Obesity is a major risk factor for hypertension. Obesity-related hypertension impacts more women than men, but the underlying mechanisms remain unclear. GLP-1, an incretin released after food intake, exerts vasculo-protective effects. Human studies have shown that GLP-1 levels are decreased in obese patients. We hypothesized that vascular GLP-1 signaling is reduced in obesity and weight loss rescues this signaling. Eight-week-old female Wistar rats were randomized into three groups: LEAN (n=9) received a chow diet (5% fat, 48.7% carbohydrate [3.2% sucrose], 24.1% protein) for 28 weeks, OBESE (n=7) received a Western diet (21% fat, 50% carbohydrate [34% sucrose], 20% protein) for 28 weeks, and reverse obese (rOBESE) (n=7) received a Western diet for 18 weeks followed by 12 weeks of chow diet. The OBESE group exhibited increased body weight (395.6 vs. 285.4g LEAN, p<0.0001) and body mass index (6.8 vs. 5.1kg/m 2 LEAN, p<0.01), while the rOBESE group lost weight (337.0 vs. 395.6g OBESE, p<0.01). Direct measurement of blood pressure (BP) using a pressure-volume catheter inserted in the carotid artery revealed increased systolic (142.8 vs. 117.2mmHg LEAN, p<0.001), diastolic (125.0 vs. 92.7mmHg LEAN, p<0.001), and mean arterial BP (130.9 vs. 107.9mmHg LEAN, p<0.001) in the OBESE group. The rOBESE group sustained elevated systolic BP (139.1 vs.117.2mmHg LEAN, p<0.05). Endothelium-dependent vasodilation studies assessed by wire myograph demonstrated that the OBESE group exhibited impaired response to acetylcholine (Emax: 82.7% vs. 97.9% LEAN, p<0.001). Similar vascular impairment was observed in the rOBESE group (EMax: 81.3% vs 97.9% LEAN, p<0.001). Strikingly, while decreased GLP-1 serum levels in the OBESE group (10.6 vs. 18.4pM/mL LEAN, p<0.05) returned to normal levels in the rOBESE group (19.4 vs.18.4pM/mL LEAN), GLP-1 receptor protein expression was reduced in both groups (24% decrease in OBESE, 52% decrease in rOBESE) as compared to LEAN. Our results support that GLP-1 signaling is implicated in obesity-related vascular dysfunction in females and weight loss does not guarantee recovery of protective GLP-1 signaling nor improvement of vasodilation. Conclusion: GLP-1 is a potential therapeutic target for obesity-related hypertension in females.

Background In the United States, consumption of a Western diet (WD), high in sugar and fat, has largely contributed to the epidemic of obesity, a major public health problem affecting both males and females. Body mass index (BMI) is a simple index of weight‐for‐height utilized to classify overweight and obesity in humans. However, this index alone is not translational in determining obesity in rodents. Moreover, sex differences in diet‐induced obesity in mice are not well‐characterized. We aim to study temporal metabolic changes in association with body weight gain caused by WD in males and females. Methods Our lab has established a model of WD‐induced obesity in mice. Adult C57BL6 male and female mice were randomized into two experimental groups. The control group (n=7) was fed a standard chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], and 24.1% protein) and the WD group (n=11) was fed a WD (40% fat, 43% carbohydrates [34% sucrose], and 17% protein) for 20 weeks. Body weight, BMI, Lee Index (calculated as the cube root of body weight (g)/body length (cm)), intraperitoneal glucose tolerance test (IPGTT), and metabolic cage studies were performed every 4 weeks to study the temporal metabolic changes caused by WD. Results While males showed significant increase in body weight after 3 weeks on WD (31.72g vs. 29.01g control, p<0.05), the obesity state was only confirmed after 8 weeks on WD as shown by increased BMI (4.04 kg/m2 vs. 3.62 kg/m2control, p<0.05). Lee index was significant after 20 weeks on WD (0.34 g/cm vs. 0.33 g/cm control, p<0.05). In comparison to males, female weight gain was delayed and significant after 5 weeks on WD (24.39g vs. 22.00g control, p<0.05). No differences in BMI (3.49 kg/m2 vs. 3.34 kg/m2 control, p=0.43) and Lee index (0.34 g/cm vs. 0.35 g/cm control, p=0.50) were observed even after 16 weeks on WD. Intriguingly, both male and female mice on WD for 20 weeks exhibited decrease in food intake (Male: 2.37 g/day vs. 3.16 g/day control, p<0.05; Female: 2.88 g/day vs. 3.81 g/day control, p<0.05) and caloric intake (Male: 8.98 kcal/day vs. 12.94 kcal/day control, p<0.05; Female: 14.81 kcal/day vs. 15.60 kcal/day control, p=0.60), as well as decreased fecal output (Male: 0.42 wet g/day vs. 2.00 wet g/day control, p<0.05; Female: 0.37 wet g/day vs. 2.17 wet g/day control, p<0.05) and urine output (Male: 1.43 ml/day vs. 2.43 ml/day control, p<0.05; Female: 0.57 ml/day vs. 0.69 ml/day control, p=0.32) compared to their respective controls. Interestingly, males showed significant intolerance to glucose after 2 months on WD (9542 ± 4375 a.u, p<0.05), while females did not show changes in glucose metabolism within 20 weeks of WD protocol. Conclusion Obesity is a complex metabolic disorder whose determination, in murine models, has been simplified to body weight gain. Our results show that significant weight gain alone may not fully characterize the obesity state in mice. Inclusion of other parameters such as BMI, Lee Index, and metabolic profile may better determine experimental obesity in a...

Background Obesity frequently leads to non‐alcoholic fatty liver disease (NAFLD), the most common liver problem affecting the American population. In fact, the prevalence of NAFLD in adult Americans ranges from 25‐46%. To date, there is no specific treatment for NAFLD. In severe cases, NAFLD can lead to fibrosis and cirrhosis which evolves into nonalcoholic steatohepatitis (NASH). Most patients with NAFLD experience no symptoms, making it a silent killer. Evidence has suggested that Toll‐like receptor 4 (TLR4) signaling, a key component of innate immunity, is implicated in the pathogenesis of NAFLD; however, the mechanisms of TLR4 activation remain unclear. We hypothesized that hyperacetylation of TLR4 leads to its overactivation in obesity, contributing to NAFLD development. Methods Eight‐week‐old male C57BL/6 mice were randomized into two experimental groups: the control group (n=4) received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], 24.1% protein) and the Western diet (WD) group (n=4) received a WD (21% fat, 50% carbohydrates [34% sucrose], and 20% protein) for 36 weeks. Body weight was obtained and intraperitoneal glucose tolerance test (IPGTT) was performed over the course of the dietary protocol at weekly and monthly intervals, respectively. During terminal experiments, liver tissue was isolated and processed for cross‐section histology. Staining with H&E and Oil‐Red‐O were used for grading of liver injury with the NAFLD Score, which involves summation of steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2) scores. A final score of 0–2 indicates no NASH, 3–4 indicates borderline NASH, and 5–8 indicates NASH. TLR4 and its downstream protein expression was detected by western blot and immunoprecipitation was utilized to determine the acetylation levels of TLR4. Results The WD group exhibited increased body weight (41.22 ± 7.71 g vs 29.94 ± 1.16 g controls, p<0.05) and liver weight (1.27 ± 0.0.07 g vs 1.27 ± 0.07 g controls, p<0.05). Results from IPGTT demonstrated that male mice develop early intolerance to glucose, within 8 weeks on WD. Hepatic histology showed ballooned hepatocytes, lobular inflammation, and steatosis, confirming presence of NAFLD with borderline NASH. Livers from the WD group exhibited increased TLR4 expression (1.4‐fold increase, p<0.05). Of note, this was accompanied by an increase in TLR4 signaling in WD livers as confirmed by elevated expression of the downstream signaling protein TNF receptor associated factor 6 (TRAF6) (0.8‐fold increase, p<0.05). Strikingly, immunoprecipitation assay revealed that TLR4 is hyperacetylated in the livers from the WD group. Conclusions Our results demonstrate for the first time that hepatic TLR4 is hyperacetylated in obesity‐related NAFLD. This hyperacetylation may serve as a trigger for the TLR4/TRAF6 signaling activation and may prove to be crucial to the development of NAFLD and NASH in obesity.

Background Overconsumption of a WD, a high‐fat and high‐sugar diet, has plagued the United States contributing to the widespread prevalence of type 2 diabetes (T2DM). Insulin resistance (IR) plays a key role in the development of T2DM and is a consistent finding among T2DM patients. The pathogenesis of IR, however, is not well understood. Based on recent evidence that inflammation precedes IR, and previous findings from our lab showing involvement of Toll‐like receptor 4 (TLR4) signaling in diabetic vascular inflammation, we hypothesize that WD leads to pancreatic inflammation through TLR4 signaling activation, contributing to IR. Methods Our lab has established a model of a WD‐induced metabolic syndrome and IR in female rats. Eight‐week old female Wistar rats were randomized into two experimental groups: Control (n = 9) and WD group (n = 16). The control group received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], and 24.1% protein), while the WD group received a WD (40% fat, 43% carbohydrates [34% sucrose], 17% protein), for 28 weeks. Body weight and food consumption were measured weekly. Lipid and glucose metabolism, and intraperitoneal glucose tolerance tests (IPGTT) were measured. Quantitative IR was assessed using Homeostasis Model Assessment of Insulin Resistance (HOMA‐IR), while functional beta cell capacity was assessed via HOMA‐B calculation. At the terminal experiments, pancreas’ were isolated for molecular (TLR4 signaling) and histological (hematoxylin and eosin, H&E) analysis. Results WD group exhibited greater body weight (415.1 ± 30.9 vs. 323.0 ± 24.0 g controls, p<0.01), and increased daily caloric consumption (130.5 ± 7.36 vs.110.7 ± 7.0 kcals controls, p<0.05) in spite of a significant reduction in daily food intake (23.7 ± 1.5 vs. 31.5 ± 1.8 g controls, p<0.01). WD negatively interfered in the glucose metabolism as evidenced by increased blood glucose area under the curve during IPGTT (17531.8 ± 574.3 vs. 12381.25 ± 1480.6 a.u. controls, p<0.001). IR in the WD group was confirmed by increased HOMA‐IR (8.09 ± 1.90 vs. 2.05 ± .042 a.u. controls, p<0.001), which was accompanied by hyperinsulinemia (0.93 ± 0.1 vs. 0.30 ± 0.09 mg/dL controls, p<0.01). No changes were observed in the HOMA‐B, suggesting that β cells remain functional. Moreover, no evidence of vacuolation, irregular outlining and size of the Langerhans islets, which are all markers of β cell dysfunction, were observed in the H&E stained pancreatic section from the WD group. Pancreatic inflammation in the WD group was confirmed by a 42% increase in TNF‐α expression (p<0.05), an inflammatory marker. Strikingly, pancreatic TLR4 (1.8‐fold increased, p<0.05) and its downstream molecule TNF‐receptor associated factor 6 (TRAF6) (3.1‐fold increased, p<0.001) were markedly upregulated in the WD group in comparison to controls, indicating TLR4/TRAF6 signaling activation in the pancreas. Conclusion Our findings suggest that a WD can induce IR along with activation of pancreatic TLR4 signaling‐related inflammation. These results...

Background Obesity has become a worldwide epidemic and is a major risk factor for the development of cardiovascular disease (CVD). Stiffening in the large arteries, such as the aorta, is a prevalent complication in obesity and frequently precedes hypertension. The mechanism by which obesity contributes to arterial stiffness remains unclear. Peroxisome proliferator‐activated receptor gamma (PPARγ) is a known vasculo‐protective factor. Post‐translational modifications of PPARγ by acetylation can regulate its function. Recently, our group identified that deacetylation of PPARγ enhances vascular endothelial function. We hypothesize that deacetylation of PPARγ protects against obesity‐related arterial stiffness. Methods A mice model of Western diet (WD) induced obesity and our model of deacetylated PPARγ mimetic knock‐in mice with a double lysine to arginine mutation (Lys268Arg, Lys293Arg, termed 2KR mice) were utilized. Adult male C57BL/6 and 2KR mice were randomized into two dietary protocols. Control groups of C57BL/6 (n=8) and 2KR mice (n=5) received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], and 24.1% protein) for 24 weeks. WD groupsof C5 7BL/6 (n=10) and 2KR mice (n= 5) received a WD (40% fat, 43% carbohydrates [34% sucrose], and 17% protein) for 24 weeks. Metabolic profiles were assessed by using single‐mouse‐sized metabolic cages. Aortic stiffness was assessed by measuring pulse wave velocity (PWV) with high‐resolution ultrasound, the gold standard for arterial stiffness. Systolic blood pressure was measured using tail cuff plethysmography. Results WD‐induced obesity was confirmed by increased body weight in both WD C57BL/6 mice (36.5g vs. 28.6g controls, p<0.001) and WD 2KR mice groups (44.7g vs. 29.86g controls, p<0.0001). Results obtained from metabolic cages revealed reduction in food, energy and water intake, and urine and fecal output in the WD C57BL/6 and WD 2KR groups as compared to their respective controls. These results indicate that weight gain in WD‐fed mice is not explained by increased energy intake, but rather a reduced metabolic rate. As expected, the WD C57BL/6 group exhibited increased aortic stiffness (5.4 m/s vs. 4.1 m/s controls, p<0.05), which was accompanied by elevated systolic blood pressure (143.8 ± 1.66 vs. 123.5 ± 4.76 mmHg controls, p<0.05). Strikingly, while WD 2KR mice developed obesity, these mice did not exhibit increased aortic stiffness (4.3 m/s vs. 4/4 m/s controls, p=0.89) nor elevated systolic blood pressures (122.4 ± 3.93 mmHg vs. x 115.2 ± 4.15 mmHg controls, p=0.26), indicating that deacetylation of PPARγ protects against aortic stiffness and increases in arterial blood pressure in obesity. Conclusions Our findings demonstrate that while deacetylation of PPARγ does not prevent the development of WD‐induced obesity, it does protect against arterial stiffness in obesity. These results indicate that PPARγ deacetylation is a potential therapeutic strategy in the prevention of obesity‐related arterial stiffness.