Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat Adipocytes by Selectively Shifting Gi 2 to Lower Density Membrane Fractions

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Abstract
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 pro- duction 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 mg/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 GH1Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant in- creases in cAMP were discernible in homogenates of GH1Dex-treated tissues, RP-cAMPS (RP-adenosine 3959-phosphothioate), a competi- tive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH1Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32 P from (g-32P)ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH1Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH1Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K 3 g or a less dense membrane pellet sedimented at 100K 3 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 3 g and 100K 3 g were solubilized and subjected to PAGE and Western analysis. GH1Dex decreased Gia2 by 44% (P , 0.02) in the 16K pellets and increased it by 52% (P , 0.01) in the 100K pellets. Gsa in the 16K pellet was unaffected by GH1Dex and was decreased (P , 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH1Dex-dependent shift of Gia2 to less dense fractions as determined by both Western analysis and (32P)NAD ribosylation cat- alyzed by pertussis toxin. No such changes were seen in the distri- bution of Gsa or 59-nucleotidase. Colchicine (100 mM) blocked the GH1Dex-dependent shift of Gia2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH1Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gia2, GH1Dex relieves some inhibition of cAMP production and conse- quently increases lipolysis. (Endocrinology 140: 1219 -1227, 1999)

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