Subcellular Distribution of and Epinephrine-induced Changes in Hormone-sensitive Lipase, Phosphorylase, and Phosphorylase Kinase in Rat Adipocytes

Abstract

Abstract A systematic study was carried out of the subcellular distribution of hormone-sensitive lipase, glycogen phosphorylase, and phosphorylase kinase in rat adipocytes and the changes in these enzymes when the cells were incubated with epinephrine. Under the conditions of homogenization used, approximately one-third of the total hormone-sensitive lipase activity was associated with the fat cake floated to the top of a homogenate by low speed centrifugation, although as discussed in detail, this value remains uncertain in view of technical problems faced in assaying lipase tightly bound to fat. Subfractionation of the fat-free homogenate showed that over 80% of the hormone-sensitive lipase was recovered in the soluble fraction and the specific activity of that fraction was considerably higher than it was in particulate fractions. The plasma membrane fraction contained less than 2% and the microsomal fraction less than 3% of the activity in the fat-free homogenate. When adipocytes were lysed, over 80% of the total lipase activity was recovered in the supernatant fraction and less than 20% in the fat cell ghosts. The distribution of lipase activity among subcellular fractions of the fat-free homogenate was not altered by previous treatment of the adipocytes with epinephrine. Lipase activity in the soluble fraction from control cells was activated by treatment with cyclic adenosine 3',5'-monophosphate-dependent protein kinase and MgATP. When the cells had been previously treated with epinephrine, the percentage of activation by protein kinase was reduced in this and in all fractions. Phosphorylase and phosphorylase kinase activity were found predominantly in the soluble fraction; small amounts (21 and 14%, respectively) were associated with the fat cake assayed after resuspension in Triton X-100. The distribution of these enzymes in cells previously treated with epinephrine was not significantly different from that in control cells. Total phosphorylase activity (assayed in the presence of 5'-AMP) was significantly increased in homogenates prepared from cells previously treated with epinephrine as was the ratio of phosphorylase activities measured in the absence and in the presence of 5'-AMP. On the other hand, there was no change in total phosphorylase kinase activity after epinephrine treatment and no change in the ratio of activities measured at low pH values (6.2 or 6.8) and high pH value (8.2). Further characterization of the phosphorylase and phosphorylase kinase systems in adipose tissue is needed to assess the regulatory role of phosphorylase kinase in this tissue.