Pathways of protein degradation in L6 myotubes.

Abstract

To assess the importance of different proteolytic pathways in anabolic and catabolic states, L6 myotubes were labeled with 3H-phenylalanine for 24 hr, then exposed for another 24 hr to control medium, alone or with one of the following: insulin (1 microgram/ml), insulin-like growth factor-I (IGF-I, 100 ng/ml), clenbuterol (100 microM), or dexamethasone (1 microM). Release of acid-soluble radioactivity and 3-methylhistidine were used to determine rates of total and myofibrillar protein degradation, respectively. Within each hormone treatment, cells were also exposed to none, one, or both of the following protease inhibitors: chloroquine (25 microM); E-64d (100 microM). Chloroquine inhibits lysosomal proteases, and E-64d is a cell-penetrating inhibitor of cysteine proteases (including the calpains). Total protein degradation was reduced by insulin (-33%) and IGF-I (-40%), increased by clenbuterol (+20%), and was unaffected by dexamethasone. Myofibrillar protein degradation was reduced by insulin (-35%), IGF-I (-55%), and clenbuterol (-22%), and was unaffected by dexamethasone. Therefore, total (mainly sarcoplasmic) and myofibrillar protein degradation were regulated independently of one another. The influence of the inhibitors was not consistent across hormone treatments. Chloroquine inhibited total but not myofibrillar proteolysis, indicating that lysosomes may be rate limiting in the former but not the latter. In contrast, E-64d had no effect on total protein degradation, but increased myofibrillar proteolysis. Interactions between anabolic and catabolic hormones and these two protease inhibitors indicate that both lysosomal and nonlysosomal proteolytic pathways are involved in regulation of total protein degradation in myotubes. In contrast, lysosomes are probably not involved in regulating the rate of myofibrillar protein degradation, the initial step likely being due to calpain activity.