Figure 2.

Up-Regulation of mTORC1 by Glucose Does Not Involve Changes in Insulin/PI3K/Akt or AMPK Signaling

INS-1 cells or freshly isolated mouse islets were chronically cultured in physiological glucose, transferred to serum-free media, and stimulated with the 11 mM glucose (12 mM in the case of islets) for 1 h, with or without inhibitor, as depicted in Figure 1C.

(A and B) Representative western blots showing the effects of acute glucose stimulation on dispersed mouse islets (A) or INS-1 cells (B) treated with or without inhibitors, as indicated. mTORC1 was measured by phosphorylation of p70S6K and S6. PI3K signaling was measured by phosphorylation of Akt. AMPK signaling was measured by phosphorylation of AMPK/Raptor.

(C–E) Glucose-induced mTORC1 signaling, as measured by S6 phosphorylation, in the absence or presence of the mTOR inhibitor, KU, or the PI3K inhibitor, Ly (C); the Akt inhibitor, MK2206 (D); the inhibitor of glucose-stimulated insulin secretion, DZ (E).

(F) Glucose-induced mTORC1 signaling, as measured by S6 phosphorylation, in the absence or presence of exogenous insulin (100 nM). Tables underneath the blots indicate the quantification of the bands (arbitrary units). Bar graphs shown are the average and standard error of at least three separate experiments in which the levels of phospho-S6 were quantified, corrected for loading, and normalized against the 11 mM glucose data point. Statistical significance was calculated by Student's t test. # indicate p values above 0.05, * indicate 0.05 > p > 0.02, ** indicate 0.02 > p > 0.01, and *** indicate 0.01 > p > 0.001.