The Role of Insulin and Growth Hormone in the Regulation of Hepatic Steroid Metabolism

The hepatic monooxygenase system catalyses the oxidative metabolism of both endogenous steroids and xenobiotics, and consists of three main components : cytochrome P-450, NADPH cytochrome P-450 reductase and lipid (reviewed by Skett 1987). Multiple pathways exist for cytochrome P-450-catalysed biotransformation in the liver, and these pathways are modulated by a large number of environmental and hormonal factors (reviewed by Mathis et al 1988). In particular, growth hormone plays a central role in the regulation of hepatic drug and steroid metabolism (Waxman 1988). Sex differences in hepatic biotransformations have been widely documented in the rat. Neonatal androgenic imprinting of cytochrome P-450 is mediated via the hypothalamo-pituitary-liver axis (Skett and Gustafsson 1979) by modulations of the GH secretory pattern (Eden 1979). Thus pulsatile GH secretion in male rats results in the predominance of oxidative drug and steroid metabolism, whilst continuous GH secretion in the female results in higher reductase activity (Mode et al 1982). Alterations of GH secretion towards a more continuous pattern have been correlated with the feminisation of hepatic steroid metabolism (Mode et al 1981). In addition, insulin has been documented to exert a "masculinisig" influence on this system (Skett 1986). The majority of studies carried out in this field have used in vivo animal models (Kramer et al 1979), isolated perfused livers (Schalch et al 1979), and liver microsomes prepared from pretreated rats (Gustafsson and Stenberg 1974). However each of these models suffer from a number of disadvantages, and their use is now becoming limited and is being largely replaced by cultured hepatocytes. This is an important technique in that it is a relatively physiological preparation, and allows the study of the effect of individual hormones in this system without the interactions caused by administration of exogenous hormones to the intact animal. The primary aim of this project was to determine whether or not GH produced its characteristic feminising effect on steroid metabolism in isolated hepatocytes, and to correlate the results obtained with the situation in vivo. Incubation of hepatocytes with GH caused no significant changes in either hepatic steroid metabolism or intracellular cyclic AMP levels. Previous reports have suggested that the lack of effects of GH in vitro may be due to the absence of additional hormonal factors that are involved in the intact animal (Colby 1980). We therefore initiated studies to determine the interactions of GH with insulin, dexamethasone and thyroxine, each of which is known to influence hepatic drug and steroid metabolism (Skett 1987). These effects are substantiated by studies in adrenalectomised, thyroidectomised or diabetic animals, where removal of a particular hormone markedly altered drug metabolism (Kato and Gillette 1965). Insulin stimulated steroid metabolism in a non-specific manner, as has been reported previously (Hussin and Skett 1987), an effect that was potently antagonised by GH. Pyerin and coworkers, in a series of articles, have documented the phosphorylation of cytochrome P-450 by both cyclic AMP-dependent and independent protein kinases concomitant with the denaturation / inactivation of cytochrome P-450 (Pyerin et al 1987). A phosphorylation mechanism appears to be involved in the action of insulin to stimulate steroid metabolism, and so altered phosphorylation of the cytochrome P-450 protein may constitute this effect. The interaction observed with GH may also involve a phosphorylation mechanism, as has been reported elsewhere (Yamada et al 1987). Dexamethasone stimulated steroid metabolism in a non-specific manner, and this effect was again antagonised by GH. The effect of dexamethasone over short-term incubations may represent a glucocorticoid effect, while the effects of long term incubation probably represents induction of cytochrome P-450PCN (gene IIIA1) (Schuetz et al 1984). The interaction with GH may involve its action to suppress cytochrome P-450 expression (Yamazoe et al 1987, Waxman et al 1988a). Incubation with thyroxine exhibited dose-related and sex-dependent effects in the short incubation studies, while no significant effects were observed over the longer incubations, except at 72h of incubation. Further incubation with GH decreased steroid metabolism markedly in the former, but enzyme activity was maintained at control levels in cultured hepatocytes over 72hr of incubation. GH modified the effects of insulin, dexamethasone and thyroxine in a non-sex-specific manner. Incubation of hepatocytes with GH in the presence of both dexamethasone and thyroxine, however, resulted in an increase in female-specific activities together with a decrease in male-specific activities, which is characteristic of the feminisation of steroid metabolism