3.  Chronic administration of the angiotensin type 2 receptor agonist C21 improves insulin action in C57BL/6 mice

Angiotensin type 2 receptor (AT2R) has been reported to have beneficial effects on insulin action providing protection against insulin resistance and type 2 diabetes. In the current study, we explored the role of AT2R in the control of insulin action. To that end, male C57Bl/6 mice were administered the non-peptidic AT2R agonist C21 for 12 weeks (1 mg/kg per day; i.p. injection; n=12); vehicle-treated animals were used as Control (n=12). On week 11, glucose (GTT) and insulin (ITT) tolerance tests were performed. To investigate the status of insulin signaling in main insulin target tissues, by the end of week 12, each group of animals (C21 and Control) was
divided into 2 subgroups and injected via cava vein, either with a solution of insulin (10 IU/kg) or with saline(for analysis of baseline values). Liver, adipose tissue (epididymal) and skeletal muscle was removed 1, 3 and 5 min after insulin injection respectively. Metabolic parameters (cholesterol, tryglicerides, glucose, insulin) were measured in blood extracted via heart puncture from saline-injected mice. Epididymal adipose tissue size was determined on hematoxylin & eosin-stained sections. Phosphorylation levels of the insulin receptor (IR), Akt, ERK1/2 and protein levels of IR, Akt and ERK1/2 were determined by western blotting (WB) in solubilized tissue samples. Adiponectin content and TNF-alpha levels were measured in adipose tissue by WB. When compared to the Control group, C21- treated mice showed: -decreased blood glucose levels while unaltered circulating
levels of cholesterol, tryglicerides and insulin. -increased insulin sensitivity and glucose tolerance as measured in ITT and GTT. -decrease mean adipocyte size - increased adiponectin content in adipose tissue - increased basal Akt phosphorylation levels at (Thr308 and Ser473) - increased insulin-stimulated levels of Akt phosphorylation at Thr308 and Ser373 in adipose tissue -increased phosphorylation levels of ERK1/2 under basal conditions in the liver -inability to respond to insulin in terms of ERK1/2 phosphorylation in either liver, adipose tissue or skeletal muscle. -unaltered phosphorylation and protein levels of the insulin receptor in any of the analyzed tissues In conclusion, long-term administration of the AT2R agonist C21 induced a marked improvement in insulin sensitivity in C57BL/6 mice that was associated with modifications in the status of insulin signaling in main insulin target tissue, together with decreased adipocyte size and increased content of adiponectin in adipose tissue. Current results indicate that the AT2R has a physiological role in the control of insulin action and glucose homeostasis in mice.