Signal Transduction Mechanisms for the Stimulation of Lipolysis by Growth Hormone: A Dissertation

Abstract

The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32 P from γ- 32 P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32 P-ribosylation of G i catalysed by pertussis toxin (PTx) suggested that the abundance of G i in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of G i α 2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of G i α 2 in the 100k pellet. No change in G s α was observed in the l6k pellet but a 35% loss of G s α was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32 P-NAD ribosylation. G i α 2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of G s α were not seen. The distribution of 32 P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32 P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of G i on adenylyl cyclase.