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. 2016
Received: 22 October 2015
Accepted: 17 February 2016
Published: 1 March 2016
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 cell-specific glucokinase haploinsufficient mice fed a diet containing a combination of sucrose and linoleic acid (SL).
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.
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.
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.
Accumulating evidence suggests that diabetic patients have a decreased life span, compared with non-diabetic subjects [1, 2]. 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 [3, 4]. 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 cell-specific glucokinase haploinsufficient (βGck+/−) diabetic mice fed a diet containing a combination of sucrose and oleic acid (SO) or sucrose and linoleic acid (SL) [7, 8]. 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 [7, 8]. 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 pre-clinical studies have also suggested the possibility that GLP-1 receptor agonists and DPP-4 inhibitors exhibit pleiotropic metabolic actions, such as cardioprotection [10, 11]. 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 inhibition . 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 [14, 15]. Hence, the effect of DPP-4 inhibition on longevity has remained controversial.
Obese diabetic db/db mice are morbidly obese and exhibit severe insulin resistance, hyperglycemia, and diabetic complications [16, 17]. High-fat-diet-loading 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 without obesity.
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 anesthetics.
The compositions of the SO and SL diets are described in Additional file 1: Table S1 [7, 8]. 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-fluoro-sitagliptin (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 . More 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
Sucrose- and linoleic acid-diet-induced early mortality in db/db mice and reduction in lethality by DPP-4 inhibition
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
DPP-4 inhibitor protected against β cell failure in db/db mice fed an SL or SO diet
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 [7, 25]. As 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
In SL-fed βGck+/− mice, DPP-4 inhibition averts adipose tissue inflammation by reducing the infiltration of CD11c+ M1 macrophages and CD8+ T cells . To 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 SL-fed 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
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 [16, 17]. 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 SL-induced 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 [7, 8]. 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 [27, 28]. 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 . Therefore, 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 [19, 31]. 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.
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 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 [34, 35]. 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 [7, 8]. 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 [38, 39]. In 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 . We 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 inter-organ 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 anti-apoptotic, anti-inflammatory, anti-oxidant, pro-angiogenic and pro-proliferative effects in ZDF rats [42, 43]. Sitagliptin 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 anti-inflammatory and anti-apoptotic properties [44–46]. These 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 [1, 47]. 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 [13–15, 19]. 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 [49–51]. 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.
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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