DPP-4 inhibition improves early mortality, β cell function, and adipose tissue inflammation in db/db mice fed a diet containing sucrose and linoleic acid
Shirakawa et al. Diabetol Metab Syndr
DPP-4 inhibition improves early mortality, β cell function, and adipose tissue inflammation in db/db mice fed a diet containing sucrose and linoleic acid
Jun Shirakawa 0 3
Tomoko Okuyama 0 3
Mayu Kyohara 0 3
Eiko Yoshida 0 3
Yu Togashi 0 3
Kazuki Tajima 0 3
Shunsuke Yamazaki 0 3
Mitsuyo Kaji 0 3
Megumi Koganei 2
Hajime Sasaki 1 2
Yasuo Terauchi 0 3
0 Department of Endocrinology and Metabolism, Graduate School of Medicine, Yokohama-City University , 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004 , Japan
1 Department of Nutritional and Life Sciences, Kanagawa Institute of Technology , Atsugi , Japan
2 Food Science Research Laboratories, R&D Division, Meiji Co., Ltd. , Odawara , Japan
3 Department of Endocrinology and Metabolism, Graduate School of Medicine, Yokohama-City University , 3-9 Fukuura, Kanazawa-ku, Yoko- hama 236-0004 , Japan
Background: Diabetes therapy that not only lowers glucose levels but also lengthens life spans is required. We previously demonstrated that DPP-4 inhibition ameliorated β cell apoptosis and adipose tissue inflammation in β cellspecific glucokinase haploinsufficient mice fed a diet containing a combination of sucrose and linoleic acid (SL). Methods: In this study, we investigated the effects of DPP-4 inhibition in obese diabetic db/db mice fed an SL diet or a control diet containing sucrose and oleic acid (SO). We also examined the effects of DPP-4 inhibition in IRS-1-deficient mice fed an SL or SO diet as a model of insulin resistance. Results: DPP-4 inhibition efficiently increases the active GLP-1 levels in db/db mice. Unexpectedly, the SL diet, but not the SO diet, markedly increases mortality in the db/db mice. DPP-4 inhibition reduces the early lethality in SL-fed db/db mice. DPP-4 inhibition improves glucose tolerance, β cell function, and adipose tissue inflammation in db/db mice fed either diet. No significant changes in glycemic control or β cell mass were observed in any of the IRS-1-deficient mouse groups. Conclusions: A diet containing a combination of sucrose and linoleic acid causes early lethality in obese diabetic db/ db mice, but not in lean and insulin resistant IRS-1 knockout mice. DPP-4 inhibition has protective effects against the diet-induced lethality in db/db mice.
Type 2 diabetes; DPP-4 inhibitor; Life span; Insulin resistance; Pancreatic β cell; Adipose tissue
Accumulating evidence suggests that diabetic patients
have a decreased life span, compared with non-diabetic
]. Therefore, promoting longevity is one of
the definitive goals for the cure and care of diabetes. The
activation of insulin signaling is a major approach for
blood glucose-lowering therapy, while the loss of insulin
receptor-mediated signaling has resulted in extensions
of the life spans in several models [
]. Dietary sugar
and fat intake influences not only metabolism in various
metabolic tissues, but also aging [
]. However, the effects
of dietary fatty acids or diabetes therapy on life span in
diabetic patients remain obscure.
Palmitic acid, oleic acid, and linoleic acid are the three
major fatty acids among plasma lipids [
]. We previously
investigated diet-induced metabolic changes in β
cellspecific glucokinase haploinsufficient (βGck+/−) diabetic
mice fed a diet containing a combination of sucrose and
oleic acid (SO) or sucrose and linoleic acid (SL) [
induced β cell apoptosis and adipose tissue
inflammation in βGck+/− mice. βGck+/− mice exhibit impaired
insulin secretion in response to glucose but have normal
insulin sensitivity. In Zucker fatty (fa/fa) rat, a model of
obesity and insulin resistance, SL diet showed β cell
failure, enhanced macrophage infiltration in adipose
tissue, and an elevated plasma tumor necrosis factor-α
concentration compared with SO diet . These results
inspired us to investigate the impacts of SL and SO diets
on metabolic tissues in obese diabetes model mice. We
also reported that treatment with a dipeptidyl
peptidase-4 (DPP-4) inhibitor ameliorated SL diet-induced
β cell apoptosis and adipose tissue inflammation in
βGck+/− mice [
]. DPP-4 inhibitors induced increases
in active incretins (GLP-1 and GIP) and other circulating
peptides by slowing enzymatic cleavage, thereby
enhancing incretin-induced glycemic control. A number of
preclinical studies have also suggested the possibility that
GLP-1 receptor agonists and DPP-4 inhibitors exhibit
pleiotropic metabolic actions, such as cardioprotection
]. A meta-analysis of randomized clinical trials
suggested that DPP-4 inhibitors reduced cardiovascular
events and all-cause mortality in patients with type 2
diabetes with a mean follow-up period of 44.1 weeks [
In an aged, high-fat-diet-induced obesity mouse model,
the survival rates were improved by chronic DPP4
]. However, the EXAMINE and SAVOR-TIMI
53 phase III/IV trials showed no significant differences
in cardiovascular events between DPP-4 inhibitors and
a placebo in type 2 diabetic patients [
]. Hence, the
effect of DPP-4 inhibition on longevity has remained
Obese diabetic db/db mice are morbidly obese and
exhibit severe insulin resistance, hyperglycemia, and
diabetic complications [
reportedly shortens the life span of db/db mice .
In the present study, we examined the effects of DPP-4
inhibitors on glucose tolerance, β cell loss, and adipose
tissue inflammation in obese diabetic db/db mice fed an
SL or SO diet. Although both of these diets contained
similar ratios of fat and saturated fatty acids, the SL diet
increased the mortality rate of db/db mice. Interestingly,
DPP-4 inhibition enabled a nearly complete
restoration of the mortality rate in SL-fed db/db mice. We also
assessed the effects of DPP-4 inhibition in IRS-1 deficient
mice fed an SL or SO diet as a model of insulin resistance
Animals and animal care
Animal study was carried out in strict accordance with
the recommendations in the Guide for the Care and Use
of Laboratory Animals of the Yokohama City
University. The protocol was approved by the Yokohama City
University Institutional Animal Care and Use
Committee (IACUC) (Permit Number: 11-29, F-A-13-043). All
experiments were performed under appropriate
anesthesia, and all efforts were made to minimize suffering.
Littermate db/db and db/+ mice aged 7 weeks were
purchased from Charles River Japan (Yokohama, Japan).
We backcrossed IRS-1−/− mice with C57Bl/6J mice more
than 10 times [
]. The mice were fed a standard-chow
diet (MF, Oriental Yeast, Japan) until 8 weeks of age and
then were given free access to the experimental diets. All
the experiments were conducted on male littermates.
Animal housing rooms were maintained at a constant
room temperature (25 °C) and a 12-h light (7:00 a.m.)/
dark (7:00 p.m.) cycle. In the survival study, mice were
monitored two times a day for clinical signs as described
in the Yokohama City University Institutional Animal
Care and Use Committee (IACUC) policy to categorize
animals as morbid or moribund. Mice were sacrificed
when distress was apparent as defined by our IACUC
approved animal protocol (hunched posture, reduced
activity, altered respiratory pattern such as abdominal
breathing, severe weight loss, or inability to stand).
Animals judged to be moribund were euthanatized with an
anesthesia (mixture of medetomidine hydrochloride,
midazolam, and butorphanol tartrate) and counted
as lethality. At the end of these experimental
procedures, the remaining mice were also euthanized with an
The compositions of the SO and SL diets are described
in Additional file 1: Table S1 [
]. The fat component of
the SO and SL diets was derived from safflower oil and
high-oleic sunflower oil blended with perilla oil,
respectively. The two diets were identical except for the type of
fat used: oleic acid was used in the SO diet, and linoleic
acid was used in the SL diet. Both diets contained similar
amounts of palmitic acids. The experimental diets were
freshly prepared weekly. A DPP-4 inhibitor
des-fluorositagliptin (DFS) was administered orally by premixing
with SO or SL to a concentration of 0.4 % . Another
DPP-4 inhibitor, MK-0626, was administered orally by
premixing with SO or SL to a concentration of 0.0045 %
]. Both the DFS and the MK-0626 used in this study
were provided by Merck & Co., Inc.
The blood glucose levels were determined using a
Glutest Neo Super, Glutest Mint (Sanwa Chemical Co.,
Japan) or Glucose Assay Kit (BioVision, CA). Serum
insulin levels and the triglyceride content in the liver
were determined using an insulin kit (Morinaga, Japan)
and the Determiner-L TG II kit (Wako Pure Chemical
Industries, Japan), respectively. The levels of plasma
alanine aminotransferase, free fatty acid, total cholesterol,
and triglyceride were assayed using enzymatic methods
(Wako Pure Chemical Industries). Serum DPP-4
activity was measured using a DPP4 Activity Assay Kit (Bio
Vision, CA). Active GLP-1 was assayed using a
Glucagon-Like Peptide-1 (Active) ELISA Kit (Millipore, MA).
An insulin tolerance test (ITT) was performed by
intraperitoneally injecting mice with human insulin (1.5 mU/g
body weight). An oral glucose tolerance test (OGTT)
was performed by withholding all food from the mice for
more than 18 h and then orally loading the mice with
glucose (1.5 mg/g body weight).
Formalin-fixed, paraffin-embedded pancreas or
adipose tissue sections were immunostained with
antibodies to insulin (Santa Cruz, sc-9168), glucagon (Abcam,
ab10988), or F4/80 (Serotec, MCA497). Biotinylated
secondary antibodies, a VECTASTAIN elite ABC kit, and a
DAB substrate kit (VECTOR) were used to examine the
sections using bright-field microscopy, and Alexa Fluor
488- and 555-conjugated secondary antibodies
(Invitrogen) were used for fluorescence microscopy. All the
images were acquired using a BZ-9000 microscope
(Keyence) or a Carl Zeiss LSM 510 confocal laser-scanning
microscope. The percent area of the pancreatic tissue
occupied by the β cells was calculated using BIOREVO
software (Keyence), as described previously [
than five tissue sections from each animal, including
representative sections of each tissue region, were analyzed.
Tissue specimens were preserved in RNAlater reagent
(QIAGEN) until the isolation of the total RNA. Total
RNA was isolated from the epididymal fat using an
RNeasy Lipid tissue kit (QIAGEN). cDNA was prepared
using the TaqMan reverse transcriptase kit (Applied
Biosystems) and was subjected to quantitative PCR
using TaqMan Gene Expression Assays (7900 real-time
PCR system; Applied Biosystems) with
THUNDERBIRD qPCR Master Mix (TOYOBO). Transcription of
each gene was detected using TaqMan Gene Expression
Assays (Thermo Fisher Scientific Inc.): F4/80 (Adgre1,
Mm00802529_m1), CD11c (Itgax, Mm00498698_
m1), TNF-α (Tnf, Mm00443258_m1), MCP-1 (Ccl2,
Mm00441242_m1), and PAI-1 (Serpine2, Mm00436753_
m1). The data was normalized according to the β-actin
(Actb, Mm02619580_g1). Each quantitative reaction was
performed in duplicate.
All the data are expressed as the mean ± S.E. and were
analyzed using an ANOVA. Differences were considered
significant if the P value was <0.05 (*, †).
A single oral dose of DPP‑4 inhibitors sufficiently suppressed DPP‑4 activity in db/db mice
To assess the effects of DPP-4 inhibitor in db/db mice
fed an SL or SO diet (Additional file 1: Table S1), we
performed an oral meal tolerance test (12 mg/g body weight)
in 8-week-old db/+ or db/db mice. The DPP-4 inhibitors
des-fluoro-sitagliptin (DFS) and MK-0626 were
separately premixed with SO or SL at a concentration of 0.4
or 0.0045 %, respectively. DPP-4 is thought to be an
adipokine that is released from adipose tissue at a higher
level in obese individuals [
]. However, the DPP-4
activities were similar between the db/+ mice and the db/db
mice fed an SO or SL diet (Fig. 1a). DFS and MK-0626
similarly inhibited the serum DPP-4 activity by
approximately 80 % in db/db mice fed an SL or SO diet (Fig. 1a).
We next measured the serum active GLP-1 concentration
after oral loading with an SO or SL meal (12 mg/g body
weight) in the presence or absence of a DPP-4 inhibitor in
standard-chow diet-fed db/+ or db/db mice. The results
showed no significant differences in serum active GLP-1
concentrations between the SO-fed and the SL-fed db/+
db/db SL + DFS
db/db SL + MK-0626
db/db SO + DFS
db/db SO + MK-0626
db/db SL + DFS
db/db SL + MK-0626
db/db SO + DFS
db/db SO + MK-0626
0 min 30 min 120 min
Fig. 1 Changes in serum DPP-4 activity and active GLP-1
concentrations in db/+ mice and db/db mice during an oral meal tolerance
test. The experiments were performed in db/+ or db/db mice fed
an SL diet, an SO diet, or a diet containing the DPP-4 inhibitor 0.4 %
des-fluoro-sitagliptin or 0.0045 % MK-0626. a Serum DPP-4 activity
was measured in mice fed the indicated diets ad libitum (n = 5).
*P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO. b Serum active GLP-1
concentration at 0 min (fasted > 20 h), 30, and 120 min after the oral
administration of each diet test meal (12 mg/g body weight) in db/+
mice and db/db mice that had been fed either the SO or SL diet
(n = 3–4). To obtain a sufficient amount of whole blood to measure
the biologically active form of GLP-1, blood was collected from the
inferior vena cava with a DPP-4 inhibitor (Millipore) at the time points
indicated. *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO
mice or db/db mice at 0, 30, or 120 min after feeding
(Fig. 1b). The serum active GLP-1 concentrations were
significantly increased by DPP-4 inhibition with DFS or
MK-0626 in db/db mice fed an SO or SL diet (Fig. 1b).
Thus, DFS and MK-0626 efficiently inhibited the DPP-4
activity and increased the active GLP-1 levels in db/db
mice. We previously reported that DFS improved β cell
ER stress, adipose tissue inflammation, and hepatic
steatosis in lean diabetic βGck+/− mice [
]. Hence, we used
DFS as the DPP-4 inhibitor for the db/db mouse model in
Sucrose‑ and linoleic acid‑diet‑induced early mortality in db/db mice and reduction in lethality by DPP‑4 inhibition
Db/+ mice and db/db mice fed an SL diet or an isocaloric
SO diet for 8 weeks were evaluated for glucose tolerance
and phenotypic changes in metabolic tissues (Fig. 2). To
evaluate the effect of a DPP-4 inhibitor as a treatment
for diet-induced metabolic dysfunction in obese diabetic
mice with severe insulin resistance, we also performed
an 8-week study comparing db/db mice fed a diet
consisting of SL or SO plus DPP-4 inhibitor (Fig. 2).
Unexpectedly, early lethality at 1–2 months after the start of
the experiments was observed in the SL-fed db/db mice,
but not in the SO-fed mice or db/db mice (Fig. 3). Over
70 % of the db/db mice died after 5–8 weeks of
SL-loading. In contrast, db/db mice fed an SL + DPP-4 inhibitor
diet exhibited more than 80 % survival at the end of the
experiment period (Fig. 3). We performed three
independent 8-week meal loading tests and combined these
results (Additional file 2: Figure S1). No apparent signs of
injury or infection were observed in the dead SL-fed db/
db mice. We also confirmed that none of the db/db mice
fed a standard chow diet died before 16 weeks of age.
Histology ITT, GTT
(14 wk) 16 week old
No significant changes in insulin sensitivity and glucose tolerance between SL‑fed db/db mice and SO‑fed db/db mice and improvement induced by DPP‑4 inhibitor
Compared with the db/+ mice, the db/db mice showed an
increased body weight, liver weight, epididymal fat weight,
blood glucose level, insulin resistance, and glucose
intolerance after feeding with either the SL or SO diet (Fig. 4a–
d; Additional file 3: Figure S2). No significant differences
in body weight, liver weight, blood glucose level, insulin
sensitivity, glucose tolerance, or insulin secretion after
glucose loading were observed between the SO group and
the SL group for either genotype (Fig. 4a–e; Additional
file 3: Figure S2). Treatment with the DPP-4 inhibitor had
no significant effect on body weight gain, liver weight, or
epididymal fat weight in db/db mice fed an SL or SO diet
(Fig. 4a; Additional file 3: Figure S2). The blood glucose
levels were decreased by the addition of the DPP-4
inhibitor to the diets until the second week of the experiment
in both groups (Fig. 4b). After the manifestation of severe
hyperglycemia in the db/db mice fed an SL or SO diet,
treatment with the DPP-4 inhibitor was no longer
capable of reducing the blood glucose levels (Fig. 4b).
However, DPP-4 inhibition significantly decreased the blood
glucose levels at 90 or 120 min in an insulin tolerance test
and at 30 min in an oral glucose tolerance test in db/db
mice after SL or SO feeding for 8 weeks (Fig. 4c, d). The
blood glucose levels at 60, 90, 120 min during glucose
tolerance test showed values over the limit of detection of
the glucometers in db/db mice fed an SL or SO diet, but
not in DPP-4 inhibitor-treated db/db mice. Furthermore,
the fasting serum insulin levels were significantly elevated
in DPP-4 inhibitor-treated db/db mice in both diet groups
(Fig. 4e). These results indicated that the DPP-4 inhibitor
provided a slight but significant improvement in insulin
sensitivity and glucose tolerance, even in obese
hyperglycemic db/db mice fed an SL or SO diet.
DPP‑4 inhibitor protected against β cell failure in db/db mice fed an SL or SO diet
SL significantly reduced the β cell mass and the β cell
proportion in islet cells through a greater increase in
apoptosis, compared with that induced by SO, in lean
diabetic βGck+/− but not wild-type mice [
diabetic db/db mice fed a normal chow diet showed a
transient increase in β cell mass and proliferation from 1 to
3 months of age, followed by a subsequent decrease with
further aging [
]. To analyze the effects of the SL and
SO diets on β cell loss in db/db mice, we investigated the
β cell mass and the β cell proportion in islet cells in
SLor SO-diet-fed 14-week-old db/db mice (Fig. 2). No
significant changes in β cell mass or β cell proportion were
observed between the SL and SO groups in both db/+
and db/db mice (Fig. 5a–d). An abnormal distribution of
db/db SO SO + DPP-4 inhibitor diet
Fig. 2 Experimental protocol. Both the db/+ and db/db mice were
fed a standard chow diet until 8 weeks of age and were then given
free access to the experimental diets. Experiments were performed
on db/+ and db/db mice after 8 weeks on the SL diet, SO diet,
SL + DPP-4 inhibitor (0.4 % des-fluoro-sitagliptin) diet, or SO + DPP-4
inhibitor (0.4 % des-fluoro-sitagliptin) diet
0 min 30 min 0 min 30 min
Fig. 4 DPP-4 inhibitor improved insulin sensitivity and glucose tolerance in SL- or SO-fed db/db mice. The experiments were performed in
16-week-old mice, as shown in Fig. 2 (n = 6–11). a Body weight gain. b Blood glucose levels. *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO. c Blood
glucose levels during insulin tolerance test (ITT). *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO. d Blood glucose levels during oral glucose tolerance
test (OGTT). *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO. e Serum insulin levels during OGTT. *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO
β cells in the islets was observed in db/db mice fed an SL
or SO diet, compared with db/+ mice (Fig. 5a, c). The
β cell mass and islet morphology in SL- or SO-fed db/
db mice were similar to those observed in normal
chowfed db/db mice of the same age (data not shown). These
results suggested that the influence of the difference in
the SL and SO diets was less than that of the intrinsic
mechanism responsible for β cell loss in db/db mice.
We previously demonstrated that DPP-4 inhibition
ameliorated β cell ER stress and apoptosis in SL-fed
βGck+/− mice, and the GLP-1 receptor agonist
liraglutide protected against reductions in β cells in neonatal
βGck−/− mice independent of insulin secretion [
expected, the treatment of SL- and SO-fed-db/db mice
with a DPP-4 inhibitor for 8 weeks produced a
significant increase in β cell mass (Fig. 5b) and the relative β cell
mass as a proportion of the total α-cell-plus-β-cell mass
(Fig. 5d); furthermore, the abnormal distribution of
pancreatic α cells was also corrected (Fig. 5c). The intensity
of the fluorescence signals of insulin was also augmented
by DPP-4 inhibition in db/db mice (Fig. 5c). Thus, DPP-4
inhibition ameliorated β cell failure in db/db mice
regardless of whether the mice were fed an SL or SO diet.
Inflamed adipose tissue was mitigated by DPP‑4 inhibition in db/db mice fed an SL or SO diet
Inflammation induced by the infiltration of macrophages
or other hemocytes into adipose tissue contributes to
obesity-related insulin resistance in db/db mice [
Compared with SO, SL increased CD11c+ M1 macrophage
infiltration into visceral adipose tissue in βGck+/− mice
]. Therefore, we analyzed the visceral adipose tissues of
Fig. 5 DPP-4 inhibitor increased β cell mass and ameliorated islet morphology in SL- or SO-fed db/db mice. The experiments were performed
in 14-week-old mice, as shown in Fig. 2. a Representative pancreatic sections stained with antibodies for insulin (brown) are shown. The scale bar
represents 100 μm. b β cell mass (n = 6–8). The β cell area is shown as a proportion of the area of the entire pancreas. c Representative pancreatic
sections stained with antibodies for insulin (green) and glucagon (red) are shown. The scale bar represents 50 μm. d Quantification of β cell mass as a
proportion of the total α-cell-plus-β-cell mass in the islet (n = 6). *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO
SL- or SO-fed db/db mice. The adipocyte area in db/db
mice fed an SL or SO diet was larger than that in SL or SO
diet-fed db/+ mice (Fig. 6a, b). An immunohistochemical
analysis revealed that the proportion of F4/80+ crown-like
structures (CLSs) in adipocytes was also increased in db/
db mice compared with db/+ mice, in both the SL and SO
groups (Fig. 6c, d). The mRNA expression levels of F4/80,
CD11c, TNF-α, MCP-1, and PAI-1 were significantly
Fig. 6 Inflamed adipose tissue was improved by DPP-4 inhibition in db/db mice fed an SL or SO diet. The experiments were performed in
14-weekold mice, as shown in Fig. 2. a Representative histogram of adipocyte size in epididymal fat. b Average size of adipocyte from indicated mice (n = 5).
c Epididymal fat tissue was stained with anti-F4/80 antibody. d The number of F4/80+ crown-like structures (CLSs) was counted as described in the
Methods (n = 5). *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db SO. e, f Assessment of the levels of expression of the indicated mRNAs in epididymal fat
as determined using real-time quantitative RT-PCR and normalization to the β-actin mRNA level (n = 5). *P < 0.05 vs. db/db SL. †P < 0.05 vs. db/db
higher among the db/db mice fed an SL or SO diet
(Fig. 6e, f ). No significant changes in the adipocyte area,
the number of F4/80+ CLSs, inflammatory gene
expressions, or serum lipid parameters were observed between
the SL and SO groups in the db/db or db/+ mice (Fig. 6a–
f; Additional file 4: Figure S3). These results indicated that
adipose tissue inflammation was not caused by the
difference in the compositions of the diets that were used
to feed the mice. In fact, we also confirmed that db/db
mice of the same age that were fed a standard diet showed
F4/80+ CLSs and inflammatory gene expressions in
adipocytes to a similar degree as that observed in db/db mice
fed an SL or SO diet (data not shown).
In SL-fed βGck+/− mice, DPP-4 inhibition averts
adipose tissue inflammation by reducing the infiltration of
CD11c+ M1 macrophages and CD8+ T cells [
evaluate the use of a DPP-4 inhibitor as a treatment for adipose
tissue inflammation in obese diabetic db/db mice with SL
or SO, we examined visceral adipose tissue inflammation
in DPP-4 inhibitor-treated db/db mice. The epididymal fat
weight and lipid parameters in the SL- or SO-fed db/db
mice were not affected by the addition of a DPP-4 inhibitor
to the diet (Additional files 3 and 4: Figures S2 and S3). The
adipocyte size tended to be decreased by the treatment
with DPP-4 inhibitor in these mice (Fig. 6a, b). DPP-4
inhibitor attenuated the proportion of F4/80+ CLSs and
macrophage-related inflammatory gene expressions in the
adipocytes of db/db mice in both diet groups (Fig. 6c–f ).
We previously demonstrated that treatment with a
DPP-4 inhibitor improved hepatic steatosis in both
SLfed and SO-fed βGck+/− mice [
]. However, no
significant differences in the hepatic triglyceride contents were
observed in SL- or SO-fed db/db mice in the presence or
absence of a DPP-4 inhibitor (Additional file 5: Figure S4).
No significant effects of an SL or SO diet and DPP‑4 inhibition in insulin‑resistant IRS‑1‑deficient mice
In db/+ or db/db mice, differential effects of an SL or SO
diet on glucose metabolism were not observed (Figs. 4, 5, 6)
]. An SL diet aggravated β cell function and adipose tissue
inflammation in lean βGck+/− mice with impaired insulin
secretion in response to glucose and normal insulin
resistance, compared with an SO diet [
]. An SL diet, but not
an SO diet, shortened the life spans of obese diabetic db/
db mice with severe insulin resistance (Fig. 3). To assess the
impact of an SL or SO diet under an insulin-resistant state,
we used an SL or SO diet with or without a DPP-4
inhibitor to feed wild-type (WT) mice and insulin receptor
substrate (IRS)-1-deficient (IRS-1−/−) mice for 27 weeks. The
IRS-1−/− mice showed normal glucose tolerance,
peripheral insulin resistance (especially in skeletal muscle),
compensatory β cell mass hyperplasia, and growth retardation
. The body weight and blood glucose levels were not
affected by the dietary composition or DPP-4 inhibition in
IRS-1−/− mice (Fig. 7a, b). An SL or SO diet containing a
DPP-4 inhibitor had no significant effects on insulin
sensitivity, glucose tolerance, or insulin secretion in response to
glucose gavage (Fig. 7c–e). No significant changes in β cell
mass or β cell proportion were observed between the SL
and SO groups with or without a DPP-4 inhibitor in
IRS1−/− mice (Fig. 7f–h). Accordingly, the aggravating effects
of an SL diet might depend on hyperglycemia or β cell
dysfunction, but not insulin resistance, in mice.
Here, we established a model in which a diet rich in
sucrose and linoleic acid induced early mortality in obese
diabetic db/db mice. Db/db mice are a well-known model
of obesity, diabetes, insulin resistance, β cell failure,
adipose tissue inflammation, diabetic complications
including heart failure, and so on [
]. The extension of the
life span is the primary goal of medical therapy for any
disease. Therefore, our model should be a good model
for studying the diet-induced exacerbation of
life-threatening disease. Short-lived SL-fed db/db mice, however,
demonstrated glucose intolerance, insulin resistance, β
cell failure, and adipose tissue inflammation to degrees
similar to those of SO-fed db/db mice. Hence, the
SLinduced shortening of the life span in db/db mice was
thought to be caused by unidentified mechanisms.
Our previous study demonstrated that an SL diet
induced β cell apoptosis and adipose tissue inflammation in
βGck+/− mice [
]. βGck+/− mice manifested
post-prandial hyperglycemia caused by impaired glucose-induced
insulin secretion and failed to proliferate β cells in response
to insulin resistance by high-fat diet loading [
Glucokinase in β cells also plays a crucial role in the
protection of β cells against ER stress-induced apoptosis through
IRS-2 dependent and independent pathways [
]. In db/
db mice, glucokinase activation by a glucokinase activator
improved the glycemic profiles [
glucokinase-mediated signals might be intact in db/db mice, and
the function of β cell glucokinase, but not insulin
resistance, could contribute to SL-induced β cell failure and
adipose tissue inflammation. Insulin resistant IRS-1−/− mice
demonstrated insulin secretory defects, hyperplastic islets,
hyperinsulinemia, and normoglycemia [
]. The results
of IRS-1−/− mice fed an SL or SO in this study showed no
significant changes in the metabolic phenotypes or β cell
mass, indicating that insulin resistance was not the cause of
β cell failure. Since db/db mice become obese and
uncontrollable hyperglycemia due to the spontaneous mutation of
leptin receptor (LepR/ObR), the β cell failure and inflamed
adipose tissue might be derived from the dysfunction of
LepR/ObR-signaling, and not from dietary components in
db/db mice fed an SL or SO diet.
IRS-1-/- SL + DPP-4i
IRS-1-/- SO + DPP-4i
IRS-1-/- SL + DPP-4i
IRS-1-/- SO + DPP-4i
Then, what is the cause of the early mortality in SL-fed
db/db mice? Due to strict ethical regulations involving
research animal studies, we are required to euthanize
animals when they become sick or injured. The most common
cause of death was therefore euthanization due to unknown
disorders. The severity of myocardial ischemia and
reperfusion injury, the development of congestive heart failure,
and the mortality after myocardial ischemia were markedly
IRS-1-/- SL + DPP-4i
IRS-1-/- SO + DPP-4i
exacerbated in db/db diabetic mice [
]. In diabetic db/db
mice, the monocyte/endothelial interaction was
accelerated by an increase in the production of 12/15 lipoxygenase
]. Linoleic acid is converted to arachidonic acid in many
animal tissues, and a linoleic acid-rich SL-diet has been
reported to increase the tissue content of arachidonic acid
], which is a precursor of prostaglandins, HETEs, and
leukotrienes. Of note, cardiac necrosis was reportedly induced
by the excessive loading of linoleic acid in STZ-induced
hyperglycemic rats [
]. However, a systematic review
and meta-analysis of prospective cohort studies indicated
that the linoleic acid intake is inversely associated with the
cardiovascular heart disease risk in a dose–response
manner . We also preliminarily evaluated the heart histology
of SL- or SO-fed db/db mice in this study, but no apparent
differences in ischemia or fibrotic lesions were observed
(Additional file 6: Figure S5). The deficiency of LepR/ObR
signaling in SL-fed db/db mice might participate in the early
mortality, because insulin resistant IRS-1 knockout mice
fed an SO or SL diet showed no abnormality in survival rate
compared with those fed normal chow. Accordingly, further
research is needed to clarify the mechanisms of SL-induced
death in db/db mice.
The results of this study also showed that DPP-4 inhibition
protected against an SL-diet-induced shortened life span in
db/db mice. DPP-4 inhibition also ameliorated glycemic
control, β cell loss, and adipose tissue inflammation in db/
db mice fed either an SL or SO diet. We previously reported
that DPP-4 inhibition improved β cell loss and adipose
tissue inflammation without inducing any changes in fatty acid
contents in the tissues of SL-fed diabetic mice [
]. So, the
protective effect of DPP-4 inhibition on life span in db/db
mice was independent of alterations in fatty acid contents in
tissues. However, although a preliminary analyses suggested
no differences in food intake among all groups, the
differences in fatty acid intake or absorption caused by diets or
DPP-4 inhibition might exist and function as contributing
factors. Evidence has suggested that postprandial
hyperglycemia is an independent risk factor for all causes of death
and cardiovascular disease . The suppression of glucose
spikes after glucose loading induced by DPP-4 inhibition
in db/db mice might be one explanation of its protective
effects on longevity. The improvement of cardiac function
after myocardial infarction through a combination of DPP-4
inhibition and G-CSF administration has been reported,
and liraglutide was able to confer cardioprotection and a
survival advantage after myocardial infarction [
addition to incretins, increased SDF-1α or BNP by DPP-4
inhibition might contribute to the protection or the
regeneration of cardiomyocytes in this study . Interestingly,
DPP-4 itself is reportedly an obesity-related adipokine that
might worsen insulin resistance [
]. In this study, however,
both SL- and SO-fed db/db mice showed similar DPP-4
activities and circulating active GLP-1 levels. Furthermore,
the efficiency of DPP-4 inhibition as measured by the DPP-4
activity and serum active GLP-1 levels showed no
significant differences between an SL diet and an SO diet in db/
db mice. Collectively, these results suggest that an
insufficiency of DPP-4 activity was not responsible for the
shortened life span of db/db mice fed an SL diet and increase of
SDF-1α, BNP, or other DPP-4 target factors by DPP-4
inhibition might be involved in longevity of those mice.
Treatment with the GLP-1 receptor agonist liraglutide improved
the decrease in the β cell mass and fatty liver independent of
insulin secretion in β cell-specific glucokinase homozygous
knockout mice but failed to prolong survival, and all the
mice died within 1 week [
]. The incretin-induced
increment in β cell mass and the prolonged longevity of db/db
mice might not be related. Recently, a report suggested that
adipose tissue inflammation increases hepatic acetyl CoA
and causes hepatic insulin resistance through
inflammatory cytokine production in high-fat-diet-fed rats [
demonstrated that DPP-4 inhibition reduced adipose
tissue inflammation and inflammatory cytokine expression,
even in db/db mice. Since the adipose tissue is the largest
endocrine organ in the body, an investigation of the
interorgan network involving the adipose tissue before and after
DPP-4 inhibition could be useful for determining the effects
of DPP-4 inhibition on life span in our model.
The dose of des-fluoro-sitagliptin and MK-0626, DPP-4
inhibitors used in this study, may be higher than a body
weight-based dose of DPP-4 inhibitors in the clinical
practice. There might be pharmacodynamic differences in the
absorption and persistent effects of a DPP-4 inhibitor in this
study because DPP-4 inhibitors are orally administrated,
not mixed in diet, in the clinical practice and the
absorbance of lipophilic drugs are elevated by bile especially in the
postprandial period. Thus, consideration should be given to
the possibility that the time- and dose-dependent effects of
DPP-4 inhibitor contributed to the outcome.
In addition to db/db mice, Zucker diabetic fatty fa/fa (ZDF)
rats, harboring a missense mutation (fatty, fa) in the leptin
receptor gene (Lepr/ObR), are also widely used as a model
of obese type 2 diabetes. In previous reports, the DPP-4
inhibitor, sitagliptin, prevented β cell dysfunction with
antiapoptotic, anti-inflammatory, anti-oxidant, pro-angiogenic
and pro-proliferative effects in ZDF rats [
also corrected the hyperglycemia, hyperlipidemia,
inflammation, and hypertension in ZDF rats . Furthermore, the
treatment of ZDF rats with sitagliptin also ameliorated
nitrosative stress, inflammation and apoptosis in retinal cells, and
prevented diabetic nephropathy progression through
antiinflammatory and anti-apoptotic properties [
protective actions of sitagliptin have important implications
for a better understanding how DPP-4 inhibition prevented
the early mortality in an obese diabetes model.
Numerous studies have demonstrated that diabetes and
obesity are related with decreased longevity in human
]. Prolonging the life span is the eventual aim of the
treatment of diabetes and obesity. Our diet-induced db/db
mice are a good model for the study of survival and
longevity in obese and diabetic subjects. The effects of DPP-4
inhibition on life span have been obscure [
The effect of DPP4 inhibition on weight is neutral, while
most other hypoglycemic agents increase weight gain [
Some reports have suggested that DPP-4 inhibition has
cardioprotective effects in humans, possibly by
enhancing left ventricular functions or myocardial regeneration
]. DPP-4 inhibition improves age-related diseases,
such as hypertension, dyslipidemia, hepatic steatosis, and
neurodegenerative diseases. DPP-4 inhibition may have a
protective effect on cardiovascular diseases, which are a
major cause of mortality in patients with diabetes. Because
the impact of DPP-4 inhibition on cardiovascular events
in diabetic patients likely depends on each patient’s
background (i.e., disease duration, age, BMI, current therapy,
glycemic control, or complications), further examination
of the hypothesis presented in this study in clinical studies
focusing on DPP-4 inhibition in humans is required.
We created a model of nutrient-induced early lethality in
an obese diabetic db/db mouse with a diet containing a
combination of sucrose and linoleic acid. We also showed
that DPP-4 inhibition ameliorated survival in those mice.
The results of the current study demonstrate the novel
therapeutic potential of DPP-4 inhibitors for the
extension of lifespan in diabetes patients.
Additional file 1: Table S1. Compositions of experimental diets.
Additional file 2: Figure S1. SL-diet-induced early mortality in db/db
mice and DPP-4 inhibition reduced lethality. Survival rates of indicated
mice in three independent cohort studies. (a) n = 12, (b) n = 8, (c) n =
7. The db/+ and db/db mice were fed the SL diet, SO diet, SL + DPP-4
inhibitor diet, or SO + DPP-4 inhibitor diet, as described in Fig. 2.
Additional file 3: Figure S2. Liver and epididymal fat weights in db/+
mice and db/db mice. The experiments were performed in db/+ or
db/db mice fed an SL diet, SO diet, SL containing DPP-4 inhibitor (0.4%
des-fluoro-sitagliptin) diet, or SO containing DPP-4 inhibitor diet for 8
weeks. (left) Liver weights as a proportion of body weight (n = 5). (right)
Epididymal fat weights as a proportion of body weight (n = 5).
Additional file 4: Figure S3. Biochemical parameters in db/+ mice and
db/db mice. Plasma alanine aminotransferase (ALT), free fatty acid (FFA),
total cholesterol (TChol), and triglyceride (TG) in the indicated groups of
mice (n = 5).
Additional file 5: Figure S4. Liver triglyceride levels in db/+ mice
and db/db mice. Concentrations of liver TG (mg/g liver) in the indicated
groups of mice (n = 5).
Additional file 6: Figure S5. Cardiac muscle morphology in db/db
mice. HE staining (upper) and Masson-Goldner staining (lower) of cardiac
muscle in the indicated of mice.
JS and Y Te contributed to the design and conception of the study. JS, TO, MK,
KT, EY, SY, Y To and M Ka performed the experiments and contributed to the
discussion. JS, OT, M Ka and Y Te analyzed the data. M Ko and HS contributed
to the design of experimental diets and provided them. JS and Y Te wrote the
manuscript. All authors gave final approval of the version to be published. All
authors read and approved the final manuscript.
The authors thank Dr. Takashi Kadowaki and Dr. Naoto Kubota (University
of Tokyo, Tokyo, Japan) for kindly gifting the IRS-1−/− mice. We thank Misa
Katayama (Yokohama City University) for secretarial assistance. The authors
thank Merck & Co., Inc. (Rahway, NJ) for providing the des-fluoro-sitagliptin
and MK-0626. This work was supported in part by Grants-in-Aid for Scientific
Research (B) 24390235 from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) of Japan (to Y Te). JS is supported by Fellowship for
Research Abroad of the Japan Society for the Promotion of Science (JSPS).
The authors declare that they have no competing interests.
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