Katsumi Toda

Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Congenitally defective aldosterone biosynthesis in humans: The involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450.

Two cDNA clones for human placental aromatase P‐450 (P‐450AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5′‐untranslated stretch and a 1336 bp 3′‐noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3′‐noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36–2355) except for one nucleotide substitution. The 3′‐noncoding region of this shorter cDNA is only 846 bp in length, but a poly(A) tract is also attached to its 3′‐terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non‐overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P‐450AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

Fenofibrate, a ligand for PPARα, inhibits aromatase cytochrome P450 expression in the ovary of mouse

Peroxisome proliferator-activated receptors (PPARs) play important roles in the metabolic regulation of lipids including steroids. In this study, we investigated whether fenofibrate, a ligand for PPARα, could influence estrogen synthesis in vivo in the ovary of mice. As reported, chronic treatment of C57BL6/J female mice with various amounts of fenofibrate as a diet reduced the serum triglycerides level and induced hepatomegaly in a dose-dependent manner. Northern blot analyses using hepatic RNA confirmed the induction of classical PPARα-target genes including acyl-CoA oxidase and lipoprotein lipase. The analyses using ovarian RNA revealed the suppression of gene expression for enzymes involved in steroidogenesis including CYP11A, CYP19, steroidogenic acute regulatory protein, and HDL receptor, but the CYP17 expression was evidently induced. Consistent with the suppression of CYP19 mRNA expression, the aromatase activity in the ovary was dose-dependently inhibited, resulting in significant decreases in the uterine size and bone mineral density. When PPARα null mice were treated with dietary fenofibrate, neither hepatomegaly nor inhibition of ovarian aromatase activity was observed, rather the activity was enhanced. These results demonstrate that fenofibrate inhibits ovarian estrogen synthesis by suppressing the mRNA expressions and that functional PPARα is indispensable for the inhibitory action of the agent in vivo.

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.

Pitavastatin ameliorates severe hepatic steatosis in aromatase-deficient (Ar-/-) mice

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Impaired hepatic FA β-oxidation in peroxisomes, microsomes, and mitochondria results in progression of massive hepatic steatosis in estrogen deficiency. This impairment, although latent, is potentially serious: About 3% of the general population in the United States is now suffering from nonalcoholic steatohepatitis associated with obesity and hyperlipidemia. Therefore, in the present study we tried to restore impaired hepatic FA β-oxidation by administering a novel statin, pitavastatin, to aromatase-deficient (Ar−/−) mice defective in intrinsic estrogen synthesis. Northern blot analysis of Ar−/− mice liver revealed a significant restoration of mRNA expression of essential enzymes involved in FA β-oxidation such as very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase. Severe hepatic steatosis observed in Ar−/− mice substantially regressed. Consistent findings were obtained in the in vitro assays of FA β-oxidation activity. These findings demonstrate that pitavastatin is capable of restoring impaired FA β-oxidation in vivo via the peroxisome proliferator-activated receptor-α-mediated signaling pathway and is potent enough to ameliorate severe hepatic steatosis in mice deficient in intrinsic estrogen.

Effects of Aromatase or Estrogen Receptor Gene Deletion on Masculinization of the Principal Nucleus of the Bed Nucleus of the Stria Terminalis of Mice

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is a sexually dimorphic nucleus, and the male BNSTp is larger and has more neurons than the female BNSTp. To assess the roles of neuroestrogen synthesized from testicular androgen by brain aromatase in masculinization of the BNSTp, we performed morphometrical analyses of the adult BNSTp in aromatase knockout (ArKO), estrogen receptor-α knockout (αERKO), and estrogen receptor-β knockout (βERKO) mice and their respective wild-type littermates. In wild-type littermates, the BNSTp of males had a larger volume and greater numbers of neuronal and glial cells than did that of females. The volume and neuron number of the BNSTp in ArKO and αERKO males and glial cell number of the BNSTp in αERKO males were significantly smaller than those of wild-type male littermates, and they were not significantly different from those in female mice with either gene knockout. In contrast, there was no significant morphological difference in the BNSTp between βERKO and wild-type mice. Next, we examined the BNSTp of ArKO males subcutaneously injected with estradiol benzoate (EB) on postnatal days 1, 2, and 3 (1.5 µg/day). EB-treated ArKO males had a significantly greater number of BNSTp neurons than did oil-treated ArKO males. The number of BNSTp neurons in EB-treated ArKO males was comparable to that in wild-type males. These findings suggested that masculinization of the BNSTp in mice involves the actions of neuroestrogen that was synthesized by aromatase and that this estrogen mostly binds to ERα during the postnatal period.

Preserved tissue structure of efferent ductules in aromatase-deficient mice.

Estrogen receptor alpha (Esr1) is proposed to play a critical role in the regulation of testicular fluid reabsorption at efferent ductules, and disruption of the Esr1 gene (Esr1(-/-)) resulted in marked dilation of the lumens of efferent ductules. This study was aimed to clarify whether disruption of the gene for aromatase (Ar), an enzyme responsible for estrogen biosynthesis, results in morphological and transcriptional alterations at efferent ductules as observed in Esr1(-/-) mice. Histology demonstrated structural preservation of the ducts in aromatase-deficient (Ar(-/-)) mice. Electron microscopic examinations reveal that endocytic apparatus and tubule-cisternal endoplasmic reticulum are present in non-ciliated cells irrespective of the genotypes. However, electron-dense and acid phosphatase-negative granules and apical tubules, which are components thought to be related to membrane recycling of endosomes, are observed only in wild-type (WT) and Ar(-/-) mice. By contrast, the Golgi complex is highly developed in Esr1(-/-) mice when compared with WT and Ar(-/-) mice. RT-PCR analysis reveals no significant differences in the expression levels of a subset of genes involved in ion transportation. Thus, from the structural and transcriptional points of view, the efferent ductules of Ar(-/-) mice are indistinguishable from those of WT mice. Moreover, data from electron microscopic examinations indicate the possible involvement of Esr1 in the regulation of vesicle recycling processes.

Protective effect of 3-methyl-1-phenyl-2-pyrazolin-5-one, a free radical scavenger, on acute toxicity of paraquat in mice

Paraquat poisoning results in damages of multiple organs including liver, kidney and lung, and antioxidants have been proven to have protective effects. As a novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was introduced to clinical use recently, its protective effect was studied on acute toxicity of paraquat in male ddY mice. When paraquat (175 mg/kg) were given orally, the survival rate was only 8% on the 6th day of paraquat ingestion in Control Group mice. Protective effect of MCI-186 was most evident and the survival rate was 42% on the 6th day and 38% on the 14th day of paraquat ingestion, respectively, when mice were treated immediately. A delay of 30 min in treatment resulted in an abrupt reduction of the survival rate. These results suggested that MCI-186 used in acute phase of paraquat intoxication might serve as a clinically available antidote for attenuating paraquat toxicity.

Estrogen deficiency results in enhanced expression of Smoothened of the Hedgehog signaling in the thymus and affects thymocyte development

Aromatase is an essential enzyme for estrogen synthesis. We investigated the role of estrogen in thymocyte development using aromatase-deficient (ArKO) mice. Like its role as a regulator of bone metabolism through regulating osteoprotegerin (OPG) production, estrogen is involved in the processes of thymocyte development although aromatase mRNA was not detectable in the thymus. Thymic regression and reduced cellularity were evident in ArKO mice. The major difficulties in thymocyte development of ArKO mice were observed during the CD44+ CD25- stage at the cortico-medullary junction and during the CD44- CD25- stage at the subcapsular region where the estrogen receptor was expressed in the stromal cells. The proportion of thymocytes during the CD44+ CD25- stage was reduced. The progression of CD44- CD25+ cells to the CD44- CD25- stage was accelerated in ArKO mice possibly due to insufficient osteoprotegerin production in estrogen-deficiency. However, the expression of Smoothened of the Hedgehog signaling was enhanced in CD4- CD8- double negative cells. This enhancement may result in impaired progression of CD44- CD25- cells to the CD4+ CD8+ double positive stage and impaired proliferation of CD4+ CD8+ double positive cells since Smoothened (Smo) is known to arrest cells as non-proliferating cells. This could be the reason why the proportion of CD3+ TCRbeta(high) cells during the late phase of thymocyte maturation was reduced in ArKO mice. From these observations, we propose that estrogen supports thymocyte development and maturation at many stages through many regulatory pathways including the sonic hedgehog- and the osteoprotegerin ligand (OPGL)-mediated signaling.