These experiments www.selleckchem.com/products/MLN-2238.html were designed to specifically focus on insulin-induced glucose metabolism in skeletal muscles and adipose tissue by infusing insulin at a rate known to suppress hepatic glucose production in all groups (Terrettaz et al., 1986). Following an overnight fast, glycaemia and insulinaemia were higher in Sirolimus- than in vehicle-treated rats (Table 1). Glycaemia was similar in both groups at the end of the clamps, while the hyperinsulinaemia reached was higher in the Sirolimus- than in the vehicle-treated group (Table 1). This resulted from the higher basal values brought about by the Sirolimus treatment, together with the fact that the same rate of insulin was infused in both groups.

Despite this higher insulinaemia, Sirolimus-treated animals displayed a marked decrease in the glucose infusion rate (GIR) compared with the vehicle-infused controls during the euglycaemic hyperinsulinaemic clamps (Figure 2C). In keeping with such a decreased GIR in Sirolimus-treated animals, the glucose utilization index measured in both red and white skeletal muscles was strongly reduced by the treatment (Figure 2D). Although the low amount of fat mass is unlikely to significantly contribute to the observed overall insulin resistance, Sirolimus treatment also significantly decreased the glucose utilization index of the epididymal and mesenteric, but not the inguinal fat depot (Figure 2E). Chronic mTOR inhibition by Sirolimus impairs Akt signalling and glucose transporter expression in skeletal muscle To gain insight into the molecular mechanisms by which Sirolimus triggers muscle insulin resistance, we analysed the expression and phosphorylation of critical insulin signalling effectors in skeletal muscle.

As muscles were collected from animals immediately at the end of the 2-DG procedure during euglycaemic hyperinsulinaemic clamps, they were stimulated with insulin before ex vivo tissue analyses. As shown in Figure 3A, the mTOR pathway was effectively blocked by Sirolimus, as evidenced by the lack of phosphorylation of S6, a substrate of the S6K, which is directly activated by mTORC1. Although phosphorylation of the insulin receptor and of Akt on Thr308 was unaffected, Akt phosphorylation on Ser473, which depends on the mTORC2 complex, was completely inhibited in Sirolimus-treated animals, in contrast to what has previously been observed in the liver of rapamycin-treated animals (Houde et al., 2010). This impaired Akt activation was further reflected by a decreased phosphorylation of AS160/TBCD4 and the glycogen synthase kinase 3 (GSK3) (Figure 3A), two important substrates of Akt controlling translocation of glucose transporters to Cilengitide the plasma membrane and glycogen synthesis.