Phorbol Ester Provokes Insulin-Like Effects on Glucose Transport, Amino Acid Uptake, and Pyruvate Dehydrogenase Activity in BC3H-1 Cultured Myocytes*

Abstract

We evaluated the possibility that diacylglycerol may function as a second messenger in insulin action. To this end, we employed 12-O-tetradecanoyl phorbol 13-acetate (TPA) to mimic diacylglycerol in BC3H-1 myocytes. Like insulin, TPA provoked rapid increases in 2-deoxyglucose transport and pyruvate dehydrogenase activity in mature insulin-responsive BC3H-1 cultured myocytes. TPA also stimulated amino acid uptake, as evidenced by uptake of alpha-methylaminoisobutyric acid; the relatively slow time course of this effect paralleled that of insulin. In contrast, the effects of TPA were not apparent in undifferentiated BC3H-1 myoblasts, which were also unresponsive to insulin. The insulin-like effects in the myocytes appeared to be specific for TPA, the biologically active phorbol diester which activates protein kinase C, as other tested phorbol derivatives were without effect. Effects of maximally effective concentrations of TPA and insulin were nonadditive. Two synthetic diacylglycerols, 1,2-diolien and 1-oleoyl-2-acetyl-sn-glycerol, also provoked insulin-like effects on 2-deoxyglucose transport. Since insulin rapidly increases diacylglycerol levels in these cells, and TPA mimics diacylglycerol biochemically, it is possible that insulin may control cellular processes through changes in diacylglycerol.

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester

“…A likely candidate would be the pyruvate dehydrogenase (PDH), a multienzymatic complex representing a rate-limiting factor for glucose oxidation. Although in two cell lines (Zajdela hepatoma cells 19 and BC3H-1 myocytes 35 ) PMA has been shown to cause an activation of PDH, it cannot be excluded that in the whole heart activation of PKC could lead to an inhibition of this enzyme. Clearly further studies are necessary to elucidate the role of PKC in the regulation of glucose oxidation in the heart of lean and obese Zucker rats.…”

Anti-inositolglycan antibodies selectively block some of the actions of insulin in intact BC3H1 cells.

“…Purified rabbit IgG (Cappel Laboratories) was used for control purposes. The data shown were obtained by using antibodies and IgG at 25 ,ug/ml. (b) Same as a using a soluble PDH/PDH phosphatase assay system.…”

Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022

“…In turn, this provides further evidence that DAG is a critical component of the signal pathway by which insulin stimulates protein synthesis and therefore supports the hypothesis [14] that this signal pathway involves a G-protein-dependent activation of phospholipase C to produce DAG which then activates protein kinase C. The present results also demonstrate that manipulation of membrane phospholipid metabolism so as to increase endogenous diacylglycerol in response to receptor activation provides an opportunity to increase the sensitivity of cell protein synthesis machinery to insulin. One would expect other responses to insulin which may possibly be controlled through DAG and protein kinase C, such as glucose transport [12,24], to be similarly affected.…”

“…The signal mechanisms by which insulin exerts these effects are only partly understood; insulin binds to a receptor in the cell plasma membrane and this leads to the generation and release from the membrane of one or more signal molecules [7,8]. Peptide mediators [9], glycophosphoinositides [10,11] and DAG [12] have Correspondence address: J.E. Hesketh, Biochemistry Division, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, Scotland been implicated in the control of fat and carbohydrate metabolism by insulin while phospholipase C activation, DAG and protein kinase C have been suggested to be involved in the signal mechanism whereby insulin stimulates protein synthesis [13,141. The aim of the present work was to use an inhibitor of DAG kinase to investigate further the role of DAG as a signal in the sequence of events by which insulin stimulates protein synthesis in 3T3 fibroblasts.…”