LEVELS OF [15N]LEUCINE AFFECT RATES AND ENRICHMENT OF [15N]ALANINE RELEASED BY L6 MUSCLE CELL CULTURES

Abstract

Leucine serves as a major nitrogen donor for de novo alanine formation in muscle. To study factors affecting nitrogen transfer, L6 muscle cells ware cultivated in DMEM -Dulbecco's MEM containing 5 mM glucose and 10% fetal bovine serum (FBS). Replicating cells were inhibited on days 9 and 11 with 1 mM cytosine arabinoside. On day 15, when cultures were fully differentiated into myotubes, experimental incubations were done for 4 hr in DMEM without glutamine and FBS. L-[15N]leucine was added at 0, 0.05, 0.2, 0.5, 1.0 or 5.0 mM. Amino acids in the media were determined as N(0,S)-heptafluorobutyryl isobutyl esters by gas chromatography and selected ion monitoring electron impact mass spectrometry. With increasing media leucine concentrations the rate of alanine release decreased from 315.1±35.0(SEM) nmol/mg cell protein per 4 hr at 0 mM to 212.1±51.5 nmol/mg at 5.0 mM. By contrast, [15N]enrichment of alanine increased from 1.9 mol% excess at 0.05 mM [15N]leucine to 21.5 mol% excess at 5.0 mM. Concomitantly, [15N]enrichment of glutamate and glutamine rose from 2.5 to 8.5%. Smaller [15N]enrichments were also found in valine (from 0.4 to 1.3%) and in isoleucine (from 1.4 to 7.2%). Under the studied culture conditions, alanine release was inhibited rather than stimulated by increasing media leucine levels. However, Jeucine became progressively, more important as a nitrogen donor.