An Investigation into the Mechanism of the Lipolytic Action of Growth Hormone in Adipose Tissue of Sheep and Rat

Doris, Rosemarie A (1996) An Investigation into the Mechanism of the Lipolytic Action of Growth Hormone in Adipose Tissue of Sheep and Rat. PhD thesis, University of Glasgow.

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The studies presented in this thesis were designed to evalulate the mechanism of action of the lipolytic effects of growth hormone in both sheep and rats. The initial study was carried out with virgin rats and involved manipulating levels of endogenous growth hormone using a specific antiserum to growth hormone (anti-rGH). The results showed that lowering levels of endogenous growth hormone resulted in an increase in the inhibition of isoprenaline-stimulated lipolysis, of isolated adipocytes, by submaximal concentrations of both PIA and PGE1. There was also an increase in the amount of the inhibitory G-protein Gi2 and these effects were reversed upon replacement of growth hormone. Therefore growth hormone in vivo, specifically suppresses the expression of alpha-Gi2, in rat adipocytes leading to decreased sensitivity to anti-lipolytic agonists. Further studies carried out with lactating and litter removed rats, again involving manipulation of endogenous growth hormone. Chronic treatment of lactating rats with growth hormone had no effect on maximum response or sensitivity to PIA, however, a diminished ability of antilipolytic agents after growth hormone treatment was seen in rats in which lactation was prematurely terminated but did not appear to involve a change in either adenosine receptor number or the amount of Gi1 plus Gi2. Therefore, it would appear that Gi in lactating rats in response to growth hormone is subject to covalent modification. That the Gi-mediated antilipolytic system is a major target of growth hormone action was investigated further by treating sheep with recombinant bovine growth hormone (Monsanto) at 10 mg per day for 7 days in vivo. Treatment of sheep with the hormone decreased the maximum inhibition of isoprenaline-stimulated lipolysis. This effect of growth hormone was not accompanied by any discernable change in the number of adenosine receptors or amounts of the various isoforms of Gi. Therefore these studies also suggested that a decrease in Gi activity was occuring. Further investigations were carried out using sheep adipose tissue in vitro. Sheep adipose tissue explants were preincubated in culture for 24 hours with no hormones and then growth hormone was added for the next 24 hours. As for the in vivo experiments, growth hormone treatment resulted in a decreased maximum response to PIA. Again there was no change in either adenosine receptor number or amounts of Gi. Further experiments were carried using this tissue culture system using a range of inhibitors to elucidate the mechanism of growth hormone action. As alphaGi2 can be phosphorylated by at least two kinases the effect of protein kinase and phosphatase inhibitors were tested in the tissue culture system. Addition of H7, a protein serine kinase inhibitor, diminished the effects of growth hormone while the protein-serine phosphatase inhibitor, okadaic acid mimicked the effect of growth hormone, and therefore suggested that the effect of growth hormone involves protein serine phosphorylation. Further experiments with the phorbol ester PMA suggested involvement of protein kinase C isoforms that can be down- regulated by PMA in growth hormone actions, but use of D609, a phosphatidylcholine phospholipase C inhibitor suggested that diacylglycerol production via this pathway is not involved. In time course experiments the maximum effect of growth hormone was reached after 24 hours demonstating that growth hormone is not simply activating a phosphorylation cascade. These results were supported by data from experiments with actinomycin D, an inhibitor of gene transcription, which was shown to knock out the effects of growth hormone. More detailed studies were carried out to elucidate the nature of the altered activity of Gj. To do this ADP-ribosylation experiments were carried out with adipocyte membranes using Pertussis toxin and PIA to determine if growth hormone altered the ability of the adenosine receptor to cause dissociation of Gi. The results obtained show that growth hormone treatment does not affect receptor coupling to Gi. Gi activity was further assessed in adipocyte membranes using the diterpene forskolin or isoprenaline to activate adenylate cyclase, and p[NH]ppG or PIA to inhibit this activation through a Gi-mediated response. The results from these experiments indicated that functional Gi was detected in membranes from control sheep adipose tissue but not in growth hormone treated tissue, indicating that the attenuation of the antilipolytic effect of adenosine may thus be due to changes in the interaction of Gi with adenylate cyclase. Thus growth hormone decreases signalling through Gi by at least two mechanisms, altering both the amount of one isoform of this key protein and also the activity of the protein. Effects of growth hormone on the system vary with physiological state and are species specific.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Miles Houslay
Keywords: Animal sciences
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-74902
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:26
Last Modified: 27 Sep 2019 15:26

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