Male KK-Ay Research Articles

Sex-specific differences in atherosclerosis, thrombospondin-1, and smooth muscle cell differentiation in metabolic syndrome versus non-metabolic syndrome mice.

Metabolic syndrome (MetS) amplifies the risks of atherosclerosis. Despite well-known sexual dimorphism in atherosclerosis, underlying mechanisms are poorly understood. Our previous findings highlight a proatherogenic protein, thrombospondin-1 (TSP-1), in hyperglycemia- or hyperleptinemia (mimicking obesity)-induced atherosclerosis. However, the role of TSP-1 in the development of atherosclerosis prompted by co-existing hyperglycemia and obesity, characteristic of MetS, is unknown. The goal of this study was to examine sex-specific differences in lesion progression in a model of combined MetS and atherosclerosis (KKAyApoE) and interrogate how these differences relate to TSP-1 expression. Male and female KKAy+/-ApoE-/- (with ectopic agouti gene expression) and age-matched non-agouti KKAy-/-ApoE-/- littermates were placed on a standard laboratory diet from 4 to 24 weeks age followed by blood and tissue harvests for biochemical, molecular, and aortic root morphometric studies. Metabolic profiling confirmed MetS phenotype of KKAy+/-ApoE-/-; however, only male genotypes were glucose intolerant with elevated VLDL-cholesterol and VLDL-triglyceride levels. Aortic root morphometry demonstrated profound lipid-filled lesions, increased plaque area, and augmented inflammatory and SMC abundance in MetS vs non-MetS males. This increase in lesion burden was accompanied with elevated TSP-1 and attenuated LMOD-1 (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in male MetS aortic vessels. In contrast, while lipid burden, plaque area, and TSP-1 expression increased in MetS and non-MetS female mice, there was no significant difference between these genotypes. Increased collagen content was noted in MetS and non-MetS genotypes, specific to female mice. Measurement of plasma testosterone revealed a link between the atherogenic phenotype and abnormally high or low testosterone levels. To interrogate whether TSP-1 plays a direct role in SMC de-differentiation in MetS, we generated KKAy+/- mice with and without global TSP-1 deletion. Immunoblotting showed increased SM contractile markers in male KKAy+/-TSP-1-/- aortic vessels vs male KKAy+/-TSP-1+/ +. In contrast, TSP-1 deletion had no effect on SM contractile marker expression in female genotypes. Together, the current study implicates a role of plasma testosterone in sex-specific differences in atherosclerosis and TSP-1 expression in MetS vs non-MetS mice. Our data suggest a sex-dependent differential role of TSP-1 on SMC de-differentiation in MetS. Collectively, these findings underscore a fundamental link between TSP-1 and VSMC phenotypic transformation in MetS.

Abstract 135: Sex-specific Differences In The Role Of TSP1 On Metabolic Syndrome-induced Atherosclerosis

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy +/- ApoE -/- ) by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Male and female age-matched MetS KKAy +/- ApoE -/- and non-MetS KKAy -/- ApoE -/- mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy +/- ApoE -/- , increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy -/- ApoE -/- mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy +/- with TSP1 -/- to generate MetS mice with and without global TSP1 deletion. Male and female KKAy +/- TSP1 +/+ (with intact TSP1) and KKAy +/- TSP1 -/- (lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy +/- TSP1 -/- aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy +/- TSP1 +/+ , suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy +/- . Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.

Abstract 204: Sex-specific Differences In The Role Of TSP1 On Metabolic Syndrome-induced Atherosclerosis

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy +/- ApoE -/- ) by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Male and female age-matched MetS KKAy +/- ApoE -/- and non-MetS KKAy -/- ApoE -/- mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy +/- ApoE -/- , increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy -/- ApoE -/- mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy +/- with TSP1 -/- to generate MetS mice with and without global TSP1 deletion. Male and female KKAy +/- TSP1 +/+ (with intact TSP1) and KKAy +/- TSP1 -/- (lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy +/- TSP1 -/- aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy +/- TSP1 +/+ , suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy +/- . Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.

Anti-diabetic effect of S-adenosylmethionine and α-glycerophosphocholine in KK-Ay mice.

Six-week-old male KK-Ay mice received drinking water with S-adenosylmethionine (SAM), α-glycerophosphocholine (GPC), or SAM+GPC for 10weeks. The serum glucose of SAM+GPC at 15weeks old, total cholesterol of GPC at 12weeks old, and triglyceride of GPC at 15weeks old and of SAM at 16weeks old were reduced. SAM+GPC reduced serum leptin and food intake. Abbreviations: SAM: S-adenosylmethionine; GPC: α-glycerophosphocholine.

The Different Effects of Sodium Glucose Cotransporter 2 Inhibitor on Obese Diabetic Mice Fed a Normal or Low-Carbohydrate Diet

Sodium glucose co-transporter 2 inhibitors (SGLT2i) are antidiabetic drugs for type 2 diabetes that lower blood glucose levels and induce weight loss. However, the efficacy of SGLT2i is known to be affected by dietary factors such as carbohydrate and fat intake. In this study, we compared the effects of the SGLT2i (dapagliflozin) in eight-week-old male KK-Ay mice (a diabetic obese model) pair-fed a normal carbohydrate diet (NCD; carbohydrate 65, protein 23, fat 12%) or a low carbohydrate diet (LCD; carbohydrate 20, protein 45, fat 35%) for 8 weeks with or without dapagliflozin (5 mg/kg p.o.) (n=5-6). Dapagliflozin reduced body weight gain and blood glucose levels in the fed state in NCD and LCD, but reduced fasting blood glucose levels in NCD only. Likewise, dapagliflozin ameliorated glucose intolerance and enhanced hepatic gluconeogenesis in NCD only. Dapagliflozin decreased serum LDL cholesterol levels and subcutaneous fat in both groups, but decreased epidydimal fat (a visceral fat) in NCD only. We then investigated the metabolic effects of dapagliflozin and their underlying mechanisms in liver using transcriptomics (DNA microarray analysis) and metabolomics (Capillary Electrophoresis-Mass Spectrometry: CE-MS and Liquid Chromatography-Mass spectrometry: LC/MS). Dapagliflozin upregulated the expression of genes related to gluconeogenesis and β-oxidation in liver in both groups. In NCD, dapagliflozin increased several kinds of amino acids, and in LCD, it increased ketone bodies. Dapagliflozin upregulated the expression of genes related to bile acid metabolism in NCD only. Both groups showed a reduction in cholesterol ester, a major component of LDL cholesterol, in liver. Our results suggest that the efficacy of SGLT2i is attenuated under LCD, and that differences in liver metabolism between NCD and LCD under dapagliflozin treatment contribute to this outcome. Disclosure F. Furuya: None. Y. Fujita: Research Support; Self; AstraZeneca, Ono Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, MSD K.K., Eli Lilly and Company. N. Matsuo: None. Y. Oguri: None. H. Minamino: None. K. Ikeda: None. S. Harashima: Advisory Panel; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Novo Nordisk Inc.. Research Support; Self; AstraZeneca. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Sanofi K.K.. Speaker's Bureau; Self; Tanabe Mitsubishi KK. Advisory Panel; Self; Tanabe Mitsubishi KK. Speaker's Bureau; Self; Novartis Pharma K.K., Eli Lilly and Company, Ono Pharmaceutical Co., Ltd.. Y. Wang: None. Y. Liu: None. N. Inagaki: Research Support; Self; Daiichi Sankyo Company, Limited, AstraZeneca, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited, Japan Tobacco Inc., MSD K.K., Sumitomo Dainippon Pharma Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Kissei Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Eli Lilly and Company, Sanwa Chemical Industrial Co., Ltd., Teijin Pharma Limited.

Increased methylmercury toxicity related to obesity in diabetic KK‐Ay mice

We examined the toxic effects of methylmercury (MeHg) in KK-Ay type 2 diabetic mice to clarify how metabolic changes associated with type 2 diabetes mellitus affect MeHg toxicity. MeHg (5 mg Hg kg (-1) day(-1) p.o.) was given to 4-week-old male KK-Ay and C57BL/6J (BL/6) mice three times per week for 6 weeks. Average body weights (BW) of vehicle-treated BL/6 and KK-Ay mice were 16.3 and 16.4 g respectively on the first day, and 24.8 and 42.3 g respectively on the last day of the experiment. MeHg-treated KK-Ay mice began to lose weight about 5 weeks after MeHg administration. Six of seven MeHg-treated KK-Ay mice showed hind-limb clasping in the final stage of the experiment. The mean blood mercury level of MeHg-treated KK-Ay mice reached a maximum of 9.8 µg ml(-1) , whereas that of the MeHg-treated BL/6 mice was 2.8 µg ml(-1) after 10 days of treatment. The average total mercury concentrations in the cerebrum and epididymal fat pad were 7.4 and 0.57 µg g(-1) , respectively, for BL/6 mice and 27 and 1.6 µg g(-1) , respectively, for KK-Ay mice. In MeHg-treated KK-Ay mice with neurological symptoms, CD204-positive macrophages were observed in the brain, kidney and spleen, indicating CD204 could be a marker for injured tissues. BW loss and significant pathological changes were not observed in other groups of mice. These results indicate that body fat gain in type 2 diabetes mellitus and low mercury accumulation in adipose tissue increased MeHg concentrations in organs and enhanced toxicity in KK-Ay mice at the same dose of MeHg per BW.

Female type 2 diabetes mellitus mice exhibit severe ischemic brain damage

Type 2 diabetes can impair the outcome of stroke as well as increase stroke risk; however, the sex difference in ischemic brain damage is not well known, and even less is known about the difference in diabetes. We therefore investigated the possible gender difference in brain damage after stroke associated with type 2 diabetes using a mouse model, KKAy. Female KKAy showed a much larger ischemic area compared with male KKAy. NADPH oxidase activity in the brain was also increased more in female than in male mice. Ovariectomy enhanced the ischemic area, and treatment with estradiol markedly attenuated the ischemic area to that in female KKAy, with a reduction of NADPH oxidase activity. Female and OVX mice showed improvement of cerebral blood flow (CBF) at 1 hour after middle cerebral artery (MCA) occlusion, but no significant difference in CBF of the ipsilateral penumbra and ipsilateral core 24 hours after MCA occlusion was observed among each group. Severe ischemic brain damage was observed in female KKAy compared with male KKAy. Estrogen showed a protective effect on the brain, at least partly from attenuation of oxidative stress in the female brain. These findings suggest that brain damage in diabetes mellitus might be more marked in women than in men.

Beneficial Effects of Sarpogrelate Hydrochloride, a 5-ht2a Receptor Antagonist, Supplemented with Pioglitazone on Diabetic Model Mice

Objective. Feeding behavior control and dietetics with consequent weight reduction can be the most efficacious and fundamental methods to normalize fasting blood glucose. However, pioglitazone treatment has been found to incrementally increase body weight. In this study, we investigated whether the combined application of a 5-HT2A receptor antagonist, sarpogrelate, with pioglitazone can provide a clinical benefit. Methods. Diabetic male KK-Ay mice were randomly assigned to four groups: those receiving 10 mg/kg/day pioglitazone treatment for 30 days (pioglitazone group, n = 7), those receiving 30 mg/kg/day sarpogrelate treatment for 30 days (sarpogrelate group, n = 7), those receiving both agents for 30 days (pioglitazone + sarpogrelate group, n = 7) and those receiving no treatment (control group, n = 7). Results. Feed intake was lower in the pioglitazone + sarpogrelate group than in the pioglitazone group. Water intake was also significantly lower in the pioglitazone, sarpogrelate and pioglitazone + sarpogrelate groups than in the control group. Combined application (pioglitazone + sarpogrelate) resulted in a 176% increase in leptin concentration compared with vehicle control. Body weight was significantly higher in the pioglitazone group, and there was a trend toward a smaller increment in body weight in the pioglitazone + sarpogrelate group. Mean values, calculated by multiplying insulin concentration and nonfasting glucose concentration, were significantly lower in the pioglitazone + sarpogrelate group than in the control group. Conclusions. These results suggest that the combined application of sarpogrelate with pioglitazone provides therapeutic benefits not only in preventing adverse effects but also in the treatment of diabetes.