Animal Models of DM2 With Reduced Pancreatic Β-cell Mass - 2006

The International Journal of Biochemistry & Cell Biology 38 (2006) 873893
Review
Animal models of type 2 diabetes with

reduced pancreatic -cell mass
Pellegrino Masiello
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia,
University of Pisa, Via Roma 55, Scuola Medica, 56126 Pisa, Italy
Available online 4 October 2005
Abstract
Type 2 diabetes is increasingly viewed as a disease of insulin deficiency due not only to intrinsic pancreatic -cell dysfunction
but also to reduction of -cell mass. It is likely that, in diabetes-prone subjects, the regulated -cell turnover that adapts cell mass
to bodys insulin requirements is impaired, presumably on a genetic basis. We still have a limited knowledge of how and when
this derangement occurs and what might be the most effective therapeutic strategy to preserve -cell mass. The animal models of
type 2 diabetes with reduced -cell mass described in this review can be extremely helpful (a) to have insight into the mechanisms
underlying the defective growth or accelerated loss of -cells leading to the -cell mass reduction; (b) to investigate in prospective
studies the mechanisms of compensatory adaptation and subsequent failure of a reduced -cell mass. Furthermore, these models
are of invaluable importance to test the effectiveness of potential therapeutic agents that either stimulate -cell growth or inhibit
-cell death.
2005 Elsevier Ltd. All rights reserved.
Keywords: Type 2 diabetes; Animal models; -cell mass; -cell growth; -cell apoptosis
Contents
0.
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Animal models of type 2 diabetes with reduced -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Spontaneous or transgenic animal models of reduced -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1. Transgenic mice deficient in factors involved in pancreas development . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.2. Transgenic mice deficient in factors involved in -cell growth and/or survival . . . . . . . . . . . . . . . . . . . . .
1.1.3. Animal models with increased -cell apoptosis due to endoplasmic reticulum (ER) stress . . . . . . . . . . .
1.1.4. Animal models with increased -cell apoptosis due to islet amyloid production . . . . . . . . . . . . . . . . . . . .
1.1.5. Animal models with increased -cell apoptosis due to gluco- and/or lipotoxicity . . . . . . . . . . . . . . . . . . .
1.1.6. Spontaneous animal syndrome of non-obese type 2 diabetes with reduction of -cell mass . . . . . . . . . .
1.2. Animal models of type 2 diabetes with experimentally induced reduction of -cell mass . . . . . . . . . . . . . . . . . . . .
1.2.1. Models with reduction of -cell mass induced by foetal growth retardation . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2. Models with surgically induced reduction of -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tel.: +39 050 221 8571; fax: +39 050 221 8557.
E-mail addresses: p.masiello@med.unipi.it,
rinomasiello@hotmail.com.
1357-2725/$ see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2005.09.007
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P. Masiello / The International Journal of Biochemistry & Cell Biology 38 (2006) 873893
Models with chemically-induced reduction of -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of treatment with exendin-4 on animal models of type 2 diabetes with reduced -cell mass . . .
Advantages and limitations of the most commonly used animal models of type 2 diabetes with -cell
mass reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.3.
1.2.4.
1.2.5.
2.
0. Introduction
Insulin insufficiency is a key feature in both type 1 and
type 2 diabetes mellitus. Type 1 diabetes is well recognized as a condition of absolute insulin deficiency due to
massive autoimmune destruction of pancreatic -cells
(Mathis, Vence, & Benoist, 2001). Type 2 diabetes is
characterized by several metabolic defects, among which
-cell secretory dysfunction and peripheral insulin resistance are considered as hallmarks of the disease in
humans (Kahn, 2003; Rizza and Butler, 1990; Weyer,
Bogardus, Mott, & Pratley, 1999). Usually, the disease
arises because of the progressive failure of endocrine
pancreas to adequately cope with the increased insulin
demand in insulin-resistant states, in particular obesity.
The results of the United Kingdom Prospective Diabetes
Study (UKPDS) clearly demonstrate that the progressive
nature of diabetes is an ongoing decline in -cell function without a change in insulin sensitivity (Kahn, 2001;
Matthews, Cull, Stratton, Holman, & Turner, 1998). It
is still debated whether this functional impairment is
due to reduced -cell mass or to an intrinsic secretory
defect of the -cells or both. Actually, it has been now
clearly established that most patients with type 2 diabetes, whether lean or obese, show a net decrease in
-cell mass (Butler et al., 2003a; Sakuraba et al., 2002;
Yoon, Ko, Cho, & Lee, 2003), but it remains uncertain
whether such reduction per se can account for inadequate insulin secretion. It is worth reminding that the
evaluation of -cell mass in man is done at autopsy and
does not derive from prospective studies. Thus, we do
not really know what is the -cell mass before the onset
of the disease.
Another intriguing aspect related to the pathogenesis of type 2 diabetes is the fact that albeit obesity is a
well known major risk factor for the development of the
disease (Burke et al., 1999; Center for Disease Control
and Prevention, 1997), two-thirds of obese subjects do
not actually develop diabetes (Mokdad et al., 2001),
likely because their -cell mass and insulin secretion can
permanently increase to compensate for the increased
metabolic load and insulin resistance. Conversely, in the
one-third of obese patients that progresses to type 2 dia-
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betes, the same metabolic conditions finally result into

-cell dysfunction and decrease in -cell mass (Lingohr,
Buettner, & Rhodes, 2002). The reasons why the same
environmental factors, i.e. nutritional excess with subsequent obesity and insulin resistance, may lead to opposite
changes in the -cell mass and function in large population groups are still unclear. In any case, the regulation
of -cell mass appears to play a pivotal role in the pathogenesis of type 2 diabetes.
Theoretically, as depicted in Fig. 1, -cell mass is
the result of the overall balance of -cell growth and
-cell loss, depending on at least four mechanisms
(Bonner-Weir, 2000a,b; Rhodes, 2005): (1) replication of
existing differentiated -cells; (2) neogenesis of -cells
from precursors usually located in the pancreatic ductal
epithelium; (3) -cell size; (4) -cell death (by apoptosis and/or necrosis). The relative contribution of each
mechanism is variable, depending on various factors,
such as species, age, metabolic load, exposure to cytotoxic factors (Rhodes, 2005). Studies mainly conducted
in rodents indicate that under normal circumstances cell growth and remodelling (this latter being marked by
apoptotic waves), occurring during foetal life (Boujendar
et al., 2002; Hanke, 2000), continue in the early postnatal period. Indeed, there is a burst of -cell replication
just after birth, followed by a rise in -cell neogenesis and a transient wave of apoptosis between 1 and 3
Fig. 1. Factors regulating -cell mass. On the left are the mechanisms
that expand the system (replication, i.e. proliferation of pre-existing
differentiated -cells; neogenesis, i.e. production of new -cells from
undifferentiated precursors located in the pancreatic duct epithelium
or scattered in the exocrine pancreas; hypertrophy, i.e. increased cell
size). On the right are the mechanisms that reduce the system (-cell
death by apoptosis or necrosis; hypotrophy, i.e. reduced cell size).
weeks of age, during which -cell mass does not increase

despite ongoing enhanced rates of -cell replication
and neogenesis (Bonner-Weir, 2000c; Finegood, Scaglia,
& Bonner-Weir, 1995; Scaglia, Cahill, Finegood, &
Bonner-Weir, 1997). This physiological wave of developmental -cell apoptosis is coincident with a decline in
the islet expression of insulin-like growth factor-II (IGFII), likely acting as an essential -cell survival factor
during foetal and neonatal life (Hill et al., 2000; Petrik,
Arany, McDonald, & Hill, 1998; Petrik et al., 1999), and
should mark for -cells a change from a proliferative,
poorly functional foetal phenotype to a fully differentiated, highly functional adult phenotype (Hellerstrom &
Swenne, 1985). In any case, the overall net effect, at least
in rodents, is a marked increase in -cell growth in the
early postnatal period and during weaning. Thereafter,
the rates of -cell replication, neogenesis and apoptosis
stabilize at low levels. In humans, although it is obviously more difficult to study in details the physiological
-cell development and turnover than in rodents, there
are nevertheless enough indications that similar phenomena occur (Bonner-Weir, 2000c; Butler et al., 2003a;
Kloppel, Lohr, Habich, Oberholzer, & Heitz, 1985),
including waves of -cell developmental apoptosis, as
reported in the third trimester of foetal life (Tornehave
& Larsson, 1997).
In the adult, -cells have an estimated life span of 60
days (Bonner-Weir, 2000b), with a very slow turnover
involving about 0.5% of the -cell population undergoing mainly self-replication (Dor, Brown, Martinez, &
Melton, 2004) and a corresponding 0.5% entering apoptosis (Bonner-Weir, 2000c; Lingohr et al., 2002). Thus,
the -cell mass remains relatively constant under physiological conditions during adult life.
A reduction in -cell mass may originate by either
an impairment of -cell replication/neogenesis or an
increased -cell apoptosis or both combined, induced
by genetic or acquired factors acting prenatally and/or
postnatally (see below). On the other hand, the plasticity of -cell mass is well known (Bernard-Kargar &
Ktorza, 2001). As a remarkable example, during pregnancy, -cell hyperplasia and hypertrophy occur, driven
by the pregnancy hormones prolactin and placental lactogen (Sorenson & Brelje, 1997), to ensure compensation
for the additional metabolic requirements of the growing foetus, while in the post-partum a decreased -cell
replication and a concomitant increase in -cell apoptosis result in prompt normalization of -cell population
(Scaglia, Smith, & Bonner-Weir, 1995).
Compensatory increases in -cell mass, depending on both replication/neogenesis and hypertrophy of
-cells, are also observed in obesity and other con-
875
ditions of insulin resistance in experimental animals,

such as Zucker fatty rats (Pick et al., 1998), and in
humans as well (Butler et al., 2003a; Kloppel et al.,
1985), likely driven by mild hyperglycaemic excursions
(Bonner-Weir, 2000c; Bernard-Kargar & Ktorza, 2001).
However, as reminded above, in one third of obese
patients, a subsequent failure of compensation occurs,
mainly due to a markedly increased frequency of -cell
apoptosis, ultimately resulting in a progressive relative
decrease of -cell mass and development of diabetes
(Butler et al., 2003a; Lingohr et al., 2002). Among the
factors which might favour such cell loss, prolonged
overstimulation of -cells may play a relevant role,
promoting apoptosis by various mechanisms, such as
protein overload and consequent increased endoplasmic
reticulum (ER) stress (Araki, Oyadomari, & Mori, 2003),
amyloid deposition (Butler et al., 2003a) and also longterm increases in cytosolic Ca2+ (Grill & Bjorklund,
2001). Furthermore, hypertrophic -cells are considered more vulnerable to stress (Bonner-Weir, 2001). It is
also well established that -cell dysfunction and loss, at
least in obesity-linked diabetes, can be due to the toxic
effects of prolonged hyperglycaemia, i.e. glucotoxicity (Donath, Gross, Cerasi, & Kaiser, 1999; Jonas et al.,
1999; Kahn, 2001), or hyperlipidaemia, i.e. lipotoxicity (McGarry & Dobbins, 1999; Shimabukuro, Zhou,
Levi, & Unger, 1998), or both, i.e. glucolipotoxicity
(El-Assaad et al., 2003; Poitout & Robertson, 2002;
Prentki & Corkey, 1996). Various downstream mechanisms driven by exposure to chronic high glucose and/or
lipid levels have been suggested to alter -cell gene
expression and metabolism and increase -cell apoptosis. These mechanisms include enhanced generation of
reactive oxygen species (Ihara et al., 1999; Laybutt et al.,
2002); -cell production of pro-apoptotic interleukin-1
with NF-kB activation and Fas signalling (Maedler et
al., 2002); FFA- or lipoprotein-induced apoptotic pathways, mediated by ceramide accumulation (Unger &
Orci, 2002) and activation of c-Jun N-terminal kinase
JNK (Roehrich et al., 2003), respectively. An intriguing link between ER stress and glucolipotoxicity, via
activation of the lipogenic transcription factor SREBP1c (steroid-response-element-binding-protein), has been
very recently reported in a -cell line and in rat islets
exposed to chronic high glucose levels (Wang, Kouri, &
Wollheim, 2005).
Alternatively or additionally to glucolipotoxicity, it
can be also hypothesized that an early reduction in cell mass, deriving from a defective prenatal or postnatal developmental -cell growth, might be a major
predisposing factor for type 2 diabetes. A simple yet
interesting possibility to explain the different final out-
876
comes of metabolic overload in different groups of obese

patients may be a different pre-existing amount of -cell
mass. In humans, a low-birth weight, most likely dependent on unfavourable intrauterine environment, has been
indeed associated with proneness to type 2 diabetes later
on in life (Barker et al., 1993; Hales & Barker, 2001).
In a smaller neonate, a proportionally smaller -cell
mass may grow, thereby having less capacity to expand
in response to increased insulin demand, as in obesity
or pregnancy, with final outcome in obesity-associated
type 2 diabetes or gestational diabetes (Hales & Barker,
2001). Furthermore, an early loss of -cell mass might
subsequently favour dysfunction of the residual -cells,
possibly due to overstimulation or toxic effects of even
mild chronic hyperglycaemia and/or hyperlipidaemia
(Donath & Halban, 2004). Of course, it would be very
helpful to monitor longitudinally the changes in -cell
mass throughout life and in various pathophysiological conditions. Unfortunately, whereas several tests exist
for the determination of insulin secretory capacity, in
vivo measurement of -cell mass in humans is currently
not possible. Target-specific imaging probes have been
recently developed in rodents by using -cell-specific
monoclonal antibodies modified for nuclear imaging,
which may allow non-invasive assessment of -cell mass
(Moore, Bonner-Weir, & Weissleder, 2001). The use
of tritiated d-mannoheptulose, administered in vivo to
preferentially label hepatocytes and insulin-producing
cells, has been also proposed, taking advantage of the
selective GLUT-2 mediated transport of this heptose
into hepatocytes and -cells, but not other cell types
(Malaisse, 2001, 2005). While waiting for such or other
methodologies to be fully established, at present we can
only rely on functional measures that indirectly reflect
-cell mass. In animal studies, for instance, correlations between in vivo functional tests (e.g., acute insulin
response) and actual -cell mass have been reported
in primates (McCulloch, Koerker, Kahn, Bonner-Weir,
& Palmer, 1991) and minipigs (Larsen, Rolin, Wilken,
Carr, & Gotfredsen, 2003b). Also in humans, functional
insulin secretory reserve as assessed by intravenous glucose and/or arginine could be a possible tool for predicting -cell mass, as reported in transplantation studies
(Teuscher et al., 1998).
In this context, animal models of type 2 diabetes
with reduced -cell mass can be extremely helpful for
a number of reasons: (a) they can reveal pathogenic
mechanisms of the -cell mass reduction; (b) they can
contribute to clarify if a reduced -cell mass is a prerequisite for type 2 diabetes development; (c) they can
contribute to assess mechanisms of compensation and
subsequent failure in the residual -cell mass; (d) they
offer opportunities to explore new approaches to treatment of diabetes.

In any case, we should be aware that none of the
animal syndromes of type 2 diabetes can reproduce the
complexity of the human disease; nevertheless, each one
can be helpful for understanding at least some aspects
of the pathogenesis and evolution of the disease. Actually, we should consider these experimental syndromes
as models of diabetogenesis rather than models of
diabetes.
This consideration appears particularly appropriate
for the diabetic syndromes with reduced beta cell mass
described in this review, as most of them lack one of
the major features of human type 2 diabetes, i.e. insulin
resistance, but at the same time provide important clues
to establish the impact of reduced -cell mass as a relevant pathogenic mechanism of the disease.
1. Animal models of type 2 diabetes with
reduced -cell mass
1.1. Spontaneous or transgenic animal models of
reduced -cell mass
These models are particularly interesting to provide
insight into the pathogenic mechanisms of -cell mass
reduction.
1.1.1. Transgenic mice decient in factors involved
in pancreas development
1.1.1.1. Pdx-1 +/ mice; -cell-specic Pdx-1 /
mice. Pancreatic duodenal homeobox factor 1 (Pdx-1),
also known as Ipf-1 in humans, is a homeodomaincontaining transcription factor required for the development of the pancreas and other foregut structures (Offield
et al., 1996). Humans and mice that do not express the
Pdx-1 gene exhibit pancreatic agenesis and congenital
diabetes (Jonsson, Carlsson, Edlund, & Edlund, 1994;
Stoffers, Zinkin, Stanojevic, Clarke, & Habener, 1997b).
Mutations of Pdx-1/Ipf-1 in the heterozygous state are
associated with one of the six known genetic forms of
maturity onset diabetes in the young (MODY), namely
MODY-4 (Stoffers, Ferrer, Clarke, & Habener, 1997a),
or with a small fraction of patients with typical adultonset type 2 diabetes (Hani et al., 1999; MacFarlane et
al., 1999). Located preferentially but not exclusively in
-cells, Pdx-1 has also a relevant role in the later differentiation of -cells, as it transcriptionally regulates
a number of -cell genes, including insulin, glucokinase, GLUT-2, prohormone convertases PC 1/3 and PC
2, as well as fibroblast growth factor receptor-1 (FGFR1) (Edlund, 1998, 2001a, 2001b). Pdx-1 mediates in
particular glucose-stimulated insulin gene transcription

(Marshak, Totary, Cerasi, & Melloul, 1996) and FGRF1 signalling that appears to be necessary for the normal expression of GLUT-2 and prohormone convertases
(Hart et al., 2000).
Transgenic mice with haploid insufficiency of Pdx-1
(Pdx-1 +/) have worsening glucose tolerance with age
and reduced insulin release in vitro. Both -cell mass and
islet number are similar to controls in young animals, but
fail to increase with age and are approximately 50% less
with respect to controls by 1 year (Johnson et al., 2003).
Pdx +/ mice show increased islet apoptosis, associated
with abnormal islet architecture and lymphocyte infiltration, whereas isolated islets and dispersed -cells are
functionally normal (Johnson et al., 2003).
The mechanism by which Pdx-1 haploinsufficiency
increases -cell apoptosis is unclear, but it might be
related to the prosurvival role of insulin receptor substrate 2 (IRS-2) signalling (see below). It has also been
suggested (Kulkarni et al., 2004) that cell death might be
secondary to a failure of Pdx-defective -cells to expand
in response to appropriate stimuli (e.g., during compensation for insulin resistance), similarly to the apoptotic
death triggered in differentiated neuronal cells following
an abortive attempt at entering the cell cycle (Becker &
Bonni, 2004).
The relevance of Pdx-1 for -cell growth and function
is confirmed by the finding that -cell-specific inactivation of Pdx-1 gene in mice results in 40% reduction
in the -cell number, impaired expression of insulin,
glucokinase and GLUT-2 in -cells with development
of diabetes at 1015 weeks of age (Ahlgren, Jonsson,
Jonsson, Simu, & Edlund, 1998; Hart, Baeza, Apelqvist,
& Edlund, 2000). In these mice, Pdx-1 / -cells also
show downregulation of FGFR-1 and prohormone convertases, leading to impaired insulin processing (Hart et
al., 2000). Interestingly, the phenotype of -cell-specific
Pdx-1 / mice could be reproduced in a transgenic
mouse line expressing a dominant-negative version of
FGRF1, in which diabetes develops at 15 weeks of age,
with reduced -cell number and impaired expression of
GLUT-2 and PC 1/3 but not glucokinase (Hart et al.,
2000).
Another transgenic mouse, with disruption of the
gene for the hepatocyte nuclear factor-1 (HNF-1),
mimics MODY-3, a rare form of MODY caused by
mutations of this gene (Fajans, Bell, & Polonsky, 2001;
Owen & Hattersley, 2001). In HFN-1 / mice, like
in MODY-3, there is a defective secretory response to
glucose and arginine, but the -cell mass, adjusted for
the reduced body weight of these animals, is not different
from wild type controls (Pontoglio et al., 1998). Thus,
877
HFN-1 appears to be mainly involved in the regulation

of -cell differentiation rather than -cell mass.
1.1.2. Transgenic mice decient in factors involved
in -cell growth and/or survival
1.1.2.1. Insulin receptor substrate-2 (IRS-2) / mice.
Disruption of murine insulin receptor substrate-1 (IRS1) or insulin receptor substrate-2 (IRS-2) causes severe
insulin resistance (Withers et al., 1998), as expected from
the role of these key adaptor molecules for insulin signalling in insulin target tissues (Rhodes & White, 2002).
However, the IRS-1 / mice do not become diabetic,
because the -cell mass increases to compensate for
insulin resistance (Withers et al., 1998). In contrast, the
IRS-2 / mice (C57Bl-6/129sv hybrid genetic background) become profoundly diabetic with fasting hyperglycaemia worsening progressively, and attaining more
than 400 mg/dl at the age of 1216 weeks (Withers et
al., 1998). Disruption of IRS-2 on a different hybrid
genetic background (C57Bl-6/CBA) confirms these features, although the severity of diabetes is less striking
(Kubota, Tobe, Terauchi, & Eto, 2000). In IRS-2 /
mice, the -cell mass fails to expand in compensation
for the insulin resistance and undergoes a progressively
increasing frequency of apoptosis (Withers et al., 1998,
1999).
It is worthwhile to note that Pdx-1 expression is
unchanged in IRS-1 / mice (Kulkarni et al., 2004),
whereas it is reduced in IRS-2 / mice (Kushner et al.,
2002), likely due to the inhibiting effect of the transcription factor Foxo-1, that in the absence of the IRS-2 signalling is not phosphorylated and is largely localized in
the nucleus (Kitamura et al., 2002). Foxo-1 is ordinarily
phosphorylated by protein kinase B (PKB, also known
as Akt) (downstream of IRS-2/phosphatidylinositol 3kinase (PI3K) pathway), thereby being retained in the
cytosol (Brazil & Hemmings, 2001). The relevance of the
link between IRS-2 and Pdx-1 is supported by the reports
that either transgenic Pdx-1 over-expression (Kushner
et al., 2002) or disruption of a single Foxo-1 allele
(Kitamura et al., 2002) rescues -cell mass and function
in IRS-2 / mice, normalizing glucose homeostasis.
On the other hand, very interestingly, the marked compensatory islet hyperplasia occurring in insulin-resistant
IRS-1 / or double IR/IRS-1 / mice is severely
restricted by Pdx-1 haploinsufficiency and an increased
-cell apoptosis is instead observed (Kulkarni et al.,
2004).
The absence of IRS-2 expression in cultured -cells
also causes marked spontaneous apoptosis (often associated with increased levels or activities of pro-apoptotic
factors like BAD), and reduction of -cell survival
878
(Lingohr et al., 2003; Withers et al., 1999). This is

consistent with the involvement of IRS-2/PI3K/PKB signalling in the phosphorylation and induction of several
anti-apoptotic substrates (activation of the ubiquitin ligase Mdm2 and subsequent degradation of p53; Mayo
& Donner, 2001), enhanced efficiency of the apoptotic
inhibitor XIAP (Dan et al., 2004), inactivation of the proapoptotic factor BAD (Chan, Rittenhouse, & Tsichlis,
1999).
Thus, IRS-2 and its downstream elements, activated
in response to not only insulin and IGF-1 (Withers et al.,
1998, 1999) but also GLP-1 signalling through cAMPresponse element binding protein (CREB)-mediated
pathways (Jhala et al., 2003), are critically important for
regulating -cell mass in adaptation to metabolic homeostasis, especially by promoting -cell survival (Dickson
& Rhodes, 2004; Hennige et al., 2003). An acquired
defect of IRS-2 signalling pathway in -cells could ultimately result into -cell loss and onset or progression of
type 2 diabetes (Rhodes & White, 2002; White, 2002).
A possible mechanism of IRS-2 acquired defect might
be an enhancement of IRS-2 ubiquitination and proteasomal degradation induced by chronic hyperglycaemia
and/or hyperlipidaemia, or by cytokines produced in
islets (Maedler et al., 2002) or derived from the expanded
adipose tissue in obese patients (Trayhurn & Wood,
2004), as recently reviewed by Dickson and Rhodes
(2004) and by Rhodes (2005).
1.1.2.2. Transgenic mice with dominant-negative CREB
in -cells. As CREB phosphorylation, induced by
glucagon-like peptide 1 (GLP-1) or other cAMP agonists, promotes insulin and IRS-2 gene expression
(Dumonteil & Philippe, 1996; Jhala et al., 2003), another
mechanism of poor IRS-2 signalling might be linked to
the impairment of this pathway. Indeed, in transgenic
mice expressing the dominant-negative CREB inhibitor
A-CREB in -cells, expression of IRS-2 is severely
blunted and a marked reduction in -cell mass secondary
to -cell apoptosis develops, with progressively increasing serum glucose and decreasing serum insulin levels
(Jhala et al., 2003).
1.1.2.3. PKB- / mice. As already mentioned, there
are evidences that of the two major signalling pathways downstream of IRS-2, PKB activation plays a
crucial role in -cell survival (Lingohr et al., 2002;
Wang et al., 2004; Wrede et al., 2002), with negligible contribution from ERK1/2 activation (Lingohr
et al., 2003). Thus, transgenic expression of a constitutively active variant of PKB in -cells prevents
FFA-induced apoptosis (Wrede et al., 2002) and exerts
a protective effect against streptozotocin-induced diabetes by enhancing -cell mass mainly through increase
of -cell survival and -cell size, without significant
effect on -cell replication or neogenesis (Tuttle et
al., 2001). Surprisingly, ablation of the PKB- isoform has not consistently reproduced the phenotype
of IRS-2 / mice with regard to -cell mass. Two
lines of PKB- / mice, established on different
genetic backgrounds, both show peripheral insulin resistance, mild/moderate hyperglycaemia and glucose intolerance, associated with basal hyperinsulinaemia (Cho
et al., 2001; Garofalo et al., 2003). However, in one of
them (C57Bl-6/129 hybrid genetic background), pancreatic -cell mass is increased, suggesting -cell compensation for insulin resistance (Cho et al., 2001),
whereas in the other (DBA/1lacJ inbred genetic background) it is decreased due to enhanced -cell apoptosis, although not as much as in IRS-2 / mice
(Garofalo et al., 2003). These data underline the complexity of the factors regulating -cell mass and might
also be indicative of the involvement of other isoforms
of PKB in -cell growth and survival (Tuttle et al.,
2001).
Many PKB substrates can affect -cell size, replication, differentiation and survival, as reviewed by Dickson
and Rhodes (2004). Here I briefly make reference to
some of them, whose ablation results in animal models
of diabetes or impaired glucose tolerance with reduction
of -cell mass.
1.1.2.4. p70S6K1 / mice. PKB is involved in
enhancement of protein synthesis in -cells through
activation of mTOR (mammalian target of rapamycin),
which in turn phosphorylates and activates two factors regulating protein synthesis, i.e. 4E-BPI (eukaryotic initiation factor-binding protein-1 or PHAS-1) and
p70S6K (the 70-kDa ribosomal subunit S6 protein kinase)
(McDaniel, Marshall, Pappan, & Kwon, 2002). PKB can
also increase protein synthesis by inactivating GSK3
and hence relieving inhibition of the protein synthesis
initiation factor eIF2B (Cohen & Frame, 2001). The
relevance of this pathway for regulation of -cell size
is confirmed by the phenotypes of two types of transgenic mice: (a) mice expressing a constitutively active
PKB specifically in -cells which actually show an
increase in -cell size (Tuttle et al., 2001); (b) p70S6K1
null mice, that have a decrease in -cell mass due to
a selective reduction of -cell size (-cells and other
endocrine cells are not affected) and not of -cell number (Diehl, Cheng, Roussel, & Sherr, 1998; Pende et al.,
2000). The p70S6K1 / mice are glucose-intolerant and
hypoinsulinaemic (Diehl et al., 1998; Pende et al., 2000);
no increase in -cell apoptosis is observed (Pende et al.,

2000).
1.1.2.5. Cyclin D2 / mice and Cdk-4 / mice. cell growth might also be mediated by PKB-dependent
inhibition of GSK3 and subsequent poor phosphorylation of cyclin D that would prevent its degradation and
promote mitogenesis (Dickson & Rhodes, 2004). Actually, transgenic disruption of either cyclin D, in particular
cyclin D2, or partner cyclin-dependent protein kinase
Cdk-4, causes insulin-deficient diabetes by a marked
decrease in -cell mass (Rane & Reddy, 2000).
Cyclin D2 / mice, obtained by homologous
recombination in embryonic stem cells, show normal prenatal development of endocrine pancreas, but
impaired post-natal replication of - and -cells, so that
by postnatal day 14, their total -cell mass is about 30%
of that of controls (Georgia & Bhushan, 2004). Animals size, body and pancreas weights and replication of
exocrine and ductal cells are not affected. The selective
replication defect in the endocrine pancreas is surprising because in most cell types upregulation of the other
D-cyclins can compensate for the loss of any particular
D-cyclin (Ciemerych et al., 2002). The failure to upregulate other D-cyclins in the -cells of cyclin D2 / mice
is limited, however, to the early postnatal period since
cyclin D1 expression is readily observed in islet cells
2 weeks after birth. The metabolic phenotype of cyclin
D2 / mice is not severe: moderate fasting hyperglycaemia, glucose intolerance and reduced post-loading
insulin peak are observed in 12-week-old animals. Thus,
impairment in cyclin D2 function might well determine
an early defect in -cell mass.
Cdk-4 / mice also display a defect in -cell proliferation, indicating that Cdk-4 acts as the requisite partner
for cyclin D2 in controlling cell cycle progression in cells (Rane et al., 1999). However, the Cdk-4 / mice,
while showing a similar insulin-deficient diabetes with a
marked decrease in -cell mass as cyclin D2 / mice,
are 40% smaller than wild type mice, suggesting that
Cdk-4 plays a more general role and is required for normal cell growth in most tissues and organs (Martin et al.,
2003). Re-expression of endogenous Cdk-4 in -cells of
Cdk-4 / mice restores -cell proliferation and normoglycaemia (Martin et al., 2003).
Independently on the lack of these physiological
factors implicated in pancreas development and -cell
growth, differentiation and survival, reduction of -cell
mass can be also the result of a marked enhancement of
-cell apoptosis due to other pathogenic mechanisms,
as indicated by a number of other interesting animal
syndromes.
879
1.1.3. Animal models with increased -cell

apoptosis due to endoplasmic reticulum (ER) stress
1.1.3.1. Akita mice. The Akita mouse harbors a spontaneous mutation in the INS2 gene that leads to the production of a mutant form of proinsulin 2 and causes earlyonset non-obese diabetes with a decreased -cell mass.
This decrease occurs progressively from birth and has
been suggested to be due to ER stress induced by retention of the mutant malfolded proinsulin 2 in the ER and
subsequent -cell dysfunction and death (Oyadomari et
al., 2002; Wang et al., 1999). The ER of affected -cells
distends and contains elevated levels of stress markers,
such as the molecular chaperone Bip/Grp78 and activated CHOP, which may induce apoptosis (Oyadomari
et al., 2002). Hyperglycaemia and falling insulin production correlate with a progressive decrease in -cell
mass. When the Akita mutation was introduced into
a CHOP / background, islet cell destruction and
hyperglycaemia were delayed in onset (Oyadomari et
al., 2002).
A similar condition is supposed to occur in the Wolfram syndrome, a rare genetic form of human diabetes,
due to a mutant ER resident protein that might alter ER
homeostasis and -cell integrity (Inoue et al., 1998).
1.1.3.2. PERK / mice. The WolcottRallison syndrome of infantile diabetes with early destruction of
pancreatic -cells, pancreatic hypoplasia and osteodystrophy is caused by homozygous mutation in the
EIF2AK3/PERK gene (Delepine et al., 2000), which
encodes for pancreatic ER kinase (PERK), a major ER
stress transducer in mammalian cells, involved in protective inhibition of protein synthesis. Disruption of PERK
produces the same phenotype in mice. In PERK /
mice, islets are normal at birth, but afterwards -cells
undergo a progressive destruction. It is supposed that
the absence of PERK interferes with the tightly regulated
balance of protein synthesis and folding ER capacity in
-cells, thereby favouring protein overload through the
ER (Harding et al., 2001).
Various apoptotic pathways (e.g. CHOP induction,
JNK and caspase 12 activation; Nakagawa et al., 2000;
Oyadomari et al., 2002; Urano et al., 2000), could be
triggered by ER stress and lead to -cell loss. Thus,
as reviewed by Harding and Ron (2002), ER-stressinduced apoptosis could be a major mechanism by
which prolonged stimulation and overload of -cells
under conditions of increased insulin demand (obesity,
insulin resistance, long-term treatment with sulphonylureas) might determine the decompensation phase often
termed pancreatic -cell exhaustion (Araki et al.,
2003).
880

apoptosis due to islet amyloid production
1.1.4.1. Mice and rats with overexpression of human
islet amyloid polypeptide (h-IAPP); h-IAPP/Agouti
mice. Amyloid deposits, derived from the physiological islet amyloid polypeptide (IAPP), co-stored and
co-released with insulin, are present in almost all individuals with type 2 diabetes and are associated with
decreased -cell mass (Butler et al., 2003a; Clark et
al., 1988; Hull, Westermark, Westermark, & Kahn,
2004), but the role of this lesion in the pathogenesis
of type 2 diabetes remains controversial (Hull et al.,
2004). Several spontaneous or genetically manipulated
animal models of islet amyloid formation are known.
Two longitudinal studies performed in primates, such
as Macaca nigra (Howard, 1986) and Macaca mulatta
(de Koning, Bodkin, Hansen, & Clark, 1993) show that
overtly diabetic animals develop extensive islet amyloid deposits, which precede elevation of blood glucose
and are associated with islet -cell loss (de Koning et
al., 1993). Domestic cats also develop diabetes associated with an early deposition of IAPP as islet amyloid
(Ma, Westermark, Johnson, OBrien, & Westermark,
1998).
Several lines of transgenic mice with targeted expression of human IAPP (rodent IAPP is not amiloidogenic;
OBrien, Butler, Westermark, & Johnson, 1993) in cells have been established as models of islet amyloid
formation (DAlessio et al., 1994; Fox et al., 1993; Yagui
et al., 1995). These mice produce, store and release
human IAPP normally (Verchere, DAlessio, Palmiter,
& Kahn, 1994), but develop islet amyloid deposits
only in the presence of altered metabolic environment.
Indeed, amyloid formation associated with reduction
of -cell mass, fasting hyperglycaemia and impaired
insulin secretion are observed in human IAPP transgenic
mice either fed for 1 year with high-fat diet (Butler,
Janson, Soeller, & Butler, 2003b) or intercrossed with
genetic murine models of obesity, insulin resistance and
-cell dysfunction, such as ob/ob mice (Hoppener et al.,
1999) and Agouti viable yellow (Avy/a) mice (Soeller et
al., 1998). The reduction of -cell mass is due, at least in
one of these models (Butler et al., 2003b), to a striking
increase in -cell apoptosis induced by soluble cytotoxic
IAPP oligomers, that outweighs the obesity-induced cell proliferation.
Recently, a human IAPP transgenic rat (the HIP rat)
has been obtained that spontaneously develops diabetes
characterized by islet amyloid formation and decreased
-cell mass (Butler et al., 2004). Normal until 5 months
of age, the -cell mass decreases by 80% in 18-monthold HIP rats (in controls it increases by 60%), due to
an increased frequency in -cell apoptosis prior to the

development of hyperglycaemia.
Taken together, these data suggest that islet amyloid
formation can be indeed responsible for -cell mass
loss secondary to increased apoptosis. Conditions of
enhanced insulin demand and intrinsic -cell dysfunction would favour this process, at least in mice.
apoptosis due to gluco- and/or lipotoxicity
In the two models of obesity-linked type 2 diabetes
that I briefly describe here, the -cell mass is not actually
reduced below that of lean controls, but is nevertheless largely inadequate to compensate for the associated
insulin resistance.
1.1.5.1. Zucker diabetic fatty rats. The diabetes-prone
Zucker fatty rat model (Finegood et al., 2001; Pick et al.,
1998; Tokuyama et al., 1995) develops extreme obesity
because of a genetic defect in the leptin receptor (Phillips
et al., 1996; Takaya et al., 1996). Whereas the original
Zucker fatty (ZF) rats compensate for the obesity-linked
insulin resistance by increasing -cell mass and insulin
secretion, selective breeding has generated a colony of
male (not female) diabetes-prone Zucker fatty (ZDF) rats
that develop diabetes because a marked enhancement in
-cell apoptosis counteracts the increase of -cell mass
(Finegood et al., 2001; Pick et al., 1998). This colony is
supposed to carry additional genetic alterations because
in the prediabetic state (at 6 weeks of age), ZDF rats
already show an intrinsic -cell secretory defect with
respect to ZF (Pick et al., 1998). Interestingly, while
in ZF animals pancreatic Pdx-1 expression as well as
Akt activity are enhanced (Jetton et al., 2005), ZDF
rats show defective -cell Pdx-1 expression after the
development of hyperglycaemia, but not in the prediabetic state (Harmon et al., 1999). Thus, Pdx-1 deficiency in ZDF rats is not genetically determined but
most likely secondary to glucotoxicity and would exacerbate -cell dysfunction by down-regulating essential
functional genes such as insulin and GLUT-2. The mechanisms underlying the increased -cell apoptosis in ZDF
rats have been attributed to lipotoxicity caused by lipid
accumulation, possibly through activation of the proapoptotic ceramide pathway (Lee et al., 1994, 1997;
Shimabukuro et al., 1998).
1.1.5.2. Psammomys obesus gerbil. Another relevant
model is the gerbil P. obesus fed in captivity with a relatively high-energy (HE) diet (2.93 kcal/g), replacing its
low-calorie natural diet of desert saltbush plant (Adler,
Kalman, Lazarovici, Bar-On, & Ziv, 1991; Donath et
al., 1999). Upon HE diet, these rodents become obese

and, similarly to the Zucker fatty rat, selective breeding has generated two lines of animals: a diabetes-prone
(DP) line that develops overt diabetes in a few days, and
a partially diabetes-resistant (DR) line, in which most
animals exhibit normoglycaemia and persistent hyperinsulinaemia (Kalderon, Gutman, Levy, Shafrir, & Adler,
1986; Nesher, Gross, Donath, Cerasi, & Kaiser, 1999).
The diabetes-prone P. obesus gerbils show defective glucose-stimulated insulin secretion, inability to
undergo adaptive changes to keep pace with increased
insulin demand and progressive loss of -cell mass
because of an impressive increase in -cell apoptosis, mainly attributed to glucose toxicity (Donath et al.,
1999; Leibowitz et al., 2001b). Interestingly, no functional Pdx-1 gene product, at least corresponding to the
highly conserved mammalian homeodomain, has been
found in P. obesus, with consequent impaired glucoseinduced insulin gene expression, that could be restored
upon Pdx-1 gene transfer into islets (Leibowitz et al.,
2001a). However, the lack of Pdx-1 also occurs in the DR
line (Leibowitz et al., 2001a), suggesting that additional
factors are required for full development of diabetes in
this model.
1.1.6. Spontaneous animal syndrome of non-obese
type 2 diabetes with reduction of -cell mass
1.1.6.1. Goto-Kakizaki (GK) rats. The spontaneously
diabetic Goto-Kakizaki rat is a genetic lean model of type
2 diabetes originating from selective breeding over many
generations of glucose-intolerant non-diabetic Wistar
rats (Goto, Suzuki, Sasaki, Ono, & Abe, 1998). GK rats
show a prenatal reduction of -cell proliferation associated to an abnormal wave of apoptosis (Miralles &
Portha, 2001). This results in a deficit in -cell mass
from birth that becomes progressively larger as a consequence of impaired new islet formation and -cell
replication rather than increased apoptosis (Movassat,
Saulnier, Serradas, & Portha, 1997; Plachot, Movassat,
& Portha, 2001; Serradas, Gangnerau, Giroix, Saulnier,
& Portha, 1998). Interestingly, this deficit appears to
be due to impaired IGF-2 production (Serradas et al.,
2002). Despite congenital reduction of -cell mass, in
the first 34 weeks of life (i.e. the period of maximal
physiological growth of beta cells), GK rats maintain
normoglycaemia (Movassat, Saulnier, & Portha, 1995;
Tourrel et al., 2002). At an adult age, GK rats have
50% depletion of total -cell mass, stable moderate nonfasting hyperglycaemia, impaired glucose tolerance and
markedly defective insulin response to glucose in vivo
and in vitro (Briaud, Kelpe, Johnson, Tran, & Poitout,
2002; Movassat et al., 1995; Portha et al., 1994). GK
881
rats also have genetically induced alterations of insulin

secretion independent of the reduction of -cell mass
(Portha, 2003).
A synopsis of the mechanisms leading to reduction
of -cell mass in spontaneous or transgenic models of
type 2 diabetes is shown in Table 1.
1.2. Animal models of type 2 diabetes with
experimentally induced reduction of -cell mass
Reduction of -cell mass has been experimentally
induced in several animal species by nutritional, surgical or chemical means to reproduce at least some
of the features of human type 2 diabetes. Such models are particularly helpful to understand the mechanisms of compensation and decompensation of a reduced
-cell mass and assess the effectiveness of potential
therapeutics acting by enhancing -cell growth and/or
survival.
1.2.1. Models with reduction of -cell mass induced
by foetal growth retardation
1.2.1.1. Intrauterine growth retardation by uteroplacental insufciency in the rat. A rat model of uteroplacental
insufficiency has been developed, designated as IUGR
for intrauterine growth retardation, induced by bilateral
uterine artery ligation at 19 days of gestation, i.e. 3 days
before term (Simmons, Templeton, & Gertz, 2001). This
model intends to mimic the unfavourable intrauterine
environment that in humans leads to low-birth weight
and is supposed to confer high risk of development of
diabetes in adult age (Barker et al., 1993; Rich-Edwards
et al., 1999). IUGR rats have lower birth weight than controls until 7 weeks of age, but later on, they surpass the
weight of controls and by 26 weeks they become obese.
At an early age, IUGR rats are glucose-intolerant and
insulin resistant; by 7 weeks they develop mild fasting
hyperglycaemia and hyperinsulinaemia. At 26 weeks,
they are markedly hyperglycaemic.
In IUGR rats, -cell mass is normal during the first
few weeks of life, despite the 50% reduction of Pdx-1
expression observed in IUGR foetuses. However, by 7
weeks of age, -cell mass becomes lower than in controls, is reduced to 50% at 15 weeks and to one-third at
26 weeks of age (Simmons et al., 2001). It is not known
if this age-related decline is caused by decreased proliferation or increased apoptosis of -cells.
This model supports the hypothesis that an abnormal
intrauterine environment can induce permanent alterations in glucose homeostasis after birth and lead to type
2 diabetes in the adulthood.
882
Table 1
Synopsis of the mechanisms leading to reduced -cell mass in spontaneous or transgenic animal syndromes of type 2 diabetes
-Cell
size
-Cell
apoptosis
Obesity or insulin
resistance
Transgenic mice deficient in factors involved in -cell development, growth and/or survival
Pdx-1 +/
N
N
IRS-2 /
N
N
RIP A-CREB transgenic mouse
N
N
p70S6K /
N
N
Cyclin D2 /
N
CdK-4 /
N
N
N
N
N
N
N
N
No
Yes
No
No
No
No
Mice with increased -cell apoptosis due to ER stress

Akita mouse (INS-2 mutation)
PERK /
N
N
N
N
No
No
Rodents with increased -cell apoptosis due to islet amyloid production

Mouse with overexpression of h-IAPP (upon HFD)
Rat with overexpression of h-IAPP (HIP rat)
h-IAPP/Agouti mousea
N
N
Yes
No
Yes
Rodents with increased -cell apoptosis due to gluco- and/or lipotoxicity

Zucker diabetic rata
Psammomys obesus gerbila (upon HED)
Yes
Yes
Spontaneous model of decreased -cell mass

GK rat
No
Animals
-Cell
replication
N
N
-Cell
neogenesis
N, normal; , decrease; , increase; , marked increase; ER, endoplasmic reticulum; HFD, high-fat diet; HED, high energy diet; RIP A-CREB,
dominant-negative CREB transgene under control of rat insulin promoter-1.
a Relative decrease of -cell mass.
1.2.1.2. Offsprings of pregnant rats fed with low-protein

diet. That foetal malnutrition can be a relevant risk factor
for the later development of type 2 diabetes is confirmed
by the observation that offsprings of pregnant rats subjected to a low-protein (LP) isocaloric diet (8% versus
20%), show, both during foetal life and in the neonatal period, reduced body weight, altered insulin secretory capacity and reduced -cell mass resulting from
reduced -cell proliferation rate and increased apoptosis (Bertin et al., 2002; Boujendar et al., 2002; Petrik
et al., 1999; Snoeck, Remacle, Reusens, & Hoet, 1990).
This is associated with reduced foetal and neonatal islet
expression of IGF-II (Petrik et al., 1999), which acts
as -cell mitogen and protects against apoptosis (Petrik
et al., 1998). Expression of Pdx-1 is also reduced in
islets from pups of LP mothers (Arantes et al., 2002).
Interestingly, taurine supplementation is able to partially prevent -cell mass reduction in the LP model
by normalization of proliferation and apoptosis rates
in concomitance with restoration of IGF-II expression
(Boujendar et al., 2002). In adulthood, rats born from
LP mothers still have reductions in -cell mass and
insulin secretion and show glucose intolerance, but usually not overt diabetes (Dahri, Reusens, Remacle, &
Hoet, 1995; Dahri, Snoeck, Reusens, Remacle, & Hoet,

1991; Reusens & Remacle, 2000). At old age, LP offsprings may develop fasting hyperglycaemia, associated
with insulin resistance (Petry, Dorling, Pawlak, Ozanne,
& Hales, 2001).
Several variants of LP diet protocol have been used,
differing for duration, severity of protein deprivation,
association with calorie restriction, as recently reviewed
by Armitage, Khan, Taylor, Nathanielsz, and Poston
(2004). The above-described endocrine and metabolic
features of the LP model are present in most of the variants, although to a different extent. It should be reminded
that in the LP isocaloric diets, calories are balanced by
addition of fat or carbohydrates, and this makes more
complex the interpretation of the results (see Armitage
et al., 2004).
1.2.2. Models with surgically induced reduction of
-cell mass
1.2.2.1. Partially pancreatectomized rats. Partial pancreatectomy has been largely used in various animal
species as a tool to directly reduce -cell mass in healthy
animals, without any genetic background and/or previous alteration of insulin-producing cells.
In several species, quite large pancreatectomies are

required to determine mild/moderate hyperglycaemia
and insulin secretory defect (5075% in man: Kendall,
Sutherland, Najarian, Goetz, & Robertson, 1990; Porte,
1991; 6070% in the pig: Stump, Swindle, Saudek, &
Strandberg, 1988; more than 80% in the rat: Jonas et
al., 1999). It is intriguing that if a small additional pancreatic mass is removed (8090%) in pigs (Lohr et al.,
1989) and humans (Gepts & Lecompte, 1981; Kloppel
et al., 1985; Saito, Yaginuma, & Takahashi, 1979); 95%
in rats (Laybutt et al., 2003), overt diabetes with severe
insulin deficiency develops.
The different outcome of surprisingly small differences in the percentage of subtotal pancreatectomy in
rats has been recently studied in details by Laybutt
et al. (2003). Tissue removal is performed by gentle
abrasion with cotton applicators leaving the pancreas
within 12 mm of the common bile duct and extending from the duct to the first part of the duodenum.
Control rats undergo laparatomy and gentle rubbing of
the pancreas between fingers. By varying the proportion of gastric lob removed, it is possible to obtain
either 85 or 95% pancreatectomy. This slight variation resulted initially in non-fasting blood glucose levels of variable severity, confirming previous results
(Jonas et al., 1999). However, over time, glycaemia
clustered into two distinct groups. Some rats maintained nearly normal glucose levels, indicating longlasting compensatory adaptation. The other rats became
highly hyperglycaemic, with no rats in the middle range
(140250 mg/dl). These observations suggest that below
a critical value of -cell mass, when compensatory
mechanisms fail, a dysfunction in the residual -cells
is likely to occur, possibly due to overload-induced ER
stress or toxic effects of mounting hyperglycaemia and
hyperlipidaemia (Donath & Halban, 2004; Laybutt et al.,
2003).
Pancreatectomized rats appear to be a good model to
investigate the adaptive mechanisms of residual -cells
in adult animals. In 60% pancreatectomized (60% Px)
rats, that are normoglycaemic and glucose-intolerant, a
limited regeneration of the residual -cells occurs, so that
the -cell mass increases from 40 to 55% of normal, several weeks after surgery (Leahy, Bonner-Weir, & Weir,
1988). Moreover, an increase in insulin responsiveness
to intermediate glucose concentrations is observed, due
to up-regulation of -cell glycolytic flux driven by an
increased activity of glucokinase (Liu, Nevin, & Leahy,
2000), the main regulator of -cell glucose utilization
(Matschinsky et al., 1993). Similar up-regulation of islet
glucose metabolism also occurs in normoglycaemic 60%
Px mice (Martn et al., 1999). Interestingly, in hypergly-
883
caemic 90% Px rats, an increase in hexokinase, and not

glucokinase, activity is the prevalent change affecting
islet glucose metabolism (Hosokawa, Corkey, & Leahy,
1997), associated with an enhanced basal insulin secretion (Leahy, Bumbalo, & Chen, 1993).
In 90% Px rats, an initial burst of -cell regeneration has been observed in the first 710 days after
surgery (Bonner-Weir, Baxter, Schuppin, & Smith,
1993). Furthermore, -cell hypertrophy is apparent in
these hyperglycaemic pancreatectomized rats 4 weeks
after surgery (Jonas et al., 1999; Xu, Stoffers, Habener,
& Bonner-Weir, 1999) and is maintained at 14 weeks
(Laybutt et al., 2003). -Cell hypertrophy is also found
in prediabetic Zucker diabetic rats with impaired glucose tolerance (Pick et al., 1998), and in pregnancy
(Scaglia et al., 1995). Thus, -cell hypertrophy could
represent a major mechanism of compensatory response
to increased demand in terminally differentiated -cells
that may prevent, at least for a while, more serious
metabolic impairment. The up-regulation of c-Myc that
occurs in 8595% Px rats more or less rapidly, depending
on the circulating glucose levels, may play an important
role in the compensatory growth of -cells, since this factor can lead to hypertrophy in the absence of cell division
(Schuhmacher et al., 1999).
Laybutt et al. (2003) have also studied the time course
of the changes in -cell phenotypes in 8595% pancreatectomized (8595% Px) rats, by evaluating the expression of a number of -cell genes coding for transduction
factors regulating -cell differentiation (Pdx-1, HNF1, NeuroD/BETA2, Nkx61) and for key metabolic factors (GLUT-2, glucokinase, pyruvate carboxylase, mitochondrial glycerophosphate dehydrogenase). In clearly
hyperglycaemic 95% Px rats, the expression of these
genes was decreased by 50% 4 weeks after pancreatectomy and further reduced at 14 weeks, whereas in 85%
Px rats with mild hyperglycaemia, a 3040% decrease in
transcription factors was found at 14 weeks only. Correction of hyperglycaemia by a 2-week phlorizin treatment
fully reverses the changes at 4 weeks but only partially
at 14 weeks. Thus, chronic hyperglycaemia induces a
deterioration of -cell phenotype, whose extent, timing
and reversibility depend on both severity and duration of
hyperglycaemia. However, it is noteworthy that at 4 and
14 weeks, 85% Px rats, despite significant differences
in -cell phenotype, have similar mild hyperglycaemia,
likely related to a concomitant two-fold increase in cell size (Laybutt et al., 2003). This suggests that, at least
in some circumstances, a reduced -cell mass is able to
maintain successful compensation for a long time, even
in the presence of mild hyperglycaemia and -cell dysfunction.
884
1.2.3. Models with chemically-induced reduction of

-cell mass
Differently from pancreatectomy, chemical induction
of diabetes offers the advantage of preservation of both
exocrine and endocrine cell populations other than cells, thus resembling the situation in human diabetes.
Furthermore, the good conditions of the animals after
chemical induction of diabetes do not require particular
welfare measures and allow studies on the effects of highfat diet that cannot be carried out in pancreatectomized
animals.
1.2.3.1. Neonatal rats treated with streptozotocin
(n-STZ rats). Neonatal Wistar rats treated with
90100 mg/kg b.w. STZ (n-STZ) on the day of birth
(Portha, Levacher, Picon, & Rosselin, 1974) or 2 days
after birth (Weir, Clore, Zmachinski, & Bonner-Weir,
1981) undergo a transient hyperglycaemia followed by
a rapid spontaneous remission until about 68 weeks
of age. Thereafter, non-fasting chronic hyperglycaemia
develops with plasma glucose concentrations usually
ranging 170200 mg/dl for rats injected at birth and
200350 mg/dl for rats injected at 2 days of age. The
remission of initial STZ-induced hyperglycaemia is
accompanied by partial -cell and insulin stores reconstitution (Bonner-Weir, Trent, Zmachinski, Clore, & Weir,
1981; Portha et al., 1989), most of new -cells originating from undifferentiated duct cells (Dutrillaux, Portha,
Roze, & Hollande, 1982; Wang, Bouwens, & Kloppel,
1994). The residual -cell mass, which is reduced to
approximately 20% upon STZ administration, is finally
recovering up to 50% as a consequence of -cell regeneration and/or neogenesis. The moderate diabetic state
of adult n-STZ rats does not affect body weight gain nor
requires insulin treatment. However, in these rats, insulin
responsiveness to glucose and tolbutamide is lacking
(Dutrillaux et al., 1982; Giroix, Portha, Kergoat, Bailbe,
& Picon, 1983), that is not justified by the 50% reduction of -cell mass per se, but might be dependent on an
incomplete differentiation of the newly formed -cells
after the initial STZ-induced loss (Portha et al., 1989;
Weir, Leahy, & Bonner-Weir, 1986).
1.2.3.2. Streptozotocin-nicotinamide-treated adult rats
(STZ-NA rats). On the basis of previous knowledge
that suitable doses of nicotinamide (NA) could exert
a partial protection against the -cytotoxic effect of
streptozotocin (STZ), we have established a new experimental diabetic syndrome in adult rats that appears
closer to human type 2 diabetes than other available
models (e.g. neonatally STZ-injected rats; GK rats),
with regard to insulin responsiveness to glucose and
sulphonylureas (Masiello et al., 1998). In 2-month-old

Wistar rats, a dose of 200230 mg/kg b.w. nicotinamide,
given intraperitoneally 15 min before streptozotocin
administration (60 mg/kg i.v.), yields a maximum of
animals (7580%) with 40% reduction of pancreatic
-cell mass (and no change in -cell mass) and moderate
stable non-fasting hyperglycaemia (150180 mg/dl).
Interestingly, the remaining 2025% treated animals
either become severely diabetic within 23 weeks
or remain normoglycaemic, yet glucose-intolerant
(Masiello, unpublished data). Thus, as reported for
8595% Px rats (Laybutt et al., 2003), we cannot obtain
STZ-NA rats with stable hyperglycaemic levels in the
middle range (200300 mg/kg).
Two to 3 weeks after diabetes induction, intravenous
glucose tolerance tests reveal clear abnormalities in glucose tolerance and insulin responsiveness, which are
reversed by tolbutamide administration. These features
remain unchanged for a long time after induction of
diabetes. In the isolated perfused pancreas of STZ-NA
rats, insulin response to glucose elevation is clearly
present, although significantly reduced with respect to
controls. Moreover, the insulin response to tolbutamide
is similar to that observed in normal pancreases. Such
in vivo and in vitro partial insulin responsiveness to
glucose and sulphonylureas is missing in other animal
models, such as n-STZ, GK and partially pancreatectomized rats (Giroix et al., 1983; Leahy, Bonner-Weir,
& Weir, 1984; Portha et al., 1991). In islets isolated from
STZ-NA rats, compensatory adaptations occur. Indeed,
glucose oxidation and utilization are increased when
expressed per islet DNA content (Novelli, Fabregat,
Fernandez-Alvarez, Gomis, & Masiello, 2001) and
insulin release is stimulated by intermediate glucose concentrations and potentiated in presence of free fatty acids
more than in controls (manuscript in preparation). A
moderate increase of -cell neogenesis in the pancreas of
STZ-NA rats without evidence of significant -cell replication, has been observed within a few weeks after treatment (Novelli et al., 2001), but extensive studies on this
topic have not been conducted yet in this model. Thus,
the STZ-NA-induced diabetic syndrome with decreased
-cell mass and preserved insulin responsiveness to glucose and tolbutamide, may provide a particularly advantageous tool for pharmacological investigations of not
only new insulinotropic agents (Broca et al., 1999), but
also factors stimulating -cell growth, such as GLP1/exendin-4 (see below). These studies would clarify
whether such promising growth-stimulating factors that
are currently tested in neonate or very young animals,
are also able to promote -cell growth in adult animals
with reduced -cell mass. Furthermore, the STZ-NA
model appears very suitable for longitudinal studies aiming at assessing whether a reduced -cell mass is able to
cope with increased insulin demand induced by high-fat
diet/obesity/insulin resistance and what are the mechanisms underlying the compensation and the subsequent
expected failure of insulin secretory function.
We have initiated studying the effect of high-fat diet
(HFD) (40% of calories provided as saturated and monounsaturated fat) given for 3 months to STZ-NA rats.
The results show that HFD-fed STZ-NA rats become
obese, insulin resistant and slightly hyperlipidaemic, but
maintain unchanged the mild hyperglycaemia typical of
this model, while developing a significant and stable
hyperinsulinaemia. Glucose tolerance tests and in vitro
stimulation of isolated islets confirm that STZ-NA rats
develop successful adaptive responses to the increased
metabolic load, at least for 3 months (manuscript in
preparation). Experiments are currently in progress to
determine whether this adaptation continues for a longer
time and which are the underlying mechanisms.
It is of interest that in the genetic model of GK rats
(see above), the effects of a high-fat diet were markedly
different than in STZ-NA rats. In fact, in GK rats, high-fat
feeding for 6 weeks induced no compensatory response
in vivo and resulted instead in a further impairment
of glucose-stimulated insulin release in vitro, associated with UCP-2 expression (Briaud et al., 2002). The
discrepancy between the two models is likely to be
dependent on the higher basal glycaemic levels and the
presence of genetic -cell dysfunction in GK rats compared to STZ-NA rats. Both of these factors could lead
to overt decompensation when associated with hyperlipidaemia (Poitout & Robertson, 2002).
1.2.3.3. Streptozotocin-nicotinamide-treated minipigs
(STZ-NA minipigs). The rat STZ-NA model has been
recently reproduced in the Gottingen minipig, in which
the combined administration of streptozotocin and
nicotinamide results in a mild diabetes characterized by
reduced -cell mass, fasting and postprandial hyperglycaemia and mildly impaired insulin secretion (Larsen et
al., 2002). Since pigs are more resistant to the diabetogenic effect of STZ than rats, doses of 125 mg/kg of STZ
i.v. and 67 mg/kg of NA were found to be the most suitable for diabetes induction. -Cell mass was reduced
by approximately 70% (Larsen et al., 2003a). A compensatory increase in insulin secretion from the residual
-cell population was observed both in vivo and in vitro
(Larsen, Rolin, Gotfredsen, Carr, & Holst, 2004). The
STZ-NA minipig model has also been used to validate
the measurement of insulin secretory capacity and glucose tolerance to predict pancreatic -cell mass in vivo
885
(Larsen et al., 2003b), as well as to study the effect of

reduced -cell mass on the pulsatile insulin secretion
(Larsen et al., 2003a) that is impaired in type 2 diabetes
(Hollingdal et al., 2000).
Several of the animal models with reduced -cell
mass have been recently used to test the possibilities
of recovery of -cell mass and function upon treatment
with pharmacological agents potentially capable of stimulating -cell replication or neogenesis. As a relevant
example, I summarize here the results of studies on the
effects of exendin-4, a long-acting agonist of GLP-1
receptor.
1.2.4. Effect of treatment with exendin-4 on animal

models of type 2 diabetes with reduced -cell mass
A number of studies have examined the ability of
exendin-4 to increase pancreatic -cell mass in vivo, as
reviewed in Nielsen, Young, and Parkes (2004).
In 9095% pancreatectomized rats, the daily administration of exendin-4 for 10 days post-pancreatectomy
stimulated a 40% expansion of -cell mass and cell proliferation, without affecting -cell size (Xu et al., 1999).
This resulted in attenuation but not normalization of
hyperglycaemia.
In GK rats, neonatal treatment with exendin-4 for a
few days enhanced pancreatic insulin content and total
-cell mass by stimulation of both proliferation and neogenesis of -cells, without effect on -cell size (Tourrel
et al., 2002). Two months after exendin-4 treatment, cell mass in GK rats achieved 63% of the -cell mass
in the Wistar group (untreated GK have 40% of the
normal -cell mass) and plasma glucose levels were
decreased compared to untreated GK rats, but not normalized (Tourrel et al., 2002).
In n-STZ rats, the response to early exendin-4 exposure for 4 days was a rapid three-fold increase in -cell
mass, attributable to stimulation of -cell neogenesis,
that was maintained in adulthood, but resulted only in a
partial decrease of hyperglycaemia and no improvement
of insulin secretory function in vivo and in vitro (Tourrel,
Bailbe, Meile, Kergoat, & Portha, 2001).
In IUGR rats, neonatal treatment with exendin-4
for 6 days prevented the development of diabetes by
completely rescuing -cell mass from the progressive
reduction observed in untreated animals (Stoffers, Desai,
DeLeon, & Simmons, 2003). This effect was due to
enhanced -cell proliferation and Pdx-1 expression.
Interestingly, it has been reported that exendin-4 fails
to increase -cell proliferation and reduce -cell apoptosis in mice with -cell-specific inactivation of Pdx-1
(Li et al., 2005).
886
Table 2
Scheme of the main advantages and limitations of various rodent models of type 2 diabetes with reduction of pancreatic -cell mass and some
suggestions for their use in diabetes research
Model
Associated
insulin
resistance
Advantages
Disadvantages and limitations
Suggestions for use
ZDF rats
Yes
Genetic model associated with

obesity and insulin resistance;
occurrence of a prediabetic phase
Studies on the mechanisms of

-cell decompensation and
glucolipotoxicity
P. obesus gerbils
Yes
Genetic model induced by

relative nutritional excess
GK rats
No
Genetic model without obesity

derived by selective breeding of
Wistar rats; stable moderate
hyperglycaemia
Specific genetic background

(alteration of leptin receptor)
affecting appetite; intrinsic
-cell dysfunction on unknown
genetic basis; markedly
abnormal lipid metabolism
Particular genetic background
adapted to desert life (thrifty
genotype); limited availability
and high cost of animals
Intrinsic -cell dysfunction on
unknown genetic basis; poor
insulin responsiveness to
glucose in vivo and in vitro
LP IUGR rats
Yes
Easy to be induced; possibility to

introduce selected nutritional
supplementations
IUGR rats
Yes
Px rats
No
Reproduction of utero-placental
insufficiency of humans with
low-birth weight; progressive
hyperglycaemia with insulin
resistance
Previously healthy animals,
without genetic background and
-cell dysfunction; induction in
young adults; intact residual
-cells
n-STZ rats
No
Stable moderate hyperglycaemia
STZ-NA rats
No
Previously healthy animals,

without genetic background and
-cell dysfunction; easy
induction in young adults; mild
stable hyperglycaemia; residual
-cells well differentiated; in
vivo and in vitro insulin
responsiveness to glucose and
tolbutamide
Difficult choice of protocol

variants regarding severity and
duration of LP diet; variability
of effects; hyperglycaemia only
at old age
No major disadvantage
Invasive technique with

removal of exocrine pancreas
and endocrine non--cells;
need for animal welfare
measures; unsuitable for HFD
experiments; lack of insulin
tolbutamide in perfused
pancreas
Residual -cells deriving from
regeneration/neogenesis
apparently not well
differentiated; lack of insulin
tolbutamide in perfused
pancreas
Residual -cells potentially
damaged by STZ; lack of
insulin resistance, however
inducible with HFD;
hyperglycaemia only in the
non-fasting state
Studies on the mechanisms of

metabolic decompensation induced
by changes in nutritional
environment
Studies on diabetic complications
and other effects of chronic
hyperglycaemia; comparison with
non-genetic models (e.g., effects of
HFD or aging)
Studies on the late metabolic
effects of nutritional restriction and
underlying mechanisms
Studies on the late metabolic effects

of altered intrauterine environment
and underlying mechanisms
Studies of the mechanisms of

compensation/failure of a primarily
reduced healthy -cell mass;
suitable for treatment with drugs
stimulating cell growth
Studies on diabetic complications

and other effects of chronic
hyperglycaemia
Studies on long-term effects of

mild/moderate hyperglycaemia on
-cell function and gene
expression; studies on
compensation/failure upon HFD;
studies on new insulin
secretagogues or drugs stimulating
cell growth; studies on the effects
of aging in animals with reduced
-cell mass
ZDF: Zucker diabetic fatty; GK: Goto-Kakizaki; LP IUGR: low-protein intrauterine growth retardation; IUGR: intrauterine growth retardation by
uterine artery ligation in pregnancy; Px: partially pancreatectomized; n-STZ: neonatal streptozotocin; STZ-NA: streptozotocin-nicotinamide; HFD:
high-fat diet.
All the above-mentioned studies using exendin-4

have been performed in neonate or very young animals
that are likely more responsive to growth-stimulating
factors. However, 9-week-old Zucker obese rats treated
with exendin-4 (Gedulin et al., 2005) and aging glucoseintolerant rats treated with GLP-1 (Perfetti, Zhou, Doyle,
& Egan, 2000) also showed beneficial effects. Furthermore, GLP-1 and exendin-4, administered for 4 weeks to
adult STZ-NA rats, were recently reported to normalize
plasma glucose levels (Ozyazgan, Kutluata, Af, Ozda, &
Akkan, 2005).
1.2.5. Advantages and limitations of the most
commonly used animal models of type 2 diabetes
with -cell mass reduction
A scheme summarizing the main advantages, disadvantages and limitations of the most common animal
models described above is shown in Table 2.
2. Conclusions
From the analysis of the animal models described
above, it appears that among the factors involved in
pancreas development, Pdx-1 emerges for its role not
only in the maintenance of fully differentiated and functional -cells but also in their physiological postnatal expansion and their survival. It has to be underlined that Pdx-1 appears as a critical regulator of cell plasticity, given its essential contribution to ensure
appropriate -cell proliferation and/or neogenesis in
the adaptive response to insulin resistance or -cell
injury.
IRS-2 and its complex downstream signalling pathway are also relevant for -cell plasticity and survival,
likely in an integrated fashion with Pdx-1 activity. Also
cAMP-dependent CREB signalling appears to play a significant role in -cell survival and is particularly attractive as a mediator of the stimulating effect of GLP-1 and
its long-acting analog exendin-4 on -cell proliferation
and neogenesis.
Defects in specific factors involved in cell cycle progression such as cyclin D2 might have pathogenic significance for -cell mass reduction only within a small
temporal window corresponding to the period of maximal postnatal -cell growth.
The animal models of reduced -cell mass associated with amyloid formation or ER stress support the
possible intervention of these factors in the pathogenesis of type 2 diabetes. The role of ER stress as a possible
general mechanism of induction of -cell death is probably underestimated and should be further investigated
in animal models of reduced -cell mass, especially in
887
situations of increased insulin demand and consequent

-cell overstimulation.
The animal models with reduction of -cell mass
induced by nutritional, surgical or chemical means show
that compensatory adaptation takes place in the residual -cell mass. Partial recovery of the lost mass may
result from post-injury induction of -cell regeneration
and/or neogenesis and eventually from treatment with
growth-stimulating pharmacological agents. Hypertrophy and increased insulin responsiveness to glucose and
free fatty acids may also occur in residual -cells. Nevertheless, the adaptive response might be insufficient
or temporary because of incomplete differentiation of
newly formed -cells and/or acquired dysfunctions of
the residual -cells chronically exposed to a metabolically altered environment. An increased frequency of
apoptosis due to prolonged overstimulation of residual
-cells, chronic hyperglycaemia/hyperlipidaemia and/or
amyloid formation might accelerate decompensation.
Interestingly, recent data obtained in partially pancreatectomized rats and STZ-NA rats indicate that mild hyperglycaemic conditions are compatible with prolonged
successful -cell compensation from a reduced -cell
mass.
An important consideration that stems from the analysis of a number of experimental models of -cell
mass reduction is that a genetically or environmentally
induced permanent alteration of the developmental pattern of cellular proliferation and differentiation in the
endocrine pancreas may result in early-onset reduction
of -cell mass and/or inability to undergo cellular and
metabolic compensatory adaptations to increased insulin
demand. It is possible that the pathogenesis of type 2 diabetes developing in one-third of obese subjects relies on
this basis.
Further investigations are required to fully elucidate
the mechanisms of -cell compensation and subsequent
failure in animal models of reduced -cell mass. Given
our current inability to accurately determine functional
human -cell mass in a non-invasive manner, these models are also of paramount importance for longitudinal
studies aimed at correlating -cell mass and metabolic
parameters in a number of pathophysiological conditions
or upon treatment with pharmacological agents.
Thus, it is expected that animal models of type 2 diabetes with reduced -cell mass will continue to have a
relevant place in diabetes research.
Acknowledgement
The helpful assistance of Dr. Michela Novelli
and Ms. Valentina DAleo in the proper arrange-
888
ment of
ledged.
the
references
is
gratefully
acknow-
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Animal Models of DM2 With Reduced Pancreatic Β-cell Mass - 2006

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Animal Models of DM2 With Reduced Pancreatic Β-cell Mass - 2006

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The International Journal of Biochemistry & Cell Biology 38 (2006) 873893

Animal models of type 2 diabetes with

Models with chemically-induced reduction of -cell mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

betes, the same metabolic conditions finally result into

weeks of age, during which -cell mass does not increase

ditions of insulin resistance in experimental animals,

comes of metabolic overload in different groups of obese

offer opportunities to explore new approaches to treatment of diabetes.

particular glucose-stimulated insulin gene transcription

HFN-1 appears to be mainly involved in the regulation

(Lingohr et al., 2003; Withers et al., 1999). This is

no increase in -cell apoptosis is observed (Pende et al.,

1.1.3. Animal models with increased -cell

1.1.4. Animal models with increased -cell

an increased frequency in -cell apoptosis prior to the

al., 1999). Upon HE diet, these rodents become obese

rats also have genetically induced alterations of insulin

Mice with increased -cell apoptosis due to ER stress

Rodents with increased -cell apoptosis due to islet amyloid production

Rat with overexpression of h-IAPP (HIP rat)

Rodents with increased -cell apoptosis due to gluco- and/or lipotoxicity

Psammomys obesus gerbila (upon HED)

Spontaneous model of decreased -cell mass

1.2.1.2. Offsprings of pregnant rats fed with low-protein

Hoet, 1995; Dahri, Snoeck, Reusens, Remacle, & Hoet,

In several species, quite large pancreatectomies are

caemic 90% Px rats, an increase in hexokinase, and not

1.2.3. Models with chemically-induced reduction of

sulphonylureas (Masiello et al., 1998). In 2-month-old

(Larsen et al., 2003b), as well as to study the effect of

1.2.4. Effect of treatment with exendin-4 on animal

Disadvantages and limitations

Suggestions for use

Genetic model associated with

Studies on the mechanisms of

Genetic model induced by

Genetic model without obesity

Specific genetic background

Easy to be induced; possibility to

Stable moderate hyperglycaemia

Previously healthy animals,

Difficult choice of protocol

Invasive technique with

Studies on the mechanisms of

Studies on the late metabolic effects

Studies of the mechanisms of

Studies on diabetic complications

Studies on long-term effects of

All the above-mentioned studies using exendin-4

situations of increased insulin demand and consequent

Gepts, W., & Lecompte, P. M. (1981). The pancreatic islets in diabetes.

Miralles, F., & Portha, B. (2001). Early development of beta-cells

that may act as a survival factor. Endocrinology, 139, 2994

Rhodes, C. J., & White, M. F. (2002). Molecular insights into

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