Characterization of the inhibition of protein degradation by insulin in L6 cells.
In muscle cells, protein degradation occurs by lysosomal and nonlysosomal mechanisms but the mechanism by which insulin inhibits protein degradation is not well understood. Using cultured L6 myotubes, the effect of insulin on muscle cell protein degradation was examined. Cells were labeled for 18 h with [3H]leucine or [3H]tyrosine and protein degradation measured by release of TCA-soluble radioactivity. Incubation with insulin for 0.5, 1, 2, or 3 h produced 0, 6, 12, and 13% inhibition, respectively, at 10(-7) M. If the cells were incubated for 3 h prior to the addition of insulin to remove short-lived proteins, the effect of insulin was enhanced, producing 26% inhibition. Very long-lived protein degradation (cells labeled for 48 h, chased for 24 h before the addition of insulin) was only inhibited 17% by insulin. This was due to serum starvation during the chase since the addition of serum to the chase medium produced a subsequent inhibition of 38% by insulin. Thus insulin had a greater effect on the degradation of longer-lived proteins. Use of inhibitors suggested that insulin requires internalization and degradation to produce inhibition of protein degradation and acts through both the proteasome and lysosomes. There appears to be no interaction with the calpains.
Fawcett
J
J
Endocrinology Section, Carl T. Hayden VA Medical Center, Phoenix, Arizona 85012, USA. janet.fawcett@med.va.gov
Hamel
F G
FG
Duckworth
W C
WC
D000818:Animals; D002460:Cell Line; D016895:Culture Media, Serum-Free; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006207:Half-Life; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D016895:Culture Media, Serum-Free; D007328:Insulin; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex