Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids

In order to evaluate the ability of EMT6/Ro multicellular spheroids to util ize various pathways of energy production, C-13 and P-31 MRS have been empl oyed to monitor the metabolism of glucose, glutamine, acetate and propionat e. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreati ne (PCr) for up to 48 h, even in the absence of glutamine. The metabolism o f 1-C-13-glucose was almost entirely to 3-C-13-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O-2. Labeling w as also observed in other glycolytic metabolites, primarily alanine and alp ha-glycerolphosphate. A low level of C-13 labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detect ed when spheroids were perfused with 1-C-13-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always le ss than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resoluti on C-13 NMR spectra of extracts from cells or spheroids. Despite the predom inantly glycolytic metabolism of glucose, the mitochondrial substrate gluta mine (2 mM, in the presence of less than or equal to 0.5 mM glucose from fe tal bovine serum), supported stable levels of NTP and PCr in the tumor cell s for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic statu s of cells in EMT6 spheroids declined slowly but measurably, and no incorpo ration of label from 2-C-13-acetate into other metabolites was detected eit her in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-C-13-propionate re placed acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of sphero ids with 3-C-13-propionate (with natural abundance glucose and glutamine) r esulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metaboli sm. Addition of vitamin B-12, a cofactor for methylmalonyl CoA reductase, t o the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labe ling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity. Copyright (C) 2000 John Wiley & Sons, Ltd.