Angiotensin AT(2) receptor contributes towards gender bias in weight gain

Abstract

Obesity is a major disease condition, in turn leading to pathological changes collectively recognized as metabolic syndrome. Recently angiotensin receptor AT(2)R has been associated negatively with body weight (BW) gain in male mice. However, the gender differences in AT(2)R and BW changes have not been studied. To understand the gender based role of AT(2)R involving BW changes, we fed male and female wild type (WT) and AT(2)R knock out (AT(2)KO) mice with C57BL6 background with high fat diet (HFD) for 16 weeks. The male AT(2)KO had higher HFD calorie intake (WT: 1280±80; AT(2)KO:1680±80 kcal) but gained less BW compared with the WT (WT: 13; AT(2)KO: 6 g). Contrary to the male animals, the female AT(2)KO mice with equivalent caloric intake (WT: 1424±48; AT(2)KO:1456±80 kcal) gained significantly more BW than the WT mice (WT: 9 g; AT(2)KO: 15 g). The male AT(2)KO on HFD displayed lower plasma insulin level, less impaired glucose tolerance (GT), and higher plasma T3 compared with WT males on HFD; whereas the female AT(2)KO mice on HFD showed elevated levels of plasma insulin, more impaired GT, lower plasma T3 and higher free fatty acid and hepatic triglycerides compared with WT females on HFD. Interestingly, compared with WT, AT(2)KO female mice had significantly lower estrogen, which was further reduced by HFD. These results suggest that AT(2)R in female mice via potentially regulating estrogen may have protective role against BW gain and impaired glucose tolerance and lipid metabolism.

Figure 1. Total calorie intake consumed over 16 weeks by the different groups of mice on normal diet (ND) and high fat diet (HFD).

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 t 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).

Figure 2. Body weight gain in AT2KO mice on high fat diet.

Cumulative body weight change (per week) in normal diet (ND) or high fat diet (HFD) fed WT and AT₂KO (A) male and (B) female mice. Insets- Total body weight change in 16 weeks. Body weight in the different groups was measured every week for each animal on a triple beam veterinary scale and measured as change in body weight over basal, which was weight measured at the start of the dietary regimen. Data analyzed using one-way ANOVA with Newman-keuls post hoc test and Student t 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).

Figure 3. Gonadal adipose depot mass in male and female WT or AT₂KO mice on normal diet (ND) or high fat diet (HFD).

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 t 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).

Figure 4. Effect of high fat diet on glucose tolerance in AT2KO mice.

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 t 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).

Figure 5. Effect of high fat diet on hyperinsulinemia in AT2KO mice.

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 t 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).

Figure 6. Effect of high fat diet on plasma T3 in AT2KO mice.

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 t 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).

Figure 7. Effect of high fat diet on plasma hepatic free fatty acids and hepatic triglycerides in AT2KO mice.

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 t 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).

Figure 8. Effect of high fat diet on urinary estradiol in AT2KO mice.

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 t 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).