating Hormones
Masur, K. (Witten)
59 Role of Glucose Metabolism in Carcinogenesis and Cancer Progression
Gatenby, R.A. (Tampa, Fla.)
71 Glucose Transporters: Their Abnormalities and Significance in Type 2
Diabetes and Cancer
Schürmann, A. (Nuthetal)
84 The Epidemiologic Relationship between Diabetes and Cancer
Zänker, K.S. (Witten)
97 Diabetes Mellitus and Breast Cancer
Wolf, I.; Rubinek, T. (Ramat Gan/Tel Aviv)
114 Nutrition, Diabetes, and Cancer
LaValle, J.B. (Cincinnati, Ohio)
134 Diabetes and Cancer: The Road Ahead
Masur, K.; Thévenod, F.; Zänker, K.S. (Witten)

145 Subject Index

Preface

This book was made possible by the contributions of leading experimental scientists
and clinicians from newly upcoming and interdisciplinary fields of research concerning
the common molecular and clinical features of chronic diseases. Chronic disease
represents the main cause of mortality in developed countries. The increase in its
prevalence is associated with changes in lifestyle habits and related risk factors such as
tobacco use, physical inactivity, overweight and obesity, and poor nutrition.
Collectively, cardiovascular diseases, cancer and diabetes/metabolic syndrome –
ranking first among the ten leading causes of death – are responsible for more than 25
million deaths in the Western world each year. Much of this disease burden could be
prevented, however, by controlling the modifiable risk factors.
The present trend of progressively lengthening lifespan in all social groups of
Western societies reflects the changing pattern of mortality from formerly untreat-
able infectious diseases to chronic (degenerative) diseases. Predictions for the contin-
uing lengthening of the lifespan of the class of 2005 and succeeding classes may be
jeopardized by the alarming increase in obesity, for example, which worsens the inci-
dence of cardiovascular disorders, diabetes and cancer.
The recent discoveries of epidemiological and molecular links between the diabetes/
metabolic syndrome and cancer originated from interdisciplinary-oriented resea-
rchers revealing roles in biological processes that are likewise varied. The diabetes/
metabolic syndrome is like the wolf in sheep’s clothing – by the time it has been diag-
nosed, most subjects might already have an established chronic disease, like cardiovas-
cular disease and cancer. The most recent findings suggest a connection between
inflammation and chronic disease, such as insulin resistance associated with diabetes
and cancer, which had not or only inadequately been appreciated previously.
 The following distinguished authors guarantee that this book is at the forefront of
experimental and clinical research in diabetes and cancer, and offers the reader novel
insights into the interdisciplinary approaches of tomorrow: F. Thévenod (Witten,
Germany) introduces the state of the art of the pathophysiology of type 2 diabetes.
M. Gotthard (Nijmegen, The Netherlands) addresses new issues of in vivo imaging of
the ␤-cell and insulinoma. B. Gallwitz (Tübingen, Germany) reviews the most
advanced therapy strategies embarking on incretins and DPP4 inhibitors. K. Masur
(Witten, Germany) bridges on a molecular level diabetes and cancer with specific ref-
erence to glucose and glucose-regulating hormones. R. Gatenby (Moffitt Cancer
Center, Tampa, Fla., USA) clearly demonstrates that the ‘Warburg effect’ has to be
reconsidered to understand the energetic metabolism of tumor cells. A. Schürmann
(Potsdam-Rehbrücke, Germany) describes the glucose transporter systems and shows
their abnormalities and significance in type 2 diabetes and cancer. K.S. Zänker
(Witten, Germany) summarizes the epidemiology and molecular epidemiology of
type 2 diabetes and cancer. I. Wolf (Tel-Hashomer, Israel) points at the increased risk
of breast cancer in relationship to type 2 diabetes. J. LaValle (Pittsburgh, Pa., USA)
describes the metabolic spiral, which leads to chronic disease. Finally, the editors of
this book (K.M., F.T., K.S.Z) advocate the efforts of Beaglehole et al. [Lancet
2007;370:2152–2157] who have established the Chronic Disease Action Group to
encourage, support, and monitor action on the implementation of an evidence-based
effort to promote global, regional, and national action to prevent and control chronic
disease.
This book should encourage scientists and physicians – working separately on var-
ious aspects of the illnesses with the highest predicted mortality in the 21st century – to
come together and combine their therapies and strategies. Since the health problems
mentioned may be merged with the overall topic ‘metabolic syndrome’, the common
goal should be early detection at the first signs indicating the onset of a metabolic
misbalance in order to prevent the consecutive cascades which lead to metabolic syn-
drome, resulting in the so-called diseases of modern civilization – cancer, diabetes
and hypertension.
This volume of Frontiers in Diabetes, ‘Diabetes and Cancer – Epidemiological
Evidence and Molecular Links’, demonstrates why that it is necessary to reflect on the
different aspects of an illness and that it is worthwhile checking for metabolic
derangements in order to find an early therapy combining approaches devised by
specialists working in different fields.
The Editors of this book are grateful to Karger Publishers, Switzerland, and to F.M.
Matschinsky (Philadelphia, Pa., USA) and M. Porta (Turin, Italy), the Editors-in-Chief
of the long-standing and well-recognized series of Frontiers in Diabetes, for publish-
ing this volume.
Kai Masur, Witten
Frank Thévenod, Witten
Kurt S. Zänker, Witten

VIII Preface
Masur K, Thévenod F, Zänker KS (eds): Diabetes and Cancer. Epidemiological Evidence and Molecular Links.
Front Diabetes. Basel, Karger, 2008, vol 19, pp 1–18

Pathophysiology of Diabetes
Mellitus Type 2: Roles of Obesity,
Insulin Resistance and ␤-Cell
Dysfunction
Frank Thévenod
Department of Physiology and Pathophysiology, University of Witten/Herdecke,
Witten, Germany

Abstract
The past two decades have seen an explosive increase in the number of people diagnosed with diabetes
mellitus worldwide, particularly type 2 diabetes (T2D), which is found associated with modern lifestyle,
abundant nutrient supply, reduced physical activity, and obesity. Actually, between 60 and 90% of cases
of T2D now appear to be related to obesity. Numerous studies have shown that insulin resistance pre-
cedes the development of hyperglycemia in subjects that eventually develop T2D. However, it is increas-
ingly being realized that T2D only develops in insulin-resistant subjects with the onset of β -cell
dysfunction. It is therefore important to characterize the mechanisms of insulin resistance and subse-
quent pancreatic β -cell failure associated with obesity in order to better understand the pathophysiol-
ogy of T2D and develop approaches to prevent T2D. Copyright © 2008 S. Karger AG, Basel

Introduction

Diabetes mellitus (DM) encompasses a range of diseases that are characterized by ele-
vation of the blood glucose level and lead to a reduced quality of life and life
expectancy, with a greater risk of heart disease, stroke, peripheral neuropathy, renal
disease, blindness and amputation. Depending on the etiology, DM can be divided
into two principal forms, type 1 (T1D) and type 2 diabetes (T2D). T1D occurs in
childhood and is due primarily to autoimmune-mediated destruction of pancreatic
␤-cell islets, resulting in absolute insulin deficiency. People with T1D must take
exogenous insulin for survival to prevent the development of ketoacidosis. The fre-
quency of T1D is low relative to T2D, which accounts for over 90% of cases globally.
T2D is more prevalent in adulthood, though it is becoming more common in children
and adolescents. T2D is characterized by insulin resistance and/or abnormal insulin
secretion. Individuals with T2D are not dependent on exogenous insulin, but may
require it for control of blood glucose levels if this is not achieved with diet alone or
with oral hypoglycemic agents.
DM has long been considered a disease of minor significance to world health, but
is now developing into one of the main public health challenges for the 21st century.
The past two decades have seen an explosive increase in the number of people diag-
nosed with DM worldwide. This DM epidemic relates particularly to T2D, which is
taking place both in developed and developing countries. The global figure of people
with DM is set to rise from the current estimate of 150 to 220 million in 2010, and 300
million in 2025 [1]. The direct healthcare costs of the disease are also considerable,
and have been estimated at around 5% of the total annual expenditure on health in
Western societies.
About 80% of T2D patients are overweight. In fact, obesity is a primary risk factor
for ‘metabolic’ diseases, which include coronary heart disease, hypertension, but also
T2D. Knowledge of adipocyte physiology is therefore crucial for a better understand-
ing of the pathophysiological basis of obesity and T2D.

Physiology of Adipose Tissues

Adipose tissues are located throughout the body. Some of these depots are structural,
providing mechanical support but contributing little to energy homeostasis. Other
adipocytes exist in the skin as subcutaneous fat. Finally, several distinct depots are
found within the body cavity, surrounding the heart and other organs, associated
with the intestinal mesentery, and in the retroperitoneum. This visceral fat drains
directly into the portal circulation and has been linked to morbidities, such as cardio-
vascular disease and T2D. Adipose tissues modulate energy balance by regulating
both food intake and energy expenditure. They also have a considerable effect on
glucose balance, which is mediated by endocrine (mainly through the synthesis
and release of peptide hormones, the so-called ‘adipokines’) and non-endocrine
mechanisms.
Among the endocrine factors, adipocyte-derived proteins with antidiabetic action
include leptin, adiponectin, omentin and visfatin. For instance, in addition to its well-
characterized role in energy balance, leptin reverses hyperglycemia by improving
insulin sensitivity in muscles and the liver. According to the current view that intra-
cellular lipids may contribute to insulin resistance, this occurs most likely by reducing
intracellular lipid levels through a combination of direct activation of AMP-activated
protein kinase (AMPK) and indirect actions mediated through central neural path-
ways [2]. Other factors tend to raise blood glucose, including resistin, tumor necrosis
factor-␣ (TNF-␣), interleukin-6 (IL-6) and retinol-binding protein 4 (RBP4). TNF-␣
is produced in macrophages and reduces insulin action [3]. IL-6 is produced by

2 Thévenod
adipocytes, and has insulin-resistance-promoting effects as well [4]. Such ‘adipocy-
tokines’ can induce insulin resistance through several mechanisms, including c-Jun
N-terminal kinase 1 (JNK1)-mediated serine phosphorylation of insulin receptor
substrate-1 (IRS-1) (see below), I␬B kinase-␤ (IKK-␤)-mediated nuclear factor-␬B
(NF-␬B) activation, induction of suppressor of cytokine signaling 3 (SOCS3) and
production of ROS [for review, see 5]. RBP4, a secreted member of the lipocalin
superfamily, is regulated by the changes in adipocyte glucose transporter 4 (GLUT4)
levels. Studies have shown that overexpression of RBP4 impairs hepatic and muscle
insulin action, and Rbp4⫺/⫺ mice show enhanced insulin sensitivity [6]. Furthermore,
high serum RBP4 levels are associated with insulin resistance in obese humans and
patients with T2D [7]. The exact mechanisms how RBP4 impairs insulin action are,
however, not clear.
Adipocytes also release non-esterified fatty acids (NEFAs) into the circulation,
which may therefore be viewed as an adipocyte-derived secreted non-endocrine
product. They are primarily released during fasting, i.e. when glucose is limiting, as a
nutrient source for most organs. Circulating NEFAs reduce adipocyte and muscle
glucose uptake, and also promote hepatic glucose output, consistent with insulin
resistance. The net effect of these actions is to promote lipid burning as a fuel source
in most tissues, while sparing carbohydrate for neurons and red blood cells, which
depend on glucose as an energy source. Several mechanisms have been proposed to
account for the effects of NEFAs on muscle, liver and adipose tissue, including pro-
tein kinase C (PKC) activation, oxidative stress, ceramide formation, and activation
of Toll-like receptor 4 [for review, see 5, 8]. Because lipolysis in adipocytes is
repressed by insulin, insulin resistance from any cause can lead to NEFA elevation,
which, in turn, induces additional insulin resistance as part of a vicious cycle. ␤-Cells
are also affected by NEFAs, depending in part on the duration of exposure; acutely,
NEFAs induce insulin secretion (as after a meal), whereas chronic exposure to NEFAs
causes a decrease in insulin secretion [9] (see below), which may involve lipotoxicity-
induced apoptosis of islet cells [10] and/or induction of uncoupling protein-2 (UCP-
2), which decreases mitochondrial membrane potential, ATP synthesis and insulin
secretion [10, 11]. The ability to store large amounts of esterified lipid in a manner
that is not toxic to the cell or the organism as a whole may therefore be one of the
most critical physiological functions of adipocytes.

The Insulin Receptor: Transduction through Tyrosine Kinase

An understanding of insulin resistance requires knowledge of the mechanisms of

insulin action in target tissues, such as liver, muscle and adipose tissue. The net
responses to this hormone include short-term metabolic effects, such as a rapid
increase in the uptake of glucose, and longer-term effects on cellular differentiation
and growth [12]. The ␣-subunits of the insulin receptor are located extracellularly

Pathophysiology of Diabetes Type 2 3

and are the insulin-binding sites. Ligand binding promotes autophosphorylation of
multiple tyrosine residues located in the cytoplasmic portions of ␤-subunits. This
autophosphorylation facilitates binding of cytosolic substrate proteins, such as IRS-1.
When phosphorylated, this substrate acts as a docking protein for proteins mediating
insulin action. Although the insulin receptor becomes autophosphorylated on
tyrosines and phosphorylates tyrosines of IRS-1, other mediators are phosphorylated
predominantly on serine and threonine residues. An insulin second messenger, pos-
sibly a glycoinositol derivative that stimulates phosphoprotein phosphatases, may be
released at the cell membrane to account for the short-term metabolic effects of
insulin. The activated ␤-subunit also catalyzes the phosphorylation of other members
of the IRS family, such as Shc and Cbl. Upon tyrosine phosphorylation, these proteins
interact with other signaling molecules (such as p85, and Grb2-Sos and SHP-2)
through their SH2 (Src-homolog-2) domains, which bind to a distinct sequence of
amino acids surrounding a phosphotyrosine residue. Several diverse pathways are
activated, and those include activation of phosphatidylinositol 3⬘-OH kinase (PI3K),
the small GTP-binding protein Ras, the mitogen-activated protein (MAP) kinase cas-
cade, and the small GTP-binding protein TC10. Formation of the IRS-1/p85 complex
activates PI3 kinase (class 1A), which transmits the major metabolic actions of
insulin via downstream effectors such as phosphoinositide-dependent kinase 1
(PDK1), Akt and mTOR. The IRS-l/Grb2-Sos complex and SHP-2 transmit mito-
genic signals through the activation of Ras to activate MAP kinase. Once activated via
an exchange of GTP for GDP, TC10 promotes translocation of GLUT4 vesicles to the
plasma membrane of muscle and fat cells, perhaps by stabilizing cortical actin fila-
ments. These pathways coordinate the regulation of vesicle trafficking (incorporation
of GLUT4 into the plasma membrane), protein synthesis, enzyme activation and
inactivation, and gene expression [for further details, see 12, 13]. The net result of
these diverse pathways is regulation of glucose, lipid, and protein metabolism as well
as cell growth and differentiation.

Pathophysiology of Adipose Tissues: Obesity and Insulin Resistance

Lipid storage in adipose tissue represents excess energy consumption relative to

energy expenditure, which in its pathological form has been coined ‘obesity’. In recent
years, overnutrition has reached epidemic proportions in developed as well as devel-
oping countries. This reflects recent lifestyle changes, however there is also a strong
genetic component as well. While the biochemical mechanism(s) for this genetic pre-
disposition are still under investigation, the genes that control appetite and regulate
energy homeostasis are now better known. For example, adipocytes produce leptin
(see above) that suppresses appetite and was initially considered a promising target
for drug therapy. However, most overweight individuals overproduce leptin, and no
more than 2–4% of the overweight population has defects in the leptin appetite

4 Thévenod
suppression pathway [14]. In contrast, genetic predisposition to obesity and/or T2D
when excess calories are consumed is common in the population: for instance, poly-
morphisms in the peroxisome proliferator-activated receptor-␥2 (PPAR-␥2) gene may
have a broad impact on the risk of obesity and insulin resistance. A minority of peo-
ple is heterozygous for the Pro12Ala variant of PPAR-␥ and is less likely to become
overweight and less likely to develop DM when overweight than the majority of Pro
homozygotes in the population [15].
One striking clinical feature of overweight individuals is a marked elevation of
serum NEFAs, cholesterol, and triacylglycerols irrespective of the dietary intake of
fat. Obesity is obviously associated with an increased number and/or size of adipose
tissue cells. These cells overproduce hormones, such as leptin, and cytokines, such as
TNF-␣, some of which appear to cause cellular resistance to insulin [16]. At the same
time, the lipid-laden adipocytes decrease synthesis of hormones, such as adiponectin,
which appear to enhance insulin responsiveness. The insulin resistance in adipose tis-
sue results in increased activity of the hormone-sensitive lipase, which is probably
sufficient to explain the increase in circulating NEFAs [17]. The high circulating lev-
els of NEFAs may also contribute to insulin resistance in the muscle and liver (see
below). Initially, the pancreas maintains glycemic control by overproducing insulin.
Thus, many obese individuals with apparently normal blood glucose control have a
syndrome characterized by insulin resistance of the peripheral tissue and high con-
centrations of insulin in the circulation. This hyperinsulinemia appears to stimulate
the sympathetic nervous system, leading to sodium and water retention and vasocon-
striction, which increase blood pressure [18]. The excess NEFAs are carried to the
liver and converted to triacylglycerol and cholesterol. Excess triacylglycerol and cho-
lesterol are released as very-low-density lipoprotein particles, leading to higher circu-
lating levels of both triacylglycerol and cholesterol. Eventually, the capacity of the
pancreas to overproduce insulin declines which leads to higher fasting blood sugar
levels and decreased glucose tolerance (see below).

Inflammation: A Process Associated with Obesity-Induced Insulin Resistance

Adipose tissue modulates metabolism by releasing NEFAs and glycerol, hormones –

including leptin and adiponectin – and proinflammatory cytokines [19]. There is
now clear evidence that obesity associated with or without T2D is an inflammatory
state, consistent with the production of TNF-␣ and other cytokines by adipose tissue.
Chronic inflammation of white adipose tissue characterized by macrophage infiltra-
tion is thought to contribute to insulin resistance associated with obesity, and in obe-
sity, the production of many of these adipokines is increased. RBP4 induces insulin
resistance through reduced phosphatidylinositol-3-OH kinase (PI3K) signaling in
muscle and enhanced expression of the gluconeogenic enzyme phosphoenolpyruvate
carboxykinase in the liver through a retinol-dependent mechanism. By contrast,

Pathophysiology of Diabetes Type 2 5

adiponectin acts as an insulin sensitizer, stimulating fatty acid oxidation in an AMPK
and peroxisome proliferator-activated receptor-␣ (PPAR-␣)-dependent manner [for
review, see 5].
In obese animals and humans, bone-marrow-derived macrophages are recruited
to the fat pad under the influence of proteins secreted by adipocytes, including
macrophage chemoattractant protein-1 (MCP-1) [19]. In addition to adipocyte-
derived factors, increased release of TNF-␣, IL-6, MCP-1 and additional products of
macrophages that populate adipose tissue might also have a role in the development
of insulin resistance [20]. TNF-␣ and IL-6 act through classical receptor-mediated
processes to stimulate both the c-Jun aminoterminal kinase (JNK) and the I␬B
kinase-␤ (IKK-␤)/nuclear factor-␬B (NF-␬B) pathways, resulting in upregulation of
potential mediators of inflammation that can lead to insulin resistance. The
adipokine MCP-1 and its receptor CCR2 may play a role in the recruitment of
macrophages to white adipose tissue and in the initiation of an inflammatory
response in mice. Thiazolidinediones, which are PPAR-␥ agonists used clinically as
insulin sensitizers, reduce MCP-1 levels and macrophage infiltration into adipose tis-
sue [21]. Increased secretion of MCP-1 from adipocytes may thus trigger such
macrophage recruitment, and the infiltrated cells may in turn secrete a variety of
chemokines and other cytokines that further promote a local inflammatory response
and affect gene expression in adipocytes, resulting in systemic insulin resistance.

NEFAs: A Critical Factor in the Development of Insulin Resistance

The amount of adipokines secreted from adipocytes is correlated with adipocyte size,
i.e. with the amount of triglyceride stored in the cells. Such a relation implies that
adipokines directly mediate insulin resistance associated with obesity. Given that the
release of NEFAs also is correlated with adipocyte size and that an increase in the
NEFA concentration in plasma is a common feature of insulin resistance, increased
NEFA levels are also associated with the insulin resistance observed in obesity and
T2D [22]. The passage of NEFAs across the plasma membrane and into the cell,
where they are thought to exert their effects, is mediated in a specific manner by fatty
acid transport protein 1 (FATP1), a transmembrane protein that enhances the cellular
uptake of NEFAs. Interestingly, FATP1-deficient mice are protected from diet-
induced obesity and insulin resistance [23]. The cytosol of cells also contains fatty
acid-binding proteins (FABPs), which are thought to facilitate the utilization of lipids
in metabolic pathways. Mice that lack both of two related adipocyte FABPs, aP2 and
mal1, are also protected from diet-induced obesity and insulin resistance [24].
In fact, it appears that the release of NEFAs may be the single most critical factor in
modulating insulin sensitivity. Insulin resistance develops within hours of an acute
increase in plasma NEFA levels in humans [22]. Conversely, insulin-mediated glu-
cose uptake and glucose tolerance improve with an acute decrease in NEFA levels

6 Thévenod
after treatment with the antilipolytic agent acipimox [25]. Increased intracellular
NEFAs may result in competition with glucose for substrate oxidation leading to the
serial inhibition of pyruvate dehydrogenase, phosphofructokinase and hexokinase II
activity [26]. It has also been proposed that increased NEFA delivery or decreased
intracellular metabolism of fatty acids results in an increase in the intracellular con-
tent of fatty acid metabolites such as diacylglycerol (DAG), fatty acyl-coenzyme A
(fatty acyl-CoA), and ceramides, which, in turn, activate a serine/threonine kinase
cascade leading to serine/threonine phosphorylation of IRS-1 and IRS-2, and a
reduced ability of these molecules to activate PI3K [27]. Subsequently, events down-
stream of insulin-receptor signaling are diminished.
These observations thus suggest that the transport of NEFAs into cells and their
intracellular availability are important determinants of diet-induced obesity and insulin
resistance. NEFAs also bind to and activate members of the G-protein-coupled class of
receptors in the plasma membrane. Among these receptors, GPR40 is preferentially
expressed in pancreatic ␤-cells and mediates the stimulatory effect of NEFAs on insulin
secretion [28], and mice that lack GPR40 have a reduced susceptibility to the hyperin-
sulinemia, hepatic steatosis, increased hepatic glucose output, hyperglycemia, and glu-
cose intolerance induced by obesity [29].
This latter finding provides support for a more ‘␤-centric’ perspective of obesity-
induced insulin resistance, as opposed to the ‘adipo-centric’ perspective, with hyper-
insulinemia per se possibly contributing to hepatic steatosis, hepatic insulin
resistance, and hyperglycemia associated with diet-induced obesity. It remains to be
determined what kinds of signals regulate the secretion of adipokines or NEFA
release from adipocytes. Oxidative stress and endoplasmic reticulum-associated
stress may be candidates for such a signal.

Relationship between Insulin Sensitivity and Insulin Release

Fluctuations in insulin sensitivity occur during the normal life cycle, with insulin
resistance being observed during puberty, pregnancy, and with ageing. On the other
hand, increased physical activity and increased carbohydrate intake are associated
with enhanced insulin sensitivity. Hence ␤-cells are markedly adaptable in their abil-
ity to regulate insulin release in a very precise manner. Obviously, the ␤-cell is funda-
mental to ensuring that in healthy subjects, plasma glucose levels remain within a
narrow physiological range [for review, see 30].
In healthy individuals, there is a feedback loop between the insulin-sensitive tis-
sues and the ␤-cells, with ␤-cells increasing insulin supply in response to demand by
the liver, muscles and adipose tissue. The relationship between insulin sensitivity and
insulin levels is reciprocal and hyperbolic [31]. In response to changes in insulin sen-
sitivity, insulin release increases or decreases reciprocally to maintain normal glucose
tolerance. Insulin sensitivity is almost always decreased in obesity and insulin-resistant

Pathophysiology of Diabetes Type 2 7

individuals, whether lean or obese, have greater insulin responses and lower hepatic
insulin clearance than insulin-sensitive individuals. In contrast, individuals with high
risk of developing T2D display inadequate insulin release for any level of insulin sen-
sitivity at any stage of the disease and even when they have normal glucose tolerance,
suggesting that ␤-cell function has already being decreased before the development
of hyperglycemia [30]. Hence, failure of this feedback loop seems to contribute to the
development of DM.
Another important implication of this feedback loop is that assessment of ␤-cell
function requires knowledge of both insulin sensitivity and the insulin response, in
other words the interpretation of the ␤-cell’s secretory response to a given stimulus
must take into account the prevailing degree of insulin sensitivity. This ability of the
␤-cell to adapt to changes in insulin sensitivity seems to result from (1) the functional
responsiveness of the cell and (2) ␤-cell mass. In response to the insulin resistance
observed in obesity, puberty and pregnancy, human ␤-cells can increase insulin
release to levels 4- to 5-fold higher than in insulin-sensitive individuals, whereas ␤-cell
volume is only enhanced by about 50%. In individuals with normal ␤-cells, glucose
tolerance is preserved during these periods of insulin resistance as the decrease in
insulin sensitivity is matched by a compensatory increase in insulin release. In con-
trast, in groups of people with T2D and those at increased risk of developing T2D, the
decline in insulin sensitivity is not matched by a reciprocal increase in the insulin
response. Instead, the insulin response also declines, which is compatible with the
idea of ␤-cell dysfunction [30].

Adaptation of ␤-Cell Function to Insulin Resistance: Increased Insulin Release

Under physiological conditions, glucose-stimulated insulin secretion requires the

metabolism of glucose and thereby the generation of ATP. The resulting increase in
the ATP/ADP ratio triggers the closure of the ATP-sensitive potassium (KATP) chan-
nel, depolarization of the cell membrane and influx of calcium through voltage-
dependent calcium channels, resulting in insulin granule exocytosis [32]. The ␤-cell’s
adaptive response to changes in insulin sensitivity is probably mediated by increased
cellular glucose metabolism, NEFA signaling and sensitivity to incretins. Data from
animal studies suggest that the increase in ␤-cell glucose metabolism involves an
increase in the activity of glucokinase, the rate-limiting enzyme responsible for glu-
cose phosphorylation after its entry into the cell [33]. Glucose utilization rises as both
oxidation and flux of glucose are increased, the latter through pyruvate carboxylase
and the replenishment of tricarboxylic acid cycle intermediates in the mitochondria.
Increased citrate levels generated by glucose metabolism may lead to generation of
malonyl-CoA and increased long-chain acyl-CoA and diacylglycerol levels through
inhibition of carnitine palmitoyl transferase 1 [34]. This leads to PKC activation and
stimulation of insulin release. In humans, however, the role of increased glucose

8 Thévenod
levels for the adaptive increase in insulin release in response to decreased insulin sen-
sitivity is still debated [30].
NEFAs are important for normal ␤-cell function and may mediate increased ␤-cell
output in response to decreased insulin sensitivity. NEFAs potentiate insulin release
in response to glucose and non-glucose secretagogues by binding to the G-protein-
coupled receptor GPR40 on the cell membrane, resulting in the activation of phos-
pholipase C signaling and a subsequent increase in intracellular calcium and
secretory granule exocytosis [28]. Additionally, fatty acyl-CoA may also be generated,
which increases insulin release both by directly stimulating secretory granule exocy-
tosis and by PKC activation [30]. A third possible mechanism is increased sensitivity
to incretin hormones, such as glucagon-like peptide-1 (GLP-1), that are produced in
the intestinal mucosa and are responsible for the enhancement of the insulin response
observed after oral – compared with intravenous – glucose administration [35]. The
␤-cell might become more responsive to the effects of GLP-1 to modulate insulin
secretion by G-protein-coupled receptor activation involving stimulation of protein
kinase A (PKA) and the guanine nucleotide exchange factor EPAC2. The extensive
innervation of the islet by both parasympathetic and sympathetic neurons, and the
intimate involvement of the central nervous system (CNS) in the regulation of metab-
olism suggest that the CNS may also have an important role in the functional adapta-
tion to changes in insulin sensitivity [for review, see 30].

Adaptation of ␤-Cell Mass to Insulin Resistance: Mechanisms of Growth and

Proliferation

Although changes in ␤-cell function are observed under conditions of increased

secretory demand, the volume of ␤-cells also increases. In rodents fed a high-fat diet
for 12 months to induce obesity and insulin resistance, islet size increases as a result
of an increase in the number of ␤-cells rather than a change in ␤-cell size, and new
islets do not form [36]. NEFAs rather than glucose may mediate this increase in ␤-cell
mass [for review, see 30, 37]. In contrast, human studies suggest that ␤-cell volume is
increased by about 50% in healthy obese individuals, which, however seems to be
more dependent on hypertrophy of existing cells than proliferation [38, 39].
Interestingly, in the long-term increased dietary fat feeding study in rats, ␤-cell mass
increased but glucose-induced insulin release did not, which indicates a dissociation
between ␤-cell mass and the secretory function [36]. Increased signaling by insulin
and/or insulin-like growth factor 1 (IGF-1) could also underlie the modulation of
islet mass. Activation of the insulin/IGF-1 receptor leads to phosphorylation of IRS-2
and downstream signaling through pathways including PI3K/protein kinase-B
(PKB/Akt) and Ras, leading to activation of the mitogen-activated protein (MAP)
kinases ERK-1 and ERK-2 [40]. IRS-2 appears to play a key role in the cellular
processes associated with increased ␤-cell proliferation, neogenesis and survival.

Pathophysiology of Diabetes Type 2 9

Finally, the incretin GLP-1 is an insulin secretagogue but is also a ␤-cell mitogen,
capable of increasing ␤-cell proliferation and reducing ␤-cell apoptosis in animal
models through several pathways, including transactivation of the epidermal growth
factor receptor and stimulation of the IRS-2 pathway [35]. Whether GLP-1 has simi-
lar effects in humans is not known. Finally, neural signaling could also contribute to
increased ␤-cell mass.

Failed Adaptation to Insulin Resistance and ␤-Cell Failure: The ‘Natural

History’ of T2D?

Normal pancreatic ␤-cell responds to a chronic fuel excess and obesity-associated

insulin resistance with compensatory insulin hypersecretion in order to maintain nor-
moglycemia. This adaptive response to insulin resistance involves changes in both
function and mass, and is so efficient that normal glucose tolerance is maintained.
Longitudinal studies of subjects that develop T2D show a rise in insulin levels in the
normoglycemic and prediabetes phases that keep glycemia near normal despite the
insulin resistance (␤-cell compensation), followed by a decline when fasting glycemia
surpasses the upper limit of normal of 5.5 mM (␤-cell failure). T2D may develop when
pancreatic ␤-cells fail to secrete sufficient amounts of insulin to meet the metabolic
demand. Both insulin secretion and insulin action are impaired in T2D [reviewed in
41]. Their relative importance has been hotly debated, but it is now recognized that ␤-
cell dysfunction is crucial for the development of the disease [30, 37]. For example,
abnormalities in insulin secretion precede the onset of T2D and may be present even
when subjects show normal glucose tolerance [42, 43]. By the time of diagnosis,
insulin secretion is significantly reduced and it continues to diminish inevitably
throughout the course of the disease [43]. T2D can also occur in the absence of insulin
resistance [44] and, conversely, some severe forms of insulin resistance (such as those
caused by mutations in the insulin receptor) may not be accompanied by diabetes [45].
Thus, it now appears that insulin resistance only leads to diabetes if combined with a
genetically determined tendency to ␤-cell dysfunction [30, 44]. In these individuals,
however, insulin resistance plays an important role in the development of diabetes by
placing an increased demand upon the ␤-cell that it is unable to match.
The number of ␤-cells is clearly reduced by about 50% in T2D [38, 39], but this
degree of ␤-cell loss cannot fully account for the change in secretory function of exist-
ing intact ␤-cells. The ␤-cell is unable to release insulin rapidly in response to intra-
venous glucose, despite the fact that the ␤-cells in individuals with T2D clearly
contain insulin and delivery of non-glucose secretagogues can acutely increase
insulin release but does not result in equivalent responses to those seen with similar
stimulation in healthy subjects [46–48]. The ␤-cell failure and T2D that follow ␤-cell
compensation could result from both inadequate expansion of ␤-cell mass and failure
of the existing ␤-cell mass to respond to glucose, e.g. due to a defect in insulin and

10 Thévenod
IGF-1 signaling in pancreatic ␤-cells or impaired incretin signaling in the ␤-cell.
However, experimental evidence for these processes is currently lacking.
The extremely elevated blood glucose levels frequently observed in diabetes may
contribute to further disease progression through glucotoxic effects on the ␤-cell and
harmful effects on insulin sensitivity, both of which can be ameliorated by therapeu-
tically lowering the glucose level [49]. By contrast, raising the blood glucose level for
20 h in healthy subjects has exactly the opposite effect: it improves insulin sensitivity
and enhances ␤-cell function [50]. This also suggests that a pre-existing, and perhaps
genetically determined, risk is crucial for ␤-cell dysfunction to occur. It is this pre-
existing abnormality that results, with time, in a progressive impairment in insulin
release and, ultimately, an increase in glucose levels, the latter of which further aggra-
vates the situation and thereby contributes to ␤-cell failure.
Groups at increased risk of subsequently developing diabetes exhibit ␤-cell dys-
function well before they would be considered to have reduced glucose tolerance, in
keeping with the idea of a pre-existing risk. Examples include women with a history
of gestational diabetes or polycystic ovarian syndrome, older subjects, who frequently
develop hyperglycemia as they continue to age, and individuals with impaired glu-
cose tolerance [30]. First-degree relatives of individuals with T2D, who are genetically
at increased risk, also have impaired ␤-cell function, even though they may still have
normal glucose tolerance [42]. This has particularly been well studied in the Pima
Indians, in whom the prevalence of diabetes is higher than almost any other group in
the world, and also been confirmed for non-Hispanic whites, African-Americans and
Hispanics participating in the Insulin Resistance Atherosclerosis Study (IRAS)
[reviewed in 30].

Pathogenesis of T2D: Genetic Factors

Many genes interact with the environment to produce obesity and/or T2D. In the case
of obesity, the most frequent mutation is that in the melanocortin-4 receptor, which
accounts for up to 4% of cases of severe obesity. Other rare causes include mutations
in leptin and the leptin receptor, prohormone convertase 1 (PC1) and pro-opiome-
lanocortin (POMC). The gene variant most commonly associated with insulin sensi-
tivity is the P12A polymorphism in PPARg, which is associated with an increased risk
of developing T2D [15, 51]. A number of genes associated with ␤-cell dysfunction
have been identified, including two non-coding single-nucleotide polymorphisms in
transcription factor 7-like 2 (TCF7L2) and mutations in the mitochondrial genome
that are also associated with neurosensory hearing loss [51]. Work is ongoing on
many candidate genes, including calpain 10, adiponectin, PPAR-␥ coactivator 1
(PGC1) and the glucose transporter GLUT2 [51].
Polymorphisms in genes encoding ␤-cell ion channels are also causative candi-
dates for a reduction in ␤-cell function that may be associated with T2D. One that has

Pathophysiology of Diabetes Type 2 11

received much recent attention is a polymorphism (E23K) in the Kir6.2 subunit of the
KATP channel (encoded by KCNJ11), with a prevalence of 34% and between 11 and
15% of the population risk of T2D in Caucasians [51]. When heterologously
expressed in mammalian cells, the E23K polymorphism leads to a 2-fold reduction in
the ATP sensitivity of the KATP channel [52]. It is therefore expected to reduce ␤-cell
electrical activity and insulin secretion. The functional effects of the E23K polymor-
phism in man are, however, controversial [53]. Polymorphisms in ABCC8/SUR1
have also been linked to T2D [54].
Because metabolism regulates ␤-cell function, polymorphisms in metabolic genes
may also influence the ability of glucose to stimulate electrical activity and insulin
secretion. Support for this idea comes from a number of rare (1–5%) monogenic forms
of diabetes referred to as maturity-onset diabetes of the young [6] because they present
early in life [51]. The first of these to be identified (MODY2) results from inactivating
mutations in glucokinase, the high Km enzyme that phosphorylates glucose in ␤-cells
and is rate-limiting for glucose metabolism. All MODY2 patients are heterozygotes:
permanent neonatal diabetes results from homozygous mutations [55]. Other forms of
MODY are due to mutations in genes (e.g. HNF1␣, HNF4␣, IpF1) encoding a tran-
scriptional network that regulates the expression of several genes critical for glucose
sensing [51]. Mutations in mitochondrial DNA (most frequently A3243G in the
leucine tRNA gene) cause maternally inherited diabetes, probably by impairing ␤-cell
metabolism and so reducing insulin secretion [56]. These account for a further 1–2%
of diabetic cases. There is also an overlap between MODY and multifactorial T2D [51].
Mitochondrial metabolism generates substantially more ATP than glycolysis and
the production of mitochondrial ATP is critical for both glucose-dependent insulin
secretion and KATP channel closure. It is therefore pertinent that polymorphisms in
genes that regulate mitochondrial ATP production are associated with T2D. UCP2 is
an uncoupling protein that resides in the inner mitochondrial membrane and uncou-
ples electron transport from ATP synthesis [11]. This suggests that the level of UCP2
expression may influence insulin secretion in man. Consistent with this idea, a com-
mon polymorphism in the UCP2 promoter (⫺866G/A) causes a 2-fold increase in the
risk of T2D in obese white Europeans [57]. The frequency of the 866A variant in the
European population is 37% [58], suggesting it may make a significant contribution
to T2D. The ␤-cell transcription factor PAX6 preferentially binds to, and transacti-
vates, the ⫺866A/A variant in insulin-secreting (INS-1) cells and is expected to
increase UCP2 mRNA expression in islet ␤-cells, thereby reducing ATP levels, elec-
trical activity and insulin release. Recent studies further indicate that islets from
⫺866A/A homozygous had lower insulin secretion in response to glucose stimulation
as compared with ⫺866G/G and ⫺866G/A carriers [59]. A common variant in mito-
chondrial DNA itself (16189) causes a T to C transition in a region of mtDNA that lies
close to control sequences governing replication and transcription. It is associated
with increased fasting plasma insulin in several populations, maternal restraint of
fetal growth and thinness at birth and is also with T2D [60].

12 Thévenod
Pathogenesis of T2D: Interactions between Genes and the Environment

Environmental factors are largely responsible for the modern-day outbreak of obesity
and T2D. Increased caloric availability and fat consumption in the setting of decreased
physical activity lead to overnutrition, increased nutrient storage, obesity and insulin
resistance. This has important consequences if ␤-cell function is already inherently
abnormal owing to genetic susceptibility: obesity promotes insulin resistance, which
can lead to insulin insufficiency if the secretory capacity of the ␤-cell is already lower
than normal. Another proposed environmental mechanism is thought to occur in
utero and/or during the early postnatal period when poor nutrition alters metabolism,
resulting in a tissue adaptation that favors the storage of nutrients (‘thrifty phenotype
hypothesis’ of T2D) [61]. The end result of these environmental changes is a deleteri-
ous interaction with genes that predispose to the development of obesity and T2D.
Any explanation of T2D must also account for the fact that disease develops with age
and that it is enhanced by obesity. There is evidence that the interaction of such lifestyle
factors with genetic ones may also occur at the level of ␤-cell function. This may reflect
the decline in mitochondrial function with age that is believed to result from accumu-
lating mutations in mitochondrial DNA [56, 62]. Obese individuals and T2D patients
have higher circulating levels of NEFAs [22, 63], which are taken up and metabolized by
␤-cells. Chronic exposure to NEFAs leads to the accumulation of long-chain acyl CoAs
(LC-CoAs) within the ␤-cell. LC-CoAs both enhance KATP channel activity and reduce
its ATP sensitivity [64], thereby reducing glucose-dependent closure of KATP channels,
electrical activity and insulin secretion. Moreover, changes in ␤-cell metabolism as a
consequence of age and/or obesity will translate into reduced ␤-cell electrical activity
and insulin secretion, not only to glucose but also to incretins and sulfonylureas.
It is well established that T2D is a polygenic disease [51]. However, how these
polymorphisms relate to the disease phenotype, has not been established. A current
model for T2D suggests that the effect of several gene variants and lifestyle factors
combine to produce a small decrease in ␤-cell function and thus a reduction in
insulin secretion. Though the functional consequences of each individual gene vari-
ant will be small, so that a single polymorphism, by itself, is unlikely to result in dia-
betes, the cumulative effect of several such polymorphisms will increase disease risk,
and in combination with age and/or obesity lead to overt disease. ␤-Cell function
could serve as a bottleneck at which the effects of many different genes and lifestyle
factors converge. Individuals at risk of T2D may carry one or more polymorphisms in
ion channel genes, or in genes regulating their activity, membrane targeting or tran-
scription. Because the functional effect of these gene variants is small this does not
cause diabetes in early life, because glucose homeostasis is tightly controlled and the
␤-cell secretory output is adjusted. However, with age, ␤-cell metabolic function
declines, leading to a reduction in glucose sensitivity, electrical activity and insulin
secretion. In non-diabetics this does not pose a problem, as the ␤-cell adjusts its
secretory output. However, in individuals who already have genetically determined

Pathophysiology of Diabetes Type 2 13

reduced ␤-cell function, the further reduction in electrical activity means that ␤-cell
is no longer able to compensate, so that insulin secretion declines and glucose intoler-
ance, and subsequently overt diabetes, develop. Obesity exacerbates the situation
both by causing insulin resistance (increasing the demand on the ␤-cell) and by fur-
ther decreasing insulin secretion from the ␤-cell. As pancreatic ␤-cells are exposed
during ␤-cell compensation to metabolic changes associated with obesity, so factors
commonly associated with obesity – such as insulin resistance (including that in ␤-cells),
adipokines, NEFAs, reactive oxygen species, and endoplasmic reticulum-associated
stress – should also be examined as candidates for inducers of ␤-cell failure.

Current Concepts and Future Directions

Having a single mechanism to explain the link between obesity, insulin resistance and
T2D would be ideal. NEFAs induce insulin resistance and impair ␤-cell function,
making them a likely culprit [8, 22, 30, 37]. Although NEFAs are critical for normal
insulin release, chronic exposure to NEFAs in vitro and in vivo is associated with
marked impairments in glucose-stimulated insulin secretion and decreased insulin
biosynthesis [8, 22, 65]. Elevated NEFA levels produced by a lipid infusion in vivo
contribute to the development of insulin resistance and also prevent the expected
compensatory ␤-cell response in humans [66]. This dual effect makes them a good
candidate to link obesity, insulin resistance and ␤-cell dysfunction in individuals with
T2D and those at risk of the disorder. This lipotoxic effect can also act synergistically
with glucose to produce even greater deleterious effects, commonly referred to as
‘glucolipotoxicity’.
A genetically determined defect in insulin release by the ␤-cell could also be cru-
cial [30, 46–48]. Impaired insulin release could result in decreased insulin levels and
decreased signaling in the hypothalamus, leading to increased food intake and weight
gain, in disordered regulation of glucose levels by decreasing suppression of hepatic
glucose production and reducing the efficiency of glucose uptake in insulin-sensitive
tissues. Decreased insulin output could also impair adipocyte metabolism, resulting
in increased lipolysis in the adipocyte and elevated plasma NEFA levels. Thus the
process may slowly feed forward, in keeping with observations that the onset of T2D
is usually a slow process that takes many years. Even mild impairments of insulin
release may have central effects on metabolic homeostasis. Insulin acts in the hypo-
thalamus to regulate body weight, and impaired insulin signaling is associated with
changes in food intake and body weight. Thus, ␤-cell dysfunction resulting in a rela-
tive reduction in insulin release would be expected to result in decreased insulin
action in this crucial brain region and be associated with weight gain and an aggrava-
tion of insulin resistance [8].
Both of the prominent features of T2D – insulin resistance in peripheral tissues
and ␤-cell failure – may result from a defect in insulin signaling. Mice lacking insulin

14 Thévenod
receptors or a dominant negative mutant of PI3K activity in the liver exhibit insulin
resistance, glucose intolerance, and a failure of insulin to suppress hepatic glucose
production and to regulate hepatic gene expression [67, 68]. These observations sug-
gest that insulin resistance in the liver contributes to the pathogenesis of T2D, which
is not the case for muscle or adipocytes. Insulin resistance at the level of the ␤-cell
may also have a role in the pathogenesis of defective insulin release. IRS-2-deficient
mice exhibit impaired pancreatic ␤-cell function [69]. Moreover, mice that lack
insulin receptors in ␤-cells also have a defect in glucose sensing and a reduced ␤-cell
mass [70]. Although there is currently no evidence that insulin receptor mutations
are commonly associated with T2D, a reduction in insulin signaling in the ␤-cell
remains an interesting possibility in further integrating defects in insulin action into
the pathogenesis of obesity and T2D.
Another such unifying hypothesis may be that genetically determined mitochon-
drial dysfunction may cause both insulin resistance in peripheral tissues and impair-
ment of glucose-induced insulin secretion in ␤-cells [10]. However, it remains to be
proven whether altered mitochondrial oxidative phosphorylation is an underlying
genetic element of insulin resistance. In a recent study, muscle- and liver-specific AIF
ablation in mice initiated a pattern of oxidative phosphorylation deficiency closely
mimicking that of human insulin resistance, but contrary to current expectations,
resulted in increased glucose tolerance, reduced fat mass, and increased insulin sensi-
tivity [71]. This finding suggests that the moderate deficiency in oxidative phospho-
rylation that is observed in peripheral tissues of insulin-resistant humans is not a
causative factor in T2D but may instead be a compensatory response.
In summary, current concepts to explain the pathogenesis of T2D propose that
genes responsible for obesity and insulin resistance interact with environmental fac-
tors (increased fat/caloric intake and decreased physical activity), resulting in the
development of obesity and insulin resistance. These increase secretory demand on
␤-cells. If the ␤-cells are normal, their function and mass increase in response to this
increased secretory demand, leading to compensatory hyperinsulinemia and the
maintenance of normal glucose tolerance. By contrast, susceptible ␤-cells have a
genetically determined risk, and the combination of increased secretory demand and
detrimental environment result in ␤-cell dysfunction and decreased ␤-cell mass,
resulting in progression to impaired glucose tolerance, followed, ultimately, by the
development of T2D. New insights of the genetic bases of these processes, of the cel-
lular events that underlie them and of their relationship to environmental factors
should enhance our ability to devise breakthrough therapies for T2D.

Acknowledgement

I thank Prof. Patrik Rorsman (Oxford Centre for Diabetes, Endocrinology and Metabolism,
Churchill Hospital, Oxford, UK) for his continuous interest in my work.

Pathophysiology of Diabetes Type 2 15

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Frank Thévenod, MD, PhD

Department of Physiology and Pathophysiology, University of Witten/Herdecke
Stockumer Strasse 12 (Thyssenhaus), DE–58448 Witten (Germany)
Tel. ⫹49 2302 926221, Fax ⫹49 2302 926182
E-Mail [email protected]

18 Thévenod
Masur K, Thévenod F, Zänker KS (eds): Diabetes and Cancer. Epidemiological Evidence and Molecular Links.
Front Diabetes. Basel, Karger, 2008, vol 19, pp 19–29

In vivo ␤-Cell Imaging in Diabetes,

␤-Cell Hyperplasia, and Insulinoma
Martin Gotthardta ⭈ Martin Béhéb ⭈ Theo Lasserc
a
Department of Nuclear Medicine, Radboud University Nijmegen Medical Center,
Nijmegen, The Netherlands; bDepartment of Nuclear Medicine, University Hospital
Giessen and Marburg, Campus Marburg, Marburg, Germany, and cLaboratoire
d’Optique Biomédicale, Faculté des Sciences et Techniques de l’Ingénieur,
Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Abstract
A reliable method for (repeated) non-invasive quantification of β -cell mass in vivo in humans will
enhance our understanding of the pathophysiology of both type 1 and type 2 diabetes. Individual
patients with type 2 diabetes show large differences regarding the relative contribution of insulin resis-
tance or insulin deficiency to the diabetic state. Also, the deterioration of β -cell function varies. When
β -cell mass could be measured in vivo in humans, the effects of different diabetes treatments on β -cell
mass could be studied and result in a more individually-tailored therapy, based on the principle underly-
ing defect. Furthermore, quantification of β -cell mass could be used for monitoring in patients with dia-
betes type 1 undergoing islet transplantation. Recently, new strategies for imaging of β -cell in vivo have
been developed. For imaging of transplanted β -cell in vivo, the β -cells can be preloaded with super-
paramagnetic iron-oxide particles. By this approach, magnetic resonance imaging of β -cell mass was
possible in mice. For imaging of pancreatic β -cells, radiolabeled tracers are preferable due to their high
sensitivity. A tracer targeting the β -cell-specific transport molecule VMAT-2 has been used in mice for
positron emission tomography. Another promising approach is targeting of the glucagon-like peptide-1
receptor with a radiopeptide. Copyright © 2008 S. Karger AG, Basel

Clinical and Scientific Significance of Non-Invasive Determination of

the ␤-Cell Mass in Diabetes

A reliable method for (repeated) non-invasive quantification of ␤-cell mass in vivo in

humans will enhance our understanding of the pathophysiology of both type 1 and
type 2 diabetes (T1D, T2D). Progressive ␤-cell loss is characteristic for T1D, but the
natural history of ␤-cell loss remains to be determined. ␤-Cell dysfunction is a hall-
mark of T2D, but it is not known at which stage of the disease this occurs. Individual
patients show large differences regarding the relative contribution of insulin resistance
or insulin deficiency to the diabetic state. Also the deterioration of ␤-cell function
varies. Development of diabetes is thought to occur in steps [1]. At early stages, ␤-cell
mass may even be increased [2]. Development of ␤-cell mass and ␤-cell function in
the course of disease do not necessarily show a direct correlation, i.e. in particular
stages of the disease, the ␤-cell function may be impaired while the ␤-cell mass is not
significantly reduced or vice versa [1]. If a technique were available for non-invasive
quantification of ␤-cell mass, it would be possible to follow the natural history of the
decline of functional and afunctional ␤-cell mass in both T1D and T2D. A method to
non-invasively measure ␤-cell mass in vivo in humans would also enable us to study
the effects of different diabetes treatments on ␤-cell mass which may result in a more
individually-tailored therapy, based on the principle underlying defect. Such a tech-
nique would for example enable us to monitor ␤-cell mass in vivo in patients receiv-
ing antidiabetic medication thought to increase ␤-cell mass in T2D (such as
Exenatide or inhibitors of dipeptidyl peptidase IV [3]).
Quantification of ␤-cell mass could also be used for monitoring in patients with
T1D undergoing islet transplantation which is a promising method for restoration of
glucose homeostasis. To obtain a sufficient number of functioning islets, islets are
typically isolated from two cadaveric donor pancreata and transplanted. As yet, it is
unknown how many of these islets will contribute to glucose homeostasis directly
after transplantation, i.e. how many islets survive during the first weeks after trans-
plantation. Using a number of approximately 800,000–900,000 islets per patient, the
rate of insulin-independent patients is approximately 80% after 1 year, which drops to
about 65% after 2 years [4]. Monitoring of the ␤-cell mass after transplantation may
help to optimize immunosuppressive therapy regimens and thus help to increase the
rate of insulin-independent ␤-cell recipients.
In diabetes research, non-invasive methods for quantification of ␤-cell mass
(including ␤-cell loss and ␤-cell neogenesis) would help to perform longitudinal
studies in animal models addressing the questions related to ␤-cell mass mentioned
above. Such methods would allow researchers to image ␤-cell mass in vivo by small
animal imaging techniques but also to follow individual islets or subgroups of islets
in vivo. This would greatly improve monitoring of new therapies in animal models,
speed up their translation into clinical trials, and would help to reduce the number of
animals required because longitudinal studies would no longer require immunohis-
tochemical determination of ␤-cell mass in pancreatic specimen from killed animals.
Furthermore, the effects of new drugs on individual islets (with respect to blood flow,
islets biodistribution of ␤-cell markers, etc.) could be monitored.

Imaging Methods for Non-Invasive Imaging of ␤-Cell Mass

The optimal solution for non-invasive imaging of ␤-cells would be a method with
high depth penetration and without radiation exposure, offering high sensitivity and

20 Gotthardt ⭈ Béhé ⭈ Lasser

specificity combined with a resolution allowing to image single ␤-cells. Current
imaging techniques offer all these characteristics, but unfortunately no single tech-
nique offers all of them at the same time. Therefore, all imaging methods potentially
useful for in vivo ␤-cell imaging are a compromise. For a given task, such as imaging
of pancreatic ␤-cells versus imaging of transplanted ␤-cells, an individual approach
needs to be chosen dependent on the specific technical characteristics of the imaging
modality used as well as characteristics of specific tracers etc. In the following, the
most important current imaging techniques with potential for in vivo ␤-cell imaging
will be described together with their specific advantages and disadvantages.

Positron Emission Tomography

Positrons are emitted from tracers labeled with positron-emitting radionuclides.
These positrons hit electrons in their close vicinity, which results in annihilation and
simultaneous emission of a pair of high-energetic photons at an angle of 180⬚ (‘coin-
cidence’). These photons cause light flashes in a crystal ring, which are detected by
adjacent photomultipliers. The source of the photons is located on a straight line
between the positions in which the signals have been detected in the crystal ring. This
information is used to create 3-dimensional (3-D) images of the tracer distribution.
The (physical) spatial resolution of modern clinical positron emission tomography
(PET) scanners currently reaches about 2 mm.

Single Photon Emission Computed Tomography

For single photon emission computed tomography (SPECT) imaging, low-energy
photons (ca. 140–200 keV) emitted by ␥-emitting radionuclides are detected by
gamma cameras. In comparison to PET, this technique uses thinner crystals for
detection of photons. Because the photons detected are not coincident (and therefore
the information about the 3-D location of the source is missing), so-called collima-
tors are used to create an image. Collimators are lead plates with bores that let only
photons pass which hit the crystal orthogonally and eliminate all others. In compari-
son to PET, this results in lower detection sensitivity as a part of the emitted radiation
is absorbed in the collimators. Since the 3-D images have to be reconstructed from 2-D
projection images and the collimators limit the resolution by the size of the bores,
SPECT has a lower spatial resolution and quantification is more difficult. The advan-
tages of SPECT are the relative low costs and the wider availability. SPECT radionu-
clides often have longer half-lives which facilitates labeling and use of tracers.

Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) relies on the detection of spin relaxation of
excited hydrogen nuclei in a magnetic field in water and lipids. In an intensive, uni-
form magnetic field, the spins of atomic nuclei with a resulting non-zero spin have to
arrange in a particular manner with the applied magnetic field according to quantum
mechanics. Nuclei of hydrogen atoms align either parallel or antiparallel to the magnetic

␤-Cell Imaging 21
field. The magnetic dipole moment of the nuclei processes around the axial field. If
exposed to short electromagnetic impulses, some of the magnetically aligned hydro-
gen nuclei assume a temporary non-aligned high-energy state. When returning to
their prior state, a change in a weak magnetic field is induced which can be detected
by coils within the magnetic field of the scanner. These signals are then converted
into 3-D images. Besides gaining anatomical information, it is also possible for exam-
ple to measure blood flow. The spatial resolution of clinical MRI scanners reaches ca.
1 mm. The sensitivity of this technique, however, is 3–6 orders of magnitude lower
than that of PET.

Optical Imaging
Optical imaging techniques suitable for in vivo ␤-cell imaging are highly sensitive
and will most probably either rely on the detection of a fluorescent tracer or on the
reflection of light. For the first technique, a fluorescent probe attached to a suitable
tracer molecule is detected by a camera system equipped with a charged-coupled
device. An excitation light of a specific wavelength (in the visible light range of
395–600 nm) illuminates the subject which emits light of a shifted wavelength which
can be detected. Another option is extended field Fourier domain optical coherence
microscopy (xf-FDOCM), a technique with a very high sensitivity and an intrinsic 3-
D imaging modality. The underlying contrast mechanism is due to small changes in
the refractive index of the investigated tissue layers and translates into a label-free tis-
sue imaging. In addition, due to the intrinsic optical amplification of the low coher-
ence interferometry, tissue structures down to a depth of ca. 2 mm can be visualized.
The depth profile of the sample is encoded in the detected interferogram and
extracted via a fast Fourier transform. Therefore, FDOCM allows 3-D imaging but
needs only 2-D sampling in the x-y direction with an extremely high imaging speed.
xf-FDOCM combines advanced illumination concepts based on Bessel beams, which
results in a uniform depth-independent lateral resolution close to single cell resolu-
tion. FDOCT (Fourier domain optical coherence tomography) in general allows
extracting the phase signal, which can be used for fast blood flow measurements.
Molecular imaging is an additional option to merge with FDOCM based on molecu-
lar tracers with a specifically enhanced absorption. Overall, xf-FDOCM provides a
fast 3-D imaging concept with high contrast and high resolution suitable for in vivo
imaging of biological tissues and small organ features.

Tracer Development
Development of radiotracers relies on several factors, including size of the tracer mol-
ecule, specificity and affinity of binding to a given target, metabolic behavior, meta-
bolic stability, stability of the label, etc. A highly diffusible small tracer molecule
rapidly binding to a target with high specificity and affinity is optimal. At the same
time, the tracer should be cleared from the background (blood, non-target tissues) as
quickly as possible, preferably via the kidneys. Clearance via the liver would lead to

22 Gotthardt ⭈ Béhé ⭈ Lasser

high uptake into the gastrointestinal tract resulting in background activity blurring
the target on the images obtained [5]. If the tracer is taken up into the cell, metabolic
trapping is one method to obtain high target-to-background ratios. Metabolic trap-
ping means that a tracer is taken up into the cell and stays there if no metabolic path-
way for tracer degradation or externalization exists. If metabolic trapping is achieved
via the labeling method, the label is called a ‘residualizing label’. At the same time,
unbound tracer is excreted via the kidney so that the target-to-background ratio
increases over time. If a tracer is not internalized, a high affinity is required to obtain
a good target-to-background ratio. An example for such a tracer would be a radiola-
beled antibody binding with high affinity to an antigen on the surface of the target
cells. If a potential tracer molecule would lose its specificity for or binding affinity to
a target due to necessary modifications for a residualizing label, it can be labeled with
F-18 or C-11, two positron emitters. Labeling with these radionuclides does not (or to
a lesser extent) cause major changes to the tracer molecule, in the case of C-11 the
radionuclide may even be integrated into the tracer molecule without changing its
chemical properties at all. The short half-life of the radionuclides, however, requires
efficient and quick labeling procedures. For specific targeting with radiotracers, the
choice of the best approach (residualizing label, high-affinity binding, C-11 label to
preserve chemical properties) is dependent on the target, the potential tracer mole-
cules and the possible/available synthesis/labeling techniques.
Production of optical tracers can be done based on radiotracers, because the label-
ing with radiometals can often be replaced by optical dyes. In the case of MRI tracers,
paramagnetic particles (such as iron oxide) have to be packed in nanoparticles coated
with specific ligands. These ligands may need modification for coating of the nanopar-
ticles. Furthermore, the nanoparticles have to be carefully chosen and optimized con-
cerning their size as this determines diffusibility, residence time in the circulation,
and the pathways of elimination.
For clinical ␤-cell imaging, MRI, PET, and SPECT are currently the most promis-
ing imaging techniques. However, none of them is able to resolve single islets or ␤-
cells. Therefore, these techniques have to rely on the quantification of the uptake of
tracers into the ␤-cells. These tracers would have to be specific for ␤-cells and target the
␤-cells with a high affinity. Furthermore, quick targeting would allow scanning within
a short period of time after tracer injection which would make the technique more
convenient for clinical use.
For research purposes, basically the same is true as said above for MRI, PET, and
SPECT. However, other imaging techniques with a limited depth of penetration
would also be suitable for in vivo ␤-cell imaging. Optical imaging offers unique pos-
sibilities for imaging in animals. Because the penetration depth is less important in
animals than in humans, it is possible to use optical imaging techniques in animal
research. Fluorescent tracers would allow to image pancreatic ␤-cells in vivo in ani-
mals. Basically, the same tracers as used for PET, SPECT, or MRI imaging could also
be labeled with fluorescent dyes. For quantification, 3-D scanning would be preferable

␤-Cell Imaging 23
which is currently in a developmental state. This technique would also rely on the
specific accumulation of a tracer in ␤-cells and would not be able to resolve single ␤-
cells or islets. In contrast, due to the high spatial resolution and the short scanning
times, xf-FDOCM would allow to image and follow single islets over time. The limi-
tation is the lower penetration depth, which is why in vivo imaging would require
laparotomy. Therefore, this technique would for example be suitable for anatomical
imaging of islet response to certain stimuli. Furthermore, ␤-cell tracers changing the
refractory index of the tissue in which they have accumulated might also be detectable
with this technique. Whether the sensitivity of xf-FDOCM for this application is suf-
ficient will largely depend on the amount of tracer accumulation in the ␤-cells as well
as the improvement of existing techniques.

Different Approaches to in vivo ␤-Cell Imaging

A large variety of potential ␤-cell imaging agents has been tested as tracers, including
mannoheptulose, glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicoti-
namide, fluorodeoxyglucose, fluorodithizone, glyburide analogs, and antibodies
[6–9]. For most of the agents developed for ␤-cell imaging it has not been demon-
strated that they are able to precisely determine the ␤-cell mass in vivo [6, 10]. For
most agents, uptake into the pancreas was relatively low or uptake into the ␤-cells was
not sufficient in relation to uptake in the exocrine pancreas to allow sensitive and spe-
cific imaging of the ␤-cells [6, 8, 11].
In order to develop a method for more specific ␤-cell imaging, the use of radiola-
beled antibodies against pancreatic ␤-cells has been proposed [9]. It was shown that
these antibodies specifically bind to ␤-cells in vivo. So far, (small animal) in vivo
imaging has not been demonstrated using this antibody preparation. A major obsta-
cle for the use of antibodies for imaging is their long circulatory half-life.
Furthermore, the large size of antibodies also hinders diffusion into the target tissues.
Therefore, their use for nuclear medicine imaging is limited as the high blood activity
decreases the target-to-background ratios [5]. This is especially true if the target is
small, consists of solid tissue, and is surrounded by other well-perfused organs and
large blood vessels, as is the case in pancreatic ␤-cell imaging.
For imaging of transplanted ␤-cells, optical imaging methods have been successful
in animal models. As stated above, optical imaging may be of very limited use in a clin-
ical situation. For research in animal models, however, it may prove very useful. Park
et al. [12] have successfully generated transgenic mice expressing firefly luciferase under
control of the mouse insulin I promoter. In this animal model, the pancreatic ␤-cells
can be visualized by whole-body bioluminescence imaging. Whether ␤-cell imaging in
this mouse model may be helpful beyond measurement of blood glucose levels in the
evaluation of diabetes remains to be shown. Another example of successful in vivo ␤-
cell imaging is the work of Fowler et al. [13] who were able to follow transplanted islets

24 Gotthardt ⭈ Béhé ⭈ Lasser

by bioluminescence imaging for more than 8 weeks. Prior to transplantation, the islets
had been transfected with the luciferase gene by adenovirus-mediated gene transfer.

Recent Advances in in vivo Imaging of Pancreatic ␤-Cells

Recently, a compound targeting vesicular monoamine transporter-2 (VMAT-2)

specifically expressed on ␤-cells has been labeled for PET imaging of ␤-cells (dihy-
drotetrabenazine (DTBZ) labeled with 11C or 18F). In a rat model of spontaneously
developing diabetes (BB-DB rat), a significant decline in pancreatic uptake of 11C-
DTBZ anticipating the loss of glycemic control, could be found in longitudinal PET
studies [14]. Based on comparison of standardized uptake values of 11C-DTBZ and
blood glucose concentrations, loss of more than 65% of the original standardized
uptake value correlated significantly with the development of persistent hyper-
glycemia. A high pancreatic uptake was also observed in rodents and non-human pri-
mates [15]. However, after a major chemical eradication of ␤-cells, the pancreatic
uptake of DTBZ was reduced by only 30–40%, indicating that the uptake of the com-
pound might lack specificity for eventual clinical use [16]. In clinical studies with
optimized dynamic imaging protocols and analyzing several parameters (uptake,
pancreatic mass, etc.), the maximal decrease of tracer uptake in patients with long-
standing T1D with complete loss of ␤-cell mass never exceeded 45% in comparison
to healthy volunteers [17, 18]. Therefore, at this timepoint it is questionable whether
small differences in ␤-cell mass in T2D or in the early phases of T1D can be detected
with this tracer, let alone the small increase of ␤-cell mass which is anticipated under
due conditions promoting neogenesis or after pharmacological treatment.
Currently, a tracer based on the glucagon-like peptide-1 (GLP-1) analog Exendin
targeting the GLP-1 receptor is under development in our laboratory. With funding
from the National Institutes of Health, it is optimized for imaging in humans. In com-
parison to DTBZ, this compound also has a high in vivo uptake in the pancreas seems
to be more specific, resulting in reduction of pancreatic uptake by ⬎90% after chem-
ical eradication of ␤-cells as opposed to 30–40% for DTBZ [19, 20]. Therefore, this
tracer may be more suitable for clinical imaging of pancreatic ␤-cells than DTBZ.
In comparison to conventional optical imaging strategies for 3-D imaging, xf-
FDOCM offers the unique possibility of in vivo dynamic imaging. This technology is
currently optimized for the determination of islet size, ␤-cell mass, and islet blood
flow (by Doppler technique). This technique has successfully been used for in vivo
imaging of human retina including blood flow [21] on a microscopic level. First
results of imaging in pancreatic islets have been very promising. Single islets could be
measured in size and the distribution of the islets could be determined in mouse pan-
creata [M. Villiger and T. Lasser, Lausanne, Switzerland, pers. commun.]. In the
future, this technique may allow to follow islet size, islet distribution, and islet blood
flow in vivo in animal models of diabetes over time.

␤-Cell Imaging 25
Recent Advances in in vivo Imaging of Transplanted ␤-Cells

In vivo imaging of transplanted islets prelabeled with superparamagnetic iron-oxide

nanoparticles using MRI has successfully been achieved in animal models by two
groups [22, 23]. The islets are incubated with superparamagnetic iron-oxide nanoparti-
cles before transplantation. Not incorporated nanoparticles are removed by washing
before transplantation. After transplantation into the liver, the loaded islets are visible as
voids on the T2-weighted image. The signal seems to be dependent on the number of
living islets because in rejection models of islet transplantation the signal decreases with
the decreasing number of islets [24, 25]. It has been shown that the labeled islets pre-
serve their function [23]. Another major concern may be that not only living islets are
imaged but also deposits of superparamagnetic iron-oxide nanoparticles remaining
after the islets themselves have died. This theoretical problem has not been reported to
have hampered the experiments so far and localization of signal and islets as deter-
mined immunohistochemically has been reported to correlate well [23, 25]. With this
technique, living islets have been followed in vivo up to 6 months after transplantation.
Therefore, this technique may be suitable for clinical imaging of transplanted islets.
Currently, the National Institutes of Health (USA) are supporting research aiming to
make the technique of prelabeling of human islets available for clinical imaging (for fur-
ther information about the funded projects, please see ‘CRISP’ (Computer Retrieval of
Information on Scientific Projects), a searchable database of all biomedical research
projects federally funded by the USA under ‘http://crisp.cit.nih.gov/’).
PET imaging has also successfully been used to image transplanted ␤-cells. Prior
to transplantation, islets were transfected with HSV1 (herpes simplex type 1) thymin-
dine kinase (HSV1-TK) phosphorylating derivatives of thymidine and acylguano-
sine. These are retained within the cells and further metabolized by cellular kinases to
di- and triphosphates. After systemic administration of the HSV1-TK ligand 9-(4-
[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) to mice after islet trans-
plantation, islet uptake of the tracer could be quantified by PET imaging and
correlated with the number of transplanted ␤-cells [26, 27].

Recent Advances in Imaging of Insulinoma and ␤-Cell Hyperplasia

Current imaging techniques for the detection of insulinomas in patients with hyperin-
sulinemic hypoglycemia include endosonography, which is considered the most sensi-
tive imaging technique, followed by MRI or CT. Somatostatin receptor scintigraphy
(SRS) is a valuable imaging technique in most neuroendocrine tumors of the gastroin-
testinal tract. In the case of benign insulinomas, however, its value is limited due to the
fact that many insulinomas do not express the somatostatin receptor subtypes binding
octreotide resulting in a sensitivity of 10–50%. Therefore, SRS plays a more important
role in the staging and follow-up of malignant insulinomas which more commonly

26 Gotthardt ⭈ Béhé ⭈ Lasser

express the respective somatostatin receptor subtypes [28]. Despite technical improve-
ments in sonography, CT, and MRI, the detection of insulinomas in the pancreas still
remains a challenge. Kauhanen et al. [29] reported a series of 10 consecutive patients
with clinically suspected insulinoma that have been imaged by PET using 18F-DOPA
(dihydroxyphenylalanine) as tracer molecule. Because neuroendocrine tumors belong
to the group of ‘APUDomas’ (APUD – amine precursor uptake and decarboxylation),
insulinomas are able to take up and decarboxylate L-DOPA, converting it to dopamine
[30]. In all of the patients that had been included into the study, the cause of hyperin-
sulinemic hypoglycemia could be detected. In 8 cases, this was an insulinoma which
could be visualized by 18F-DOPA while CT, MRI and sonography were negative. In 2
patients, nesidioblastosis (␤-cell hyperplasia) was diagnosed. In all patients, histologi-
cal confirmation of the findings was available. This study, despite the low number of
patients caused by the rarity of the disease, impressively shows the potential power of
18
F-DOPA as tracer for the detection of neuroendocrine tumors and especially insuli-
nomas. This study also shows that a considerable part of the patients diagnosed with
hyperinsulinemic hypoglycemia may suffer from ␤-cell hyperplasia instead of insuli-
noma. These results support the finding that about 5% of adult patients with hyperin-
sulinemic hypoglycemia suffer from ␤-cell hyperplasia and not from insulinoma [31].
In two studies in children suffering from congenital hyperinsulinism, PET with 18F-
DOPA was able to differentiate those patients with focal ␤-cell hyperplasia from those
with diffuse ␤-cell hyperplasia. As a consequence, the children with focal ␤-cell hyper-
plasia underwent resection of the focus and were cured. In diffuse ␤-cell hyperplasia,
medical treatment is performed and in case of non-response, subtotal pancreatectomy
is the remaining therapeutic option [32, 33]. From 49 children included in one study,
15 were recognized as suffering from focal ␤-cell hyperplasia while from the remain-
ing 34 patients, 24 underwent partial pancreatectomy due to insufficient response to
medical treatment. From these, only 3 had focal ␤-cell hyperplasia false negative in
18
F-DOPA PET [32]. These results demonstrate that 18F-DOPA PET provides addi-
tional value in the diagnosis of insulinoma as well as ␤-cell hyperplasia. In ␤-cell
hyperplasia, it is the only imaging technique available to differentiate between focal
and diffuse disease with high sensitivity and specificity.
In preclinical studies, a new radiopharmaceutical targeting the GLP-1 receptor has
been tested for imaging of pancreatic insulinomas in a transgenic mouse model [34].
This tracer showed an unusually high specific uptake [35] and in a clinical situation
may offer advantages over 18F-DOPA because it has no uptake into the exocrine pan-
creas. Therefore, it may even become possible to image diffuse ␤-cell hyperplasia
with this tracer. This tracer molecule has also been tested for peptide receptor radio-
therapy in the same transgenic mouse model and showed very promising results [36].
Therefore, it may also offer a therapeutic option in (malignant) insulinoma and in
diffuse ␤-cell hyperplasia. Whether uptake into the exocrine pancreas is present in
humans (which may disturb quantification of uptake and detection of diffuse ␤-cell
hyperplasia) has to be evaluated in the future [37].

␤-Cell Imaging 27
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25 Evgenov NV, Medarova Z, Pratt J, Pantazopoulos P, 32 Ribeiro MJ, Boddaert N, Bellanné-Chantelot C,
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Med 2007;48:736–743.

Martin Gotthardt, MD, PhD

Department of Nuclear Medicine, Radboud University Nijmegen Medical Center
PO Box 9101, NL–6500 HB Nijmegen (The Netherlands)
Tel. ⫹31 24 36 14048, Fax ⫹31 24 36 18942
E-Mail [email protected]

␤-Cell Imaging 29
Masur K, Thévenod F, Zänker KS (eds): Diabetes and Cancer. Epidemiological Evidence and Molecular Links.
Front Diabetes. Basel, Karger, 2008, vol 19, pp 30–43

Incretin-Based Therapies for

the Treatment of Type 2
Diabetes – DPP-4 Inhibitors
and Incretin Mimetics
Baptist Gallwitz
Department of Medicine IV, Eberhard Karls University, Tübingen, Germany

Abstract
The current treatment options for type 2 diabetes do not achieve the glycemic goals. Improving islet
function by incretin hormone action is a novel and attractive therapeutic approach. There are two differ-
ent approaches to utilize incretin action in the treatment of type 2 diabetes: dipeptidyl-peptidase IV
(DPP-4) inhibitors inhibit the degradation of the incretins glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropic peptide. The DPP-4 inhibitors sitagliptin and vildagliptin are orally active and
have been shown to be efficacious and safe. They reduce hemoglobin A1c (HbA1c), fasting and post-
prandial glucose by glucose-dependent stimulation of insulin secretion and inhibition of glucagon
secretion. They are weight neutral. Indirect measures show an improvement of β -cell function. DPP-4
inhibitors do not cause a higher rate of hypoglycemia in comparison to metformin or placebo. The sec-
ond option is using GLP-1 receptor agonists, called incretin mimetics. Exenatide is available for subcuta-
neous injectable therapy, liraglutide is in phase III clinical trials. Both compounds are peptides. They
reduce HbA1c sustainedly, lead to weight loss and also show an improvement in β -cell function in man
and an increase in β -cell mass in animal models. Copyright © 2008 S. Karger AG, Basel

Introduction

Current Therapies in Type 2 Diabetes

The prevalence of type 2 diabetes is rising dramatically consecutively leading to an
increase of complications of the disease. It is predicted that the total number of peo-
ple with diabetes may be 370 million worldwide by the year 2030, along with a sub-
stantial rise in prediabetic conditions [1]. Since type 2 diabetes is increasing and
most patients do not reach their therapeutic goals, novel treatment options are
needed.
 While insulin resistance is constant in the course of type 2 diabetes, islet function
continuously declines over time. Disease progression of type 2 diabetes is characterized
by the loss of islet function. Hyperglycemia, free fatty acids, cytokines, adipokines
and toxic metabolic products may lead to a loss of
-cell function and
-cell mass in
the islets. The  cells in the islet additionally develop a disturbance of glucagon secre-
tion. In healthy subjects, glucagon secretion is suppressed under hyperglycemic con-
ditions, whereas in type 2 diabetes glucagon secretion is elevated, leading to excessive
glucose production by the liver [2].
The therapeutic options currently available do not address the problem of islet-cell
dysfunction. Sulfonylureas and glinides both exclusively stimulate insulin secretion
from the
cells; metformin and glitazones act on insulin resistance, and -glucosi-
dase delays the breakdown of complex carbohydrates. Exogenous insulin replaces the
endogenous secretory insulin deficit, although it potentially causes weight gain and
hypoglycemia. The progressive loss of islet function observed in type 2 diabetes is not
ameliorated by any of the current therapeutic options [3].

Glucagon-Like Peptide-1
The gastrointestinal hormones ‘glucose-dependent insulinotropic peptide’ (GIP) and
‘glucagon-like peptide-1 (GLP-1)’ stimulate insulin secretion after a meal. They are
responsible that orally administered glucose evokes a greater insulin response than an
intravenously administered glucose infusion calculated to lead to identical serum
glucose excursions. The difference in the insulin response was called the ‘incretin
effect’ [4]. GIP and GLP-1 are important ‘incretins’. The incretin effect is reduced or
even absent in patients with type 2 diabetes [5].
The promising therapeutic potential of GLP-1 as a pharmacological tool for treat-
ing type 2 diabetes was discovered in the 1990s. In contrast to other insulinotropic
agents, e.g. the sulfonylureas, the insulinotropic effect of GLP-1 depends even more
closely on the actual glucose concentration providing the possibility of glucose nor-
malization without the risk of hypoglycemias. In patients with type 2 diabetes, exoge-
nous GLP-1 increases insulin secretion and normalizes both fasting and postprandial
blood glucose. It further has the ability to restore the blunted first phase of insulin
secretion in type 2 diabetes [6].
Besides the glucose-lowering effects, GLP-1 has a variety of additional ‘non-
insulinotropic’ physiological actions that may be advantageous in type 2 diabetes therapy: it
suppresses glucagon secretion from the  cells and slows gastric emptying. It therefore
contributes to satiety and to a slower passage and resorption of carbohydrates. Additi-
onally, GLP-1 acts as a mediator of satiety in the hypothalamus, where it is also found as
neurotransmitter [3]. Patients with type 2 diabetes having received GLP-1 as a continu-
ous infusion have lost body weight [7]. Furthermore, GLP-1 stimulates
-cell formation
from precursor cells and also inhibits their apoptosis leading to an increase in
-cell mass
and to an improvement in
-cell function [8]. Table 1 summarizes the biological effects
of GLP-1 that are favorable in view of the pathophysiolgical findings in type 2 diabetes.

Incretin-Based Therapies for the Treatment of Type 2 Diabetes 31

Exploring the Variety of Random
Documents with Different Content
Cain finds that he killed himself more than his brother. We should
never sin if our foresight were but as good as our sense ; the issue
of sin would appear a thousand times more horrible than the act is
pleasant.
 10 OF THE DELUGE. [Book I CONTEMPLATION V. — OF
THE DELUGE. The world was grown so foul with sin, that God saw it
was time to wash it with a flood : and so close did wickedness cleave
to the authors of it, that when they were washed to nothing, yet it
would not off; yea, so deep did it stick in the very grain of the earth,
that God saw it meet to let it soak long under the waters. So, under
the law, the very vessels that had touched unclean water, must
either be rinsed or broken. Mankind began but with one ; and yet he
that saw the first man, lived to see the earth peopled with a world of
men ; yet man grew not so fast as wickedness. One man could soon
and easily multiply a thousand sins — never man had so many
children : so that when there M'ere men enough to store the earth,
there were as many sins as would reach up to heaven ; whereupon
the waters came down from heaven, and swelled up to heaven
again. If there had not been so deep a deluge of sin, there had been
none of the waters ; from whence, then, was this superfluity of
iniquity ? Whence but from the unequal yoke with infidels ? These
marriages did not oeget men so much as wickedness ; from hence
religious husbands both lost their pietj-, and gained a rebellious and
godless generation. That which was the first occasion of sin, was the
occasion of the increase of sin : A woman seduced Adam — women
betrayed the sons of God : the beauty of tlie apple betrayed the
woman — the beauty of these women betrayed this holy seed: Eve
saw, and lusted — so did they; this also was a forbidden fruit — they
lusted, tasted, sinned, died. The most sins begin at the eyes ; by
them commonly Satan creeps into the heart : that soul can never be
in safety that hath not covenanted with his eyes. God needed not
have given these men any warning of his judgment ; they gave liim
no warning of their sins, no respite ; yet that God might approve his
mercies to the very wicked, he gives them an hundred and twenty
years' respite of repenting. How loath is God to strike, that threats
so long ! He that delights in revenge surprises his adversary ;
whereas he that gives long warnings desires to be prevented. If we
were not wilful, we should never smart. Ne'ther doth he give them
time onlv, but a faithful teacher. It is a happy thing when he that
teacheth others is righteous. Noah's hand taught them as much art
his tongue. His business in building the ark was a real sermon to the
world, wherein at once were taught mercy and life to the believer,
and to the rebellious, destruction. Methinks I see those monstrous
sons of Lamech coming to Noah, and asking him what he means by
that strange work ? whether he meant to sail upon the dry land? To
whom, when he reports God's purpose and his, they go away
laughing at his idleness, and tell one another in sport, that too much
holiness hath made him mad : yet cannot they all flout Noah out of
his faith ; he preaches, and builds, and finishes. Doubtless more
hands went to this work than his. Many a one wrought upon the ark,
which yet was not saved in the ark. Our outward works cannot save
us without our faith ; we may help to save others, and perish
ourselves. What a wonder of mercy is this that I here see! One poor
family called out of a world, and, as it were, eight grains of corn
fanned from a whole barnful of chaff. One hypocrite was saved with
the rest, for Noah's sake ; not one righteous man was swept away
for company: for these few was the earth preserved still under the
waters, and all kinds of creatures upon the waters ; which else had
been all destroyed. Still the world stands for their sakes for whom it
was preserved, else fire should consume that which could not be
cleansed by water. This difference is strange : I see the savagest of
all creatures, lions, tigers, bears, by an instinct from God, come to
seek the ark (as we see swine, foreseeing a storm, run home crying
for shelter), — men I see not : reason once dcl)auched is worse
than brutishness. God hath use even of these fierce and cruel
beasts, and glory by them ; even thev, being created for man, must
live by him, though to his punishment. How gently do they offer and
submit themselves to their preserver ! renewing that obeisance to
this repairer of the M-orld, which they, before sin, ^nelded to him
that first stored the world. He that shut them into the ark when they
were entered, shut their mouths also when they did enter. The lions
fawn upon Noah and Daniel. What heart cannot the Maker of them
mollify ! The unclean beasts God would have to live, the clean to
multiply ; and therefore he sends to Noah seven of the clean, of the
unclean two. He knew the one would annoy man with their
multitude, the other would enrich him. Those things are worthy of
most respect, which are of most use. But why seven ? Surely that
God, that created seven days in the week, and made
 CONT. v.] OF THE DELUGE. 11 one for himself, did here
preserve, of seven clean beasts, one for himself for sacrifice. He
gives us six for one in earthly things, that in spiritual we should be
all for him. Now the day is come, all the guests are entered, the ark
is shut, and the windows of heaven opened. I doubt not but many of
those scoffers, when they saw the violence of the waves descending
and ascending, according to Noah's prediction, came wading middle-
deep unto the ark, and importunately craved that admittance which
they once denied ; but now, as they formerly rejected God, so are
they justly rejected of God. Ere vengeance begin, repentance is
seasonable ; but if judgment be once gone out, we cry too late.
\Vhile the gospel solicits us, the doors of the ark are open ; if we
neglect the time of grace, in vain shall we seek it with tears. God
holds it no mercy to pity the obstinate. Others, more bold than they,
hope to overrun the judgment ; and, climbing up to the high
mountains, look down upon the waters with more hope than fear.
And now when they see their hills become islands, they climb up
into the tallest trees ; there with paleness and horror at once look
for death, and study to avoid it, whom the waves overtake at last,
half dead with famine and half with fear. Lo ! now from the tops of
the mountains they descry the ark floating upon the waters, and
behold with envy that which before they beheld with scorn. In vain
doth he fly whom God pursues. There is no way to fly from his
judgments, but to fly to his mercy by repentance. The faith of the
righteous cannot be so much derided, as their success is magnified.
How securely doth Noah ride out this uproar of heaven, earth, and
waters ! He hears the pouring down of the rain above his head ; the
shrieking of men, and roaring and bellowing of beasts on both sides
of him ; the raging and threats of the waves under him ; he saw the
miserable shifts of the distressed unbelievers ; and, in the
meantime, sits quietly in his dry cabin, neither feeling nor fearing
evil. He knew that he which owned the waters would steer him ;
that he who shut him in would preserve him. How happy a thing is
faith ! what a quiet safety, what an heavenly peace doth it work in
the soul, in the midst of all the inundation of evil ! Now, when God
hath fetched again all the life which he had given to his unworthy
creatures, and reduced the world unto its first form, wherein waters
were over the face of the earth, it was time for a renovation of all
things to succeed this destruction. To have continued this deluge
long, had been to punish Noah that was righteous. After forty days,
therefore, the heavens clear up ; after an hundred and fifty, the
waters sink down. How soon is God weary of punishing, which is
never weary of blessing ! But may not the ark rest suddenly? If we
did not stay some while un. der God's hand, we should not know
how sweet his mercy is, and how great our thankfulness should be.
The ark, though it was Noah's fort against the waters, yet it was his
prison ; he was safe m it, but pent up : he that gave him life by it,
now thinks time to give him liberty out of it. God doth not reveal all
things to his best servants. Behold, he that told Noah, an hundred
and twenty years before, what daj' he should go into the ark, yet
foretells him not now in the ark what day the ark should rest upon
the hills, and he should go forth. Noah therefore sends out his
intelligencers, the raven and the dove, whose wings in that vaporous
air might easily descry further than his sight. The raven, of quick
scent, of gross feed, of tough constitution ; no fowl was so fit for
discovery : the likeliest things always succeed not. He neither will
venture far into that solitary world for fear of want, nor yet come
into the ark for love of liberty, but hovers about in uncertainties.
How many carnal minds fly out of the ark of God's church, and
embrace the present world ; rather choosing to feed upon the
unsavoury carcases of sinful pleasures, than to be restrained within
the strait lists of Christian obedience! The dove is sent forth, a fowl
both swift and simple. She, like a true citizen of tne ark, returns, and
brings faithful notice of the continuance of the waters, by her
restless and empty return; by her olive-leaf, of the abatement. How
worthy are those messengers to be welcome, which with innocence
in their lives, bring glad tidings of peace and salvation in their
mouths ! <> Noah rejoices and believes ; yet still he waits seven
days more. It is not good to devour the favours of God too greedily ;
but so take them in, that we may digest them. O strong faith of
Noah, that was not weary with this delay! Some man would have so
longed for the open air, after so long closeness, that, upon the first
notice of safety, he would have uncovered and voided the ark. Noah
stays seven day? ere he will open, and well-near two months ere he
will forsake the ark ; and not then unless God that commanded to
enter, had bidden him depart. There is no action good without faith ;
no faith witiiout a word,
 12 OF NOAH. [Boor IL Happy is that man -whicli in all
things (neglecting the counsels of flesh and blood) depends upon
the commission of his Maker! BOOK II. CONTEMPLATION I. — OF
NOAH. /^ No sooner is Noah come out of the ark, • but he builds an
altar : not an house for himself, but an altar to the Lord. Our faith
will ever teach us to prefer God to ourselves : delayed thankfulness
is not worthy of acceptation. Of those few creatures that are left,
God must have some ; they are all his : yet his goodness will have
man know that it was he, for whose sake they were preserved. It
was a privilege to those very brute creatures, that they were saved
from the waters, to be offered up in fire unto God. What a favour is
it to men, , to be reserved from common destructions, I to be
sacrificed to their Maker and Rel .-udeemer. *^ Lo, this little fire of
Noah, through the virtue of his faith, purged the world, and
ascended up into those heavens from which the waters fell, and
caused a glorious rainbow to appear therein for his security : all tne
sins of the former world were not so unsavoury unto God, as this
smoke was pleasant. No perfvmie can be so sweet as the holy
obedience of the faithful. Now God that was before annoyed with
the ill savour of sin, smells a sweet savour of rest. Behold here a
new and second rest! First, God rested from making the world, now
he rests from destrojnng it ; even while we cease not to offend, he
ceases from a public revenge. His word was enough ; yet withal he
gives a sign, which may speak the truth of his promise to the very
eyes of men. Thus he doth still in his blessed sacraments, which are
as real words to the soul. The rainbow is the pledge of our safety,
which even naturally signifies the end of a shower : all the signs of
God's in'.) stitution are proper and significant. But who would look,
after all this, to have found righteous Noah, the father of the new
world, lying drunken in his tent ! Who could think that wine should
overthrow him that was preserved from the waters ! that he, who
could not be tainted with the sinful examples of the former world,
should begin the example of a new sin of his own ! Wliat are we
men if we be but ourselves ! While God upholds us, no temptation
can move us : when he leaves us, no temptation is too weak to
overthrow us. What living man had ever so noble proofs of the
mercy, of the justice of God : Mercy upon himself, justice upon
others ! What man had so gracious approbation from his Maker ?
Behold, he oi whom in an unclean world, God said, Thee only have I
found righteous, proves now unclean when the world was purged.
The preacher of righteousness unto the former age, the king, priest,
and prophet of the world renewed, is the first that renews the sins
of that world which he had reproved, and which he saw condemned
for sin. God's best children have no fence for sins of infirmity. Which
of the saints have not once done that, whereof they are ashamed ?
God, that lets us fall, knows how to make as good use of the sins of
his holy ones, as of their obedience. If we had not such patterns,
who could choose but despair at the sight of his sins? Yet we find
Noah drunken but once. One act can no more make a good heart
unrighteous, than a trade of sin can stand with regeneration. But
when I look to the efTect of this sin, I cannot but blush and wonder.
Lo ! this sin is worse than sin : other sins move shame, but hide it ;
this displays it to the world. Adam had no sooner sinned, but he saw
and abhorred his own nakedness, seeking to hide it even with
bushes. Noah had no sooner sinned, but he discovers his nakedness,
and hath not so much rule of himself as to be ashamed. One hour's
drunkenness betrays that which more than six hundred years'
sobriety had modestly concealed. He tliat gives himself to wine, is
not his own : what shall we think of this vice, which robs a man of
himself, and lays a beast in his room ? Noah's nakedness is seen in
wine. It is no unusual quality, in this excess, to disclose secrets.
Drunkenness doth both make imperfections, and show those we
have to others' eyes : so would God have it, that we might be
doubly ashamed both of those weaknesses which we discover, and
of that weakness which moved us to discover. Noah is uncovered but
in the midst of his own tent : it had been sinful, though no man had
seen it. Unknown sins have their guilt and shame, and are justly
attended with known punishments. Ungracious Cham saw it and
laughed : his father's shame should have been his ; the deformity of
those parts from which he had his being, should have begotten in
him a secret horror and dejection. How many graceless men make
sport at the causes of their
 CONT. IL] OF BABEL. 13 humiliation! Twice had Noah given
him life ; yet neither the name of a father and f)reserver, nor age
nor virtue, could shield lim from the contempt of his own. I see that
even God's ark may nourish monsters. Some filthy toads may lie
under the stones of the temple : God preserves some men in
judgment. Better had it been for Cham to have perished in the
waters, than to live unto his father's curse. Not content to be a
witness of this filthy sight, he goes on to be a proclaimer of it. Sin
doth ill in the eye, but worse in the tongue. As all sin is a "work of
darkness, so it should be buried in darkness. The report of sin is
ofttiraes as ill as the commission ; for it can never be blazoned
without uncharitableness ; seldom without infection. Oh the
unnatural, and more than Chamish impiety of those sons, which
rejoice to publish the nakedness of their spiritual parents, even to
their enemies ! Yet it was well for Noah that Cham could tell it to
none but his own ; and those, gracious and dutiful sons. Our shame
is the less, if none know our faults but our friends. Behold how love
covereth sins ! These good sons are so far from going forward to see
their father's shame, that they go backward to hide it. The cloak is
laid on both their shoulders ; they both go back with equal paces,
and dare not so much as look back, lest they should unwillingly see
the cause of their shame, and will rather adventure to stumble at
their father's body, than to see his nakedness. How did it grieve
them to think, that they, which had so often come to their holy
father with reverence, must now in reverence turn their backs upon
him ! that they must now clothe him in pity, which had so often
clothed them in love ! And, which adds more to their dut}', they
covered him and said nothing. This modest sorrow is their praise,
and our example. The sins of those we love and honour, we must
hear of with indignation, fearfully and unwillingly believe,
acknowledge with grief and shame, hide with honest excuses, and
bury in silence. How equal a regard is this both of piety and
disobedience ! Because Cham sinned against his father, therefore he
shall be plagued in his children : Japheth is dutiful to his father, and
finds it in his posterity. Because Cham was an ill son to his father,
therefore his sons shall be servants to his brethren : because
Japheth set his shoulder to Shem's, to bear the cloak of shame,
therefore shall Japheth dwell in the tents of Shem, partaking with
him in blessings OS in duty. When we do but what we ought, j'et
God is thankful to us ; and re- j wards that, which we should sin if
we did not. Who could ever yet show me a man rebelliously
undutiful to his parents, that hath prospered in himself, and his
seed? CONTEMPLATION 11. — OF BABEL. "' How soon are men and
sins multiplied ! within one hundi'ed years, the world is as full of
both, as if there had been no deluge. Though men could not but see
the fearful monuments of the ruin of their ancestors, yet how quickly
had they forgotten a flood ! Good Noah lived to see the world both,
populous and wicked again : and doubtless ofttimes repented to
have been the preserver of some, whom he saw to traduce the vices
of the former world to the renewed. It could not but grieve him to
see the destroyed giants revive out of his own loins, and to see them
of his flesh and blood tyrannise over themselves. In his sight
Nimrod, casting off the awe of his holy grandfather, grew imperious
and cruel, and made his own kinsmen servants. How easy a thing it
is for a great spirit to be the head of a faction, when even brethren
will stoop to servitude ! And now, when men are combined together,
evil and presumptuous motions find encouragment in multitudes,
and each man takes a pride in seeming forwardest : we are the
cheerfuller in good, when we have the assistance of company ;
much more in sinning, by how much we are more prone to evil than
good. It was a proud word — " Come, let us build a city and a tower,
whose top may reach to heaven." They were newly come down from
the hills unto the plains, and now think of raising up of an hill of
building in the plain. When their tents were pitched upon the
mountains of Armenia, they were as near to heaven as their tower
could make them ; but their ambition must needs aspire to an height
of their own raising. Pride is ever discontented, and still seeks matter
of boasting in her own works. How fondly do men reckon without
God ! " Come let us build ;" as if there had been no stop but in their
own will ; as if both earth and time had been theirs. Still do all
natural men build Babel, forecasting their own plots so resolutely, as
if there were no power to countermand them. It is just with God,
that peremptory determinations seldom prosper : whereas those
things, which are fearfully and modestly undertaken, commonly
succeed. " Let us build us a city." If they had
 14 OF BABEL. [Book II. taken God with tliam, it had been
commendable ; establishing of societies is pleasing to him that is the
God of order : but a tower whose top may reach to heaven, was a
shameful arrogance, an impious presumption. Who would think, that
we little ants, that creep upon this earth, should think of climbing up
to heaven, by multiplying of earth ? Pride ever looks at the highest.
The first man would know as God ; these would dwell as God :
covetousness and ambition know no limits. And what if they had
reached up to heaven ? Some hills are as high as they could hope to
be, and yet are no whit the better ; no place alters the condition of
nature. An angel is glorious, though he be upon earth ; and man is
but earth though he be above the clouds. The nearer they had been
to heaven, the more subject they had been to the violences of
heaven, to thunders, lightnings, and those other higher
inflammations : what had this been, but to thrust themselves into
the hands of the revenger of all wicked insolences ! God loves that
heaven should be looked at, and affected with all humble desires,
with the holy ambitions of faith, not with the proud imaginations of
our own achievements. But wherefore was all this ? not that they
loved so much to be neighbours to heaven, as to be famous upon
earth. It was not commodity that was here sought, not safety, but
glory. Whither doth not thirst of fame carry men, whether in good or
evil ? It makes them seek to climb to heaven ; it makes them not
fear to run down headlong to hell. Even in the best things, desire of
praise stands in competition with conscience, and brags to have the
more clients. One builds a temple to Diana, in hope of glory,
intending it for one of the great wonders of the world ; another, in
hope of fame, burns it. He is a rare man that hath not some Babel of
his own, whereon he bestows pains and cost, only to be talked of. If
they had done better things in a vain-glorious purpose, their act had
been accursed : if they had built houses to God, if they had
sacrificed, prayed, lived well ; the intent poisons the action : But
now both the act and the purpose are equally vain, and the issue is
as vain as either. God hath a special indignation at pride above all
sins, and will cross our endeavours, not for that they are evil, (what
hurt could be in lapng one brick upon another ?) but for that they
are proudly undertaken. He could have hindered the laying of the
first stone, and might as easily have made a trench for the
foundation, the grave of the builders ; but he loves to see what
wicked men would do, and to let fools run themselves out of breath.
What monument should they have had of their own madness, and
his powerful interruption, if the walls had risen to no height ? To
stop them, then, in the midst of theii course, he meddles not with
either theii hands or their feet, but their tongues ; not by pulling
them out, not by loosing their strings, not by making them say
nothing, but by teaching them to say too much. Here is nothing
varied but the sound of letters ; even this frustrates the work, and
befools the workmen. How easy is it for God ten thousand ways to
correct and forestall the greatest projects of men ! He that taught
Adam the first words, taught them words that never were. One calls
for brick, the other looks him in the face, and wonders what he
commands, and how and why he speaks such words as were never
heard, and instead thereof brings him mortar, returning him an
answer as little understood ; each chides with other, expressing his
choler, so as he only can understand himself. From heat they fall to
quiet entreaties, but still with the same success. At first every man
thinks his fellow mocks him ; but now perceiving this serious
confusion, their only answer was silence, and ceasing : they could
not come together, for no man could call them to be understood ;
and if they had assembled, nothing could be determined, because
one could never attain to the other's purpose : no, they could not
have the honour of a general dismission, but each man leaves his
trowel and station, more like a fool than he undertook it : so
commonly actions begun in glory shut up in shame. All external
actions depend upon the tongue. No man can know another's mind,
if this be not the interpreter. Hence, as there were many tongues
given to stay the building of Babel, so there were as many given to
build the New Jerusalem, the evangelical church. How dear hath
Babel cost all the world ! At the first, when there was but one
language, men did spend their time in arts ; (so was it requisite at
the first settling of the world, and so came earlj' to perfection) : but
now we stay so long (of necessity) upon the shell of tongues, that
we can hardly have time to chew the sweet kernel of knowledge.
Surely men would have grown too proud, if there had been no
Babel. It falls out ofttimes that one sin is a remedv of a greater.
Division of tongues must
 CONT. III.] OF ABRAHAM. 15 needs slacken any work.
Multiplicity of languages had not been given by the Holy Ghost, for a
blessing to the church, if the world had not been before possessed
with multiplicity of languages for a punishment. Hence it is, that the
building of our Sion rises no faster, because our tongues are di-
^ided. Happy were the church of God, if we all spake but one
language : while we differ, we can build nothing but Babel ;
difference of tongues caused their Babel to cease, but it builds ours.
CONTEMPLATION III. OF ABRAHAM. It was fit that he which sliould
be the father and pattern of the faithful, should be thoroughly tried ;
for in a set copy every fault is important, and may prove a rule of
error. Often trials which Abraham passed, the last was the sorest. No
son of Abraham can hope to escape temptations, while he sees that
bosom in which he desires to rest, so assaulted with difficulties.
Abraham must leave his country and kindred, and live amongst
strangers. The calling of God never leaves men where it finds them.
The earth is the Lord's, and all places are alike to the wise and
faithful. If Chaldea had not been grossly idolatrous, Abraham had
not left it ; no bond must tie us to the danger of infection. But
whither must he go ? To a place he knew not, to men that knew not
him. It is enough comfort to a good man, wheresoever he is, that he
is acquainted with God : we are never out of our way, while we
follow the calling of God. Never any man lost by his obedience to the
Highest. Because Abraham yielded, God gives him the possession of
Canaan. I wonder more at his faith in taking this possession, than in
leaving his own. Behold, Abraham takes possession for that seed
which he had not ; which in nature he was not like to have : of that
land whereof he should not have one foot, wherein his seed should
not be settled for almost five himdred years after. The power of faith
can prevent time, and make future things present. If we be the true
sons of Abraham, we have already (while we sojourn here on earth)
the possession of our land of promise ; while we seek our country,
we have it. Yet even Canaan doth not afford him bread, which yet he
must believe shall flow with milk and honey to his seed. Sense must
yield to faith. Woe were us, if we must judge of our future estate by
the present. Egj^pt gives relief to Abraham, when Canaan cannot.
In outward things, God's enemies may fare better than his friends.
Thrice had Egypt preserved the church of God ; in Abraham, in
Jacob, in Christ. God ofttimes makes use of the world for the behoof
of his, though without their thanks ; as contrarily he uses the wicked
for scourges to his own inheritance, and burns them ; because in his
good they intended evil. But what a change is this ! Hitherto hath
Sarah been Abraham's wife ; now Egypt hath made her his sister ;
fear hath turned liim from a husband to a brother : no strength of
faith can exclude some doublings. God hath said, I will make thee a
great nation : Abraham saith, the Egyptians will kill me. He that lived
by his faith, yet shrinketh and sinneth. How vainly shall we hope to
believe without all fear, and to live without infirmities ! Some little
aspersions of unbehef cannot hinder the praise and power of faith.
Abraham believed, and it was imputed to him for righteousness. He
that through inconsiderateness doubted twice of his own life,
doubted not of the hfe of his seed, even from the dead and dry
womb of Sarah ; yet it was more difficult that his posterity should
five in Sarah, than that Sarah's husband should five in Egypt : this
was above nature, yet he believes it. Sometimes the believer sticks
at easy trials, and yet breaks through the greatest temptations
without fear. Abraham was old, ere this promise and hope of a son,
and still the older, the more incapable ; yet God makes him wait
twenty -five years for performance. No time is long to faith, which
hath learned to defer hopes without fainting and irksomeness.
Abraham heard this news from the angel, and laughed ; Sarah heard
it, and laughed : they did not more agree in their desire, than differ
in their affection. Abraham laughed for joy ; Sarah for distrust.
Abraham laughed, because he believed it would be so ; Sarah,
becasue she believed it could not be. The same act varies in the
manner of doing, and the intention of the doer. Yet Sarah laughed
but within herself, and is bewrayed. How God can find us out in
secret sins ! How easily did she now think, that he, which could
know of her inward laughter, could know of her conception ! and
now she that laughed, and believed not, believeth and feareth. What
a lively pattern do I see in Abraham, and Sarah, of a strong faith,
and a weak ; of strong in Abraham, and weak in Sarah ! She to
make God good of his word
 16 OF ISAAC SACRIFICED. [Book II. to Abraham, knowing
her own barrenness, substitutes an Hagar ; and, in an ambition of
seed, persuades to polygamy. Abraham had never looked to obtain
the promise by any other than a barren womb, if his own wife had
not importuned Mm to take another. When our own apparent means
fail, wealc faith is put to the shifts, and projects strange devices of
her own, to attain her end. She will rather conceive by another
womb, than be childless. When she hears of an impossibility to
nature, she doubteth, and yet hides her diffidence ; and, when she
must believe, feareth, because she did distrust. Abraham hears and
believes, and expects and rejoices : he saitli not, I am old and weak
; Sarah is old and barren : where are the many nations that shall
come from these withered loins ? It is enough to him that God hath
said it : he sees not the means, he sees the promise. He knew that
God would rather raise him up seed from the very stones that he
trode upon, than himself should want a large and happy issue. There
is no faith where there is either means or hopes. Difficulties and
impossibilities are the true objects of behef. Hereupon God adds to
his name, that which he would fetch from his loins, and made his
name as ample as his posterity. Never any man was a loser by
believing : faith is ever recompensed with glory. Neither is Abraham
content only to wait for God, but to smart for him. God bids liim cut
his own flesh ; he willingly sacrifices this parcel of his skin and blood
to him that was the owner of all. How glad he is to carry this painful
mark of the love of his Creator ! How forward to seal this covenant
with blood, betwixt God and him ! not regarding the soreness of his
body, in comparison of the confirmation of his soul. The wound was
not so grievous as a signification was comfortable. For herein he
saw, that from his loins should come that blessed seed, which should
purge his soul from all corruption. Well is that part of us lost which
may give assurance of the salvation of the whole. Our faith is not yet
sound, if it have not taught us to neglect pain for God, and more to
love his sacraments than our own flesh. CONTEMPLATION IV. — OF
ISAAC SACRIFICED. But all these are but easy tasks of faith : all
ages have stood amazed at the next ; not knowing whether they
should more •wonder at God's command, or Abraham's obedience.
Many years had that good patriarc'n waited for his Issac ; now at
last he hath joyfully received him, and that with this gracious
acclamation, " In Isaac shall thy seed be called, and all nations
blessed." Behold the son of his age, the son of his love, the son of
his expectation ; he that might not endure a mock from his brother,
must now endure the kmife of his father : " Take thine only son
Isaac whom thou lovest, and get thee to the land of Moriah, and
offer him there for a burntoffering." Never any gold was tried in so
hot a fire. Who but Abraham would not have expostulated with God
? What ! doth the God of mercies now begin to delight in blood ? Is
it possible that murder should become piety ? Or if thou wilt needs
take pleasure in a human sacrifice, is there none but Isaac fit for
thine altar ? none but Abraham to offer him ? Shall these hands
destroy the fruits of mine own loins ? Can I not be faithfiJ, unless I
be unnatural ? Or if I must needs be the monster of all parents, will
not Ishmael j-et be accepted ? O God! where is thy mercy? where is
thy justice' Hast thou given me but one only son, and must I now
slay him ? Wh}'^ did I wait so long for him? Why didst thou give
him me ? Why didst thou promise me a blessing in him ? What will
the heathen say, when they shall hear of this infamous massacre ?
How can thy name, and my profession, escape a perpetual
blasphemy ? With what face shall I look upon my wife Sarah, whose
son I have murdered ? How shall she entertain the executioner of
Isaac ? Or who will beheve that I did this from thee? How shall not
all the world spit at this holy cruelt}', and say. There goes the man
that cut the throat of liis own son ! Yet if he were an ungracious or
rebellious child, his deserts might give some colour to this violence :
but to lay hands on so dear, so dutiful, so hopeful a son, is incapable
of all pretences. But grant that thou, which art the God of nature,
mayest either alter or neglect it ; what shall I say to the truth of thy
promises ? Can thy justice admit contradictions ? Can thy decrees be
changeable ? Canst thou promise and disappoint ? Can these two
stand together — Isaac shall live to be the father of nations, and
Isaac shall now die by the hand of his father ? When Isaac is once
gone, where is my seed, where is my blessing ? O God, if thy
commands and purposes be capable of alteration, alter this bloody
sentence, and let thy first word stand.
 CONT. IV.] OF ISAAC SACRIFICED. 17 These would have
been the thoughts of a weak heart. But God knew that he spake to
an Abraham, and Abraham knew that he had to do with a God : faith
had taught him not to argue but obey. In a holy wilfulness he either
forgets nature or despises her : he is sure that what God commands
is good, that what he promises is infallible ; and therefore is careless
of the means, and trusts to the end. In matters of God, whosoever
consults with flesh and blood, shall never offer up his Isaac to God.
There needs no counsellor when we know God is the commander ;
here is neither grudging, nor deliberating, nor delaying ; his faith
would not suffer him so much as to be sorry for that he must do.
Sarah herself may not know of God's charge and her husband's
purpose, lest her affection should have overcome her faith ; lest her
weakness, now grown importunate, should have said. Disobey God,
and die. That which he must do, he will do ; he that hath learned
not to regard the life of his son, had learned not to regard the
sorrow of his wife. It is too much tenderness to respect the censures
and constructions of others, when we have a direct word from God.
The good patriarch rises early, and addresses himself to his sad
journey. And now must he travel three whole days to this execution ;
and still must Isaac be in his eye, whom all this while he seems to
see bleeding upon the pile of wood which he carries. There is
nothing so miserable as to dwell under the expectation of a great
evil. That misery which must be, is mitigated with speed, and
aggravated with delay. All this while, if Abraham had repented him,
he had leisure to return. There is no small trial, even in the very time
of trial. Now, when they are come within sight of the chosen
mountain, the servants are dismissed. What a devotion is this that
will abide no witnesses ! He will not suffer two of his own vassals to
see him do that, which soon after all the world must know he hath
done ; yet is not Abraham afraid of that piety, which the beholders
could not see without horror, without resistance, which no ear could
hear of without abomination. What stranger could have endured to
see the father carry the knife and fire, instruments of that death
which he had rather suffer than inflict ; the son securely carrying
that burden which must carry Iiim ? But if Abraham's heart could
have known how to relent, that question of his dear, innocent, and
religious son had melted it into compassion : " My father, behold the
fire and the wood, but where is the sacrifice ?" I know not whether
that word (my father) did not strike Abraham as deep as the knife of
Abraham could strike his son : yet doth he not so much as think, O
miserable man, that may not at once be a son to such a God, and
father to such a son ! Still he persists, and conceals ; and, where he
meant not, prophesies, " My son, God shall provide a lamb for the
burntoffering." The heavy tidings were loath to come forth. It was a
death to Abraham to say what he must do. He knows his own faith
to act this ; he knows not Isaac's to endure it. But now when Isaac
hath helped to build the altar, whereon he must be consumed, he
hears (not without astonishment) the strange command of God, the
final will of his father : My son, thou art the lamb, which God hath
pro\'ided for this burnt-offering. If my blood would have excused
thee, how many thousand times had I rather to give thee my own
life, than take thine ! Alas ! I am full of days, and now, of long, lived
not but in thee. Thou mightest have preserved the life of thy father,
and have comforted his death ; but the God of us both hath chosen
thee. He, that gave thee unto me miraculously, bids me, by an
unusual means, return thee unto him. I need not tell thee that I
sacrifice all my worldly joys, yea and myself, in thee ; but God must
be obeyed: neither art thou too dear for him that calls thee. Come
on, my son, restore the life that God hath given thee by me. Offer
thyself willingly to these flames ; send up thy soul cheerfully unto
thy glory ; and know, that God loves thee above others, since he
requires thee alone to be consecrated in sacrifice to himself. Who
cannot imagine with what perplexed mixtures of passions, with what
changes of countenance, what doubts, what fears, what
amazement, good Isaac received this sudden message from the
mouth of his father ! how he questioned, how he pleaded ! But
when he had somewhat digested his thoughts, and considered that
the author was God, the actor Abraham, the action a sacrifice, he
now approves himself the son of Abraham : now he encourages the
trembling hands of his father, with whom he strives in this praise of
forwardness and obedience: now he offers his hands and feet to the
cords, his throat to the knife, his body to the altar ; and, growing
ambitious of the sword and fire, entreats his father to do that which
he would have
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