We have examined various aspects of hormonal control of adipocyte metabolism with isolated rat epididymal adipocytes as a model system. (A) In 32-Pi-loaded cells, the phosphorylation of a 65 KDa protein, found associated with the lipid fraction of adipocyte homogenates, was examined under conditions of unchanging, steady- state cAMP-dependent protein kinase (A-kinase) activity. Steady- state phosphorylation was achieved following a pulse, or overshoot, of phosphate incorporation, indicating that increases in A-kinase are accompanied by increased phosphate activity, and that the concerted action of both kinase and phosphatase provide the cell with a means to produce graded responses to graded increases in cellular cAMP. In a manner independent of A-kinase activity. Insulin also leads to the removal of phosphate from this protein. Insulin stimulates the phosphorylation of an abundant 62 KDa protein, also located exclusively in the lipid fraction: phosphorylation of this protein is abolished by increased A-kinase activity. Such data reveal a tight interplay, or crosstalk, between adenylate cyclase-linked receptors and the insulin receptor. (B) A high affinity antibody against hormone-sensitive lipase (HSL) was raised and used to probe a fat cell cDNA library. Also, a probe for the HSL gene has been produced from a peptide sequence derived from HSL purified in this laboratory. (C) Analysis of fat cell G proteins showed that fat cells contain 3 different subspecies of Gi and 2 different Gs proteins, all located primarily on plasma membranes. However, intracellular membranes contain a large """"""""Gs-like"""""""" protein of 55 KDa that is ADP-ribosylated by cholera toxin, both in vivo and in vitro, and recognized by affinity purified antibodies against Gs.
