Clinical Science (2011) 120, 73–80 (Printed in Great Britain) doi:10.1042/CS20100372 73

Sitagliptin prevents the development
of metabolic and hormonal disturbances,

increased β-cell apoptosis and liver steatosis
induced by a fructose-rich diet in normal rats

Bárbara MAIZTEGUI1, Marı́a I. BORELLI1, Viviana G. MADRID1,
Héctor DEL ZOTTO1, Marı́a A. RASCHIA, Flavio FRANCINI, Marı́a L. MASSA,
Luis E. FLORES, Oscar R. REBOLLEDO and Juan J. GAGLIARDINO
CENEXA – Centro de Endocrinoloǵıa Experimental y Aplicada (UNLP-CONICET LA PLATA, Centro Colaborador OPS/OMS),
Facultad de Ciencias Médicas UNLP, 60 y 120 (1900) La Plata, Argentina

A B S T R A C T

The aim of the present study was to test the effect of sitagliptin and exendin-4 upon metabolic
alterations, β-cell mass decrease and hepatic steatosis induced by F (fructose) in rats. Normal
adult male Wistar rats received a standard commercial diet without (C) or with 10% (w/v)
F in the drinking water (F) for 3 weeks; animals from each group were randomly divided into
three subgroups: untreated (C and F) and simultaneously receiving either sitagliptin (CS and FS;
115.2 mg/day per rat) or exendin-4 (CE and FE; 0.35 nmol/kg of body weight, intraperitoneally).
Water and food intake, oral glucose tolerance, plasma glucose, triacylglycerol (triglyceride), insulin
and fructosamine concentration, HOMA-IR [HOMA (homoeostasis model assessment) for insulin
resistance], HOMA-β (HOMA for β-cell function) and liver triacylglycerol content were measured.
Pancreas immunomorphometric analyses were also performed. IGT (impaired glucose tolerance),
plasma triacylglycerol, fructosamine and insulin levels, HOMA-IR and HOMA-β indexes, and liver
triacylglycerol content were significantly higher in F rats. Islet β-cell mass was significantly lower
in these rats, due to an increase in the percentage of apoptosis. The administration of exendin-4
and sitagliptin to F animals prevented the development of all the metabolic disturbances and the
changes in β-cell mass and fatty liver. Thus these compounds, useful in treating Type 2 diabetes,
would also prevent/delay the progression of early metabolic and tissue markers of this disease.

INTRODUCTION

T2DM (Type 2 diabetes mellitus) represents a serious
health problem worldwide due to its high and growing
incidence and to the negative impact of its associated
complications upon quality of life and healthcare costs
[1]. To cope with this problem, two different approaches

have been implemented: the improvement of care quality
and the development of new therapeutic tools, and the
primary prevention of the disease in people at risk [2].

The development of incretin mimetic and incretin
enhancer drugs represents a promising therapeutic tool
to achieve treatment goals [3–5], considering that people
with T2DM have a significant decrease of incretin

Key words: β-cell mass, exendin-4, hypertriglyceridaemia, insulin resistance, pre-diabetes, sitagliptin.
Abbreviations: AUC, area under the curve; DPP-4, dipeptidyl peptidase-4; F, fructose; GLP-1, glucagon-like peptide-1; HOMA,
homoeostasis model assessment; HOMA-β, HOMA for β-cell function; HOMA-IR, HOMA for insulin resistance; IGT, impaired
glucose tolerance; IR, insulin resistance; OGTT, oral glucose tolerance test; PCNA, proliferating cell nuclear antigen; T2DM, Type 2
diabetes mellitus; Vvi, volume density.
1 These authors contributed equally to the study.
Correspondence: Dr Juan J. Gagliardino (email cenexa@speedy.com.ar).

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74 B. Maiztegut and others

effect [6]. GLP-1 (glucagon-like peptide-1), a prominent
active compounds of the incretin family, modulates
many processes in pancreatic islet: it potentiates insulin
synthesis and secretion [5], inhibits glucagon secretion
[7], increases islet cell proliferation, and decreases cell
apoptosis [8,9]; further, exendin-4 (a GLP-1 receptor
agonist), reverses the loss of islet mass grafts after
transplantation [10]. The mechanisms whereby GLP-1
may exert these effects have been reviewed by Drucker
and Nauck [3].

The pleiotropic effects of GLP-1 suggest that it
could potentially slow down or prevent the continuous
deterioration of β-cell mass and function characteristic of
T2DM and it could potentially prevent its development
in people at risk. While many reports support the
usefulness of incretin mimetic and incretin enhancer
drugs for the effective treatment of T2DM, there is not
sufficient evidence of their effectiveness to prevent/delay
its development.

To provide such evidence, it would be useful to have
an animal model with clinical/metabolic characteristics
similar to those of people at high risk for T2DM. In this
regard, it has been proved that F (fructose) administration
to normal rats for different time periods sequentially
induces IGT (impaired glucose tolerance) and T2DM
[11–16]. These rats also have fatty liver and progressively
decreased β-cell mass and function due to an increase of
β-cell apoptosis, thus resembling the changes described
in people with T2DM [17,18].

In an attempt to elucidate this issue, we currently
studied the effect of sitagliptin, a DPP-4 (dipeptidyl
peptidase-4) inhibitor, and exendin-4 upon the metabolic,
β-cell mass and hepatic changes induced by F administra-
tion to normal rats for 21 days. Results demonstrate that
the simultaneous administration of sitagliptin or exendin-
4 with F prevented the development of all the recorded
metabolic disturbances induced by this diet and their
negative impact upon β-cell mass.

MATERIALS AND METHODS

Chemicals and drugs
Collagenase was obtained from Serva Feinbiochemica;
BSA fraction V and other reagents were from Sigma.
F was obtained from Corn Products International.
Exendin-4 was obtained from Bachem, and sitagliptin
was provided by Process Research, Merck Research
Laboratories.

Experimental groups
Normal male Wistar rats (180–200 g) were divided into
two groups: a control group that was fed with a standard
commercial diet (C), and another group that received the
same diet plus 10 % (w/v) F in the drinking water for
3 weeks. Animals from C and F were randomly divided

into three subgroups (n = 20 each group): untreated
(C and F), treated with sitagliptin (CS and FS), or
treated with exendin-4 (CE and FE). All the animals
were housed in a room with controlled temperature
(25 ◦C) and lighting (12 h light/dark cycle). CE and FE
rats were injected with exendin-4 (0.35 nmol/kg of body
weight, intraperitoneally) twice a day, while sitagliptin
was administered orally (115.2 mg/day per rat) premixed
with the milled pellet at 0.6 % (w/w). Water and food
intake were measured daily, while individual body weight
was recorded once a week.

Animal experiments and handling were performed
according to the “Ethical principles and guidelines for
experimental animals” of the Swiss Academy of Medical
Sciences (3rd Edition 2005).

OGTT (oral glucose tolerance test)
OGTT was performed in 12-h fasted rats from each ex-
perimental group 24 h prior to sacrifice. Glucose (1 g/kg
of body weight in saline solution) was given through a
gavage tube placed into the stomach and blood samples
were obtained from the retro-orbital plexus under light
pentobarbital anaesthesia (48 mg/kg of body weight) at
0, 30, 60 and 120 min following the glucose load. In these
samples, glucose concentration was measured with test
strips (One touch Ultra; Lifescan) and plasma insulin
by RIA using a specific antibody against rat insulin, rat
insulin standard (Linco Research) and highly purified
porcine insulin labelled with 125I [19]. Results for glucose
were expressed as the AUC (area under the curve) in
mmol · l− 1 · min− 1. HOMA-IR [HOMA (homoeostasis
model assessment) for insulin resistance] was calculated
according to the formula: insulin (μ-units/ml)×glucose
(mmol/l)/22.5; the formula for HOMA-β (HOMA for
β-cell function) was: insulin (μ-units/ml)×20/glucose
(mmol/l) − 3.5 [20]. Values correspond to fasting serum
glucose and insulin levels. Fasting plasma triacylglycerol
(triglyceride) and fructosamine levels were measured
using commercial kits (BioSystems) implemented in an
automated clinical analyser.

Liver triacylglycerol content
Liver triacylglycerol extraction was performed following
the Schwartz and Wolins protocol [21]; concentration
was measured with a commercial enzymatic assay kit
(TG Color GPO/PAP AA, Wiener Laboratory, Rosario,
Argentina).

Immunohistochemical studies
The whole pancreas from three animals of each
experimental group was carefully dissected and removed
and its wet weight was recorded; thereafter, a piece
of the tail of each pancreas was obtained, fixed in
10 % formaldehyde and embedded in paraffin. Serial
sections of each one of the fixed pancreases (5 μm)

C© The Authors Journal compilation C© 2011 Biochemical Society



Prevention of metabolic and tissue alterations 75

were obtained from three different depths of the
blocks with a rotatory microtome and mounted on
silanized slides (3-amino-propyltriethoxy-silane; Sigma).
Sections were deparaffinized, incubated for 30 min in
3 % (v/v) hydrogen peroxide in methanol to block the
endogenous peroxidase activity and rehydrated in a
descending ethanol series, followed by incubation in
2.5 % porcine serum to reduce non-specific binding.
The slides were then incubated for 24 h at 4 ◦C in a
humidified chamber with our own anti-guinea pig insulin
antibody (1:20 000 dilution) [16]. The final staining was
performed by incubating the slides for 30 min with
appropriately diluted streptavidin–biotin complex (1:40
and 1:20 dilution respectively; Sigma); thereafter, the
sections were stained with haematoxylin.

Cell replication
Sequential double-staining of sections was performed
using these combinations: (i) PCNA (proliferating cell
nuclear antigen) antibody (1:4000 dilution; Sigma;) [22],
and (ii) β-cells (our own guinea pig-insulin antibody;
1:20 000 dilution). First, we performed PCNA staining as
described above using carbazole as chromogen; the same
section was then immunostained for β-cells, except that
alkaline phosphatase and Fast Blue (Sigma) were used as
chromogens. Alkaline phosphatase conjugate was applied
to each section for 30 min at room temperature (25 ◦C);
the sections were then washed and alkaline phosphatase
was applied for a further 30 min. Within a given cell
type, the replication rate was quantified and expressed
as the percentage of PCNA-labelled cells among the total
β-cells counted (no less than 3000 cells of each type).
The immunohistochemical staining has been previously
validated [23]. For every immunostaining performed,
simple controls were done by omitting the primary
antibody.

β-Cell apoptosis
The propidium iodide technique was used to identify
apoptotic bodies [24]. For this purpose, deparaffinized–
rehydrated pancreas sections were incubated for 30 min
in a dark humidified chamber with a propidium
iodide (4 μg/ml; Sigma) and RNase A (100 μg/ml;
Sigma) solution. Then, the sections pretreated with
guinea pig non-immune sera diluted in Tris-buffered
saline (pH 7.4) were incubated for 1 h with the insulin
antibody. To measure the fluorescence labelling of
the primary antibody, the slides were incubated at
room temperature for 45 min in the dark chamber
with the IgG-specific, fluorescein-conjugated, affinity-
purified guinea pig antibody (against heavy and light
IgG chains; Santa Cruz Biotechnology). The sections
were then washed with PBS and mounted in Tris-
glycerol (pH 8.4) for analysis with an immunofluorescent
Zeiss Axiolab epifluorescence microscope equipped
with an HBO50 mercury lamp and two different

filters. For the quantitative assessment of apoptosis,
positively immunofluorescence-labelled β-cells were
counted under a ×40 objective lens in sections obtained
from different levels of the paraffin blocks. The number
of apoptotic β-cells was expressed as the percentage of
the total number of β-cells counted.

Morphometric analysis
Morphometric analysis was performed by videomicro-
scopy using a Jenamed 2 Carl Zeiss light microscope
and a RGB CCD Sony camera, together with
the OPTIMAS software (Bioscan). The following para-
meters were measured: total pancreatic area (excluding
connective tissue), insulin-positive ductal cell and islet
β-cell area [Vvi (volume density)]. To estimate islet and
ductal cell mass, we multiplied the respective Vvi by the
weight of the total pancreas [25].

The tissue sections were examined by two blinded
independent observers who were oblivious to which
study group the section came from. In case the inter-
observer measurement of any variable in any sample was
higher than 10 %, they independently re-evaluated the
section and rescored the variable.

Statistical analysis
The experimental data were analysed using variance
analysis (one-way ANOVA) and post-hoc Bonferroni
and Tamhane tests for multiple comparisons. Since insulin
levels and the HOMA-IR and HOMA-β indexes did not
have a normal distribution, the natural logarithm of the
values was used to perform the analysis. Differences were
considered significant when P < 0.05.

RESULTS

Body weight, food consumption and
water intake
Comparable body weight and body increase were
recorded in C and F animals after the 3-week
treatment period (Table 1). Exendin-4 but not sitagliptin
administration reduced significantly body weight gain in
both C and F rats.

F animals drank a larger volume of water than C ones,
while the ingested amount of solid food was significantly
larger in the latter group (Table 1). This fact resulted
in a different percentage of daily intake of nutrients by
F compared with C rats (carbohydrates/proteins/lipids,
59:32:9 compared with 45:43:12 respectively), with a
comparable caloric intake (54.1 +− 0.70 compared with
61.4 +− 3.22 kcal/day respectively). Neither exendin-4
nor sitagliptin affected the calorie intake in C and F
animals (CE: 56.8 +− 7.2; CS: 52.5 +− 2.5; FE: 48.9 +− 0.8;
FS: 52.9 +− 1.8 kcal/day).

C© The Authors Journal compilation C© 2011 Biochemical Society



76 B. Maiztegut and others

Table 1 Changes in body weight, food consumption and water intake in the experimental groups
Values are means+− S.E.M. (n = 20). ∗P < 0.01 compared with F; †P < 0.01 compared with C.

Body weight (g)

Group Initial Final �g Food consumption (g) Water intake (ml)

C 206+− 6.7 278+− 7.9 72 +− 4.0 21.3+− 1.1 25.9 +− 1.2
CE 207+− 2.9 255+− 4.1 47 +− 6.0† 19.6+− 2.5 28 +− 3.0
CS 217+− 5.2 300+− 5.6 83 +− 4.9 21.6+− 0.8 28 +− 1.8
F 208+− 8.6 278+− 10.7 70 +− 4.2 15.1+− 0.1† 49.8+− 5.9†
FE 202+− 4.3 251+− 3.3 49 +− 2.1∗ 14.3+− 0.3 38.6 +− 3.4
FS 211+− 2.3 281+− 2.5 70 +− 1.4 15.1+− 0.6 45.6 +− 8.8

Table 2 Blood glucose, triacylglycerol, insulin and fructosamine levels, and AUC during the OGTT in the experimental
groups
Values are means+− S.E.M. (n = 20). ∗P < 0.03, ∗∗P < 0.01, ∗∗∗P < 0.003, ∗∗∗∗P < 0.001 and ∗∗∗∗∗P < 0.0001; †P < 0.02, ††P < 0.01 and
†††P < 0.0001 compared with C.

Group Glucose (mmol/l) Triacylglycerol (mmol/l) Insulin (ng/ml) Fructosamine (μmol/l) AUC (mmol · l− 1 · min− 1)

C 4.48+− 0.12 0.47+− 0.01 0.30 +− 0.02 175.1+− 8.5 1.51 +− 0.08
CE 4.17+− 0.09 0.48+− 0.06 0.25 +− 0.03 150.6+− 6.3 1.49 +− 0.14
CS 4.45+− 0.16 0.63+− 0.09 0.27 +− 0.03 190.7+− 10 1.35+− 0.17
F 4.94+− 0.22 1.14+− 0.12††† 1.09 +− 0.28† 218.6+− 8.2†† 3.05+− 0.23†††
FE 4.19+− 0.18 0.72+− 0.09∗∗∗ 0.30 +− 0.05∗ 154.6+− 8.1∗∗∗∗∗ 1.37 +− 0.15∗∗∗∗∗

FS 4.68+− 0.27 0.67+− 0.05∗∗∗∗ 0.31 +− 0.05∗ 172.5+− 6.8∗∗ 1.54 +− 0.07∗∗∗∗∗

OGTT
The AUC during the OGTT was significantly higher in
F as compared with C animals (Table 2). These values
decreased significantly to almost control levels in F rats
treated with exendin-4 or sitagliptin.

Blood glucose, triacylglycerol, insulin and
fructosamine levels
While blood glucose levels were comparable in all the
groups, triacylglycerol, fructosamine and insulin levels
were significantly higher in F compared with C rats
(Table 2).

The HOMA-IR and HOMA-β indexes were also
significantly higher in F animals, indicating the presence
of an IR (insulin resistance) state and a β-cell attempt
to cope with the increased insulin demand in these rats
(Figures 1A and 1B). All these high levels decreased to
almost control values in F rats treated with either exendin-
4 or sitagliptin. Neither exendin-4 nor sitagliptin affected
those parameters in C animals (Table 2 and Figures 1A
and 1B).

Liver triacylglycerol content
F rats had higher liver triacylglycerol content than C
animals (476.4 +− 29.9 compared with 186.8 +− 11.8 μg/
100 mg of tissue respectively; P < 0.001). While neither
exendin-4 nor sitagliptin affected hepatic triacylglycerol
content in C animals, both drugs decreased it significantly

in F rats (FE: 374.7 +− 29.9 μg/100 mg of tissue, P < 0.05;
FS: 299.8 +− 16.7 μg/100 mg of tissue; P < 0.001).

Morphometric analysis and β-cell
apoptosis
Pancreas sections from C, F, FE and FS rats are shown
in Figures 2(A)–2(D) respectively. It can be seen that
β-cell mass markedly decreased in F rats, but this mass
recovered a normal profile after exendin-4 and sitagliptin
administration.

The morphometric analysis demonstrates that, in
fact, islet β-cell mass was significantly lower (37 %)
in F rats (Table 3). Exendin-4 and sitagliptin did not
markedly affect this parameter in C animals; however,
both compounds increased significantly islet β-cell mass
in F rats (201 % and 68 % respectively), attaining values
close to those measured in C rats.

Insulin-positive duct-cell mass also increased in F rats,
suggesting a possible neogenesis reaction, but it was not
affected by exendin-4 or sitagliptin administration.

Although no significant differences were recorded
in PCNA labelling among groups, higher values were
recorded in F rats.

The β-cell apoptotic percentage was significantly
higher (167 %) in F rats (Figures 2E and 2F, and
Table 3). Such increased percentage of apoptosis fell
to almost control values after exendin-4 or sitagliptin

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Prevention of metabolic and tissue alterations 77

Table 3 Morphometric analysis and β-cell apoptosis in the experimental groups
Values are means+− S.E.M. from three different levels of the block corresponding to three animals from each group. ∗P < 0.01 and ∗∗P < 0.001 compared with F;
†P < 0.01 and ††P < 0.001 compared with C. ND, not detectable.

Group Islet β-cell mass (mg) Insulin-positive duct cell mass (mg) β-Cell replication (% PCNA) Apoptotic β-cell (%)

C 5.00+− 0.45 ND 1.81 +− 1.31 0.18 +− 0.02
CE 6.59+− 1.32 ND 2.29+− 0.7 0.19 +− 0.04
CS 4.54+− 0.5 ND 2.79+− 0.76 0.14 +− 0.02
F 3.17+− 0.37† 0.02+− 0.007 2.43 +− 0.95 0.48 +− 0.02††
FE 9.54+− 1.8∗ 0.02+− 0.005 1.65 +− 0.77 0.14 +− 0.03∗∗

FS 5.32+− 0.7∗ 0.02+− 0.008 1.16 +− 0.27 0.18 +− 0.02∗∗

Figure 1 HOMA-IR (A) and HOMA-β (B) indexes in the experimental groups
Values are means+− S.E.M. (n = 20 in each case). †P < 0.05 and ††P < 0.01 compared with C; ∗P < 0.05 and ∗∗P < 0.02 compared with F.

administration (71 % and 63 % decrease respectively;
Figures 2G and 2H, and Table 3).

DISCUSSION

As previously shown, F administration to normal
rats for 21 days induces hypertriglyceridaemia, IR
(hyperinsulinaemia, high insulin/glucose molar ratio
and increased HOMA-IR), IGT, increased protein
glycosylation (high serum fructosamine levels) and a
significant 37 % decrease in β-cell mass; the latter could be
mainly ascribed to an increased percentage of apoptosis
(167 %) [16] that was not compensated for by a non-
significant increase in β-cell replication percentage and
an apparent neogenesis reaction (increased percentage
of insulin-positive duct cells). Two other organs were
also markedly affected in these rats: (i) the mass of
abdominal adipose tissue increased significantly, showing
a higher proportion of saturated fatty acid composition
together with a higher release of these fatty acids, leptin
and adiponectin, and significant changes in glycoxidative
stress markers [15,26], and (ii) the liver showed an
abnormal glucose metabolism shifted towards lipid
synthesis and deposit [27]. Altogether, these changes
resemble those described in human pre-diabetes, T2DM
and the metabolic syndrome [28]. We have postulated

that the underlying mechanism of all these changes was
the development of a glycoxidative stress that probably
starts in abdominal adipose tissue and in the liver [15,26].

In the current study, exendin-4 injection to F rats
maintained the triacylglycerol, fructosamine and insulin
plasma levels within the range of values recorded in
C rats. The HOMA-IR and HOMA-β indexes also
remained within normal range, indicating that IR was
not developed in these animals and thus preventing the
consequent β-cell function overload. β-Cell apoptosis
percentage and mass values also lay within the range of
those measured in C rats. These changes were associated
with the normalization of IGT and liver triacylglycerol
content recorded in F rats.

Similar and even more pronounced changes were
observed when F rats were treated with sitagliptin.
Conversely, administration of either exendin-4 or
sitagliptin to C rats did not modify significantly any of
the above mentioned parameters.

The beneficial effects of GLP-1 receptor agonists
and DPP-4 inhibitors upon hyperglycaemia and
β-cell mass present in different models of T2DM
have been largely and consistently documented [8,29–
36]. Conversely, the available evidence of their effect
upon IGT is scarce and not conclusive, and under
such metabolic condition the incretin effect upon β-
cell mass depends apparently on the experimental

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78 B. Maiztegut and others

Figure 2 Representative sections of the pancreas from the
experimental groups showing β-cell mass (A–D) and β-cell
apoptosis (E–H)
(A–D) Pancreas sections from C (A), F (B), FE (C) and FS (D) rats. The β-cell
mass appears markedly decreased in F as compared with C, while exendin-4
and sitagliptin gave β-cell mass a profile comparable with that observed in C
rats. Magnification, ×10. (E–H) β-cell apoptosis in C (E), F (F), FE (G) and FS
(H). It greatly increased in F compared with C. After exendin-4 and sitagliptin
administration, the profile of this process was comparable with that observed in
C rats. Magnification, ×40.

model used. Winzell and Ahrén, using mice with IGT
induced by administration of a high fat diet, improved
such impairment by increasing the serum insulin
levels [37]; they did not mention however, any effect
upon β-cell mass. Alogliptin (another DPP-4 inhibitor)
administered alone to prediabetic db/db mice produced
neither robust hyperglycaemia prevention nor sufficient
β-cell preservation, but, associated with voglibose (an
α-glucosidase inhibitor), it improved diabetic indexes
and prevented diabetes development [38]. Although
these authors did not perform quantitative analysis,
they mentioned that combined treatment preserved
pancreatic β-cells and islet structure [38]. In this study,
the combined treatment did not affect significantly
plasma triacylglycerol or total cholesterol levels.
On the other hand, administration of P32/98 (a
DPP-4 inhibitor) for 3 weeks to fatty Zucker rats

with manifest IGT improved their day–night blood
glucose profile, reduced significantly their triacylglycerol
and non-esterified fatty acids levels, and increased
the glucose-induced insulin release, without affecting
β-cell Vvi [39]. Xu et al. also showed that exendin-4
administration improved IGT developed in rats with 90–
95 % pancreatectomy and increased β-cell mass due to
an increase in their percentage of replication/neogenesis;
however, they did not measure β-cell apoptosis [29].
Conversely, other authors showed no significant changes
in β-cell area or islet morphology [40–42].

In our study, performed in an animal model that
portrays characteristics similar to those recorded in
human pre-diabetes, exendin-4 and sitagliptin adminis-
tration induced similar and significant beneficial effects
upon the multiple F-induced metabolic disturbances and
decreased β-cell mass. The latter effect was due to a
significant decrease in β-cell apoptosis. These two agents
also decreased liver steatosis.

We did not currently study the molecular mechanism
by which sitagliptin decreased F-induced β-cell apop-
tosis. In this regard, Maida et al. [43] have recently
reported that sitagliptin administration for 60 days to
streptozotocin diabetic mice significantly reduced plasma
DPP-4 activity and increased the percentage of β-cell
area. The latter increase was associated with significantly
lower levels of cleaved caspase-3 immunopositivity
within the islets. These authors also reported that
sitagliptin increased the pancreatic mRNA levels of IGF-
1 and Akt-1, two genes involved in the control of
β-cell survival [44]. We could thus assume that in our F
model, sitagliptin exerted its beneficial effects upon β-cell
apoptosis and mass using similar intracellular pathways.

We have previously shown that F animals portray
increased markers of glycoxidative stress in abdominal
adipose tissue and in the liver [15,26]. Based on the uni-
fying hypothesis proposed by Brownlee [45], we could
assume that such stress is the underlying common cause
of IR and of the increased rate of non-enzymatic protein
glycosylation observed in our F rats. Complementarily,
the F load would shift liver carbohydrate metabolism
towards fat deposition and triacylglycerol release, thus
promoting fatty liver, hypertriglyceridaemia and IR. The
combination of glycoxidative stress, lipotoxicity (high
plasma triacylglycerol levels) and increased demand of
insulin (IR) would stimulate β-cell apoptosis [46], with
the consequent decrease in its functional mass; IGT
would be the consequence of such β-cell dysfunction. The
beneficial effect of both exendin-4 and sitagliptin upon
the overall metabolic disturbances and β-cell dysfunction
strongly suggests that both compounds can successfully
counteract the mentioned pathogenic events. We cannot
at the moment define, however, the possible specific
metabolic target/s on which these compounds work.

In brief, our data show for the first time that
administration of exendin-4 and sitagliptin prevents, to a

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Prevention of metabolic and tissue alterations 79

similar extent, F-induced hypertriglyceridaemia, IR and
IGT; these two drugs also prevent the decrease in β-cell
mass through a main decrease in their apoptosis. Since
these results were obtained in an animal model with
metabolic as well as islet and liver characteristics that
resemble those observed in human pre-diabetes, exendin-
4 and sitagliptin could be useful therapeutic tools to
prevent the progression of this metabolic state towards
T2DM.

AUTHOR CONTRIBUTION

Bárbara Maiztegui, Marı́a Borelli, Héctor Del Zotto
and Juan Gagliardino conceived and designed the
study, performed the statistical analyses and drafted
the manuscript. Bárbara Maiztegui, Marı́a Borelli
and Háctor Del Zotto carried out the oral glucose
tolerance experiments and metabolic determinations.
Viviana Madrid and Héctor Del Zotto performed
the immunohistochemical studies and morphometric
analyses. Marı́a Massa and Flavio Francini carried out
the liver triacylglycerol content determinations. Morı́a
Massa, Luis Flores and Oscar Rebolledo handled the
experimental animals and carried out the biochemical
determinations. All authors read and approved the final
manuscript.

ACKNOWLEDGEMENTS

We thank Adrián Dı́az for animal handling and insulin
assays, César Bianchi for technical assistance, Jorge
Elgart for his contribution in statistical analysis, and
Adriana Di Maggio for careful manuscript editing prior
to submission.

FUNDING

This study was partially supported by an unrestricted
grant provided by Merck, Sharp & Dohme, Argentina and
the National Research Council [grant number R1550/07].

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Received 13 July 2010/19 August 2010; accepted 26 August 2010
Published as Immediate Publication 26 August 2010, doi:10.1042/CS20100372

C© The Authors Journal compilation C© 2011 Biochemical Society