Norie Murayama

Cytochrome P450 in livers of diabetic rats: regulation by growth hormone and insulin.

The effects of pituitary and pancreatic hormones on the change in hepatic cytochrome P450s were studied in alloxan- or streptozotocin-induced male rats. In two major sex-specific forms, P450-male and P450(6 beta-1), the former was decreased in chronic (5 week) diabetes to only less than one-third of controls and the latter was also reduced in early (1 week) diabetes. In contrast, a main phenobarbital-inducible form, P450b, was enhanced 25- to 30-fold in these diabetic rats. 3-Methylcholanthrene-inducible P448H was also elevated 3-fold in alloxan-induced diabetes. These changes in hepatic contents of P450-male, P450-6 beta-1, and P450b, which are under the regulation of pituitary growth hormone, associated well with the reported results of time-dependent changes in growth hormone levels in diabetes (G.S. Tannenbaum (1981) Endocrinology 108, 76-82), suggesting that the change in growth hormone level is a factor responsible for alterations in hepatic cytochrome P450s. Normalizing effects of insulin on these forms were also studied. Treatment of diabetic rats with insulin reversed the decreased amounts of both P450-male protein and mRNA. Insulin also normalized hepatic contents of P450b, P4506 beta-1, and P448H. However, the treatment of hypophysectomized rats with insulin had no effect, and treatment of diabetic rats with growth hormone or a suppressing agent of somatostatin, cysteamine, showed trivial effects on P450-male and P450b. These results suggest that insulin does not act directly as a substitute of growth hormone, but exerts its effect indirectly through the normalization of a growth hormone-mediated process(es) in diabetic rats.

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2

Stereoselective 4′‐hydroxylations of R‐(−)‐mephenytoin and S‐(+)‐mephenytoin and 3′‐hydroxylation of R‐(‐)‐hexobarbital and S‐(+)‐hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P‐450 human‐2 assessed by Western blots was correlated to microsomal S‐(+)‐mephenytoin 4′‐hydroxylation, R‐(−)‐hexobarbital 3′ α‐hydroxylation, and S‐(+)‐hexobarbital 3′ ß‐hydroxylation, and was less correlated to R‐(−)‐mephenytoin 4′‐hydroxylation, R‐(−)‐hexobarbital 3′ ß‐hydroxylation, and S‐(+)‐hexobarbital 3′ α‐hydroxylation. Antibodies raised against P‐450 human‐2 inhibited microsomal S‐(+)‐mephenytoin 4′‐hydroxylation efficiently but was less efficient on R‐(−) ‐mephenytoin 4′‐hydroxylation in extensive metabolizers and on 4′‐hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R‐(−)‐hexobarbital 3′ α‐hydroxylation and S‐(+)‐hexobarbital 3′ ß‐hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4′‐hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P‐450 human‐2 is a typical S‐(+)‐mephenytoin 4′‐hydroxylase and a major hexobarbital 3′‐hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P‐450 human‐2 complementary dexoyribonucleic acid‐derived protein in yeast microscomes.

Thyroid hormone suppression of hepatic levels of phenobarbital-inducible P-450b AND P-450e and other neonatal P-450S in hypophysectomized rats

Mechanism of developmental suppression of cytochrome P-450 (P-450) in rat livers was studied using Western blots. The contents of phenobarbital (PB)-inducible P-450b and P-450e, expressed constitutively in livers, were higher in neonate than in adult rats. The contents were also 10 approximately 50 fold higher in hypophysectomized than in intact adult male rats. Administration of L-triiodothyronine (T3, 50 micrograms/kg) or human growth hormone (4 U/kg) reversed almost completely the increased amounts of P-450b and P-450e. T3-induced suppression was also observed on two other neonatal P-450s (P-450 6 beta-1 and P-448-H), which are expressed in neonatal periods in livers. The postnatal developmental profiles of hepatic P-450b were correlated inversely with that of serum free T3 level in rats reported (Walker et al. (1980) Pediat. Res. 14, 249). These results suggest, in addition to pituitary growth hormone (Yamazoe et al. (1987) J. Biol. Chem. 262, 7423), the possible involvement of T3 on the suppressive regulation of PB-inducible and other neonatal P-450s.

Alteration of hepatic drug metabolizing activities and contents of cytochrome P-450 isozymes by neonatal monosodium glutamate treatment.

By the treatment of newborn male rats with monosodium glutamate (MSG), microsomal benzo[a]pyrene hydroxylation, propoxycoumarin O-depropylation, and testosterone (T) 6 beta- and 2 beta-hydroxylations in the adult rats were decreased significantly, while microsomal aniline and T 7 alpha-hydroxylations were increased. However, the treatment of newborn female rats did not significantly alter any of the drug-metabolizing activities examined, except that T 6 beta-hydroxylation and androstenedione formation were slightly increased. The hepatic contents of male-specific cyt. P-450, P-450-male and P-4506 beta, which show high catalytic activities on respective T 16 alpha/2 alpha-, and T 6 beta/2 beta-hydroxylations, decreased in MSG-treated male rats. The level of the female specific enzyme, P-450-female, slightly decreased in the MSG-treated female rats, whereas higher phenobarbital (PB)-induction of PB-inducible isozymes, P-450b and P-450e, was observed in MSG-treated than in control female rats. These results are consistent with the idea that disruption of a pulsatile secretion of growth hormone, which is induced by the neonatal MSG treatment, leads to changes in drug metabolizing activities through the alteration of the levels of sex-specific cyt. P-450s, but also indicate that MSG-treated rats are not an animal model equivalent to hypophysectomized rats.

Suppression of clofibrate-induction of peroxisomal and microsomal fatty acid-oxidizing enzymes by growth hormone and thyroid hormone in primary cultures of rat hepatocytes

Using primary cultures of rat hepatocytes on a matri-gel, effects of peroxisome proliferator and omega-hydroxydodecanoic acid on cellular levels of acyl-CoA oxidase and CYP4A have been studied to determine the hormonal influence in serum-free media. Peroxisomal acyl-CoA oxidation, microsomal CYP4A content and laurate omega-hydroxylation were increased in rat hepatocytes by the addition of 100 microM clofibrate or Wy14,643 for two days. omega-Hydroxydodecanoic acid (100 microM) also increased peroxisomal acyl-CoA oxidation, but had no clear effect on microsomal CYP4A level and laurate omega-hydroxylation. CYP4A-mediated laurate omega-hydroxylation in hepatocytes was suppressed by the addition of pituitary growth hormone (0.05 mU/ml), but was not altered by the addition of triiodothyronine (30 nM). In contrast, clofibrate-mediated induction of acyl-CoA oxidase activity was decreased by the addition of either one of the hormones in hepatocytes. Suppression by those hormones was also observed with omega-hydroxydodecanoic acid-mediated induction of acyl-CoA oxidase activity. These results indicate the possibility that GH and T3 exert the suppressive effects on peroxisomal acyl-CoA oxidation through plural mechanisms with and without the alteration of CYP4A levels in livers.

KP-10, a novel protein kinase C substrate in intact mouse epidermal cells, is phosphorylated by novel protein kinase Cη and/or ξ

Abstract Recently this group found an endogenous substrate protein for Ca 2+ -independent novel protein kinase C (nPKC), i.e. KP-10 (pI 4.7/25,500 M r ), in primary cultured mouse epidermal cells [NIshikawa, K. et al. (1992) Cell. Signal. 4 , 757–776]. In the present study, the nPKC isozymes which phosphorylate KP-10 in these cells were determined. Western blot analysis revealed that PKCα, η and ξ were present in the epidermal cell 105,000 g supernatants and that the content of PKCξ was much higher than those of PKCα and η. Neither PKCβ, δ nor ϵ was detected in the 105,000 g supernatants. Phosphatidylserine and phorbol 12-myrisate 13-acetate (PMA)-dependent KP-10 phosphorylating activity was immunoprecipitated by anti-PKCη and ξ antibodies, but not by antiPKCα antibody. These results suggest that PKCη and/or ξ phosphorylate KP-10 and play pivotal roles in intracellular signal pathways in intact epidermal cells.

Decrease in the Metabolic Activating Capacities of Arylamines in Livers Bearing Hyperplastic Nodules: Association with the Selective Changes in Hepatic P‐450 Isozymes

The mechanism of the alteration in carcinogenic arylamine‐activating capacities in livers bearing pre‐neoplastic (or hyperplastic) nodules induced by the Solt‐Farber protocol was investigated in relation to the changes in hepatic cytochrome P‐450 isozymes. In the Salmonella mutagenesis test, the numbers of revertants induced with 2‐amino‐3‐methylimidazo[4,5‐f]quinoline and 2‐aminofluorene were significantly lower in the presence of microsomes of nodule‐bearing livers than of control livers. A similar tendency was also observed with another heterocyclic arylamine, 2‐amino‐6‐methyldipyrido‐[1,2‐a:3′,2′‐d]imidazole. In Western blots using specific antibodies against 5 different forms of cytochrome P‐450, hepatic contents of P‐450‐male (a main constitutive form) and P‐450b (a main phenobarbital‐inducible form) were decreased in the livers with hyperplastic nodules to 63% and 35% of the corresponding controls, while no significant decrease was observed in the contents of P‐448‐H (a main 3‐methylcholanthrene‐inducible form), P‐4506β‐1 (testosterone 6β‐hydroxylase) and P‐450e (a phenobarbital‐inducible form). In accordance with the reduction in P‐450‐male, capacities for microsomal 16α‐ and 2α‐hydroxylations, but not 6β‐hydroxylation, of testosterone were decreased in the livers with hyperplastic nodules. Although P‐448‐H has higher capacities for the activation of arylamines than does P‐450‐male, the hepatic content of P‐450‐male is more than ten‐fold higher than that of P‐448‐H in both normal and nodule‐bearing livers. These results indicate that the selective decrease in hepatic content of P‐450‐male is likely to be a main cause of the decrease in arylamine metabolic activating capacities in livers with hyperplastic nodules.