ANGIOTENSIN AT2R ACTIVATION INCREASES ACE2/ANG1-7/MASR AXIS AND PREVENTS FATTY-DIET-INDUCED OBESITY

Purpose: Adipose tissue, the obligatory site of fat accumulation causing obesity, expresses local renin angiotensin system (RAS). RAS is commonly known to regulate blood pressure. However recently, we observed that angiotensin type 2 receptor (AT2R) activation prevents high-fat diet (HFD)-induced adiposity and hyperinsulinemia. Although, the mechanism(s) is(are) not known. There is evidence that other RAS components namely, the angiotensin type 1 receptor (AT1R) positively regulates while the angiotensin converting enzyme 2 (ACE2), which catalyzes the production of Mas receptor (MasR) peptide agonist Ang (1-7), inversely regulates adiposity and hyperinsulinemia. In light of the RAS inter-regulatory features, we hypothesize that AT2R activation causes a decrease in AT1R expression and an increase in the ACE2/Ang (1-7)/MasR expression in adipose tissue exerting beneficial effects on HFD-induced adiposity. Method: Male C57BL/6 mice (12-weeks old) were pretreated with AT2R agonist (C21, 0.3 mg/kg, daily i.p.) for 4 days. Thereafter, the animals were placed on normal chow diet (ND) or HFD with concurrent drug treatment for next 10 days. Results: The HFD increased the epididymal white adipose tissue (eWAT) weight, plasma free fatty acid (FFA), triglyceride (TG) and insulin levels. The increase in these parameters was prevented by C21 treatment. Western blot analysis demonstrated that HFD increased the protein expressions of AT1R and ACE, but decreased Ang (1-7) peptide level as measured by LC/MS analysis in eWAT. The C21 treatment under HFD condition caused a significant decreased in protein expressions of eWAT AT1R and ACE, but an increase in the expressions of MasR, ACE2 and ANG(1-7) peptide level. Conclusions: The pharmacological activation of AT2R with C21 affects adipose RAS components i.e., an increase in ACE2/Ang(1-7)/MasR levels and a decrease in ACE/AT1R expression, improving lipid metabolism and hyperinsulinemia under HFD condition. We propose that the pharmacological activation of AT2R may serve as therapeutic target for controlling obesity and associated metabolic disorders. Supported in part by grant from the NIH (RO1 DK-61578) to Dr. Hussain. Key Words: angiotensin receptors, angiotensin converting enzyme 2, obesit

Role of Angiotensin II Type 2 Receptor in Regulation of Lipid Metabolism and Adiposity

Obesity is becoming increasingly common in the United States and worldwide. Abundant supplies of cheap foods and sedentary life styles are predisposing human beings to obesity which is an underlying risk factor for metabolic syndrome and cardiovascular diseases. Adipose tissue is the obligatory site of fat accumulation which causes obesity. Moreover, adipose tissue expresses local renin angiotensin system (RAS) which has been implicated in obesity. Recent studies using angiotensin type 2 receptor (AT2R) knockout mice suggested that AT2R contributes to adiposity and obesity in male mice while prevents adiposity and obesity in female mice. On the other hand, deficiency of AT2R in Apolipoprotein E knockout mice (a model of atherosclerosis) exaggerated adiposity in male mice suggesting that AT2R prevents adiposity in this animal model. Thus, role of AT2R on adiposity in knockout studies was in-conclusive and also indicated a gender specific role of AT2R. Furthermore, the studies showed that the relative expression of AT2R deceases after birth through adolescence and adulthood. Also, there are reports suggesting that estrogen (E2) positively regulates AT2R expression and AT2R affects levels of urinary E2 which is known regulator of adiposity. Hence, we designed the present study to investigate the effect of pharmacological activation of AT2R on adiposity and to study whether the said effect is reduced at adolescence. Male C57BL/6 mice (5 and 12 weeks old) were pretreated with the AT2R agonist C21 (0.3 mg/kg, daily i.p.) for 4 days. Thereafter the animals were placed on normal chow diet (ND) or high-fat diet (HFD) with concurrent drug treatment for the next 10 days. The HFD significantly increased epididymal WAT (eWAT) weight, adipocyte size, plasma free fatty acid (FFA) and triglyceride (TAG) in these mice. The pharmacological activation of AT2R reduced the HFD-induced increase in eWAT weight, adipocyte size, plasma FFA and TAG. Thus, we observed that the pharmacological activation of AT2R reduced the HFD-induced adiposity and plasma parameters in male mice, both in young and adults. To explore the effects of C21 on lipid metabolism we investigated the adipose lipid metabolism (lipogenic and lipolytic) regulators in the eWAT of 5 weeks old male mice only. We observed that HFD increased the protein expressions (measured by western blot) of eWAT lipogenic regulators, namely adipose fatty acid binding protein (FABP4) and fatty acid synthase (FASN). On the other hand, HFD caused a decrease in the protein expression of eWAT adipose triglyceride lipase (ATGL) and an increase in hormone-sensitive lipase (HSL) protein expression 5 weeks old male mice. Interestingly, C21 treatment altered HFD-induced changes in lipogenic and lipolytic regulators. To explore further whether C21 directly impacts eWAT lipogenic/lipolytic regulators or impacts them due to its effect on fatty acid uptake in adipocytes, we performed an in vitro study using isolated primary epididymal adipocytes from 5 weeks old male mice. We observed that C21 acting on epididymal adipocyte AT2R via a NOS/GC/PKG-dependent pathway reduced fatty acid transport in adipocytes. Thus, pharmacological activation of AT2R reduced the fatty acid transport in adipocytes and possibly in consequence prevented HFD-induced adiposity in the male mice. Our studies with AT2R knock out female mice demonstrated that genetic deletion of AT2R causes an increase in adiposity with a parallel decrease in urinary E2 in female mice. Thus to explore whether the pharmacological activation of AT2R using C21 prevents adiposity in female mice and whether the E2 plays a role in C21-mediated effect on adiposity, female C57BL/6 ovary-intact (Ovi) and ovariectomized (Ovx) mice were used. These mice were treated with C21 (0.3 mg/kg, daily i.p.) with(out) E2 (5 µg/day) supplementation and placed on HFD for 10 days. We observed that the C21 treatment prevented the HFD-induced adiposity and plasma parameters, namely FFA, TAG and insulin in female mice independent of E2. In the liver, C21 treatment increased the CPT1A expression (an index of β-oxidation) and prevented liver weight increase under HFD. Interestingly, we also observed that C21 treatment increased the physical activity of female mice. Thus, this study demonstrated that pharmacological activation of AT2R increased physical activity of the female mice and prevented the HFD-induced adiposity in these mice. Overall these studies demonstrate that the pharmacological activation of AT2R reduces fatty acid uptake in adipocytes. This reduction in fatty acid uptake possibly prevents HFD-induced changes in lipid metabolism, adiposity and plasma parameters in mice. Moreover, pharmacological activation of AT2R also increases physical activity which may increase utilization of the fatty acid via oxidation and thus remove fatty acid from the circulation. In light of these studies, we propose that AT2R activation may serve as a potential therapeutic target to control obesity which in turn protects against obesity-associated metabolic disorders.Pharmacological and Pharmaceutical Sciences, Department o

Total calorie intake consumed over 16 weeks by the different groups of mice on normal diet (ND) and high fat diet (HFD).

<p>Weekly 100 g of food in the form of pallet was placed in the food slot of the mice cages. At the end of the week, remaining food was weighed and the amount subtracted from the initial 100 g to account for the food consumed. The food intake in the different groups was calculated in terms of calorie consumed. Data analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 13; WTHFD = 15; AT₂KOND = 7; AT₂KOHFD = 11); n for females (WTND = 7; WTHFD = 20; AT₂KOND = 10; AT₂ KOHFD = 17).</p

Effect of high fat diet on plasma T3 in AT2KO mice.

<p>Plasma T3 levels in (A) male and (B) female of WT or AT₂KO mice normal diet (ND) and high fat diet (HFD). Plasma T3 levels in different groups were determined using enzyme-linked immunosorbent assay (ELISA) kit as per the manufacturer’s instructions. Data analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 8; WTHFD = 8; AT₂KOND = 8; AT₂KOHFD = 8); n for females (WTND = 8; WTHFD = 8; AT₂KOND = 8; AT₂KOHFD = 8).</p

Effect of high fat diet on hyperinsulinemia in AT2KO mice.

<p>Plasma insulin levels in (A) male and (B) female WT and AT₂KO fed on normal diet (ND) and high fat diet (HFD). Plasma insulin levels in different groups were determined using ELISA kit as per the manufacturer’s instructions. Data were analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 20; WTHFD = 8; AT₂KOND = 6; AT₂KOHFD = 6); n for females (WTND = 19; WTHFD = 20; AT₂KOND = 16; AT₂KOHFD = 16).</p

Effect of high fat diet on glucose tolerance in AT2KO mice.

<p>Glucose tolerance test (GTT) curves in (A) male and (B) female WT or AT₂KO mice on normal diet (ND) or high fat diet (HFD). (C) Peak values and (D) area under curve (AUC) of GTT male and female WT and AT₂KO on ND or HFD. In week 16 of the treatment, the mice of different groups were fasted for 6 hours and given 25 mmol of glucose through IP injection. The blood glucose was measured by pricking the tail of conscious animals at 0, 15, 30, 60, 120 and 180 minutes using a glucometer. Data were analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 5; WTHFD = 4; AT₂KOND = 4; AT₂KOHFD = 5); n for females (WTND = 4; WTHFD = 4; AT₂KOND = 5; AT₂KOHFD = 6).</p

Gonadal adipose depot mass in male and female WT or AT₂KO mice on normal diet (ND) or high fat diet (HFD).

<p>At the end of 16-weeks treatment, following sacrifice, paired gonadal adipose depot (surrounding the epididymus and testes in males, and uterus and ovaries in females), were removed, pat dry on tissue paper and weighed. Data were analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 4; WTHFD = 3; AT₂KOND = 3; AT₂KOHFD = 4); n for females (WTND = 4; WTHFD = 3; AT₂KOND = 6; AT₂KOHFD = 3).</p

Effect of high fat diet on plasma hepatic free fatty acids and hepatic triglycerides in AT2KO mice.

<p>Plasma FFA (A) and Hepatic triglycerides (B) levels in male and female WT or AT₂KO mice either on normal diet (ND) or high fat diet (HFD). Plasma FFA level was measured by colorimetric method using FFA quantification kit. Hepatic TG level was measured by colorimetric method using TG quantification kit. Data analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05). In plasma FFA, n for males (WTND = 3; WTHFD = 3; AT₂KOND = 3; AT₂KOHFD = 3); n for females (WTND = 7; WTHFD = 3; AT₂KOND = 3; AT₂KOHFD = 3). For hepatic triglycerides, n for males (WTND = 4; WTHFD = 3; AT₂KOND = 4; AT₂KOHFD = 3); n for females (WTND = 3; WTHFD = 3; AT₂KOND = 3; AT₂KOHFD = 4).</p

Effect of high fat diet on urinary estradiol in AT2KO mice.

<p>Urinary estradiol levels of (A) male and (B) female WT or AT₂KO female mice. The urinary 17-β-estradiol was measured using a commercial EIA (Enzyme immune assay) kit as per manufacturer’s instruction. Data analyzed using one-way ANOVA with Newman-keuls post hoc test and Student <i>t</i> test (p<0.05); n for males (WTND = 6; WTHFD = 6; AT₂KOND = 6; AT₂KOHFD = 6); n for females (WTND = 6; WTHFD = 8; AT₂KOND = 13; AT₂KOHFD = 13).</p