Sitagliptin lowers glucagon and improves glucose tolerance in prediabetic obese SHROB rats.

Abstract

The SHROB (spontaneously hypertensive rat - obese strain) is a model of prediabetes and metabolic syndrome with insulin resistance, glucose intolerance and hypertension. Inhibitors of dipeptidyl dipeptidase IV (DPP-IV) are effective hypoglycemic agents in type 2 diabetes through potentiation of incretin hormones that act in the pancreas to increase insulin and decrease glucagon release. We sought to determine whether the DPP-IV inhibitor sitagliptin might be effective in prediabetes relative to standard therapy with the sulfonylurea glyburide, by using the SHROB model. SHROB show normal fasting glucose but are insulin resistant and hyperglucagonemic. SHROB were treated for six weeks with vehicle, sitagliptin (30 mg/kg/d) or glyburide (1 mg/kg/d) and compared with untreated lean spontaneously hypertensive rats. Body weight, food intake and fasting glucose were all unchanged in all three SHROB groups, but glucagon was reduced by 33% by sitagliptin while remaining unchanged following glyburide or vehicle. In oral glucose (6 g/kg) tolerance testing, both sitagliptin and glyburide lowered plasma glucose. Both sitagliptin and glyburide shifted peak insulin secretion earlier (30 min for glyburide and 60 min for sitagliptin but 240 min for vehicle). Only sitagliptin significantly enhanced insulin secretion. Sitagliptin is effective in normalizing excess glucagon levels and delaying exaggerated insulin secretion in response to a glucose challenge in a prediabetic model.

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Original Research
Sitagliptin lowers glucagon and improves glucose tolerance
in prediabetic obese SHROB rats
Brian Chen, Alex Moore, Liza V S Escobedo, Matthew S Koletsky, Diana Hou,
Richard J Koletsky and Paul Ernsberger
Department of Nutrition, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 41106, USA
Corresponding author: Paul Ernsberger. Email: pre@cwru.edu
Abstract
The SHROB (spontaneously hypertensive rat – obese strain) is a model of prediabetes and metabolic syndrome with insulin
resistance, glucose intolerance and hypertension. Inhibitors of dipeptidyl dipeptidase IV (DPP-IV) are effective hypoglycemic
agents in type 2 diabetes through potentiation of incretin hormones that act in the pancreas to increase insulin and decrease
glucagon release. We sought to determine whether the DPP-IV inhibitor sitagliptin might be effective in prediabetes relative to
standard therapy with the sulfonylurea glyburide, by using the SHROB model. SHROB show normal fasting glucose but are
insulin resistant and hyperglucagonemic. SHROB were treated for six weeks with vehicle, sitagliptin (30 mg/kg/d) or glyburide
(1 mg/kg/d) and compared with untreated lean spontaneously hypertensive rats. Body weight, food intake and fasting
glucose were all unchanged in all three SHROB groups, but glucagon was reduced by 33% by sitagliptin while remaining
unchanged following glyburide or vehicle. In oral glucose (6 g/kg) tolerance testing, both sitagliptin and glyburide lowered
plasma glucose. Both sitagliptin and glyburide shifted peak insulin secretion earlier (30 min for glyburide and 60 min for
sitagliptin but 240 min for vehicle). Only sitagliptin significantly enhanced insulin secretion. Sitagliptin is effective in
normalizing excess glucagon levels and delaying exaggerated insulin secretion in response to a glucose challenge in a
prediabetic model.
Keywords: genetic obesity, insulin resistance, metabolic syndrome, DPP-IV inhibitors, sitagliptin, sulfonylureas, glyburide
Experimental Biology and Medicine 2011; 236: 309–314. DOI: 10.1258/ebm.2010.010161
Introduction
A recently introduced therapy for type 2 diabetes is the inhi-
bition of dipeptidyl dipeptidase IV (DPP-IV) in order to
enhance the effectiveness of endogenous insulin-releasing
gut hormones, especially glucagon-like peptide-1 (GLP-1)
but also gastric inhibitory peptide (GIP).1,2GLP-1 has mul-
tiple actions that contribute to improved glucose homeosta-
sis. These include promotion of insulin release in response
to food intake, inhibition of glucagon release, delay of
gastric emptying, and hypothalamic effects that include pro-
motion of satiety and reduced food intake.3The selective
and highly potent DPP-IV inhibitor sitagliptin was the
first agent to be introduced clinically and has been shown
effective in lowering levels of glycemia in type 2 diabetes.
Sitagliptin and other DPP-IV inhibitors lower glycosylated
hemoglobin HbA1cby about 1%, which means that com-
bined treatment with other agents is usually required to
achieve glucose control.4However, little is known about
the effect of these agents on the progression to type 2 dia-
betes, especially in its initial stages. There are reports that
sitagliptin is ineffective in lowering fasting glucose in pre-
diabetes.5Furthermore, little information is available on
the effects of sitagliptin on metabolism apart from plasma
glucose levels.
The SHROB (spontaneously hypertensive rat – obese
strain) show many of the characteristics of human metabolic
syndrome, a common precursor to type 2 diabetes.6,7These
traits include insulin resistance, glucose intolerance, hyper-
lipidemia affecting triglycerides more than cholesterol,
fatty liver and hypertension. The SHROB are spontaneously
hypertensive rats (SHRs) with a naturally occurring knock-
out of the leptin receptor, which causes dramatic weight
gain and metabolic disturbances. SHROB differ from the
better-known Zucker rats in having a more complete dis-
ruption of the leptin body weight regulatory system, result-
ing from a naturally occurring knockout of the leptin
receptor as opposed to the amino acid substitution caused
by the mutation in Zucker rats.8Furthermore, SHROB
have a hypertensive genetic background. The SHROB
model shows normal fasting plasma glucose levels, but
ISSN: 1535-3702
Copyright # 2011 by the Society for Experimental Biology and Medicine
Experimental Biology and Medicine 2011; 236: 309–314

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exhibits severe insulin resistance and hyperglycemia follow-
ing an oral glucose load. SHROB also show elevated gluca-
gon levels in both fasting and postprandial states and
impaired suppression of glucagon in response to an oral
glucose challenge.9SHROB show delayed and exaggerated
insulin secretion in response to an oral load. We hypoth-
esized that increased levels of GLP-1 and GIP induced by
sitagliptin treatment may reverse the abnormalities in
secretionof insulinand
Furthermore, if these pancreatic abnormalities contribute
to other aspects of the metabolic syndrome, then sitagliptin
may have additional therapeutic actions in metabolic syn-
drome. For example, the distribution of body fat in meta-
bolic syndrome favors accumulation of fat within the
abdomen. Antidiabetic agents in the thiazolidinedione
class induce remodeling of adipose tissue to favor subcu-
taneous depots.10The effects of DPP-IV inhibitors on fat dis-
tribution are not known, but glucagon may have actions on
lipolysis that could redistribute fat depots.11DPP-IV inhibi-
tors increase lipolysis in human subcutaneous adipocytes
through unknown mechanisms.12
glucagonin the SHROB.
Methods
Materials
Sitagliptin was a gift from Merck (Rahway, NJ, USA).
Glyburide and other chemicals were obtained from Sigma
Chemical (St Louis, MO, USA) or Fisher (Pittsburgh, PA,
USA) and were of analytical grade.
Animal procedure
Female SHROB and lean SHRs were obtained from Charles
River laboratories at six weeks of age and maintained under
standard laboratory conditions. Female animals were used
because of the greater cardiovascular relative risk in
women with diabetes.13In addition, female SHROB are
acyclic and sterile and show no significant differences in
glucose and insulin metabolism relative to male SHROB.6
Animals were housed individually and were provided
food (Teklad 8664, Harlan Teklad, South Easton, MA,
USA) and water ad libitum prior to drug treatment.
Animals were on a 12:12-h light–dark cycle (lights on
from 7:00 to 19:00) and were maintained at a constant temp-
erature of 218C. These procedures were carried out with the
approval of the Case Western Reserve University Animal
Care and Use Committee.
Chronic drug treatment
Agents were supplied in drinking water. The vehicle drinking
solution consisted of 0.7% sorbitol, 250 ppm sodium sacchar-
ine and 100 ppm sodium benzoate as a preservative. The sor-
bitol was necessary to solubilize the glyburide, and along
with the saccharin maintained palatability and ensured ade-
quate fluid intake. Fluid intake was allowed ad libitum and
monitored continuously so that adjustments could be made
to drug concentrations as necessary. Sitagliptin was added
at 500 ppm, which provided 30 mg/kg/d at the average
fluid intake of approximately 60 mL/kg body weight. Pilot
studies showed that this dose was about equally effective in
improving glucose tolerance as 1 mg/kg/d glyburide. The
dose of 30 mg/kg/d is considerably higher than the human
dose. However, sitagliptin has a half-life of two hours in
rats14versus 13 h in humans.2The short half-life also necessi-
tated continuous administration through drinking water in
place of the once-a-day dosing used in humans. Glyburide
was provided at 15 ppm, which provided 1 mg/kg/d, a
dose previously reported to control type 2 diabetes in the
rat.15Control untreated SHROB were provided with vehicle
solution to drink. Body weight and food intake were deter-
mined twice a week, and fasting tail blood samples (0.4 mL)
were obtained in heparinized capillary tubes at 9:00 every
other week. Lean SHRs were provided with plain water
and sacrificed at the same age as the SHROB to provide a
metabolically normal comparison. They were fasted in paral-
lel with the SHROB prior to blood collection and sacrifice.
Oral glucose tolerance test
After 60 d of treatment, an oral glucose tolerance test
(OGTT) (6 g/kg glucose by oral gavage) was conducted
after an 18-h fast. As previously described,16rats were
given by oral gavage a 50% glucose solution at a dose of
6 g/kg body weight. Blood (0.2 mL) was obtained from
the tail of conscious animals at baseline and 30, 60, 120,
240 and 360 min after the glucose load. Blood glucose was
determined at each time point from 2.0 mL fresh whole
blood from the tail using a clinical meter (One Touch Ultra,
Lifescan, Milpitas, CA, USA). Area under the curve was deter-
mined for each parameter for quantification of the OGTT.
Plasma biochemical measurements
Blood samples were chilled on ice, centrifuged for 20 min at
5000g at 48C, and the plasma frozen at 2708C until assayed
for plasma insulin and glucagon by ELISA, using rat insulin
and glucagon standards and antibodies directed against
rat insulin and glucagon (ALPCO, Salem, NH, USA).
Triglycerides were measured enzymatically using a stan-
dard kit (Sigma, St Louis, MO, USA). Triglycerides in
5 mL plasma were first hydrolyzed by lipoprotein lipase to
liberate glycerol, which was then phosphorylated by gly-
cerol kinase. Glycerol-1-phosphate was then oxidized by
glycerol phosphate oxidase to liberate hydrogen peroxide.
Peroxidase then catalyzed the coupling of O2 with 4-
aminoantipyrine and sodium N-ethyl-N-(3-sulfopropyl)
m-anisidine to produce a quinoneimine dye that shows an
absorbance maximum at 540 nm proportional to the trigly-
ceride content.
Terminal studies
At the end of the experiment, animals were anesthetized
with enflurane and a 5 mL blood sample was obtained by
cardiac puncture. At autopsy, we measured fat pad
weights. These were classified as visceral fat: retroperito-
neal, mesenteric and gonadal (epididymal/myometrial),
andassubcutaneous fat:subscapulardepot.Also
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determined were weights of the following organs: liver,
heart and kidneys.
Liver glycogen determination
The method was adapted from Lo et al.17Samples of frozen
liver (about 0.3 g) were homogenized in 2.5 mL of 30% KOH
solution for one minute using a polytron on its highest
setting (Tekmar Tissuemizer, Mason, OH, USA). The
samples were then boiled at 988C for 1–2 h, after which
the samples were chilled on ice. Five milliliter of 100%
ethanol was then added along with 10 mL of 4 mol/L
lithium chloride as a carrier to each sample in order to
precipitate glycogen. After four hours, samples were spun
at 5000g at 48C (Sorvall RT 600B Centrifuge, Thermo
Scientific, Asheville, NC, USA) for 30 min. The supernatant
was then poured off and the glycogen-enriched pellet was
resuspended in 0.5 mL of 4 N HCl. This was then boiled
again for one hour at 988C to achieve hydrolysis. Samples
were neutralized and re-centrifuged as before. The super-
natant was analyzed for glucose by the glucose oxidase
(Trinder, Sigma Chemical) method adapted for 96-well
plates. Standards of 40, 20, 10, 5, 2.5, 1.25 and 0 mmol/L
glucose were used in parallel with the samples (20 mL)
and 200 mL of color reagent. The plates were incubated for
10 min at room temperature and read at 505 nm in a plate
spectrophotometer (Tecan Rainbow, Durham, NC, USA).
Data analysis
Results are presented as mean+standard error of the mean.
Comparisons between groups were made using one- or
two-way analysis of variance (ANOVA). Changes over
time during the treatment interval were compared by
ANOVA with repeated measures (REMANOVA). We used
Prism 5.0 (Graph Pad Software, San Diego, CA, USA)
with post hoc analyses by Neuman–Keuls test. Standard
curves were analyzed by fitting a logistic equation in
Prism 5.0. Glycogen was calculated as (glucose)/(liver
weight ? 100) ¼ glycogen (in mg/g liver).
Results
Body weight increased in all three groups of SHROB, reach-
ing about 500 g by the end of the experiment, more than
double the body weight of lean SHR littermates (Table 1).
Neither drug treatment had any effect on body weight or
on food or water intake (data not shown). Consistent with
the absence of any effect on body weight, total depot fat
recovered at autopsy was also not changed (Table 1).
Interestingly, the ratio of visceral to subcutaneous fat mass
was significantly shifted by sitagliptin treatment in favor
of subcutaneous relative to visceral fat. Fat distribution
Table 1Characteristics of SHROB treatment groups and untreated SHRs at study termination
Group SHROB vehicle (N 5 14) SHROB sitagliptin (N 5 11)SHROB glyburide (N 5 9)SHR‡untreated (N 5 8)
Final body weight (g)
Total dissectible fat (g)
Visceral fat (g)
Subcutaneous fat (g)
V/S ratio
Heart weight (g)
Liver weight (g)
Kidney weight (g)
507+27
63+6
28+3
32+4
0.89+0.09
1.29+0.04
17.6+1.0
2.27+0.12
499+21
65+6
25+3
33+4
0.77+0.04?
1.29+0.05
17.3+1.8
2.48+0.13
495+11
59+5
32+3
29+2
1.13+0.15
1.36+0.09†
16.9+1.2
2.38+0.18
244+6
9.0+1.3
9.0+1.3
,1
.10
1.09+0.05
8.3+0.5
1.63+0.06
SHR, spontaneously hypertensive rat; SHROB, spontaneously hypertensive rat – obese strain; V/S, visceral to subcutaneous fat mass
Data are mean+SEM
?Significantly different from glyburide-treated SHROB, P , 0.05 by Newman–Keuls
†Significantly different from vehicle-treated SHROB, P, 0.05 by Newman–Keuls
‡Each of the values for lean SHR is significantly different from all of the SHROB groups
Figure 1
treatment with vehicle (N ¼ 14), sitagliptin (N ¼ 6) and glyburide (N ¼ 5).
Values for an age-matched sample of lean SHR (N ¼ 8) are shown at day
zero for comparison. All values exceed the lean SHR group except for
final valuesfor thesitagliptin
SHR, spontaneously hypertensive rat
Progression of fasting plasma glucagon levels over time during
group(P , 0.05,Newman–Keuls).
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was shifted favorably by sitagliptin relative to both the
vehicle- and glyburide-treated groups.
Heart weights were increased by glyburide treatment
(Table 1). This apparent cardiac hypertrophy remained sig-
nificant after correction for body weight.
Fasting plasma glucose levels were within the normal
range and remained relatively constant during treatment
and did not differ between groups (week 4 values; vehicle
81+2 mg/dL, sitagliptin 87+4 mg/dL and glyburide
88+6 mg/dL). Fasting insulin levels in all groups markedly
elevated relative to lean SHRs.
Fasting glucagon levels were within the previously
reported range, and were approximately two-fold elevated
relative to lean SHRs of the same age (Figure 1). By five
weeks of treatment, but not before, sitagliptin reduced glu-
cagon levels to within the range of lean SHRs. Because
glucose values determined in the same plasma samples
were unchanged, changes in glucose cannot account for
the difference in glucagon.
SHROB showed marked glucose intolerance relative to lean
SHRs, confirming previous results. Sitagliptin and glyburide
both reduced postprandial glucose levels. Glucose levels did
not differ between drug-treated groups at any time point
(Bonferronitestsfollowing
However, the overall area under the curve was reduced to a
greater extent by sitagliptin than by glyburide (vehicle
35,713 mg min/dL, sitagliptin 13,655 mg min/dL and glybur-
ide 22,788 mg min/dL).
In response to an oral glucose load, vehicle-treated
SHROB showed a delayed and exaggerated rise in plasma
insulin (Figure 2). Both sitagliptin and glyburide normalize
the insulin response to a glucose challenge by shifting the
peak of secretion earlier. Insulin peaks at 30–60 min after
two-way REMANOVA).
Figure 2
over time in minutes following the glucose load is shown. Where error bars are not visible, the error is smaller than the size of the symbol. Sitagliptin and glyburide
are equally effective in lowering the glucose response to oral challenge. Sitagliptin but not glyburide restores the first phase of insulin secretion in the oral tol-
erance test. SHR, spontaneously hypertensive rat
Oral glucose tolerance test results for glucose (a) and insulin (b). Fasting values are shown at time zero, and then the progression of glucose and insulin
Figure 3
glyburide in comparison to untreated SHRs. SHR, spontaneously hyperten-
sive rat; SHROB, spontaneously hypertensive rat – obese strain
Liver glycogen content in SHROB treated with vehicle, sitagliptin or
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glucose ingestion, whereas in controls insulin secretion con-
tinues to rise through 240 min. Only sitagliptin significantly
enhanced insulin secretion at time points up to 120 min. The
overall area under the curve was slightly reduced by sita-
gliptinrelative tovehicle
10,316 ng min/mL) but markedly reduced by glyburide
(2557 ng min/mL).
Consistent with previous findings, plasma triglycerides
were significantly higher in SHROB vehicle controls
(186+20 mg/dL) than in lean SHRs matched for age and
sex (79+21 mg/dL). Treatment with sitagliptin had no
effect on triglycerides (205+26 mg/dL). Glyburide likewise
had no effect (217+29 mg/dL).
As expected, fasted liver samples from lean SHRs showed
very low levels of glycogen (0.36+0.14 mg/g). Vehicle-
treated SHROB showed much higher levels of glycogen
(16.2+1.4 mg/g), suggesting that mobilization of glycogen
in the fasting state is impaired. Neither drug treatment
affected liver glycogen levels (Figure 3).
control(7569versus
Discussion
We have shown that the DPP-IV inhibitor sitagliptin has sig-
nificant effects on glucose metabolism in a rat model of
metabolic syndrome and prediabetes. Sitagliptin is at least
as effective as the sulfonylurea compound glyburide in nor-
malizing glucose tolerance following an oral glucose load.
Sitagliptin also restored the first phase of insulin secretion
in response to oral glucose more effectively than glyburide.
Fasting glucagon levels, which are elevated in the SHROB
model, were normalized after five weeks of treatment. The
reason for the delayed fall in glucagon is not known. The
target hormone GLP-1 is known to have trophic effects as
well as immediate effects on the endocrine pancreas,18and
slowly developing effects might contribute to the effects of
sustained treatment. Maximum effectiveness in humans is
known to require several weeks of treatment.
Previous studies of sitagliptin in rodent models have
shown reduced glycemia in insulinopenic models such as
streptozotocin-treated rats or mice.19,20In contrast, the
present study used a severely hyperinsulinemic model.
Sitagliptin was efficacious, despite preserved and even elev-
ated pancreatic insulin secretion. This study is the first to
demonstrate that chronic sitagliptin can lower fasting gluca-
gon levels, an effect that is predicted based on the glucagon
lowering actions of endogenous GLP-1 and GIP.21In human
type 2 diabetes, excess hepatic production of glucose during
fasting is a major component of fasting hyperglycemia, a
process under the control of glucagon.22Reducing levels
of glucagon in the postprandial period could conceivably
delay the onset of diabetes in prediabetics.
Sitagliptin can acutely reduce postprandial levels of
plasma triglyceride.23In the present study, we found no
effect of chronic sitagliptin on fasting triglycerides.
In a rat model of amyloidal degeneration of the pancreas,
sitagliptin induced tissue damage to the exocrine pancreas
even while protecting the endocrine pancreas.24In the
present study, careful examination of the pancreas at
autopsy revealed no visible pathology in any of the SHROB
groups. Moreover, all rats tolerated the drug treatments well
and showed no change in rate of weight gain or food intake.
While insulinopenic diabetes prevents the activation of
glycogen synthesis by insulin, insulin-resistant type 2 dia-
betic patients showed elevated levels of glycogen after
fasting.25
Glycogen levels are markedly increased in
SHROB livers, presumably reflecting the influence of pro-
found fasting hyperinsulinemia. The persistence of liver gly-
cogen after a fast is all the more remarkable when
considering the elevated levels of glucagon present.
Neither sitagliptin nor glyburide lowered the fasting levels
of insulin, so it is perhaps not surprising that they failed
to normalize elevated liver glycogen in the SHROB model.
Unexpectedly, glyburide was found to be associated with
increased cardiac mass. In at least one report, type 2 diabetic
patients treated with glyburide show greater left ventricular
mass than those treated with other agents.26This may reflect
the influence of cardiac ATP-sensitive potassium channels.
We conclude that sitagliptin is effective in lowering
fasting glucagon, improving glucose tolerance, restoring
early phase insulin secretion and favorably affecting fat dis-
tribution in a prediabetic model of metabolic syndrome. If
these drug actions extend to human prediabetics, then sita-
gliptin might delay the onset of diabetes.
Author contributions: BC prepared the first draft of the
manuscript and carried out the liver glycogen analysis and
assisted with the other studies. AM, LVSE, MSK and DH
all worked with the animals and carried out all the assays.
RJK edited the manuscript, contributed to the initial
concept, and worked with the animals including all of the
glucose tolerance tests. PE designed the study, worked with
the animals and carried out assays, analyzed the data, pre-
pared the graphs, and prepared the final manuscript.
ACKNOWLEDGEMENTS
This study was supported by an unrestricted grant from
Merck to PE.
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