20b-Hydroxysteroid Dehydrogenase Catalyzes Ketone- Reduction of Acetohexamide, an Oral Antidiabetic Drug, in Liver Microsomes of Adult Male Rats

We examined the catalytic properties and physiological function of an enzyme responsible for the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of adult male rats. Progesterone, 17a-hydroxyprogesterone, cortisone and cortisol, which have a ketone group at 20-position of C21-steroids, were potent inhibitors for ketone-reduction of acetohexamide in liver microsomes of adult male rats. Progesterone was also found to inhibit competitively the ketone-reduction of acetohexamide, suggesting that the ketone-reduction of acetohexamide and progesterone is catalyzed by the same enzyme. When progesterone was used as a substrate, 20b-hydroxysteroid dehydrogenase present in liver microsomes of adult rats, such as acetohexamide reductase, exhibited a male-specific and androgen-dependent activity. Furthermore, a significant correlation was observed between the activities of 20b-hydroxysteroid dehydrogenase and acetohexamide reductase in liver microsomes of individual male rats at various ages. Based on all results, we conclude that 20bhydroxysteroid dehydrogenase catalyzes the ketone-reduction of acetohexamide in liver microsomes of adult male rats. Acetohexamide, 4-acetyl-N-(cyclohexylcarbamoyl)benzenesulfonamide, is an oral antidiabetic drug that has a ketone group within its chemical structure. This drug is mainly reduced to the corresponding alcohol metabolite, (2)hydroxyhexamide, through enzymatic system in humans and animals (McMahon et al., 1965; Imamura et al., 1989). We have demonstrated that the activity of acetohexamide-reducing enzyme (acetohexamide reductase) in liver microsomes of adult rats is much higher in the males than in the females, and is regulated by androgens (Imamura et al., 1987, 1993). Our previous paper (Imamura et al., 1993) has also shown that even in male rats, the activity of the microsomal acetohexamide reductase is not detectable until 4 wk of age after birth, although it markedly increases during pubertal period to reach the adult level. It is most likely that a male-specific and androgen-dependent enzyme, which is responsible for biosynthesis or metabolism of endogenous carbonyl compounds, has the ability to reduce acetohexamide. 20b-HSD (EC 1.1.1.53) is known to catalyze the stereoselective reduction of C21-steroids with a ketone group at 20position such as progesterone (4-pregnene-3,20-dione) and 17a-hydroxyprogesterone (4-pregnene-17a-ol-3,20-dione). For example, 4-pregnene-17a,20b-diol-3-one (17a,20b-dihydroxy-4-pregnen-3-one) is produced from 17a-hydroxyprogesterone by 20b-HSD (Nakajin et al., 1988, 1989). Interestingly, 4-pregnene-17a,20b-diol-3-one has been identified as maturation-inducing hormone in oocytes of several fish species (Nagahama and Adachi, 1985; Young et al., 1986; Nagahama, 1997), and the detailed molecular mechanisms of oocyte maturation induced by this steroid have been proposed (Nagahama, 1997). These reports demonstrate that in fish species, 20b-HSD plays an important role in the induction of oocyte maturation. Furthermore, 20b-hydroxy-C21-steroids have been found in various organs of mammalian species (Tanaka et al., 1992). However, the physiological function of 20b-HSD present in various organs of mammalian species is poorly understood. Recently, 20b-HSD present in liver microsomes of adult rats, as well as acetohexamide reductase present in liver microsomes of adult rats described above, has been reported to exhibit a male-specific activity (Apanovitch et al., 1992; Apanovitch and Walz, 1996). In our study, we describe evidence that the ketone-reduction of acetohexamide in liver microsomes of adult male rats is catalyzed by 20b-HSD. Materials and Methods Materials. Acetohexamide was a gift from Shionogi Co. (Osaka, Japan). Hydroxyhexamide was synthesized from acetohexamide according to the method of Girgis-Takla and Chroneos (1979). ProgesReceived for publication March 26, 1998. ABBREVIATIONS: 20aand 20b-HSD, 20aand 20bhydroxysteroid dehydrogenase; CYP, cytochrome P450; ANOVA, analysis of variance; HPLC, high-performance liquid chromatography. 0022-3565/98/2872-0504$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:504–507, 1998 504 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from terone (4-pregnene-3,20-dione), 4-pregnene-20b-ol-3-one and 4-pregnene-20a-ol-3-one were purchased from Sigma Chemical Co. (St. Louis, MO). Testosterone propionate was obtained from Nacalai Tesque (Kyoto, Japan). Other steroids, which were used as inhibitors, were purchased from Sigma. NADP, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were purchased from Oriental Yeast Co. (Tokyo, Japan). S-Warfarin was obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). All other chemicals were of