The suitability of an established myogenic line (L6) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days a t which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized a t a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate a t an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10-24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose-derived pyruvate enters mitochondria1 pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.

A B S T R A C T Leucinie metabolism in

Rat myoblasts grown in culture for 5 days responded acutely to insulin (10 mU/ml) over a 3 hour incubation period by stimulating C14-glucose incorporation into glycogen by 260%. When these cells were grown in the presence of insulin (10 mU/ml) for the first 4 days (no exposure to insulin during the final 24 hours), insulin had no significant acute effect. These data provide direct evidence that insulation itself may induce antagonism under certain conditions.

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.

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.