11β-Hydroxysteroid dehydrogenase is an exclusive 11β-reductase in primary cultures of rat hepatocytes: Effect of physicochemical and hormonal manipulations

Abstract

11β-Hydroxysteroid dehydrogenase (11βHSD) catalyzes the conversion of corticosterone to inert 11-dehydrocorticosterone, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11βHSD-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 11βHSD-1 in liver, its function, regulation, and indeed even reaction direction, has been hampered by the lack of clonal hepatic cell lines that express 11βHSD-1. Studies of nonhepatic cell lines have suggested that 11βHSD-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 11βHSD-1 in hepatocytes, we defined conditions for primary culture of adult rat hepatocytes that maintain high 11βHSD-1 messenger RNA expression. In intact primary hepatocytes over a wide range of steroid concentrations (2.5-250 nM), 11β-reduction was the predominant reaction direction [33.5 ± 0.5% conversion of 11-dehydrocorticosterone (25 nM) to corticosterone after 30 min], with undetectable 11β-dehydrogenation. However, homogenates of hepatocyte cultures showed plentiful 11β- 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 11β-reductase or 11β-dehydrogenase activity. High concentrations of KCN (10 mM) modestly increased 11β- reductase activity (32.1 ± 1.7% to 48.8 ± 0.5%), whereas 11β- 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 11β-reductase activity from 23.4 ± 0.7% to 35.5 ± 1.5% and 11βHSD-1 messenger RNA expression (207% rise), an action inhibited by insulin (10-7 M). GH, estradiol, and T3 had no effect. These results demonstrate that 11βHSD-1 functions as an 11β-reductase in intact rat hepatocytes in culture. The cosubstrate ratio only weakly affects reaction direction. Glucocorticoid and insulin regulation of hepatic 11βHSD-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 11β-reductase activity and its implications for glucocorticoid-regulated hepatic functions.