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

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the conve rsion of corticosterone to inert 11-dehydrocorticosterone, thus regula ting glucocorticoid access to intracellular receptors. The type 1 isof orm (11 beta HSD-1) is a bidirectional NADP(H)-dependent enzyme in vit ro and is highly expressed in liver, where it is regulated by glucocor ticoids, thyroid hormones, estrogen, and GH in vivo. In humans in vivo , enzyme inhibition alters glucose homeostasis, an effect thought to b e mediated in the liver. However, detailed investigation of the biolog y of 11 beta HSD-1 in liver, its function, regulation, and indeed even reaction direction, has been hampered by the lack of clonal hepatic c ell 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 dire ction varies between cell types, possibly reflecting intracellular cos ubstrate (NADP(+)/NADPH) ratios or pH. To investigate reaction directi on and gene regulation of 11 beta HSD-1 in hepatocytes, we defined con ditions for primary culture of adult rat hepatocytes that maintain hig h 11 beta HSD-1 messenger RNA expression. In intact primary hepatocyte s over a wide range of steroid concentrations (2.5-250 nM), 11 beta-re duction was the predominant reaction direction [33.5 +/- 0.5% conversi on of 11-dehydrocorticosterone (25 nM) to corticosterone after 30 min] , with undetectable 11 beta-dehydrogenation. However, homogenates of h epatocyte cultures showed plentiful 11 beta-dehydrogenase activity. Tr eatment of hepatocyte cultures with the metabolic inhibitors sodium az ide (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, res pectively, but had no effect on 11 beta-reductase or 11 beta-dehydroge nase activity. High concentrations of KCN (10 mM) modestly increased 1 1 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. Dexa methasone (10(-7) M) induced hepatocyte 11 beta-reductase activity fro m 23.4 +/- 0.7% to 35.5 +/- 1.5% and 11 beta HSD-1 messenger RNA expre ssion (207% rise), an action inhibited by insulin (10(-7) M). GH, estr adiol, and T-3 had no effect. These results demonstrate that 11 beta H SD-1 functions as an 11 beta-reductase in intact rat hepatocytes in cu lture. The cosubstrate ratio only weakly affects reaction direction. G lucocorticoid and insulin regulation of hepatic 11 beta HSD-1 is direc tly mediated, but other hormonal controls are either lost in culture o r mediated indirectly. This primary hepatocyte culture system will all ow investigation of the control of 11 beta-reductase activity and its implications for glucocorticoid-regulated hepatic functions.