Hormonal Regulation of Adipocyte Enzymes THE EFFECTS OF EPINEPHRINE AND INSULIN ON THE CONTROL OF LIPASE, PHOSPHORYLASE KINASE, PHOSPHORYLASE, AND GLYCOGEN SYNTHASE

Abstract The effects of epinephrine and insulin on three enzyme systems in adipocytes were investigated with regard to the role of adenosine 3',5'-monophosphate (cyclic AMP) in their control and the possibility that differential regulatory mechanisms might operate beyond cyclic AMP. When rat adipocytes were incubated with increasing concentrations of epinephrine (0.01 to 100 µm) elevation of cyclic AMP and glycerol production, activation of phosphorylase and deactivation of glycogen synthase appeared to be closely correlated. Activatability of hormone-sensitive lipase in cell extracts by cyclic AMP-dependent protein kinase decreased with increasing concentrations of epinephrine indicating conversion of the nonactivated to the activated form during incubation of the cells. Activation of lipase in cell-free extracts by cyclic AMP and MgATP was promptly arrested by addition of protein kinase inhibitor. This finding, together with results of previous studies, rules against the involvement of a "lipase kinase" analogous to that of phosphorylase kinase in phosphorylase activation. Phosphorylase kinase activities measured between pH 6.0 and 9.2 were unaffected at any concentration of epinephrine. Moreover, activation of phosphorylase in cell-free extracts was not inhibited by addition of protein kinase inhibitor. When adipocytes were incubated in Ca2+-free Krebs-Ringer bicarbonate medium, activation of phosphorylase in response to epinephrine was not impaired. Phosphorylase kinase assayed in cell-free extracts did not require Ca2+ for the activation of endogenous phosphorylase. However, some Ca2+ dependence was shown when exogenous skeletal muscle phosphorylase b was used as substrate. Thus, Ca2+ does not appear to regulate the catalytic activity of adipocyte phosphorylase kinase, nor could any change in the activation state of this enzyme in response to epinephrine be observed. These results distinguish adipocyte phosphorylase kinase from that in muscle and in heart which requires Ca2+ and is clearly convertible to an activated form by epinephrine. Incubation of adipocytes with glucose (10 mm) significantly reduced the phosphorylase activity ratio (activity in the absence of 5'-AMP divided by that in its presence). Epinephrine stimulated increases in lipolysis and phosphorylase activity in the presence or absence of glucose, but its effect on glycogen synthase was apparent only in the presence of glucose. The insulin-induced increase in glycogen synthase activity ratio (activity in the absence of glucose 6-phosphate divided by that in its presence) was also dependent on glucose. Insulin (0.4 nm, 50 microunits per ml), in the presence of glucose, almost completely antagonized the epinephrine-induced increases in lipolysis, activation of phosphorylase and deactivation of glycogen synthase. The antilipolytic action of insulin in cells previously exposed to epinephrine was associated with reversion of the activated lipase to its nonactivated form. However, insulin antagonized the elevation of cyclic AMP induced by epinephrine only marginally. This dissociation between the antagonism of insulin and epinephrine on the activities of the three enzymes on the one hand and the concentration of cyclic AMP on the other hand makes it seem unlikely that the action of insulin is mediated by a reduction in cyclic AMP concentration.