Phorbol ester inhibition of insulin-stimulated deoxyribonucleic acid synthesis in BC3H-1 myocytes.

Abstract

Insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA) each acutely stimulates hexose transport, amino acid uptake, and pyruvate dehydrogenase activity in the BC3H-1 myocyte in a nonadditive fashion, suggesting that the acute effects of insulin and TPA are mediated through a common mechanism of action. Here we have demonstrated that while chronic incubation with insulin stimulated DNA synthesis by 3- to 6-fold, TPA, in contrast, did not stimulate DNA synthesis and, indeed, caused a 70% inhibition of insulin-stimulated DNA synthesis in a dose-dependent fashion. In differentiated myocytes, insulin maximally stimulated hydroxyurea-sensitive [3H]thymidine incorporation into DNA at 200-400 nM with an ED50 of 5-8 nM, suggesting that insulin stimulates DNA synthesis via the insulin receptor rather than through growth factor receptors. Phorbol ester inhibition of insulin-stimulated DNA synthesis was specific for the active tumor-promoting phorbols and the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol. Maximal TPA inhibition of insulin-stimulated DNA synthesis was observed at 100 nM with an ID50 of 30 nM TPA, values analogous to those required for TPA stimulation of hexose transport in the myocyte. Chronic incubation with TPA did not inhibit insulin-stimulated protein synthesis, acute K+ flux, K+ accumulation, cytosolic thymidine levels, or insulin binding, indicating that TPA inhibits a specific intracellular event mediating DNA synthesis and suggesting that the acute and chronic effects of insulin in BC3H-1 myocytes are regulated by distinct pathways.