Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells.

Summary Kinase Suppressors of Ras 1 and 2 (KSR1 and KSR2) function as molecular scaffolds to potently regulate the MAP kinases ERK1/2 and affect multiple cell fates. Here we show that KSR2 interacts with and modulates the activity of AMPK. KSR2 regulates AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryo fibroblasts and glycolysis in a neuronal cell line. Disruption of KSR2 in vivo impairs AMPK-regulated processes affecting fatty acid oxidation and thermogenesis to cause obesity. Despite their increased adiposity, ksr2-/- mice are hypophagic and hyperactive, but expend less energy than wild type mice. In addition, hyperinsulinemic-euglycemic clamp studies reveal that ksr2-/- mice are profoundly insulin resistant. The expression of genes mediating oxidative phosphorylation is also down regulated in the adipose tissue of ksr2-/- mice. These data demonstrate that ksr2-/- mice are highly efficient in conserving energy, revealing a novel role for KSR2 in AMPK-mediated regulation of energy metabolism.

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