Figure 4 Chronic mTOR inhibition by rapamycin alters Akt activation by insulin in L6 myotubes. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10�C100 nM rapamycin (RAPA) before analyses. (A) Representative Western blots of total and phosphorylated … Finally and in contrast thing to Akt, ERK1/2 activation by insulin was unaffected by rapamycin. Although S6K has been shown to trigger IRS1 degradation through its phosphorylation on serine residues, we also did not observe a higher protein expression of IRS1 or IRS2 in L6 myotubes treated with rapamycin, despite the fact that IRS2 mRNA expression was significantly upregulated (Figure 4E,F).

Rapamycin prevents insulin-induced glucose uptake and glycogen synthesis in L6 cells by altering the expression of GLUT transporters and GLUT4 translocation to the plasma membrane To further delineate the molecular mechanisms by which mTOR inhibition affects skeletal muscle glucose metabolism in vivo, we investigated the effects of rapamycin on glucose uptake and glycogen synthesis in cultured rat L6 myotubes exposed for 48 h to 10�C100 nM rapamycin. In control myotubes, basal glucose uptake was increased by 1.89 �� 0.22-fold following incubation with insulin (Figure 5A). In contrast, 48 h exposure to 10�C100 nM rapamycin strongly decreased the basal, as well as the insulin-stimulated glucose uptake (Figure 5A). Similarly, insulin stimulation increased glycogen synthesis in control myotubes by 2.57 �� 0.33-fold (Figure 5B). However, following 48 h exposure to rapamycin, basal glycogen synthesis was reduced, and the insulin-induced glycogen synthesis was completely inhibited (Figure 5B).

Figure 5 Chronic mTOR inhibition by rapamycin impairs glucose uptake, glycogen synthesis, GLUT transporters expression and translocation to the plasma membrane in response to insulin in L6 cells. L6 myotubes were exposed for 48 h to either DMSO (CTL) or 10�C100 … We then assessed whether an altered expression Anacetrapib or translocation to the plasma membrane of GLUT transporters could contribute to the inhibition of glucose uptake and glycogen synthesis in L6 cells. Consistent with our ex vivo observations (Figure 3D), rapamycin treatment downregulated the protein expression of both GLUT1 and GLUT4 in L6 myotubes. However, at the mRNA level, only GLUT1 was decreased, suggesting that rapamycin treatment down-regulates GLUT4 at a post-transcriptional level (Figure 5C,D). More importantly, rapamycin prevented almost completely the translocation of GLUT4 to the plasma membrane following insulin stimulation (Figure 5E).