11-BETA-HYDROXYSTEROID DEHYDROGENASE IS AN EXCLUSIVE 11-BETA-REDUCTASE IN PRIMARY CULTURES OF RAT HEPATOCYTES - EFFECT OF PHYSIOCHEMICAL AND HORMONAL MANIPULATIONS

P M JAMIESON, K E CHAPMAN, C R W EDWARDS, J R SECKL

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Abstract

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the conversion of corticosterone to inert 11-dehydrocorticosterone, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11 beta HSD-1) is a bidirectional NADP(H)-dependent enzyme in vitro and is highly expressed in liver, where it is regulated by glucocorticoids, thyroid hormones, estrogen, and GH in vivo. In humans in vivo, enzyme inhibition alters glucose homeostasis, an effect thought to be mediated in the liver. However, detailed investigation of the biology of 11 beta HSD-1 in liver, its function, regulation, and indeed even reaction direction, has been hampered by the lack of clonal hepatic cell lines that express 11 beta HSD-1. Studies of nonhepatic cell lines have suggested that 11 beta HSD-1 is directly regulated by hormones, and transfection of nonhepatic cell lines has shown that reaction direction varies between cell types, possibly reflecting intracellular cosubstrate (NADP(+)/NADPH) ratios or pH.

To investigate reaction direction and gene regulation of 11 beta HSD-1 in hepatocytes, we defined conditions for primary culture of adult rat hepatocytes that maintain high 11 beta HSD-1 messenger RNA expression. In intact primary hepatocytes over a wide range of steroid concentrations (2.5-250 nM), 11 beta-reduction was the predominant reaction direction [33.5 +/- 0.5% conversion of 11-dehydrocorticosterone (25 nM) to corticosterone after 30 min], with undetectable 11 beta-dehydrogenation. However, homogenates of hepatocyte cultures showed plentiful 11 beta-dehydrogenase activity. Treatment of hepatocyte cultures with the metabolic inhibitors sodium azide (5 mM) and KCN (1 mM) altered cellular NADP(+)/NADPH ratios from 0.244 +/- 0.042 in controls to 0.020 +/- 0.001 and 0.152 +/- 0.009, respectively, but had no effect on 11 beta-reductase or 11 beta-dehydrogenase activity. High concentrations of KCN (10 mM) modestly increased 11 beta-reductase activity (32.4 +/- 1.7% to 48.8 +/- 0.5%), whereas 11 beta-dehydrogenation remained at the limit of detection. Manipulation of culture medium pH (6.2-8.0) had no effect on enzyme activity. Dexamethasone (10(-7) M) induced hepatocyte 11 beta-reductase activity from 23.4 +/- 0.7% to 35.5 +/- 1.5% and 11 beta HSD-1 messenger RNA expression (207% rise), an action inhibited by insulin (10(-7) M). GH, estradiol, and T-3 had no effect.

These results demonstrate that 11 beta HSD-1 functions as an 11 beta-reductase in intact rat hepatocytes in culture. The cosubstrate ratio only weakly affects reaction direction. Glucocorticoid and insulin regulation of hepatic 11 beta HSD-1 is directly mediated, but other hormonal controls are either lost in culture or mediated indirectly. This primary hepatocyte culture system will allow investigation of the control of 11 beta-reductase activity and its implications for glucocorticoid-regulated hepatic functions.

Original languageEnglish
Pages (from-to)4754-4761
Number of pages8
JournalEndocrinology
Volume136
Issue number11
Publication statusPublished - Nov 1995

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