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Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model
Psychoneuroendocrinology 73 (2016) 186–195
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Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model Maimaiti Yisireyili a , Kyosuke Takeshita (MD, PhD, FAHA) (A/Prof.) a,b,∗ , Motoharu Hayashi a , Hongxian Wu a , Yasuhiro Uchida a , Koji Yamamoto c , Ryosuke Kikuchi b , Chang-Ning Hao a , Takayuki Nakayama e , Xian Wu Cheng a , Tadashi Matsushita b,c , Shigeo Nakamura d , Toyoaki Murohara a a
Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan c Department of Blood Transfusion, Nagoya University Hospital, Nagoya, Japan d Department of Pathology, Nagoya University Hospital, Nagoya, Japan e Department of Blood Transfusion, Aichi Medical University Hospital, Nagakute, Japan b
a r t i c l e
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Article history: Received 14 April 2016 Received in revised form 4 July 2016 Accepted 2 August 2016 Keywords: Restraint stress Adipose tissue inflammation DPP-4 Inhibitor Reactive oxygen species Insulin resistance
a b s t r a c t Background: Stress evokes lipolytic release of free fatty acid (FFA) and low-grade inflammation in visceral adipose tissue, mediated by increased adipokine secretion, and contributes to glucose metabolism disorder and prothrombotic state. We tested the hypothesis that alogliptin, a dipeptidyl peptidase-4 inhibitor, can ameliorate the biological effects of chronic stress in mice. Method and results: C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle or alogliptin (dose: 15 or 45 mg/kg/day). Plasma levels of lipids, proinflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-␣, and interleukin-6), and 8-hydroxydeoxyguanosine were measured with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was examined by CD11b-positive cell count and mRNA expression of CD68 and F4/80 was examined by immunohistochemistry and RTPCR, respectively. The mRNA levels of the above-mentioned proinflammatory cytokines, NADPH oxidase 4, adiponectin, and coagulation factors (plasminogen activation inhibitor-1 and tissue factor) in WAT were also assessed with RT-PCR. Glucose metabolism was assessed by glucose and insulin tolerance tests, plasma levels of DPP-4 activity, glucagon-like peptide-1, expression of DPP-4, insulin receptor substrate-1 and glucose transporter 4 in WAT and skeletal muscle. Alogliptin administration suppressed stressinduced FFA release, oxidative stress, adipose tissue inflammation, DPP-4 activation, and prothrombotic state in a dose-dependent manner, and improved insulin sensitivity in stressed mice. Conclusions: The results indicate that alogliptin improves stress-induced prothrombotic state and insulin resistance; suggesting that alogliptin could have beneficial therapeutic effects against cardiovascular complications in diabetic patients under stress. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: 8-OHdG, 8-hydroxy-deoxyguanosine; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; MetS, metabolic syndrome; NPYN, neuropeptide Y; PAI-1, plasminogen activator inhibitor-1; RAS, renin-angiotensin system; ROS, reactive oxygen species; SNS, sympathetic nervous system; TF, tissue factor; TNF-␣, tumor necrosis factor-␣; VAT, visceral adipose tissue. ∗ Corresponding author at: Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Nagoya, Aichi 466-8550, Japan. E-mail address: [email protected] (K. Takeshita). http://dx.doi.org/10.1016/j.psyneuen.2016.08.004 0306-4530/© 2016 Elsevier Ltd. All rights reserved.
Epidemiological studies have demonstrated that chronic psychological stress in modern lifestyle is closely linked to the incidence of metabolic syndrome (MetS), diabetes mellitus, and thromboembolism (Chandola et al., 2006). This type of stress can activate various stress pathways, including the hypothalamicpituitary-adrenal axis and sympathetic nervous system (SNS), as well as elicit physiological responses, resulting in stress-related disorders.
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We identified recently visceral adipose tissue (VAT) as a target of psychological stress and the development of various disorders including MetS, and demonstrated that two-week intermittent restraint stress in a murine model evoked chronic inflammation in the adipose tissue followed by lipolysis in VAT with free fatty acid (FFA) release and toll-like 4 receptor stimulation (Uchida et al., 2012). Indeed, stress-induced low-grade inflammation of the adipose tissue is associated with the production of various inflammatory adipokines, including tumor necrosis factor-␣ (TNF␣), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), as well as accumulation of monocytes, giving rise to impaired insulin sensitivity and prothrombotic state with increases in tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) (Uchida et al., 2012) as well as perpetuation of the pathophysiological process of MetS (Tamura et al., 2008). Based on the
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above findings [2], we concluded that the stress-induced adipose inflammation can be a therapeutic target, and showed indeed that irbesaltan, an angiotensin receptor blocker, suppressed MCP1 production, reduced adipose tissue inflammation, and improved insulin sensitivity and prothrombotic state (Hayashi et al., 2014). There is a growing evidence to suggest that chronic psychological stress promotes the production of reactive oxygen species (ROS) throughout the body. Chronic psychological stress can induce oxidative stress in various body tissues, including the brain and peripheral blood cells, and these effects can be partially reversed by anxiolytic agents (Miller and Sadeh, 2014). As ROS accumulate in the tissue and dysfunctional fat instigates metabolic dysfunctions (Murdolo et al., 2013), it is assumed that ROS production in VAT is involved in stress-induced adipose inflammation.
Fig. 1. Alogliptin suppresses stress-induced monocyte accumulation in inguinal adipose tissue. Inguinal adipose tissues from stressed and control (non-stressed) mice were analyzed by H&E staining (a), CD11b immunostaining (b and c), and quantitative RT-PCR for CD68 and F4/80 (D and E). a: Accumulation of mononuclear cells in inguinal adipose tissues following the 2-week restraint stress. Top panel, ×40 magnification, bar = 250 m. Inset, ×200 magnification, bar=50 m. b: Accumulation of CD11b-positive cells (monocytes) in adipose tissue of stressed mice (×200 magnification, bar=50 m). c: Quantitative analysis of CD11b-positive cells relative to total nuclear number. Data are mean ± SD of 7 mice per group. * P<0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d and e: Quantitative analysis of F4/80 (d) and CD68 (e) expression levels in adipose tissue. Values are expressed relative to the vehicle-treated control mice. Data are mean ±SD of 7 mice per group. * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice (both by Student’s t-test).
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The dipeptidyl peptidase-4 (DPP-4) enzyme, which is highly expressed in VAT and immunocytes, cleaves various substrates, such as incretins and cytokines (Rohrborn et al., 2015). DPP-4 is considered an adipokine because its upregulation is involved in lipolysis and chronic low-grade inflammation in patients with Mets and type 2 diabetes (Rohrborn et al., 2015; Lamers et al., 2011). DPP-4 inhibitors exert their hypoglycemic effects by preventing the breakdown of short-lived endogenous incretins, such as glucagonlike peptide-1 (GLP-1), resulting in increased insulin levels and inhibition of glucagon release (Rohrborn et al., 2015). Furthermore, DPP-4 inhibitors have anti-inflammatory properties that are independent of the blood glucose-lowering effect. Alogliptin, a potent and highly selective DPP-4 inhibitor, is reported to suppress the expression of inflammatory chemokines, reduce vascular lesion in atherogenic low-density lipoprotein receptor-deficient mice (Akita et al., 2015), and suppress ROS production in a murine abdominal aortic aneurysmal model (Bao et al., 2014). The present study was designed to determine the role of chronic stress on MetS, DPP-4 activity, ROS production, and inflammatory changes in VAT. We also evaluated the outcome of treatment with alogliptin in a murine model of chronic stress, with special emphasis on the suppression of stress-induced adipose inflammation and improvement in insulin resistance and thrombotic state. 2. Materials and methods For a complete description of the materials and methods, see the Supplementary material.
2.3. Quantitative PCR Total RNA extraction, reverse-transcription, and quantitative PCR were performed as described previously (Takeshita et al., 2002). The sequences of the primers used in this study are listed in Supplementary Table. The amount of each RNA was normalized to that of -actin mRNA. 2.4. Histological analysis Inguinal adipose (VAT) tissue samples were harvested and subjected to immunohistochemistry using antibodies for CD11b (1:100; Abcam plc, Cambridge, UK) and 8-hydroxydeoxyguanosine (8-OHdG) (1 g/ml; Japan Institute for the Control of Aging, Fukuroi, Japan) and the standard protocols described previously (Hayashi et al., 2014; Uchida et al., 2012). Two investigators blindly and independently counted the number of CD11b- and 8OHdG-positive and −negative cells under a microscope at ×200 magnification. Ten microscopic fields were chosen in three different sections per mouse for examination. 2.5. Biochemical assays Plasma levels of cytokines and hormones were quantified using proper enzyme-linked immunosorbent assay kits for 8-OHdG, MCP-1, TNF-␣, IL-6, insulin, GLP-1 and DPP-4 activity, as described previously (Takeshita et al., 2007; Uchida et al., 2012; Hayashi et al., 2014). The kits used in this study are listed in the Supplemental Material.
2.1. Animals 2.6. Intraperitoneal glucose and insulin tolerance tests Eight-week-old male C57BL/6J mice (Chubu Kagaku Shizai Co, Nagoya, Japan) were housed two per cage under standard conditions (23 ± 1 ◦ C, 50 ± 5% humidity), with a 12-h light/dark cycle (lights on at 7:30 a.m.) in a viral pathogen-free facility in the Division for Research of Laboratory Animals, Nagoya University Graduate School of Medicine (Uchida et al., 2012; Yamamoto et al., 2002). The study protocol was approved by the Institutional Animal Care and Use Committee of Nagoya University (Protocol Number 27009), and the study was performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
After two weeks of daily stress, mice were subjected to an intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) using standard methods (Uchida et al., 2012). Briefly, for GTT, mice were fasted overnight and then challenged with D-glucose at 2 g/kg body weight (Sigma-Aldrich, St. Louis, MO), followed by serial assessment of blood glucose up to 120 min using a blood glucose level monitor (Glutest Ace, Sanwa Kagaku Kenkyusho Co, Nagoya, Japan). For ITT, mice were fasted for 16 h before testing. Insulin (0.75 U/kg, Actrapid Penfill, NovoNordisk, Copenhagen, Denmark) was injected intraperitoneally, and blood glucose was measured.
2.2. Restraint stress protocol 2.7. Statistical analysis Mice were randomly assigned to the control (n = 20) or the stress group (n = 30). Control mice were left undisturbed and allowed contact with each other, while stressed mice were isolated in individual cages and subjected to 2 h/day (between 10:00 a.m. and 12:00 p.m., 6 days/week) of immobilization stress over a period of two weeks as described in detail previously (Uchida et al., 2012; Yamamoto et al., 2002; Takeshita et al., 2004). Within each group, mice were randomly assigned to be orally fed either a drug-free CE-2 diet (vehicle) or a CE-2 diet containing alogliptin (15 or 45 mg/kg/day, a generous gift from Takeda Pharmaceutical Company), 10 mice per group, for 2 weeks. The control animals were given either the vehicle or high dose of alogliptin. The stressed animals were given either the vehicle, low, or high dose of alogliptin. Body weight and food intake were monitored during the 2-week period. Animals were anesthetized (intraperitoneal sodium pentobarbital, 150 mg/kg) and euthanized in the morning next to the last restraint stress. After euthanasia, biological samples were collected for total RNA extraction, analysis of plasma lipid profile, expression levels of biological markers, and pathological examination (Aoyama et al., 2009).
Data are expressed as mean ± SD. Differences between groups were assessed by Student’s t-test or one-way ANOVA followed by Fisher’s test, and considered significant at P < 0.05. 3. Results 3.1. Alogliptin prevents stress-induced adipose tissue inflammation As reported previously (Uchida et al., 2012), significant increase in mononuclear cell infiltration was noted in white adipose tissue (WAT) from the 8-week-old stressed and vehicle-treated mice compared to the non-stressed mice (Fig. 1a). Analysis of expression of macrophage surface markers demonstrated that the stress procedure significantly increased CD11b-positive cells and mRNA expression levels of monocyte/macrophage cell surface markers (F4/80 and CD68) in WAT (Fig. 1b–d). Alogliptin markedly reduced monocyte accumulation and mRNA expression levels of monocyte surface markers in WAT of stressed mice in a dose-dependent
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Fig. 2. Alogliptin restores stress-induced decrease in weight gain and reduces adipose tissue weight. a: Body weight gain and food intake in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.03 compared with the vehicle-treated and stressed mice, † P < 0.01, compared with the vehicle-treated and stressed mice (by Student’s t-test). b: Plasma fat and fatty acid composition in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. ** P < 0.01, compared with the vehicle-treated control mice, ** P < 0.01, compared with the vehicle-treated and stressed mice (both by Student’s t-test). c: Inguinal adipose tissue weight in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. *P < 0.002, compared with the vehicle-treated control mice, ** P < 0.04, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d: Images of inguinal fat pad. The dot line marks adipose tissue.
manner. The high dose of alogliptin did not alter monocyte accumulation in control mice.
stress (Fig. 3d). Alogliptin reduced 8-OHdG expression in WAT, plasma 8-OHdG concentrations, and adipose Nox-4 expression in a dose-dependent manner (Fig. 3).
3.2. Alogliptin reduces stress-induced lipolysis and FFA release As described previously (Uchida et al., 2012), the 2-week-stress period significantly reduced body weight gain in vehicle-treated mice (Fig. 2a), and this effect was abrogated by alogliptin in a dose-dependent manner (Fig. 2a). The high dose alogliptin did not alter body weight gain of non-stressed mice after the two-week treatment (Fig. 2a). Food consumption was almost similar in the different groups (Fig. 2a). Stress and alogliptin treatment did not change total cholesterol or triglyceride levels (Fig. 2b), but stress increased FFA concentration while alogliptin significantly reduced these concentrations in a dose-dependent manner (Fig. 2b). Stress also significantly reduced inguinal WAT weight compared to non-stress, and this decrease recovered after alogliptin treatment (Fig. 2c). Indeed, alogliptin restored stress-induced shrinkage of inguinal fat pad (Fig. 2d). These results suggest that alogliptin treatment reduces stressinduced lipolysis and FFA release. 3.3. Alogliptin suppresses stress-induced free radical production Stress increased the expression of 8-OHdG in adipose tissues (Fig. 3a), including adipocytes, monocytes. Stress increased 8OHdG-positive cells in WAT by two-fold, compared with the control (Fig. 3b). Stress also increased plasma 8-OHdG concentrations by two-fold, relative to the control (Fig. 3c). NADPH oxidase 4 (Nox4) mRNA expression was also upregulated in WAT after restraint
3.4. Alogliptin reduces inflammatory adipokine levels in stressed mice The 2-week restraint stress significantly upregulated MCP-1, TNF-␣, and IL-6 mRNA expression levels in adipose tissues, and these changes were suppressed in a dose-dependent manner by alogliptin (Fig. 4). Alogliptin also lowered the stress-induce rise in MCP-1, TNF-␣, and IL-6 plasma levels. The stress-induced decrease in adiponectin mRNA expression was abrogated by alogliptin (Fig. 4d). However, no changes in the expression levels of these adipokines were noted in adipose tissue of control mice treated with vehicle or high-dose alogliptin. 3.5. Alogliptin suppresses stress-induced DPP-4 activation In the vehicle-treated stressed mice, DPP-4 mRNA expression in WAT was more than double that of the vehicle-treated control mice (Fig. 5a). Plasma DPP-4 activity was also significantly increased in the vehicle-treated stressed mice (Fig. 5b). In parallel with these changes, plasma GLP-1 levels were significantly reduced in stressed mice (Fig. 5c). Alogliptin reduced DPP-4 activity and increased plasma GLP-1 levels in non-stressed animals (Fig. 5b and c). Alogliptin significantly reduced both WAT DPP-4 mRNA level and plasma DPP-4 activity in stressed animals in a dose-dependent manner (Fig. 5a, b). Furthermore, alogliptin also restored plasma
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Fig. 3. Alogliptin suppresses stress-induced free radical production. To examine free radical generation in inguinal white adipose tissue, and plasma from stressed and control (non-stressed) mice treated with or without 15 or 45 mg/kg/day alogliptin, were subjected to immunohistochemistry and ELISA for 8-OHdG, and quantitative RT-PCR for NOX-4. Data are mean ± SD. n = 6–7 mice per group. a. Increased 8-OHdG expression in inguinal adipose tissues (a) from stressed mice ( × 200 magnification, bar = 50 m). 8-OHdG was expressed in monocytes and adipocytes in WAT of stressed mice. Alogliptin suppressed the expression. b: Quantitative analysis of 8-OHdG-positive cells relative to total nuclear number in inguinal adipose tissues. Data are mean ± SD of 7 mice per group. *P < 0.001, compared with the vehicle-treated control mice, **P < 0.002, compared with the vehicle-treated and stressed mice (both by Student’s t-test). c: Quantitative analysis of plasma 8-OHdG levels. Data are mean ± SD of 7 mice per group. *P < 0.01, compared with vehicle-treated control mice, **P < 0.01, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d. Quantitative analysis of NOX-4 expression level in adipose tissue. Values are expressed relative to the vehicle-treated control mice Data are mean ± SD of 7 mice per group. *P < 0.002, compared with the vehicle-treated control mice, **P < 0.002, compared with the vehicle-treated and stressed mice (both by Student’s t-test).
GLP-1 levels in a similar manner (Fig. 5c). Plasma insulin levels were measured 30 min after intraperitoneal administration of 2 g/kg body weight D-glucose in overnight fasting mice. Alogliptin increased plasma insulin levels both in non-stressed and stressed mice (Fig. 5d).
3.6. Alogliptin rescues stress-induced decline in insulin sensitivity and prothrombotic state Alogliptin significantly improved glucose and insulin tolerance after stress (Fig. 6a). Furthermore, it restored the mRNA expression levels of insulin receptor substrate-1 (IRS-1) and glucose transporter-4 (GLUT-4) in inguinal WAT (Fig. 6b), but not in skeletal muscle (adductor muscles). Stress significantly increased the expression levels of PAI-1 and TF in WAT, and plasma PAI-1 levels (Fig. 6c), while alogliptin abrogated these changes, compared with vehicle, similar to the changes in monocyte accumulation and induction of proinflammatory cytokines (Fig. 6c). Considered together, the above findings indicate that alogliptin suppresses stress-induced lipolysis and adipose inflammation to improve insulin resistance and prothrombotic state.
4. Discussion The main finding of this study was that alogliptin, a DPP-4 inhibitor, suppressed stress-induced adipose tissue inflammation and improved insulin resistance and thrombotic state in a dosedependent manner. In the present study, two-week intermittent restraint stress resulted in low-grade inflammation and induction of PAI-1 and TF in WAT, in agreement with previous findings (Uchida et al., 2012). We have previously shown that single acute restraint stress transiently increased TNF-␣ expression and mononuclear cells in WAT within 2 h, and that the repetitive stress within 1 week hardly induced inflammatory changes in WAT (Uchida et al., 2012; Yamamoto et al., 2002). Therefore, we recognized these finding on adipose tissue inflammation as a consequence of chronic stress. Stress also increased ROS production and DPP-4 expression in WAT, and plasma DPP-4 activity. Alogliptin administered at 15 or 45 mg/kg/day markedly suppressed stress-induced WAT inflammation and ROS production, resulting in reduction of TF and PAI-1. Furthermore, alogliptin suppressed stress-induced DPP-4 activation, improved glucose tolerance with an increase in plasma GLP-1,
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Fig. 4. Alogliptin reduces expression of stress-induced proinflammatory adipokines and restores adiponectin expression in adipose tissue. Inguinal white adipose tissues from control mice treated with vehicle or 45 mg/kg/day alogliptin, and stressed mice treated with vehicle or 15 or 45 mg/kg/day alogliptin were analyzed by quantitative RT-PCR for MCP-1 (a), TNF-␣ (b), IL-6 (c), and adiponectin (d). Values are expressed relative to vehicle-treated control mice. Plasma levels of MCP-1, TNF-␣, and IL-6 from these groups were also measured (a-d, respectively). Data are mean ± SD of 6 mice per group. (a) * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice, † P < 0.02 compared with vehicle-treated mice, †† P < 0.004 compared with stressed mice (all by Student’s t-test). (b) * P < 0.001, compared with vehicle-treated control mice, ** P < 0.001, compared with vehicle-treated and stressed mice, † P < 0.03 compared with vehicle-treated mice, †† P < 0.005, compared with stressed mice (all by Student’s t-test). (c) * P < 0.001, compared with vehicle-treated control mice, ** P < 0.002, compared with vehicle-treated and stressed mice, † P < 0.01, compared with vehicle-treated control mice, †† P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (d) * P < 0.001, compared with vehicle-treated control mice, † P < 0.002 compared with vehicle-treated mice, †† P < 0.001, compared with stressed mice (all by Student’s t-test).
and increased insulin sensitivity with restoration of adipose IRS-1 and GLUT-4. We demonstrated recently that the same type of stress activated the renin-angiotensin system (RAS) with increased expression of adipose angiotensinogen (Hayashi et al., 2014). Both NADH/NADPH oxidase activation by RAS and inflammation-related ROS formation synergistically increased ROS levels in VAT after stress (Fig. 3). ROS accumulation in adipose tissue exacerbates organ dysfunction, resulting in impaired glucose homeostasis due to insulin resistance (Figs. 5 and 6). We reported previously that adipose-derived PAI-1 and TF, which stabilize thrombi and initiate the coagulation cascade, respectively, participate in the stress-induced thrombus formation in aged mice (Yamamoto et al., 2014; Yamamoto et al., 2002). Furthermore, we showed that proinflammatory adipokines, including MCP-1 and TNF-␣, induce prothrombotic factors in adipose tissues after stress (Uchida et al., 2012). Both ROS generation and inflammatory adipokines in adipose tissue synergistically induced PAI-1 and TF in adipose tissue (Fig. 6) (Takeshita and Murohara, 2014; Yamamoto et al., 2014). Previous studies showed that DPP-4 inhibition suppresses ROS accumulation in kidney in animal models of type 1 and 2 diabetes, and in patients with type 2 diabetes (Avogaro and Fadini, 2014). Alogliptin-induced suppression of ROS generation in murine aortic aneurysmal model involves the GLP-1 signaling pathway (Bao et al., 2014). Our finding
of abrogation of stress-induced ROS accumulation by alogliptin is consistent with the above observations, and could serve to improve stress-induced prothrombotic state. DPP-4 is highly expressed in lymphocytes and monocytes, and is associated with immunoregulatory functions (Iwata et al., 1999; Augustyns et al., 1999). DPP-4 expression in VAT increases according to inflammatory cell accumulation. Indeed mRNA expression of DPP-4 in VAT increased after the stress period, and was decreased by anti-inflammatory effects of DPP-4 inhibitor (Fig. 5a). DPP-4 expression would be induced by inflammatory stimuli. As promoter of DPP-4 contains Stat3 binding site to augment DPP-4 expression (Bauvois et al., 2000), activation of IL-6 signaling, containing Jak-Stat signaling, can increase DPP-4 expression. Inversely, anti-inflammatory effects of DPP-4 inhibitor suppress production of inflammatory cytokines including IL-6 to reduce DPP-4 expression. Thus inflammatory status would alter DPP-4 expression in VAT. DPP-4 plays an important role in the immune and inflammatory responses in dysmetabolic conditions (Avogaro and Fadini, 2014). Since DPP-4 expression is increased in VAT of obese subjects with MetS and promotes MetS-related processes, it is considered as a MetS-related adipokine that can produce insulin signal disruption (Lamers et al., 2011; Rohrborn et al., 2015). We demonstrated previously that low-grade inflammation in VAT is the common mechanism linking stress-induced disor-
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Fig. 5. Alogliptin suppressed stress-induced DPP-4 activation. DPP-4 expression in inguinal white adipose tissue and DPP-4 activity and GLP-1 concentration in plasma were measured in stressed and control (non-stressed) mice without or with 15 or 45 mg/kg/day alogliptin by quantitative RT-PCR for DPP-4 (a), DPP-4 activity assay (b), and ELISA for plasma GLP-1 (c). Samples were collected after overnight fasting. Plasma insulin levels (d) were also measured to investigate the effects of DPP-4 inhibition at 30 min after intraperitoneal injection of 2 g/kg D-glucose followed by overnight fast. Data are mean ± SD of 6 mice per group. Values in (a) and (b) are expressed relative to vehicle-treated control mice. (a) * P < 0.001, compared with the vehicletreated control mice, †P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (b) * P < 0.001, compared with vehicle-treated control mice, †P < 0.005, compared with vehicle-treated control mice. #P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (c) * P < 0.01 and †P < 0.04, compared with vehicle-treated control mice. #P < 0.05, compared with vehicle-treated and stressed mice. (d) * P < 0.001, compared with vehicle-treated control mice. †P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test).
ders and MetS (Uchida et al., 2012). We also reported that DPP-4 is involved in stress-induced dysmetabolic conditions. Inflammatory cytokines, including TNF-␣, induce DPP-4 expression to regulate the immune and inflammatory responses (Rohrborn et al., 2015). Conversely, the anti-inflammatory effects of DPP-4 inhibitors have been demonstrated in inflammatory-related disease models, such as diabetes and cardiovascular disease (Avogaro and Fadini, 2014; Rohrborn et al., 2015). DPP-4 inhibitors lower DPP-4 activity by 70–90% (Avogaro and Fadini, 2014). In the present study, we also showed that alogliptin suppressed DPP-4 activity in parallel with reduction of inflammatory adipokines and monocyte accumulation (Figs. 4 and 5). DPP-4 has been also suggested to be a biomarker for the diagnosis of major depression and other stress-related diseases (Maes et al., 2009). Recently soluble and membrane DPP-4 has been highlighted to catabolize Neuropeptide Y (NPY) in blood circulation (Wagner et al., 2016). NPY and its receptors are widely expressed in cerebral areas such as the cortex, hippocampus, and amygdala to regulate anxiety, mood, cognition and stress resilience (Farzi
et al., 2015). Indeed overexpression of NPY reduced anxiety-like and depression-like behavior (Thorsell et al., 2000). Augmentation of NPY by DPP-4 inhibition or DPP-4 deficiency proved to exert antistress effects in animal models (El Yacoubi et al., 2006; Karl et al., 2003; Lautar et al., 2005). In the present study, pharmacological DPP-4 inhibition would synergically reduce stress-induced effects via central nerve system and anti-inflammatory effects. In stressed subjects, stress-induced cortisol release and adipose SNS activation initiate lipolysis to increase FFA concentrations (Uchida et al., 2012). This stress-induced lipolysis and FFA release can initiate stress-induced adipose inflammation via activation of toll like receptor-4, and subsequent TNF-␣ receptor activation in adipocytes accelerates stress-induced lipolysis to propagate a vicious cycle (Uchida et al., 2012). Vildagliptin, a DPP-4 inhibitor, suppressed diet-induced lipolysis (Ahren et al., 2011). In the present study, alogliptin also reduced monocyte accumulation and TNF-␣ induction, and broke the vicious circle between stressinduced lipolysis and adipose tissue inflammation (Figs. 2 and 3).
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Fig. 6. Alogliptin ameliorates stress-induced insulin resistance and prothrombotic state. (a) Glucose tolerance (top panel) and insulin tolerance (lower panel) showed significant recovery in alogliptin (45 mg/kg/day)-treated and stressed mice. Data are mean ± SD of 6 mice per group. * P < 0.001 compared with vehicle-treated mice, ** P < 0.04, compared with stressed mice (both by Student’s t-test). (b) Quantitative analysis of IRS-1 and GLUT4 expression in inguinal white adipose tissue and skeletal muscle (adductor muscle) of stressed mice treated with vehicle or alogliptin (45 mg/kg/day). Data are mean ± SD of 6 mice per group. * P < 0.05, compared with vehicle-treated and stressed mice (by Student’s t-test). (c) Quantitative analysis of TF and PAI-1 expression in inguinal white adipose tissue, and plasma PAI-1 levels of control and stressed mice treated with vehicle or alogliptin (15 or 45 mg/kg/day). Data are mean ± SD of 6–7 mice per group. *P < 0.001, compared with vehicle-treated and stressed mice, ** P < 0.001, compared with vehicle-treated and stressed mice (by Student’s t-test). †P < 0.002, compared with vehicle-treated and stressed mice, ‡P < 0.03, compared with vehicle-treated and stressed mice (by Student’s t-test).
As mentioned above, DPP-4 inhibitors have antidiabetic properties with anti-inflammatory effects via GLP-1 signaling (Avogaro and Fadini, 2014). In the present study, alogliptin improved both glucose and insulin sensitivity (Fig. 6). Alogliptin-induced restoration of GLP-1 level improved glucose tolerance in stressed mice, and alogliptin normalized insulin sensitivity by modulating IRS-1 and GLUT4 expression levels in WAT through significant suppression of adipose TNF-␣ (Ruan et al., 2002). The anti-inflammatory effect of alogliptin should improve insulin resistance because any decrease in plasma IL-6 is also expected to functionally improve insulin signaling in skeletal muscles at IRS-1 function, which is independent from IRS-1 and GLUT4 expression (Benito, 2011).
A theoretical review in a population cohort study suggests that psychological stress precedes the onset of diabetes (Pouwer et al., 2010). Accumulating evidence suggests the association of disturbed psychophysiological responses with cardiovascular risk factors, including subclinical measures of atherosclerosis, such as endothelial dysfunction, hypertension, and impaired glucose and lipid metabolism (Hamer and Malan, 2010). Diabetic patients under persistent stress are prone to thrombotic disease (Austin et al., 2013). Treatment with alogliptin could improve insulin resistance in at least a subgroup of diabetics under stress and might improve clinical outcome beyond glucose metabolism control. Several limitations exist in the presentation and analysis of the current data. We did not investigate the effect of corticosterone
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on stress-induced adipose tissue inflammation although corticosterone release induces lipolysis and FFA release in the downstream of hypothalamic-pituitary-adrenal axis activation (Arnaldi et al., 2010; Ziemssen and Kern, 2007). We could not identify the effect of basal DPP-4 activity on adipose inflammation because the finding of adipose tissue inflammation was subtle in non-stressed mice. The aim of this current study was to investigate the effect of DPP4 inhibitor on stress-induced adipose inflammation. While these information would be useful in future experiments in exploring the mechanism of stress-induced adipose tissue inflammation further, it is beyond the scope of the current study. In conclusion, we demonstrated that alogliptin inhibited stressinduced ROS generation and adipose inflammation to break the vicious cycle between lipolysis and inflammation. The antiinflammatory effects of alogliptin restored insulin sensitivity and improved the prothrombotic state in stressed mice. Role of the funding sources This work was supported by grants from the Takeda Medical Research Foundation, Suzuken Memorial Foundation, Kondo Memorial Foundation to KT, and a Grant-in-Aid for Scientific Research (Kakenhi 25461336 and 16K15411). MY is a recipient of JSPS doctoral fellowship for overseas researchers (JSPS KAKENHI Grant Number 15J00397). Conflicts of interest The authors declare no duality of interest associated with this work. Acknowledgments We thank Dr. Issa F.G., Word-Medex Pty Ltd, for the careful reading and editing of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.psyneuen.2016. 08.004. References Ahren, B., Schweizer, A., Dejager, S., Villhauer, E.B., Dunning, B.E., Foley, J.E., 2011. Mechanisms of action of the dipeptidyl peptidase-4 inhibitor vildagliptin in humans. Diabetes Obes. Metab. 13, 775–783. Akita, K., Isoda, K., Shimada, K., Daida, H., 2015. Dipeptidyl-peptidase-4 inhibitor, alogliptin, attenuates arterial inflammation and neointimal formation after injury in low-density lipoprotein (LDL) receptor-deficient mice. J. Am. Heart Assoc. 4, e001469. Aoyama, T., Takeshita, K., Kikuchi, R., Yamamoto, K., Cheng, X.W., Liao, J.K., Murohara, T., 2009. Gamma-secretase inhibitor reduces diet-induced atherosclerosis in apolipoprotein E-deficient mice. Biochem. Biophys. Res. Commun. 383, 216–221. Arnaldi, G., Scandali, V.M., Trementino, L., Cardinaletti, M., Appolloni, G., Boscaro, M., 2010. Pathophysiology of dyslipidemia in Cushing’s syndrome. Neuroendocrinology 92 (Suppl (1)), 86–90. Austin, A.W., Wissmann, T., von Kanel, R., 2013. Stress and hemostasis: an update. Semin. Thromb. Hemost. 39, 902–912. Augustyns, K., Bal, G., Thonus, G., Belyaev, A., Zhang, X.M., Bollaert, W., Lambeir, A.M., Durinx, C., Goossens, F., Haemers, A., 1999. The unique properties of dipeptidyl-peptidase IV (DPP IV / CD26) and the therapeutic potential of DPP IV inhibitors. Curr. Med. Chem. 6, 311–327. Avogaro, A., Fadini, G.P., 2014. The effects of dipeptidyl peptidase-4 inhibition on microvascular diabetes complications. Diabetes Care 37, 2884–2894. Bauvois, B., Djavaheri-Mergny, M., Rouillard, D., Dumont, J., Wietzerbin, J., 2000. Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells. Oncogene 19, 265–272. Bao, W., Morimoto, K., Hasegawa, T., Sasaki, N., Yamashita, T., Hirata, K., Okita, Y., Okada, K., 2014. Orally administered dipeptidyl peptidase-4 inhibitor
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Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model Maimaiti Yisireyili a , Kyosuke Takeshita (MD, PhD, FAHA) (A/Prof.) a,b,∗ , Motoharu Hayashi a , Hongxian Wu a , Yasuhiro Uchida a , Koji Yamamoto c , Ryosuke Kikuchi b , Chang-Ning Hao a , Takayuki Nakayama e , Xian Wu Cheng a , Tadashi Matsushita b,c , Shigeo Nakamura d , Toyoaki Murohara a a
Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan c Department of Blood Transfusion, Nagoya University Hospital, Nagoya, Japan d Department of Pathology, Nagoya University Hospital, Nagoya, Japan e Department of Blood Transfusion, Aichi Medical University Hospital, Nagakute, Japan b
a r t i c l e
i n f o
Article history: Received 14 April 2016 Received in revised form 4 July 2016 Accepted 2 August 2016 Keywords: Restraint stress Adipose tissue inflammation DPP-4 Inhibitor Reactive oxygen species Insulin resistance
a b s t r a c t Background: Stress evokes lipolytic release of free fatty acid (FFA) and low-grade inflammation in visceral adipose tissue, mediated by increased adipokine secretion, and contributes to glucose metabolism disorder and prothrombotic state. We tested the hypothesis that alogliptin, a dipeptidyl peptidase-4 inhibitor, can ameliorate the biological effects of chronic stress in mice. Method and results: C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle or alogliptin (dose: 15 or 45 mg/kg/day). Plasma levels of lipids, proinflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-␣, and interleukin-6), and 8-hydroxydeoxyguanosine were measured with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was examined by CD11b-positive cell count and mRNA expression of CD68 and F4/80 was examined by immunohistochemistry and RTPCR, respectively. The mRNA levels of the above-mentioned proinflammatory cytokines, NADPH oxidase 4, adiponectin, and coagulation factors (plasminogen activation inhibitor-1 and tissue factor) in WAT were also assessed with RT-PCR. Glucose metabolism was assessed by glucose and insulin tolerance tests, plasma levels of DPP-4 activity, glucagon-like peptide-1, expression of DPP-4, insulin receptor substrate-1 and glucose transporter 4 in WAT and skeletal muscle. Alogliptin administration suppressed stressinduced FFA release, oxidative stress, adipose tissue inflammation, DPP-4 activation, and prothrombotic state in a dose-dependent manner, and improved insulin sensitivity in stressed mice. Conclusions: The results indicate that alogliptin improves stress-induced prothrombotic state and insulin resistance; suggesting that alogliptin could have beneficial therapeutic effects against cardiovascular complications in diabetic patients under stress. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: 8-OHdG, 8-hydroxy-deoxyguanosine; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; MetS, metabolic syndrome; NPYN, neuropeptide Y; PAI-1, plasminogen activator inhibitor-1; RAS, renin-angiotensin system; ROS, reactive oxygen species; SNS, sympathetic nervous system; TF, tissue factor; TNF-␣, tumor necrosis factor-␣; VAT, visceral adipose tissue. ∗ Corresponding author at: Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Nagoya, Aichi 466-8550, Japan. E-mail address: [email protected] (K. Takeshita). http://dx.doi.org/10.1016/j.psyneuen.2016.08.004 0306-4530/© 2016 Elsevier Ltd. All rights reserved.
Epidemiological studies have demonstrated that chronic psychological stress in modern lifestyle is closely linked to the incidence of metabolic syndrome (MetS), diabetes mellitus, and thromboembolism (Chandola et al., 2006). This type of stress can activate various stress pathways, including the hypothalamicpituitary-adrenal axis and sympathetic nervous system (SNS), as well as elicit physiological responses, resulting in stress-related disorders.
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We identified recently visceral adipose tissue (VAT) as a target of psychological stress and the development of various disorders including MetS, and demonstrated that two-week intermittent restraint stress in a murine model evoked chronic inflammation in the adipose tissue followed by lipolysis in VAT with free fatty acid (FFA) release and toll-like 4 receptor stimulation (Uchida et al., 2012). Indeed, stress-induced low-grade inflammation of the adipose tissue is associated with the production of various inflammatory adipokines, including tumor necrosis factor-␣ (TNF␣), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), as well as accumulation of monocytes, giving rise to impaired insulin sensitivity and prothrombotic state with increases in tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) (Uchida et al., 2012) as well as perpetuation of the pathophysiological process of MetS (Tamura et al., 2008). Based on the
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above findings [2], we concluded that the stress-induced adipose inflammation can be a therapeutic target, and showed indeed that irbesaltan, an angiotensin receptor blocker, suppressed MCP1 production, reduced adipose tissue inflammation, and improved insulin sensitivity and prothrombotic state (Hayashi et al., 2014). There is a growing evidence to suggest that chronic psychological stress promotes the production of reactive oxygen species (ROS) throughout the body. Chronic psychological stress can induce oxidative stress in various body tissues, including the brain and peripheral blood cells, and these effects can be partially reversed by anxiolytic agents (Miller and Sadeh, 2014). As ROS accumulate in the tissue and dysfunctional fat instigates metabolic dysfunctions (Murdolo et al., 2013), it is assumed that ROS production in VAT is involved in stress-induced adipose inflammation.
Fig. 1. Alogliptin suppresses stress-induced monocyte accumulation in inguinal adipose tissue. Inguinal adipose tissues from stressed and control (non-stressed) mice were analyzed by H&E staining (a), CD11b immunostaining (b and c), and quantitative RT-PCR for CD68 and F4/80 (D and E). a: Accumulation of mononuclear cells in inguinal adipose tissues following the 2-week restraint stress. Top panel, ×40 magnification, bar = 250 m. Inset, ×200 magnification, bar=50 m. b: Accumulation of CD11b-positive cells (monocytes) in adipose tissue of stressed mice (×200 magnification, bar=50 m). c: Quantitative analysis of CD11b-positive cells relative to total nuclear number. Data are mean ± SD of 7 mice per group. * P<0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d and e: Quantitative analysis of F4/80 (d) and CD68 (e) expression levels in adipose tissue. Values are expressed relative to the vehicle-treated control mice. Data are mean ±SD of 7 mice per group. * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice (both by Student’s t-test).
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The dipeptidyl peptidase-4 (DPP-4) enzyme, which is highly expressed in VAT and immunocytes, cleaves various substrates, such as incretins and cytokines (Rohrborn et al., 2015). DPP-4 is considered an adipokine because its upregulation is involved in lipolysis and chronic low-grade inflammation in patients with Mets and type 2 diabetes (Rohrborn et al., 2015; Lamers et al., 2011). DPP-4 inhibitors exert their hypoglycemic effects by preventing the breakdown of short-lived endogenous incretins, such as glucagonlike peptide-1 (GLP-1), resulting in increased insulin levels and inhibition of glucagon release (Rohrborn et al., 2015). Furthermore, DPP-4 inhibitors have anti-inflammatory properties that are independent of the blood glucose-lowering effect. Alogliptin, a potent and highly selective DPP-4 inhibitor, is reported to suppress the expression of inflammatory chemokines, reduce vascular lesion in atherogenic low-density lipoprotein receptor-deficient mice (Akita et al., 2015), and suppress ROS production in a murine abdominal aortic aneurysmal model (Bao et al., 2014). The present study was designed to determine the role of chronic stress on MetS, DPP-4 activity, ROS production, and inflammatory changes in VAT. We also evaluated the outcome of treatment with alogliptin in a murine model of chronic stress, with special emphasis on the suppression of stress-induced adipose inflammation and improvement in insulin resistance and thrombotic state. 2. Materials and methods For a complete description of the materials and methods, see the Supplementary material.
2.3. Quantitative PCR Total RNA extraction, reverse-transcription, and quantitative PCR were performed as described previously (Takeshita et al., 2002). The sequences of the primers used in this study are listed in Supplementary Table. The amount of each RNA was normalized to that of -actin mRNA. 2.4. Histological analysis Inguinal adipose (VAT) tissue samples were harvested and subjected to immunohistochemistry using antibodies for CD11b (1:100; Abcam plc, Cambridge, UK) and 8-hydroxydeoxyguanosine (8-OHdG) (1 g/ml; Japan Institute for the Control of Aging, Fukuroi, Japan) and the standard protocols described previously (Hayashi et al., 2014; Uchida et al., 2012). Two investigators blindly and independently counted the number of CD11b- and 8OHdG-positive and −negative cells under a microscope at ×200 magnification. Ten microscopic fields were chosen in three different sections per mouse for examination. 2.5. Biochemical assays Plasma levels of cytokines and hormones were quantified using proper enzyme-linked immunosorbent assay kits for 8-OHdG, MCP-1, TNF-␣, IL-6, insulin, GLP-1 and DPP-4 activity, as described previously (Takeshita et al., 2007; Uchida et al., 2012; Hayashi et al., 2014). The kits used in this study are listed in the Supplemental Material.
2.1. Animals 2.6. Intraperitoneal glucose and insulin tolerance tests Eight-week-old male C57BL/6J mice (Chubu Kagaku Shizai Co, Nagoya, Japan) were housed two per cage under standard conditions (23 ± 1 ◦ C, 50 ± 5% humidity), with a 12-h light/dark cycle (lights on at 7:30 a.m.) in a viral pathogen-free facility in the Division for Research of Laboratory Animals, Nagoya University Graduate School of Medicine (Uchida et al., 2012; Yamamoto et al., 2002). The study protocol was approved by the Institutional Animal Care and Use Committee of Nagoya University (Protocol Number 27009), and the study was performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
After two weeks of daily stress, mice were subjected to an intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) using standard methods (Uchida et al., 2012). Briefly, for GTT, mice were fasted overnight and then challenged with D-glucose at 2 g/kg body weight (Sigma-Aldrich, St. Louis, MO), followed by serial assessment of blood glucose up to 120 min using a blood glucose level monitor (Glutest Ace, Sanwa Kagaku Kenkyusho Co, Nagoya, Japan). For ITT, mice were fasted for 16 h before testing. Insulin (0.75 U/kg, Actrapid Penfill, NovoNordisk, Copenhagen, Denmark) was injected intraperitoneally, and blood glucose was measured.
2.2. Restraint stress protocol 2.7. Statistical analysis Mice were randomly assigned to the control (n = 20) or the stress group (n = 30). Control mice were left undisturbed and allowed contact with each other, while stressed mice were isolated in individual cages and subjected to 2 h/day (between 10:00 a.m. and 12:00 p.m., 6 days/week) of immobilization stress over a period of two weeks as described in detail previously (Uchida et al., 2012; Yamamoto et al., 2002; Takeshita et al., 2004). Within each group, mice were randomly assigned to be orally fed either a drug-free CE-2 diet (vehicle) or a CE-2 diet containing alogliptin (15 or 45 mg/kg/day, a generous gift from Takeda Pharmaceutical Company), 10 mice per group, for 2 weeks. The control animals were given either the vehicle or high dose of alogliptin. The stressed animals were given either the vehicle, low, or high dose of alogliptin. Body weight and food intake were monitored during the 2-week period. Animals were anesthetized (intraperitoneal sodium pentobarbital, 150 mg/kg) and euthanized in the morning next to the last restraint stress. After euthanasia, biological samples were collected for total RNA extraction, analysis of plasma lipid profile, expression levels of biological markers, and pathological examination (Aoyama et al., 2009).
Data are expressed as mean ± SD. Differences between groups were assessed by Student’s t-test or one-way ANOVA followed by Fisher’s test, and considered significant at P < 0.05. 3. Results 3.1. Alogliptin prevents stress-induced adipose tissue inflammation As reported previously (Uchida et al., 2012), significant increase in mononuclear cell infiltration was noted in white adipose tissue (WAT) from the 8-week-old stressed and vehicle-treated mice compared to the non-stressed mice (Fig. 1a). Analysis of expression of macrophage surface markers demonstrated that the stress procedure significantly increased CD11b-positive cells and mRNA expression levels of monocyte/macrophage cell surface markers (F4/80 and CD68) in WAT (Fig. 1b–d). Alogliptin markedly reduced monocyte accumulation and mRNA expression levels of monocyte surface markers in WAT of stressed mice in a dose-dependent
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Fig. 2. Alogliptin restores stress-induced decrease in weight gain and reduces adipose tissue weight. a: Body weight gain and food intake in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.03 compared with the vehicle-treated and stressed mice, † P < 0.01, compared with the vehicle-treated and stressed mice (by Student’s t-test). b: Plasma fat and fatty acid composition in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. ** P < 0.01, compared with the vehicle-treated control mice, ** P < 0.01, compared with the vehicle-treated and stressed mice (both by Student’s t-test). c: Inguinal adipose tissue weight in control mice treated without or with 45 mg/kg/day alogliptin and stressed mice treated without or with 15 or 45 mg/kg/day alogliptin. Data are mean ± SD of 7 mice per group. *P < 0.002, compared with the vehicle-treated control mice, ** P < 0.04, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d: Images of inguinal fat pad. The dot line marks adipose tissue.
manner. The high dose of alogliptin did not alter monocyte accumulation in control mice.
stress (Fig. 3d). Alogliptin reduced 8-OHdG expression in WAT, plasma 8-OHdG concentrations, and adipose Nox-4 expression in a dose-dependent manner (Fig. 3).
3.2. Alogliptin reduces stress-induced lipolysis and FFA release As described previously (Uchida et al., 2012), the 2-week-stress period significantly reduced body weight gain in vehicle-treated mice (Fig. 2a), and this effect was abrogated by alogliptin in a dose-dependent manner (Fig. 2a). The high dose alogliptin did not alter body weight gain of non-stressed mice after the two-week treatment (Fig. 2a). Food consumption was almost similar in the different groups (Fig. 2a). Stress and alogliptin treatment did not change total cholesterol or triglyceride levels (Fig. 2b), but stress increased FFA concentration while alogliptin significantly reduced these concentrations in a dose-dependent manner (Fig. 2b). Stress also significantly reduced inguinal WAT weight compared to non-stress, and this decrease recovered after alogliptin treatment (Fig. 2c). Indeed, alogliptin restored stress-induced shrinkage of inguinal fat pad (Fig. 2d). These results suggest that alogliptin treatment reduces stressinduced lipolysis and FFA release. 3.3. Alogliptin suppresses stress-induced free radical production Stress increased the expression of 8-OHdG in adipose tissues (Fig. 3a), including adipocytes, monocytes. Stress increased 8OHdG-positive cells in WAT by two-fold, compared with the control (Fig. 3b). Stress also increased plasma 8-OHdG concentrations by two-fold, relative to the control (Fig. 3c). NADPH oxidase 4 (Nox4) mRNA expression was also upregulated in WAT after restraint
3.4. Alogliptin reduces inflammatory adipokine levels in stressed mice The 2-week restraint stress significantly upregulated MCP-1, TNF-␣, and IL-6 mRNA expression levels in adipose tissues, and these changes were suppressed in a dose-dependent manner by alogliptin (Fig. 4). Alogliptin also lowered the stress-induce rise in MCP-1, TNF-␣, and IL-6 plasma levels. The stress-induced decrease in adiponectin mRNA expression was abrogated by alogliptin (Fig. 4d). However, no changes in the expression levels of these adipokines were noted in adipose tissue of control mice treated with vehicle or high-dose alogliptin. 3.5. Alogliptin suppresses stress-induced DPP-4 activation In the vehicle-treated stressed mice, DPP-4 mRNA expression in WAT was more than double that of the vehicle-treated control mice (Fig. 5a). Plasma DPP-4 activity was also significantly increased in the vehicle-treated stressed mice (Fig. 5b). In parallel with these changes, plasma GLP-1 levels were significantly reduced in stressed mice (Fig. 5c). Alogliptin reduced DPP-4 activity and increased plasma GLP-1 levels in non-stressed animals (Fig. 5b and c). Alogliptin significantly reduced both WAT DPP-4 mRNA level and plasma DPP-4 activity in stressed animals in a dose-dependent manner (Fig. 5a, b). Furthermore, alogliptin also restored plasma
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Fig. 3. Alogliptin suppresses stress-induced free radical production. To examine free radical generation in inguinal white adipose tissue, and plasma from stressed and control (non-stressed) mice treated with or without 15 or 45 mg/kg/day alogliptin, were subjected to immunohistochemistry and ELISA for 8-OHdG, and quantitative RT-PCR for NOX-4. Data are mean ± SD. n = 6–7 mice per group. a. Increased 8-OHdG expression in inguinal adipose tissues (a) from stressed mice ( × 200 magnification, bar = 50 m). 8-OHdG was expressed in monocytes and adipocytes in WAT of stressed mice. Alogliptin suppressed the expression. b: Quantitative analysis of 8-OHdG-positive cells relative to total nuclear number in inguinal adipose tissues. Data are mean ± SD of 7 mice per group. *P < 0.001, compared with the vehicle-treated control mice, **P < 0.002, compared with the vehicle-treated and stressed mice (both by Student’s t-test). c: Quantitative analysis of plasma 8-OHdG levels. Data are mean ± SD of 7 mice per group. *P < 0.01, compared with vehicle-treated control mice, **P < 0.01, compared with the vehicle-treated and stressed mice (both by Student’s t-test). d. Quantitative analysis of NOX-4 expression level in adipose tissue. Values are expressed relative to the vehicle-treated control mice Data are mean ± SD of 7 mice per group. *P < 0.002, compared with the vehicle-treated control mice, **P < 0.002, compared with the vehicle-treated and stressed mice (both by Student’s t-test).
GLP-1 levels in a similar manner (Fig. 5c). Plasma insulin levels were measured 30 min after intraperitoneal administration of 2 g/kg body weight D-glucose in overnight fasting mice. Alogliptin increased plasma insulin levels both in non-stressed and stressed mice (Fig. 5d).
3.6. Alogliptin rescues stress-induced decline in insulin sensitivity and prothrombotic state Alogliptin significantly improved glucose and insulin tolerance after stress (Fig. 6a). Furthermore, it restored the mRNA expression levels of insulin receptor substrate-1 (IRS-1) and glucose transporter-4 (GLUT-4) in inguinal WAT (Fig. 6b), but not in skeletal muscle (adductor muscles). Stress significantly increased the expression levels of PAI-1 and TF in WAT, and plasma PAI-1 levels (Fig. 6c), while alogliptin abrogated these changes, compared with vehicle, similar to the changes in monocyte accumulation and induction of proinflammatory cytokines (Fig. 6c). Considered together, the above findings indicate that alogliptin suppresses stress-induced lipolysis and adipose inflammation to improve insulin resistance and prothrombotic state.
4. Discussion The main finding of this study was that alogliptin, a DPP-4 inhibitor, suppressed stress-induced adipose tissue inflammation and improved insulin resistance and thrombotic state in a dosedependent manner. In the present study, two-week intermittent restraint stress resulted in low-grade inflammation and induction of PAI-1 and TF in WAT, in agreement with previous findings (Uchida et al., 2012). We have previously shown that single acute restraint stress transiently increased TNF-␣ expression and mononuclear cells in WAT within 2 h, and that the repetitive stress within 1 week hardly induced inflammatory changes in WAT (Uchida et al., 2012; Yamamoto et al., 2002). Therefore, we recognized these finding on adipose tissue inflammation as a consequence of chronic stress. Stress also increased ROS production and DPP-4 expression in WAT, and plasma DPP-4 activity. Alogliptin administered at 15 or 45 mg/kg/day markedly suppressed stress-induced WAT inflammation and ROS production, resulting in reduction of TF and PAI-1. Furthermore, alogliptin suppressed stress-induced DPP-4 activation, improved glucose tolerance with an increase in plasma GLP-1,
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Fig. 4. Alogliptin reduces expression of stress-induced proinflammatory adipokines and restores adiponectin expression in adipose tissue. Inguinal white adipose tissues from control mice treated with vehicle or 45 mg/kg/day alogliptin, and stressed mice treated with vehicle or 15 or 45 mg/kg/day alogliptin were analyzed by quantitative RT-PCR for MCP-1 (a), TNF-␣ (b), IL-6 (c), and adiponectin (d). Values are expressed relative to vehicle-treated control mice. Plasma levels of MCP-1, TNF-␣, and IL-6 from these groups were also measured (a-d, respectively). Data are mean ± SD of 6 mice per group. (a) * P < 0.001, compared with the vehicle-treated control mice, ** P < 0.001, compared with the vehicle-treated and stressed mice, † P < 0.02 compared with vehicle-treated mice, †† P < 0.004 compared with stressed mice (all by Student’s t-test). (b) * P < 0.001, compared with vehicle-treated control mice, ** P < 0.001, compared with vehicle-treated and stressed mice, † P < 0.03 compared with vehicle-treated mice, †† P < 0.005, compared with stressed mice (all by Student’s t-test). (c) * P < 0.001, compared with vehicle-treated control mice, ** P < 0.002, compared with vehicle-treated and stressed mice, † P < 0.01, compared with vehicle-treated control mice, †† P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (d) * P < 0.001, compared with vehicle-treated control mice, † P < 0.002 compared with vehicle-treated mice, †† P < 0.001, compared with stressed mice (all by Student’s t-test).
and increased insulin sensitivity with restoration of adipose IRS-1 and GLUT-4. We demonstrated recently that the same type of stress activated the renin-angiotensin system (RAS) with increased expression of adipose angiotensinogen (Hayashi et al., 2014). Both NADH/NADPH oxidase activation by RAS and inflammation-related ROS formation synergistically increased ROS levels in VAT after stress (Fig. 3). ROS accumulation in adipose tissue exacerbates organ dysfunction, resulting in impaired glucose homeostasis due to insulin resistance (Figs. 5 and 6). We reported previously that adipose-derived PAI-1 and TF, which stabilize thrombi and initiate the coagulation cascade, respectively, participate in the stress-induced thrombus formation in aged mice (Yamamoto et al., 2014; Yamamoto et al., 2002). Furthermore, we showed that proinflammatory adipokines, including MCP-1 and TNF-␣, induce prothrombotic factors in adipose tissues after stress (Uchida et al., 2012). Both ROS generation and inflammatory adipokines in adipose tissue synergistically induced PAI-1 and TF in adipose tissue (Fig. 6) (Takeshita and Murohara, 2014; Yamamoto et al., 2014). Previous studies showed that DPP-4 inhibition suppresses ROS accumulation in kidney in animal models of type 1 and 2 diabetes, and in patients with type 2 diabetes (Avogaro and Fadini, 2014). Alogliptin-induced suppression of ROS generation in murine aortic aneurysmal model involves the GLP-1 signaling pathway (Bao et al., 2014). Our finding
of abrogation of stress-induced ROS accumulation by alogliptin is consistent with the above observations, and could serve to improve stress-induced prothrombotic state. DPP-4 is highly expressed in lymphocytes and monocytes, and is associated with immunoregulatory functions (Iwata et al., 1999; Augustyns et al., 1999). DPP-4 expression in VAT increases according to inflammatory cell accumulation. Indeed mRNA expression of DPP-4 in VAT increased after the stress period, and was decreased by anti-inflammatory effects of DPP-4 inhibitor (Fig. 5a). DPP-4 expression would be induced by inflammatory stimuli. As promoter of DPP-4 contains Stat3 binding site to augment DPP-4 expression (Bauvois et al., 2000), activation of IL-6 signaling, containing Jak-Stat signaling, can increase DPP-4 expression. Inversely, anti-inflammatory effects of DPP-4 inhibitor suppress production of inflammatory cytokines including IL-6 to reduce DPP-4 expression. Thus inflammatory status would alter DPP-4 expression in VAT. DPP-4 plays an important role in the immune and inflammatory responses in dysmetabolic conditions (Avogaro and Fadini, 2014). Since DPP-4 expression is increased in VAT of obese subjects with MetS and promotes MetS-related processes, it is considered as a MetS-related adipokine that can produce insulin signal disruption (Lamers et al., 2011; Rohrborn et al., 2015). We demonstrated previously that low-grade inflammation in VAT is the common mechanism linking stress-induced disor-
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Fig. 5. Alogliptin suppressed stress-induced DPP-4 activation. DPP-4 expression in inguinal white adipose tissue and DPP-4 activity and GLP-1 concentration in plasma were measured in stressed and control (non-stressed) mice without or with 15 or 45 mg/kg/day alogliptin by quantitative RT-PCR for DPP-4 (a), DPP-4 activity assay (b), and ELISA for plasma GLP-1 (c). Samples were collected after overnight fasting. Plasma insulin levels (d) were also measured to investigate the effects of DPP-4 inhibition at 30 min after intraperitoneal injection of 2 g/kg D-glucose followed by overnight fast. Data are mean ± SD of 6 mice per group. Values in (a) and (b) are expressed relative to vehicle-treated control mice. (a) * P < 0.001, compared with the vehicletreated control mice, †P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (b) * P < 0.001, compared with vehicle-treated control mice, †P < 0.005, compared with vehicle-treated control mice. #P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test). (c) * P < 0.01 and †P < 0.04, compared with vehicle-treated control mice. #P < 0.05, compared with vehicle-treated and stressed mice. (d) * P < 0.001, compared with vehicle-treated control mice. †P < 0.001, compared with vehicle-treated and stressed mice (all by Student’s t-test).
ders and MetS (Uchida et al., 2012). We also reported that DPP-4 is involved in stress-induced dysmetabolic conditions. Inflammatory cytokines, including TNF-␣, induce DPP-4 expression to regulate the immune and inflammatory responses (Rohrborn et al., 2015). Conversely, the anti-inflammatory effects of DPP-4 inhibitors have been demonstrated in inflammatory-related disease models, such as diabetes and cardiovascular disease (Avogaro and Fadini, 2014; Rohrborn et al., 2015). DPP-4 inhibitors lower DPP-4 activity by 70–90% (Avogaro and Fadini, 2014). In the present study, we also showed that alogliptin suppressed DPP-4 activity in parallel with reduction of inflammatory adipokines and monocyte accumulation (Figs. 4 and 5). DPP-4 has been also suggested to be a biomarker for the diagnosis of major depression and other stress-related diseases (Maes et al., 2009). Recently soluble and membrane DPP-4 has been highlighted to catabolize Neuropeptide Y (NPY) in blood circulation (Wagner et al., 2016). NPY and its receptors are widely expressed in cerebral areas such as the cortex, hippocampus, and amygdala to regulate anxiety, mood, cognition and stress resilience (Farzi
et al., 2015). Indeed overexpression of NPY reduced anxiety-like and depression-like behavior (Thorsell et al., 2000). Augmentation of NPY by DPP-4 inhibition or DPP-4 deficiency proved to exert antistress effects in animal models (El Yacoubi et al., 2006; Karl et al., 2003; Lautar et al., 2005). In the present study, pharmacological DPP-4 inhibition would synergically reduce stress-induced effects via central nerve system and anti-inflammatory effects. In stressed subjects, stress-induced cortisol release and adipose SNS activation initiate lipolysis to increase FFA concentrations (Uchida et al., 2012). This stress-induced lipolysis and FFA release can initiate stress-induced adipose inflammation via activation of toll like receptor-4, and subsequent TNF-␣ receptor activation in adipocytes accelerates stress-induced lipolysis to propagate a vicious cycle (Uchida et al., 2012). Vildagliptin, a DPP-4 inhibitor, suppressed diet-induced lipolysis (Ahren et al., 2011). In the present study, alogliptin also reduced monocyte accumulation and TNF-␣ induction, and broke the vicious circle between stressinduced lipolysis and adipose tissue inflammation (Figs. 2 and 3).
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Fig. 6. Alogliptin ameliorates stress-induced insulin resistance and prothrombotic state. (a) Glucose tolerance (top panel) and insulin tolerance (lower panel) showed significant recovery in alogliptin (45 mg/kg/day)-treated and stressed mice. Data are mean ± SD of 6 mice per group. * P < 0.001 compared with vehicle-treated mice, ** P < 0.04, compared with stressed mice (both by Student’s t-test). (b) Quantitative analysis of IRS-1 and GLUT4 expression in inguinal white adipose tissue and skeletal muscle (adductor muscle) of stressed mice treated with vehicle or alogliptin (45 mg/kg/day). Data are mean ± SD of 6 mice per group. * P < 0.05, compared with vehicle-treated and stressed mice (by Student’s t-test). (c) Quantitative analysis of TF and PAI-1 expression in inguinal white adipose tissue, and plasma PAI-1 levels of control and stressed mice treated with vehicle or alogliptin (15 or 45 mg/kg/day). Data are mean ± SD of 6–7 mice per group. *P < 0.001, compared with vehicle-treated and stressed mice, ** P < 0.001, compared with vehicle-treated and stressed mice (by Student’s t-test). †P < 0.002, compared with vehicle-treated and stressed mice, ‡P < 0.03, compared with vehicle-treated and stressed mice (by Student’s t-test).
As mentioned above, DPP-4 inhibitors have antidiabetic properties with anti-inflammatory effects via GLP-1 signaling (Avogaro and Fadini, 2014). In the present study, alogliptin improved both glucose and insulin sensitivity (Fig. 6). Alogliptin-induced restoration of GLP-1 level improved glucose tolerance in stressed mice, and alogliptin normalized insulin sensitivity by modulating IRS-1 and GLUT4 expression levels in WAT through significant suppression of adipose TNF-␣ (Ruan et al., 2002). The anti-inflammatory effect of alogliptin should improve insulin resistance because any decrease in plasma IL-6 is also expected to functionally improve insulin signaling in skeletal muscles at IRS-1 function, which is independent from IRS-1 and GLUT4 expression (Benito, 2011).
A theoretical review in a population cohort study suggests that psychological stress precedes the onset of diabetes (Pouwer et al., 2010). Accumulating evidence suggests the association of disturbed psychophysiological responses with cardiovascular risk factors, including subclinical measures of atherosclerosis, such as endothelial dysfunction, hypertension, and impaired glucose and lipid metabolism (Hamer and Malan, 2010). Diabetic patients under persistent stress are prone to thrombotic disease (Austin et al., 2013). Treatment with alogliptin could improve insulin resistance in at least a subgroup of diabetics under stress and might improve clinical outcome beyond glucose metabolism control. Several limitations exist in the presentation and analysis of the current data. We did not investigate the effect of corticosterone
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on stress-induced adipose tissue inflammation although corticosterone release induces lipolysis and FFA release in the downstream of hypothalamic-pituitary-adrenal axis activation (Arnaldi et al., 2010; Ziemssen and Kern, 2007). We could not identify the effect of basal DPP-4 activity on adipose inflammation because the finding of adipose tissue inflammation was subtle in non-stressed mice. The aim of this current study was to investigate the effect of DPP4 inhibitor on stress-induced adipose inflammation. While these information would be useful in future experiments in exploring the mechanism of stress-induced adipose tissue inflammation further, it is beyond the scope of the current study. In conclusion, we demonstrated that alogliptin inhibited stressinduced ROS generation and adipose inflammation to break the vicious cycle between lipolysis and inflammation. The antiinflammatory effects of alogliptin restored insulin sensitivity and improved the prothrombotic state in stressed mice. Role of the funding sources This work was supported by grants from the Takeda Medical Research Foundation, Suzuken Memorial Foundation, Kondo Memorial Foundation to KT, and a Grant-in-Aid for Scientific Research (Kakenhi 25461336 and 16K15411). MY is a recipient of JSPS doctoral fellowship for overseas researchers (JSPS KAKENHI Grant Number 15J00397). Conflicts of interest The authors declare no duality of interest associated with this work. Acknowledgments We thank Dr. Issa F.G., Word-Medex Pty Ltd, for the careful reading and editing of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.psyneuen.2016. 08.004. References Ahren, B., Schweizer, A., Dejager, S., Villhauer, E.B., Dunning, B.E., Foley, J.E., 2011. Mechanisms of action of the dipeptidyl peptidase-4 inhibitor vildagliptin in humans. Diabetes Obes. Metab. 13, 775–783. Akita, K., Isoda, K., Shimada, K., Daida, H., 2015. Dipeptidyl-peptidase-4 inhibitor, alogliptin, attenuates arterial inflammation and neointimal formation after injury in low-density lipoprotein (LDL) receptor-deficient mice. J. Am. Heart Assoc. 4, e001469. Aoyama, T., Takeshita, K., Kikuchi, R., Yamamoto, K., Cheng, X.W., Liao, J.K., Murohara, T., 2009. Gamma-secretase inhibitor reduces diet-induced atherosclerosis in apolipoprotein E-deficient mice. Biochem. Biophys. Res. Commun. 383, 216–221. Arnaldi, G., Scandali, V.M., Trementino, L., Cardinaletti, M., Appolloni, G., Boscaro, M., 2010. Pathophysiology of dyslipidemia in Cushing’s syndrome. Neuroendocrinology 92 (Suppl (1)), 86–90. Austin, A.W., Wissmann, T., von Kanel, R., 2013. Stress and hemostasis: an update. Semin. Thromb. Hemost. 39, 902–912. Augustyns, K., Bal, G., Thonus, G., Belyaev, A., Zhang, X.M., Bollaert, W., Lambeir, A.M., Durinx, C., Goossens, F., Haemers, A., 1999. The unique properties of dipeptidyl-peptidase IV (DPP IV / CD26) and the therapeutic potential of DPP IV inhibitors. Curr. Med. Chem. 6, 311–327. Avogaro, A., Fadini, G.P., 2014. The effects of dipeptidyl peptidase-4 inhibition on microvascular diabetes complications. Diabetes Care 37, 2884–2894. Bauvois, B., Djavaheri-Mergny, M., Rouillard, D., Dumont, J., Wietzerbin, J., 2000. Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells. Oncogene 19, 265–272. Bao, W., Morimoto, K., Hasegawa, T., Sasaki, N., Yamashita, T., Hirata, K., Okita, Y., Okada, K., 2014. Orally administered dipeptidyl peptidase-4 inhibitor
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