Figure 5.

Exposure to Excess Glucose Results in mTORC1 Hyperactivation owing to Enhanced Mitochondrial Pyruvate Metabolism

(A–E) INS-1 cells were kept in physiological glucose or transiently exposed to excess glucose for 20–24 h. Cells were then returned to 5 mM glucose in serum-free media and stimulated with the indicated glucose dose for 1 h (acute stimulation). (A) Diagram depicting the media composition and glucose concentrations throughout the different stages of the experimental timeline for (B)–(E). (B) Representative western blot showing glucose dose-dependent activation of mTORC1 signaling, as measured by S6 phosphorylation, in cells kept in physiological glucose (left lanes) or exposed to excess glucose (right lanes) and stimulated with glucose in the presence or absence of pyruvate or UK5099, as indicated. Blot shown is representative of four separate experiments. (C–E) Bar graphs showing the average and standard error of four independent experiments (identical to the experiment shown in [B]) in which the levels of phospho-S6 were quantified, corrected for loading, and normalized against the 11 mM glucose data point. Cells kept in physiological glucose are shown in light gray bars and cells pre-exposed to excess glucose are shown in dark gray bars. In (D), cells were treated with UK5099 at the time of glucose stimulation. In (E), cells were transferred to serum-free media without pyruvate before treatment with the indicated glucose dose. 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 > 0.003.