Phosphatidylinositol-3-kinase in Isolated Rat Adipocytes

lated cells, 75% of anti-Tyr(P)-immunoprecipitableP1- 3-kinase activity was found in the low density micro- somes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecip- itable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in re- sponse to insulin nor was it anti-Tyr(P)-immunopre- cipitable. These data demonstrate that insulin in- creases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma mem- branes. Phosphatidylinositol-3-kinase (PI-3-kinase)' has been im- plicated in the signaling mechanism of various growth factors and oncogenes that act through tyrosine kinases (1-3). Stud- ies in cell lines expressing mutant platelet-derived growth factor (PDGF) receptors or middle T proteins (4-6) suggest that PI-3-kinase is involved in the regulation of cell growth and proliferation. However, the presence of PI-3-kinase in nonproliferating cells, such as platelets (7) and neutrophils (8), suggests it has additional roles. Insulin increases anti-Tyr(P)-immunoprecipitable PI-3-ki- nase activity (9, lo), as well as the levels of PI-3,4-P2 and PIP3 (9) in CHO cells transfected with insulin receptor (CHO- IR). These and other data (11) have been taken to suggest that PI-3-kinase is activated by tyrosine phosphorylation, and that it may be a substrate for the insulin receptor kinase. When PI-3-kinase is activated by PDGF, the majority of the stimulated enzyme is associated with activated PDGF recep- tor (5). In contrast, only a small percentage of anti-Tyr(P)- immunoprecipitable PI-3-kinase is associated with the insulin receptor in CHO-IR cells stimulated with insulin (9,lO). The major physiological targets of insulin action are terminally differentiated tissues that respond to insulin by changes in intermediary metabolism rather than cell growth, and the effect of insulin on PI-3-kinase in these tissues has not been investigated. The purpose of the present investigation was to examine the effect of insulin on the subcellular distribution of PI-3- kinase activity in a mammalian insulin target tissue. Isolated rat adipocytes are ideally suited for this purpose since they are a major site of insulin action, they are one of the primary sites of insulin resistance in diabetes and obesity, and methods have been developed for the preparation of well characterized subcellular fractions of these cells. Furthermore, the effects of insulin on fat cells are manifested by changes in interme- diary metabolism that are subject to counter-regulation by other hormones. For the purposes of this study, anti-Tyr(P)- immunoprecipitable activity and total PI-3-kinase activity were measured in the different subcellular fractions in control and insulin-stimulated adipocytes. EXPERIMENTAL PROCEDURES