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.
Critical Amino Acid Residues Determine the Binding Affinity and the Ca2+ Release Efficacy of Maurocalcine in Skeletal Muscle Cells