Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2C12 myoblasts: Modulation by eicosapentaenoic acid

The effect of a proteolysis inducing factor (PIF) on protein synthesis and degradation and the modulation of this effect by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), have been examined using a surrogate mod el system, C2C12 myoblasts in vitro. After 90 min of incubation, PIF produc ed a significant inhibition of protein synthesis in a dose-dependent manner , with maximal inhibition at a concentration of 4 nM. The effect was attenu ated both by treatment with a monoclonal antibody to PIP and by treatment w ith insulin at physiological concentrations (1 nM) and below (0.1 nM), but not by EPA (50 mu M) The inhibitory effect on protein synthesis was transit ory and was not seen after prolonged incubation with PIF. An increased rate of protein degradation was observed in C2C12 myoblasts after addition of P IP, which was also maximal at a concentration of PIF of 4 nM. Higher concen trations of PIP did not produce an increase in protein degradation. Unlike the effect on protein synthesis, the enhanced protein degradation was compl etely abolished by pretreatment with 50 mu M EPA, suggesting that the two e ffects are mediated by different mechanisms. PIP produced an increased rele ase of [H-3]arachidonic acid from prelabeled myoblasts with a dose-response curve parallel to that of protein degradation and with a maximum at 4 nM P IF. Release of [H-3] arachidonic acid was completely blocked in cells pretr eated with 50 mu M EPA, suggesting that the effect was related to protein d egradation. The [H-3]arachidonic acid was rapidly metabolized to prostaglan dins E-2 and F-2 alpha and to 5-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs). Production of all eicosanoids was attenuated in cells pretreated with EPA. Of all of the metabolites, only 15-HETE produced a significant in crease in protein degradation in C2C12 myoblasts with a maximal effect at 3 0 nM and with a bell-shaped dose-response curve similar to that produced by PIP. These results suggest that PIF enhances protein degradation as a resu lt of an increased production of 15-HETE.