Effects of glutamine deprivation on glucose and amino acid metabolism in tissue culture.

Abstract

The effects of glutamine deprivation on cultured skeletal muscle cells were analyzed by incubating 10-day-old myotube preparations in glutamine free Dulbecco's modified Eagle medium containing 10% fetal calf serum for up to 48 h. Under these conditions net glutamine production was not observed, but active ammonia production (average rate = 1.0 nmol/min . mg protein) continued despite glutamine withdrawal. Glutamine deprivation was associated with a progressive depletion of intracellular aspartate and glutamate. Maximal aspartate depletion correlated with a 15-fold increase in the intracellular lactate:pyruvate ratio and a 3-fold decrease in the estimated intracellular glutamate:(alpha-ketoglutarate) (ammonia) ratio. Despite wide shifts in cell metabolite concentrations, the mass action ratios of alanine and aspartate aminotransferase approximated the expected equilibria constants. These results suggest that 1) glutamine deprivation is associated with a marked reduction of aspartate, and the maintenance of aspartate depletion is due in part to the tendency of aspartate aminotransferase to maintain the metabolites of this reaction at a near equilibrium level; 2) the transport of reducing equivalents from the cytosolic to the mitochondrial compartments via the malate-aspartate shuttle may be limited under conditions of aspartate depletion.

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells

“…However, a significant increase in medium lactate/ pyruvate ratio was observed (Table II) in association with a decrease in cell aspartate (Table III). A similar inverse relationship between the medium lactate/pyruvate ratio and cell aspartate has been observed previously (14,16). No significant differences in medium lactate/pyruvate ratio or cell aspartate were observed at 1 or 2 h of incubation; e.g., at 2 h the lactate/pyruvate ratio vas 8±1 in control cells and 9±1 in treated cells, and the cell aspartate level was 5.3±0.3 nmol/mg protein in control cells and 5.9±0.5 nmol/mg protein in treated cells.…”

“…Experimental incubations for the determination of metabolite levels were initiated by discarding old medium and adding to 100-mm dishes 3 ml of Dulbecco's modified Eagle's medium containing no fetal calf serum and adjusted to 0.4 mM pyruvate, 2.5 mM lactate, and 0.5 mM glutamine; the levels of other nutrients in this medium have been listed previously (12). Dishes with or without clofibric acid (2 mM) were incubated up to 4 h. At the end of the incubation, neutralized perchloric acid extracts of cold-washed cells and of medium were prepared as reported previously, with special storage precaution under acid pH for the a-keto-acids, pyruvate and a-ketoglutarate (12,16).…”

“…All metabolite levels were determined in duplicate with enzymatic fluorometric or spectrophotometric methods as reported previously (12,16). Clofibrate levels in the medium were determined spectrophotometrically (17).…”

Glutaminolysis in Animal Cells

“…Although alanine production was similar in degree to adult skeletal muscle, the L6 myotubes exhibited a net utilization of glutamine (Pardridge et at., 1978) and production of ammonia much in contrast to normal muscle, which is a site of net glutamine production (Garber et at., 1976). Depletion of glutamine in the medium resulted in a marked reduction of cellular aspartate, and a lesser reduction in cellular glutamate, alanine, and malate (Pardridge and Casenello-Ertl, 1979). Surprisingly, there was no reduction in ammonia production.…”

“…Pardridge et at. (1978) and Pardridge and Casenello-Ertl (1979) have examined aspects of glutamine metabolism in a myogenic cell line, L6, that displayed some properties of skeletal muscle tissue. In contrast to tumor cells and proliferating normal fibroblasts, which converted nearly stoichiometric amounts of glucose to lactate, only 35 to 40% of metabolized glucose could be accounted for as lactate in L6 myotubes.…”