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Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat Adipocytes by Selectively Shifting Giα2 to Lower Density Membrane Fractions*

Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat... AbstractGH, 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 μg/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[γ -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 × g or a less dense membrane pellet sedimented at 100K × 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 × g and 100K × g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Giα2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gsα 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 Giα2 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 Gsα or 5′-nucleotidase. Colchicine (100 μm) blocked the GH+Dex-dependent shift of Giα2 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 Giα2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat Adipocytes by Selectively Shifting Giα2 to Lower Density Membrane Fractions*

Yip, Rupert Guk-Chor; Goodman, H. Maurice
Endocrinology , Volume 140 (3) – Mar 1, 1999
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References (61)

Publisher
Oxford University Press
Copyright
Copyright © 1999 by The Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
DOI
10.1210/endo.140.3.6580
pmid
10067847
Publisher site
See Article on Publisher Site

Abstract

AbstractGH, 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 μg/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[γ -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 × g or a less dense membrane pellet sedimented at 100K × 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 × g and 100K × g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Giα2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gsα 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 Giα2 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 Gsα or 5′-nucleotidase. Colchicine (100 μm) blocked the GH+Dex-dependent shift of Giα2 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 Giα2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Journal

Endocrinology – Oxford University Press

Published: Mar 1, 1999

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