INSULIN PRODUCES A GROWTH HORMONE-LIKE INCREASE IN INTRACELLULAR FREECALCIUM-CONCENTRATION IN OKADAIC ACID-TREATED ADIPOCYTES

In vivo, GK and insulin usually produce opposing effects on carbohydra te and lipid metabolism in adipocytes, even though their signal transd uction pathways overlap. However, when added to rat adipocytes that ha ve been made GH deficient, GH briefly produces responses that are qual itatively like those of insulin. Subsequently, GH induces refractorine ss to this acute insulin-like response, in a sense restricting its eff ects to a unique subset of possible physiological actions. Okadaic aci d is an inhibitor of type I and IIa phosphoprotein phosphatases and af fects glucose metabolism in fat cells in a manner that is reminiscent of GH. Okadaic acid initially mimics the actions of insulin, and subse quently, even after it has been removed by thorough washing, blunts th e ability of adipocytes to accelerate glucose metabolism in response t o insulin or GH. Because refractoriness to the insulin-like effect of GH is associated with GH-induced increases in intracellular free calci um concentrations ([Ca2+](i)), we examined the effects of insulin on [ Ca2+](i) in okadaic acid-treated adipocytes. Adipocytes were incubated with 0.25 mu M 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 mu U/ml) a ffected [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 in crease in [Ca2+](i) produced by insulin were identical to those previo usly reported for GH. The effect of insulin was mimicked by the dihydr opyridine calcium channel activator BayK 5552 or depolarization of the cell membrane with 30 mM KCI and was blocked by the dihydropyridine c alcium 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 calpho stin), and D609, an inhibitor of phospholipase C, as reported for GH. Acquisition of the ability to increase [Ca2+](i) in response to insuli n required a lag period of at least 2 h after removal of okadaic acid and was prevented by inhibitors of RNA and protein synthesis. Adipocyt es that were incubated with inhibitors of protein kinase A (KT-5720), or protein kinase C (staurosporine) along with okadaic acid also faile d 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 sub strates of protein kinases A and C may mediate the transcriptional eve nt(s) that enable adipocytes to activate L-type Ca2+ channels in respo nse to insulin. Blockade of protein kinases A or C or removal of calci um from the incubation medium did not restore the ability of okadaic a cid-treated adipocytes to increase glucose metabolism in response to i nsulin, nor did pretreatment of adipocytes with dibutyryl cAMP or phor bol 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 the ir transduction pathways.