Figure 4.

Metabolic Regulation of mTOR Signaling

(A) Pimonidazole (green) is merged with anti-GLS2 (red) in left images, and right images depict tumors anti-GLS2 only (red) in control untreated tumors (top) and sunitinib-treated tumors (bottom). The arrow indicates GLS2-negative staining, while the arrowhead indicates GLS2-positive staining in hypoxic cells. The scale bars represent 25 μm.

(B) βTC3 cells, acclimated to low glucose as in Figure 3, were cultured in 2 mM glutamine (Gln), or 2 mM Gln + 20 mM lactate ± selective metabolic inhibitors, and assessed for p-S6 levels. Both βTC3s and lactate avid SiHa cells (Figure S2C) markedly upregulated p-S6 when cultured in lactate + Gln versus Gln alone; this upregulation could be reversed by 100 nM rapamycin treatment, or partially reversed using 40 μM DON, a competitive inhibitor of glutaminase. In addition, blocking lactate uptake with the MCT1 inhibitors 1 mM CHC or 10 μM 7ACC2 also reversed this upregulation, as did 200 μM AOA, which blocks the transamination of pyruvate + glutamate to alanine + α-ketoglutarate. The bottom images depict experiments performed with the selective GLS1 inhibitor, 50 μM BPTES, which failed to reverse the lactate induced pS6 upregulation, in contrast to DON, CHC, or 7ACC2. Below the blot is a graphic depicting the net consumption or production of lactate from time = 0.

(C) Graphic depicting the relative production of α-ketoglutarate in Gln + lactate versus Gln-only conditions. The samples were normalized by protein concentration.