COMPOUND 21 STIMULATES THE ACTIVATION OF AKT AND ERK1/2 IN MOUSE ADIPOSE TISSUE IN VIVO

Abstract

Objective: To analyze signaling events triggered by in vivo stimulation of the AT2 receptor. Design and method: Male B57Bl/6 mice were used as model at the age of 3 month. Mice were subjected to a bolus in vivo intravenous injection (cava vein) of saline buffer containing either the AT2 agonist compound 21 (0.25 mg/Kg), a mixture of C21 (0.25 mg/Kg) and the AT2 antagonist PD123319 (2.5 mg/Kg) or saline for baseline assesment under anesthesia. A group of animals that received insulin (0.25 mg/Kg) were used as positive control. The heart and the inguinal adipose tissue were removed after 1 and 3 min respectively, flash-frozen and kept at -70 C until analysis. Proteins from heart and adipose tissue were solubilized in a buffer containing 1% Triton together withprotease and phosphatase inhibitors. Protein content was measured by the BCA method and homogenates were subjected to Western Blotting using specific anti-phospho Akt or anti-phospho ERK1/2 antibodies. The content of Akt and ERK content was detected through incubation with either anti-Akt or anti-ERK1/2 antibodies. Specific bands were detected by chemiluminescence. Protein loading control was checked by coomassie blue staining of every PVDF membrane employed. Results: Injection of C21 resulted in the activation of both Akt and ERK1/2 in mouse adipose tissue. The extent of activation of Akt and ERK1/2 attained was equivalent to approximately 30–40% of the levels of activation of these enzymes obtained after an injection of a dose of insulin known to generate maximum activation of these signaling components. Co-infusion of C21 and PD123319 abolished the in vivo activation of Akt and ERK1/2, indicating that these events were mediated by the AT2 receptor. Conclusions: In vivo administration of the AT2 receptor agonist C21 resulted in the stimulation of the phosphorylation of both Akt and ERK1/2 at stimulatory sites indicative of activation of these enzymes. Co-infusion of C21 with the specific AT2 receptor antangonist blunted this effect. It is concluded that the AT2 receptor is capable of recruiting both Akt as ERK1/2 as signaling molecules in mouse adipose tissue in vivo.