Prolonged incubation of skeletal muscle in vitro: prevention of increases in glucose transport

Abstract

During experiments involving prolonged incubation of skeletal muscle, we observed large increases in glucose transport activity. The basal rate of 3-O-methylglucose (3-MG) transport increased two- to fourfold in rat epitrochlearis muscles incubated for 9 h without insulin in Krebs-Henseleit buffer supplemented with 8 mM glucose. The stimulatory effect of a low concentration of insulin (30 microU/ml, added during the final 30 or 60 min of incubation) on glucose transport activity was enhanced 2.5-fold after 6 h and approximately 5-fold after 9 h of incubation. Exposure of muscles to 100 microU/ml of insulin for the first 8 h inhibited slightly but significantly the increase in insulin-stimulated 3-MG transport over a 9-h incubation period. Incubation of muscles in minimal essential medium (MEM) for 9 h inhibited the time-dependent rise in basal and insulin-stimulated transport by approximately 45%. The effect of MEM was reproduced with MEM essential, but not nonessential, amino acids. Incubation of muscles with MEM plus 100 microU/ml of insulin for the first 8 h prevented the increases in 3-MG transport activity measured after a 9-h incubation period. Muscles incubated for 9 h maintained ATP and phosphocreatine concentrations, and changes in glycogen concentrations were small. Thus we have defined conditions for long-term incubation of skeletal muscle under which a progressive increase in glucose transport is prevented.