Refractoriness to the insulin-like effects of growth hormone depends upon calcium

GH produces an acute but transient insulin-like response in adipocytes that have been deprived of GH for at least 3 h. The insulin-like response is followed by a period of refractoriness during which a second insulin-like response to GH cannot be elicited. These studies were undertaken to evaluate the role of calcium in the insulin-like response and refractoriness. Methionyl human GH (hGH) (100 ng/ml) increased the incorporation of D-[3-3H] glucose into lipid by 50-100% in fat cells that preincubated for 3 h without hormone and usually by less than 10% in fat cells that were made refractory by exposure to 100 ng/ml of hGH in the first hour of incubation. Insulin (100 microU/ml) increased lipogenesis by 3-5-fold whether fat cells were sensitive or refractory to GH. To determine whether calcium plays a role in either the insulin-like response to GH or the refractory phenomenon, we examined the effects of trifluoroperazine (20 or 50 microM) and calmidazolium (1.0 microM) which block calmodulin, as well as verapamil (30 microM), which blocks calcium channels. These agents did not interfere with stimulation of incorporation of D-[3-3H]glucose into lipid by GH (100 ng/ml) or insulin (100 microU/ml) but restored sensitivity to the insulin-like effects of GH in otherwise refractory cells. When freshly isolated, and hence refractory, cells were incubated for 1 h in calcium-free medium that contained 0.5 mM EGTA, GH stimulated lipogenesis by 30% (P less than 0.001) even though the response to insulin was markedly decreased in the absence of calcium. Conversely, when added to sensitive cells in the presence of normal extracellular Ca2+ concentrations during the 4th h of incubation, the calcium ionophore, A23187 (1.0 microM) produced an apparent refractoriness to the insulin-like effect of GH but had no effect on the response to insulin. The data indicate that maintenance of refractoriness to the insulin-like action of GH depends upon a calcium-calmodulin-sensitive process and suggest that, in producing refractoriness, GH may increase availability of intracellular calcium perhaps by increasing the rate of calcium entry into adipocytes. Since changes in responsiveness to GH were not paralleled by changes in responsiveness to insulin, it is suggested that the calcium dependent process responsible for refractoriness must lie at an early step in the signaling pathway.