A B S T R A C T Leucinie metabolism in

Skeletal muscle is known to contain arginase, but, because this enzyme is also present in erythrocytes, the exact origin of arginine-derived ornithine in peripheral tissues is uncertain. In the present studies, skeletal muscle cells obtained from regenerating hindlimb muscle of adult rats were grown in primary tissue culture for approximately 3 wk and then studied in regard to changes in medium amino acid concentrations over a 48-h period. The consumption of arginine and serine was observed in parallel with the production of ornithine, proline, citrulline, glycine, and urea. Medium threonine and methionine concentrations were relatively constant over 48 h. Incubation of muscle cells with [U-14C]arginine resulted in the formation of [14C]ornithine and [14C]proline at rates at least 10-fold greater than could be accounted for by enzyme constituents of fetal calf serum. In addition, [guanido-14C]arginine was converted to [14C]urea and [U-14C]serine was converted to [14C]glycine. These studies indicate that cultured skeletal muscle cells contain a high arginase capacity and actively synthesize ornithine and urea from arginine.

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The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2 i-0.2 to 1.5 & 0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06 2 0.01 to 0.82 ? 0.04 mM between 0 and 48 hours and decreased to 0.72 2 0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD+ ratio, as represented by the intracellular lactateipyruvate ratio (r = 0.78-0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70 & 0.01 to 0.30 2 0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD+ redox state. ( 3 ) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production. line was shown to share several aspects of intermediary metabolism with skeletal muscle in vivo, e.g., active mitochondria1 metabolism of glucose-derived pyruvate, stable ATP and citrate levels for up to 96 hours of incubation, and active rates (up to 60 nmolilOO mg proteidmin) of alanine production (Pardridge and Davidson, '79). However, the L,; line differed from adult skeletal muscle in at least three respects: (1) active rates of glutamine consumption by the L, cells were observed, as opposed to adult skeletal muscle in which net glutamine production is the norm (Ruderman and Berger, '74; Garber et al., '76); moreover, the glutamine dependency of the LIj line was manifested by gross abnormalities in the cellular NADH redox state under conditions of glutamine deprivation (Pardridge and Casanello-Ertl, '79); (2) low to nonexistent levels of phosphocreatine were observed for the L, line, as opposed to normal skeletal muscle, which is characterized by high intracellular concentrations of phosphocreatine (Garber et al., '761, and (3) high concentrations of intracellular glucose were observed in the L,; cells, as opposed to adult muscle, wherein the intracellular glucose space is zero (Goodman, e t al., '74).The above differences between...

Well-differentiated cultured skeletal muscle cells (myotubes) obtained from adult rats were incubated for up to 48 h in Dulbecco's modified Eagle's medium. Medium glucose decreased from 4.9 +/- 0.1 mM at 0 h to 13 +/- 1 microM by 24 h; approximately 60% of glucose was converted to lactate. Pyruvate, alanine, and citrate were continuously produced, even during the period of 24-48 h when no glucose or lactate utilization was observed. Branched-chain amino acid utilization increased more than fourfold during the incubation period of 24-48 h; during this time, intracellular ATP, pyruvate, alpha-ketoglutarate, malate, and citrate levels were constant despite the absence of glucose or lactate consumption. Incubation of muscle cells with 2 mM clofibric acid resulted in a 76% inhibition of leucine metabolism. Coincident with the drug-induced inhibition of a branched-chain amino acid utilization, alanine and citrate production was blocked, and cell levels of pyruvate, alpha-ketoglutarate, malate, and citrate were markedly reduced. These studies suggest branched-chain amino acids contribute significantly to anaplerotic pathways in cultured skeletal muscle cells and that these pathways lead to the net production of alanine and citrate during periods of minimal carbohydrate utilization.