No abstract

The role of insulin during GH-stimulated adipogenesis of 3T3-F442A fibroblasts was investigated. Adipogenesis in defined medium (DM), as quantified by the level of glycerol-3-phosphate dehydrogenase activity, revealed that there existed a strict requirement for both insulin and GH during adipogenesis. The concentration of insulin required to elicit half-maximal adipogenesis was approximately 20 nM. Insulin-like growth factor I was less effective than insulin in promoting adipogenesis, indicating that insulin action during differentiation was most likely mediated through the insulin receptor. Cellular viability was not compromised by the absence of insulin, as judged by colony-forming efficiency or trypan blue exclusion. Deletion of insulin from DM supplemented with 1 nM recombinant human GH reduced glycerol-3-phosphate dehydrogenase activity to uninduced levels. Removal of other individual DM constituents did not have this effect. The growth factors fibroblast growth factor, platelet-derived growth factor, and bombesin did not substitute for insulin during GH-stimulated adipogenesis. The characteristic increase in cell number observed during serum-based differentiation, reflecting clonal expansion of young adipocytes, did not occur in DM supplemented with insulin, and insulin-like growth factor I were necessary for this event. These results suggest that insulin functions in concert with GH as a coinducer of the differentiating signals.

The cytotoxic effect of crotoxin, a heterodimeric phospholipase A2 from the venom of Crotalus durissus terrificus, was examined on murine erythroleukemia cells in vitro. Crotoxin cytocidal effect on cell growth had an EC50 of approximately 0.1-0.2 microM (3.0-5.0 micrograms/ml) in serum-free medium. Cytotoxicity was independent of cell growth since both quiescent and proliferating cells had similar sensitivities to the toxin. Dissociation of the crotoxin complex and phospholipase A2 activity of its subunit B are required for cytotoxicity, since the covalently linked crotoxin complex or the specific alkylation of the active site on the subunit B abolish the cytotoxic activity on murine erythroleukemia cells. Specific interaction between crotoxin and murine erythroleukemia cells appears to be required since the homologous phospholipase A2 from Crotalus atrox venom, with a higher phospholipase A2 specific activity than crotoxin, was 86-fold less potent than crotoxin. The data in this report show that the cytotoxic effect of crotoxin on murine erythroleukemia cells is consistent with the specific binding of the toxin resulting in cytocidal action mediated by the phospholipase A2 activity of crotoxin subunit B.