GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 microg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, Rp-cAMPS (Rp-adenosine 3'5'-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from [gamma-32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K x g or a less dense membrane pellet sedimented at 100K x g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K x g and 100K x g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Gi alpha2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gs alpha in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Gi alpha2 to less dense fractions as determined by both Western analysis and [32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gs alpha or 5'-nucleotidase. Colchicine (100 microM) blocked the GH+Dex-dependent shift of Gi alpha2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gi alpha2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Growth hormone (GH) produces insulin-like effects in rat adipocytes that have been deprived of GH for at least 3 h. The effect of a saturating concentration of GH is qualitatively and quantitatively similar to that produced by 2-4 ng/ml insulin but differs from that of insulin in the respect that adipocytes become refractory to prolonged or repeated stimulation with GH. Since activation of tyrosine kinase is an early event in the action of both hormones, we investigated the possibility that GH stimulation of tyrosine phosphorylation of some protein in the insulin transduction cascade might result in the similar effect of the two hormones. Adipocytes were preincubated for 3 h in the absence of hormones and then reincubated without or with 500 ng/ml GH or 4-400 ng/ml insulin for 10 min. The cells were lysed with an equal volume of buffer containing 1% SDS and preheated to 100 degrees C. Proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes, and tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antiserum coupled to horseradish peroxidase and reagents to produce chemiluminescence. The faint band seen at 185 kDa in control lanes was increased by GH treatment in five independent experiments. Insulin produced a similar effect at a concentration of 4 ng/ml, and phosphorylation increased in a dose-related manner in cells treated with higher concentrations of insulin. A prominent approximately 95-kDa band that is probably not the beta subunit of the insulin receptor was also seen in GH-treated cells. The beta subunit of the insulin receptor has similar electrophoretic mobility to the 95-kDa protein, but was not phosphorylated to an extent that allowed detection when insulin was added at concentrations below 400 ng/ml. Phosphorylation of the 185- and 95-kDa bands was evident within 1 min after addition of GH, persisted for at least 30 min, and was equally prominent in sensitive and refractory cells. Antiserum to IRS-1 immunoprecipitated the tyrosine-phosphorylated 185-kDa protein. The data suggest that IRS-1 is a substrate for a GH-activated tyrosine kinase, possibly JAK-2, which may account for the insulin-like effects of GH. The data further suggest that refractoriness to insulin-like stimulation by GH may result from an additional GH-dependent action that is distinct from phosphorylation of IRS-1.