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In vivo, GH and insulin usually produce opposing effects on carbohydrate and lipid metabolism in adipocytes, even though their signal transduction pathways overlap. However, when added to rat adipocytes that have been made GH deficient, GH briefly produces responses that are qualitatively like those of insulin. Subsequently, GH induces refractoriness to this acute insulin-like response, in a sense restricting its effects to a unique subset of possible physiological actions. Okadaic acid is an inhibitor of type I and IIa phosphoprotein phosphatases and affects glucose metabolism in fat cells in a manner that is reminiscent of GH. Okadaic acid initially mimics the actions of insulin, and subsequently, even after it has been removed by thorough washing, blunts the ability of adipocytes to accelerate glucose metabolism in response to insulin or GH. Because refractoriness to the insulin-like effect of GH is associated with GH-induced increases in intracellular free calcium concentrations ([Ca2+]i), we examined the effects of insulin on [Ca2+]i in okadaic acid-treated adipocytes. Adipocytes were incubated with 0.25 microM okadaic acid for 1 h, washed, and reincubated without okadaic acid for 2 h before measurement of [Ca2+]i using fura-2 as a calcium indicator. Neither GH (500 ng/ml) nor insulin (100 microU/ml) affected [Ca2+]i in cells in which glucose metabolism was stimulated, but both hormones rapidly increased [Ca2+]i in adipocytes that were refractory to insulin-like stimulation. The characteristics of the increase in [Ca2+]i produced by insulin were identical to those previously reported for GH. The effect of insulin was mimicked by the dihydropyridine calcium channel activator BayK 5552 or depolarization of the cell membrane with 30 mM KCl and was blocked by the dihydropyridine calcium channel blocker, nimodipine (100 nM), implicating activation of voltage-sensitive L-type Ca2+ channels. The increase in [Ca2+]i was also mimicked by sn-1,2-dioctanoylglycerol and blocked by inhibitors of protein kinase C (staurosporine, chelerythrine chloride, and calphostin), and D609, an inhibitor of phospholipase C, as reported for GH. Acquisition of the ability to increase [Ca2+]i in response to insulin required a lag period of at least 2 h after removal of okadaic acid and was prevented by inhibitors of RNA and protein synthesis. Adipocytes that were incubated with inhibitors of protein kinase A (KT-5720), or protein kinase C (staurosporine) along with okadaic acid also failed to increase [Ca2+]i in response to insulin. Conversely, adipocytes that were incubated with dibutyryl cAMP, methylisobutyl xanthine, or phorbol ester instead of okadaic acid increased [Ca2+]i when treated with insulin 2 h later. These results suggest that phosphorylated substrates of protein kinases A and C may mediate the transcriptional event(s) that enable adipocytes to activate L-type Ca2+ channels in response to insulin. Blockade of protein kinases A or C or removal of calcium from the incubation medium did not restore the ability of okadaic acid-treated adipocytes to increase glucose metabolism in response to insulin, nor did pretreatment of adipocytes with dibutyryl cAMP or phorbol ester decrease insulin-induced stimulation of glucose metabolism. The failure of insulin to increase glucose metabolism in okadaic acid-treated adipocytes thus cannot be ascribed to the increase in [Ca2+]i. These findings indicate that just as GH can produce an insulin-like response, so too can insulin produce a GH-like response, and highlight the need to understand how specificity of hormone action is achieved in cells that respond to different hormones that share elements of their transduction pathways.

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.

The sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM.