Fat tissue produces a variety of secreted proteins (adipocytokines) with important roles in metabolism. We isolated a newly identified adipocytokine, visfatin, that is highly enriched in the visceral fat of both humans and mice and whose expression level in plasma increases during the development of obesity. Visfatin corresponds to a protein identified previously as pre-B cell colony-enhancing factor (PBEF), a 52-kilodalton cytokine expressed in lymphocytes. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice. Mice heterozygous for a targeted mutation in the visfatin gene had modestly higher levels of plasma glucose relative to wild-type littermates. Surprisingly, visfatin binds to and activates the insulin receptor. Further study of visfatin's physiological role may lead to new insights into glucose homeostasis and/or new therapies for metabolic disorders such as diabetes.

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Bisphenol A (4,4Ј-isopropylidenediphenol, BPA) is widely used in the chemical industry in the manufacturing of epoxy, polycarbonate, and polyester-styrene resins, and trace levels of BPA leach from polycarbonate plasticware and resins used for food packaging materials. This compound is suspected to be an endocrine disrupter. 2) Additionally, it has been reported that exposure of male rats and mice to BPA may be associated with increased incidence of cancers of the hematopoietic system and that high doses of BPA cause reproductive toxicity and affect cellular development in these species. 3,4) However, the effect of BPA in vivo and its mechanism of action are still unclear.Cytochrome P450 (CYP) comprises a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals including drugs, carcinogens, and steroids including sex hormones. [5][6][7] In the rat, BPA is metabolized to DNA-reactive bisphenol-o-quinone through 5-hydroxybisphenol and bisphenol semiquinone, 4) and the formation of the DNA adducts in a microsomal activation system is markedly decreased by CYP inhibitors, 8) suggesting that CYPs are closely associated with the metabolism and toxicity of BPA in rats. However, there are few reports on BPA metabolism in humans and on the human CYP(s) that metabolizes BPA.Recently, we reported that BPA inhibits human hepatic CYP-mediated drug-metabolizing activities including aminopyrine N-demethylation, especially by CYP2C8 and CYP2C19. 9) Additionally, Hanioka et al. reported that the administration of BPA to male rats decreased the catalytic activities and protein levels of male-specific CYP isoforms (such as CYP2C11 and CYP3A2) in rat liver microsomes, 10) and that BPA inhibited the drug-metabolizing activities of rat hepatic CYP1A2, CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1, and CYP3A2. 11) CYP17 is found in the endoplasmic reticulum of the adrenal cortex and gonads, and mediates both 17a-hydroxylase and 17,20-lyase reactions of pregnenolone and progesterone, and is thus involved in the biosynthesis of glucocorticoids and sex hormones. 12) Therefore endocrine disrupters (sex hormone-like compounds) may affect the activity of CYP17. Recently, we reported that the aminopyrine Ndemethylase activity of CYP17 was comparable with that of CYP3A4, a dominant CYP in human liver. 13) This paper describes an in vitro investigation of BPA metabolism by human hepatic CYPs and steroidogenic CYP17 based on the disappearance rate of parent compounds from an incubation mixture and the inhibitory effect of BPA on the progesterone 17a-hydroxylase activity of CYP17. MATERIALS AND METHODS MaterialsBisphenol A was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Progesterone, 17a-hydroxyprogesterone, and estrone were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Human NADPH-cytochrome P450 reductase (fp 2 ) and hydrocortisone acetate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of the highest purity commercially available. Expression o...

The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

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ABSTRACT:The amino acid residues affecting the substrate specificity of human cytochrome P450 CYP2C9 and CYP2C19 for their metabolic activities were investigated using chimeras and mutant enzymes, which were constructed by replacing the corresponding residues. Although CYP2C19 showed nearly the same tolbutamide hydroxylase activity as CYP2C9, the activities for the CYP2C19(H99I) mutant and the chimeras that replaced residues 1-212 were much lower than those for CYP2C19. The activities of the CYP2C19(H99I) mutant and the chimeras that replaced residues 228-340 were lower than those for CYP2C19 toward S-mephenytoin, aminopyrine, and testosterone. These results suggest that residues in substrate recognition site (SRS) 3 and 4 are important for the substrate specificity, whereas His99 is important in the substrate binding of

Endocrine disrupters are exogenous substances which can influence endocrine function in humans and animals, and a number of these chemicals, termed "xenoestrogens", have estrogenic activity. Artificial xenoestrogens include the alkylphenols, nonylphenol and octylphenol, and bisphenol A. 2,3) Alkylphenol polyethoxylates are widely used as detergents, emulsifiers, and wetting agents in industry. In the environment, nonylphenol occurs predominantly as a degradation product of nonylphenol ethoxylate. 4)Cytochrome P450s (CYP) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals including drugs and carcinogens, and steroids including sex hormones. 5-7) Lee et al. 8) reported that the metabolism of nonylphenol by rat hepatic microsomes was induced by phenobarbital; the activity was inhibited by 4-amino-2,6-dinitro-1-t-butylxylene, a specific CYP2B inhibitor, and that human CYP2B6 metabolized nonylphenol. Additionally, nonylphenol inhibited progesterone 6b-hydroxylase activity and 7-ethoxyresorufin O-deethylase activity by hepatic microsomes from dexamethazone-and b-naphthoflavone-treated rats, respectively. 9,10) However, there are no reports describing the effect of nonylphenol on human hepatic CYP-mediated drug-metabolizing activity.CYP17 is found in the endoplasmic reticulum of the adrenal cortex and gonads, and mediates both 17a-hydroxylase and 17,20-lyase reactions of pregnenolone and progesterone, thus being involved in the biosynthesis of glucocorticoids and sex hormones.11) Therefore, endocrine disrupters (sex hormone-like compounds) may affect the activity of CYP17.Recently the heterologous expression systems for human CYPs have been developed in Escherichia coli, in the yeast Saccharomyces cerevisiae, and in human Hep G2 cells, and these genetic technologies have been shown to be very useful in determining the metabolism of xenobiotics by different CYPs. [12][13][14] We reported that the aminopyrine N-demethylation process is metabolized by most of the human hepatic CYPs measured, 15) indicating that this is useful to investigate the effect of inhibitors on human hepatic CYPs.16) Additionally, bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylation by CYP2C8, a mixed-type inhibition of S-mephenytoin 4Ј-hydroxylation by CYP2C19, and a competitive-type inhibition of progesterone 17a-hydroxylation by CYP17 with K i values of 97, 113, and 71 mM, respectively. 16,17) In the present study, we investigated the inhibitory effect of nonylphenol on hepatic CYPs and steroidogenic CYP17 using the cDNA-expressed P450s. MATERIALS AND METHODSMaterials n-Nonylphenol, aminopyrine, hydrocortisone acetate and human NADPH-cytochrome P450 reductase (fp 2 ) were purchased from Wako Pure Chemical Industries, Ltd. Estrone, progesterone and 17a-hydroxyprogesterone were obtained from Sigma Chemical Co. 4Ј-Hydroxydiclofenac, S-mephenytoin and 4Ј-hydroxymephenytoin were purchased from Sumika Chemical Analysis Service, Ltd., and diclofenac was from Ultrafi...