Figure 3. Alterations in cellular metabolism induced by mTORC1 activation.

A) Placement of the mTORC1-induced metabolic genes within their metabolic pathways. Genes found to be regulated by mTORC1 are shown in red, with those in the most stringent set indicated (*).

B) mTORC1 signaling stimulates glucose uptake. MEFs were serum starved for 16 hours in the presence of vehicle or rapamycin (20 nM). Glucose uptake was measured as the incorporation of 2-deoxy-D-[³H]-glucose over 4 minutes and normalized to cell number. Levels are presented as mean±SD relative to Tsc2^+/+ cells from four independent experiments. *p<0.002 versus Tsc2^+/+; **p<0.009 versus Tsc2^−/−.

C) mTORC1 signaling increases lactate production. Cells were grown as in (B), and lactate secretion into the media was measured over the final hour. Lactate concentrations normalized to cell number are presented as mean±SD relative to Tsc2^+/+ cells from three independent experiments. *p<0.00001 versus Tsc2^+/+; **p<0.006 versus Tsc2^−/−.

D) mTORC1 signaling stimulates de novo lipid biosynthesis. MEFs were serum starved for 24 h in the presence of vehicle or rapamycin (20 nM) and were incubated with D-[6-¹⁴C]-glucose for the final 4 h. ¹⁴C incorporation into the lipid fraction was measured and is presented as mean±SD relative to Tsc2^+/+ cells. These data are representative of three independent experiments. *p<0.006 versus Tsc2^+/+; **p<0.004 versus Tsc2^−/−.

E) Metabolomic profiling demonstrates that mTORC1 activation increases metabolites of glycolysis and the pentose phosphate pathway. MEFs were grown as in (B) and metabolites were extracted and profiled by LC-MS. Relative levels of specific metabolites, normalized to cell number, from three independent samples for each cell line and treatment are shown in the heat map. Metabolites showing a pattern consistent with mTORC1 regulation are indicated (*). The complete metabolomic profile is provided as supplemental Figure S3 and Table S4.

F) mTORC1 signaling increases flux through glycolysis. MEFs were grown as in (B) and incubated with [1,2-¹³C]-glucose for 15 min prior to metabolite extraction and LC-MS analysis. Levels of dually ¹³C-labeled glycolytic intermediates, normalized to cell number, are presented as mean±SD over three independent samples.

G) mTORC1 signaling increases flux through the oxidative branch of the pentose phosphate pathway. The ratio of singly (1×) and doubly (2×) ¹³C-labeled to unlabeled (¹²C) pentose phosphate pathway metabolites were measured by LC-MS in the samples from (F). P-values for pair-wise comparisons in (F) and (G) are listed in the accompanying table.