Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and pos... more Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and postnatal testes, respectively. In mice, FLCs emerge in the interstitial space of the testis as early as embryonic day 12.5 and thereafter increase in number during the fetal stage. We previously established a transgenic mouse line in which FLCs are labeled with EGFP and demonstrated that the EGFP-labeled FLCs were present even in adult testes. However, the characteristics of FLCs during postnatal stages remained unclear. In the present study, a comparison of the transcriptomes of FLCs from prenatal and postnatal testes and of ALCs from adult testes revealed that FLCs gradually alter their characteristics across developmental stages and come to roughly resemble ALCs. Many cholesterogenic genes simultaneously expressed a unique alternation pattern, while many oxidative phosphorylation and β-oxidation (both mitochondrial functions) genes showed a different unique pattern. These metabolic gene expression alterations might be triggered by milieu changes, such as nutrient and oxygen supply, from the prenatal to the postnatal period.
Ad4BP/SF-1 (NR5A1), a member of the nuclear receptor superfamily, is known to play crucial roles ... more Ad4BP/SF-1 (NR5A1), a member of the nuclear receptor superfamily, is known to play crucial roles in the regulation of steroidogenesis in the gonads and adrenal cortex, and many studies have demonstrated that all steroidogenic genes are direct targets of Ad4BP/SF-1. In addition, in vivo KO studies demonstrated that no steroidogenic organs formed in gene-disrupted mice, strongly suggesting that Ad4BP/SF-1 is essential for the development of the gonads and adrenal gland. However, it remains unclear how Ad4BP/SF-1 regulates the development of the steroidogenic organs and which additional non-steroidogenic genes are targeted.
American Journal of Physiology-endocrinology and Metabolism, Feb 1, 2021
Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes ... more Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes are interrelated with each other and associated with systemic metabolism, the details of which, however, are largely unknown. Eicosapentaenoic acid (EPA) is a promising nutrient against sarcopenia and has multifaceted effects on systemic metabolism. In this study, we hypothesized that the aging process in skeletal muscle can be intervened by the administration of EPA. Seventy-five-week-old male mice were assigned to groups fed an EPA-deprived diet (EPA−) or an EPA-enriched diet with 1 wt% EPA (EPA+) for 12 wk. Twenty-four-week-old male mice fed with normal chow were also analyzed. At baseline, the grip strength of the aging mice was lower than that of the young mice. After 12 wk, EPA+ showed similar muscle mass but increased grip strength compared with EPA−. EPA+ displayed higher insulin sensitivity than EPA−. Immunohistochemistry and gene expression analysis of myosin heavy chains (MyHCs) revealed fast-to-slow fiber type transition in aging muscle, which was partially inhibited by EPA. RNA sequencing (RNA-Seq) analysis suggested that EPA supplementation exerts pathway-specific effects in skeletal muscle including the signatures of slow-to-fast fiber type transition. In conclusion, we revealed that aging skeletal muscle in male mice shows lower grip strength and fiber type changes, both of which can be inhibited by EPA supplementation irrespective of muscle mass alteration.NEW & NOTEWORTHY This study demonstrated that the early phenotype of skeletal muscle in aging male mice is characterized by muscle weakness with fast-to-slow fiber type transition, which could be ameliorated by feeding with EPA-enriched diet. EPA induced metabolic changes such as an increase in systemic insulin sensitivity and altered muscle transcriptome in the aging mice. These changes may be related to the fiber type transition and influence muscle quality.
Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yea... more Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.
cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H... more cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.
A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially d... more A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially digested by EcoRI. Bovine P-450(11 beta) cDNA, pcP-450(11 beta)-2 [Morohashi et al. (1987) J. Biochem. 102,559-568], was used as a probe for screening the genomic library. Ten clones carrying P-450(11 beta) genomic DNA were isolated from 8 x 10(4) colonies and classified into five groups (CB11 beta-1, CB11 beta-3, CB11 beta-7, CB11 beta-20, and CB11 beta-21) according to differences in the restriction endonuclease sites. Nucleotide sequences of amino acid coding regions of the five clones were determined by the dideoxy sequencing method using synthetic nucleotides corresponding to various parts of the cDNA as primers. The nucleotide sequences revealed that three clones, CB11 beta-1, CB11 beta-3, and CB11 beta-21, were pseudogenes. Amino acid sequences coded by the other two clones, CB11 beta-7 and CB11 beta-20, were identical with that coded by a previously described cDNA, pcP-450(11 beta)-3 [Kirita et al. (1988) J. Biochem. 104, 683-686]. The promoter regions of the five clones were introduced in front of chloramphenicol acetyltransferase (CAT) gene of pSV00CAT and used to examine P-450(11 beta) gene regulation in cultured cells. The five recombinant plasmids showed cAMP-responsive CAT activities in Y-1 cells, a cell strain derived from adrenal tumor. The induction rates of the recombinant plasmids carrying the promoters of normal genes, CB11 beta-7 and -20, were larger than those of pseudogenes, CB11 beta-1, -3, and -21. CAT activities expressed by the promoter regions of the normal genes in the presence or absence of cAMP in Y-1 cells were almost equal to that by the promoter region of human P-450(SCC) gene. Though the promoter of the P-450(SCC) gene also showed cAMP-responsive CAT activity in I-10 cells, a cell strain derived from Leyding cell tumor, P-450(11 beta) gene promoter did not express the activity in I-10 cells.
We isolated three genomic clones of rat P-450(11 beta) genes (CYP11B). Two of them corresponded t... more We isolated three genomic clones of rat P-450(11 beta) genes (CYP11B). Two of them corresponded to 11 beta-hydroxylase gene (CYP11B1) and aldosterone synthase gene (CYP11B2), respectively. The third one was a novel gene resembling both CYP11B1 and CYP11B2, and was named CYP11B3 gene (CYP11B3). CYP11B2 and CYP11B3 are located tandemly in the genome in the same direction approximately 24 kb apart. These three genes were highly homologous in their amino acid coding regions, with 88% (CYP11B1 to CYP11B2), 89% (CYP11B2 to CYP11B3), 96% (CYP11B1 to CYP11B3) nucleotide identity. The numbers and the locations of the exons of these three genes also exactly corresponded to each other. However, the nucleotide sequences of the 5' upstream regions of CYP11B1 and CYP11B2 were significantly different, suggesting different transcriptional regulations. CYP11B3 had almost the same sequence as CYP11B1 gene in the 5' upstream region. A putative Ad4 site, a cis-acting element present in the promoter regions of all the steroidogenic P-450s so far reported [Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T. (1992) J. Biol. Chem. 267, 17913-17919], was found in the promoter regions of both CYP11B1 and CYP11B2. Gel retardation analysis showed the binding of Ad4BP purified from bovine adrenal cortex to these two Ad4 sites. We analyzed the relative abundance of the mRNAs corresponding to these three genes by the generation of RT-PCR libraries from rat adrenal total RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
Using pcP-450(11 beta)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a ... more Using pcP-450(11 beta)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a different cDNA clone, pcP-450(11 beta)-3, was isolated from a cDNA library of bovine adrenal cortex. The restriction enzyme map of pcP-450(11 beta)-3 was highly homologous but not identical with that of pcP-450(11 beta)-2. Nucleotide sequence determination revealed the substitutions of 14 nucleotides and 3 amino acids between pcP-450(11 beta)-2 and -3. Blotting analysis involving two different oligonucleotide probes specific to these two cDNAs indicated that at least two kinds of P-450(11 beta) mRNA were expressed in individual animals and that at least two kinds of P-450(11 beta) genes exist in the bovine genome.
Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were iso... more Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.
Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and pos... more Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and postnatal testes, respectively. In mice, FLCs emerge in the interstitial space of the testis as early as embryonic day 12.5 and thereafter increase in number during the fetal stage. We previously established a transgenic mouse line in which FLCs are labeled with EGFP and demonstrated that the EGFP-labeled FLCs were present even in adult testes. However, the characteristics of FLCs during postnatal stages remained unclear. In the present study, a comparison of the transcriptomes of FLCs from prenatal and postnatal testes and of ALCs from adult testes revealed that FLCs gradually alter their characteristics across developmental stages and come to roughly resemble ALCs. Many cholesterogenic genes simultaneously expressed a unique alternation pattern, while many oxidative phosphorylation and β-oxidation (both mitochondrial functions) genes showed a different unique pattern. These metabolic gene expression alterations might be triggered by milieu changes, such as nutrient and oxygen supply, from the prenatal to the postnatal period.
Ad4BP/SF-1 (NR5A1), a member of the nuclear receptor superfamily, is known to play crucial roles ... more Ad4BP/SF-1 (NR5A1), a member of the nuclear receptor superfamily, is known to play crucial roles in the regulation of steroidogenesis in the gonads and adrenal cortex, and many studies have demonstrated that all steroidogenic genes are direct targets of Ad4BP/SF-1. In addition, in vivo KO studies demonstrated that no steroidogenic organs formed in gene-disrupted mice, strongly suggesting that Ad4BP/SF-1 is essential for the development of the gonads and adrenal gland. However, it remains unclear how Ad4BP/SF-1 regulates the development of the steroidogenic organs and which additional non-steroidogenic genes are targeted.
American Journal of Physiology-endocrinology and Metabolism, Feb 1, 2021
Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes ... more Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes are interrelated with each other and associated with systemic metabolism, the details of which, however, are largely unknown. Eicosapentaenoic acid (EPA) is a promising nutrient against sarcopenia and has multifaceted effects on systemic metabolism. In this study, we hypothesized that the aging process in skeletal muscle can be intervened by the administration of EPA. Seventy-five-week-old male mice were assigned to groups fed an EPA-deprived diet (EPA−) or an EPA-enriched diet with 1 wt% EPA (EPA+) for 12 wk. Twenty-four-week-old male mice fed with normal chow were also analyzed. At baseline, the grip strength of the aging mice was lower than that of the young mice. After 12 wk, EPA+ showed similar muscle mass but increased grip strength compared with EPA−. EPA+ displayed higher insulin sensitivity than EPA−. Immunohistochemistry and gene expression analysis of myosin heavy chains (MyHCs) revealed fast-to-slow fiber type transition in aging muscle, which was partially inhibited by EPA. RNA sequencing (RNA-Seq) analysis suggested that EPA supplementation exerts pathway-specific effects in skeletal muscle including the signatures of slow-to-fast fiber type transition. In conclusion, we revealed that aging skeletal muscle in male mice shows lower grip strength and fiber type changes, both of which can be inhibited by EPA supplementation irrespective of muscle mass alteration.NEW & NOTEWORTHY This study demonstrated that the early phenotype of skeletal muscle in aging male mice is characterized by muscle weakness with fast-to-slow fiber type transition, which could be ameliorated by feeding with EPA-enriched diet. EPA induced metabolic changes such as an increase in systemic insulin sensitivity and altered muscle transcriptome in the aging mice. These changes may be related to the fiber type transition and influence muscle quality.
Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yea... more Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.
cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H... more cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.
A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially d... more A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially digested by EcoRI. Bovine P-450(11 beta) cDNA, pcP-450(11 beta)-2 [Morohashi et al. (1987) J. Biochem. 102,559-568], was used as a probe for screening the genomic library. Ten clones carrying P-450(11 beta) genomic DNA were isolated from 8 x 10(4) colonies and classified into five groups (CB11 beta-1, CB11 beta-3, CB11 beta-7, CB11 beta-20, and CB11 beta-21) according to differences in the restriction endonuclease sites. Nucleotide sequences of amino acid coding regions of the five clones were determined by the dideoxy sequencing method using synthetic nucleotides corresponding to various parts of the cDNA as primers. The nucleotide sequences revealed that three clones, CB11 beta-1, CB11 beta-3, and CB11 beta-21, were pseudogenes. Amino acid sequences coded by the other two clones, CB11 beta-7 and CB11 beta-20, were identical with that coded by a previously described cDNA, pcP-450(11 beta)-3 [Kirita et al. (1988) J. Biochem. 104, 683-686]. The promoter regions of the five clones were introduced in front of chloramphenicol acetyltransferase (CAT) gene of pSV00CAT and used to examine P-450(11 beta) gene regulation in cultured cells. The five recombinant plasmids showed cAMP-responsive CAT activities in Y-1 cells, a cell strain derived from adrenal tumor. The induction rates of the recombinant plasmids carrying the promoters of normal genes, CB11 beta-7 and -20, were larger than those of pseudogenes, CB11 beta-1, -3, and -21. CAT activities expressed by the promoter regions of the normal genes in the presence or absence of cAMP in Y-1 cells were almost equal to that by the promoter region of human P-450(SCC) gene. Though the promoter of the P-450(SCC) gene also showed cAMP-responsive CAT activity in I-10 cells, a cell strain derived from Leyding cell tumor, P-450(11 beta) gene promoter did not express the activity in I-10 cells.
We isolated three genomic clones of rat P-450(11 beta) genes (CYP11B). Two of them corresponded t... more We isolated three genomic clones of rat P-450(11 beta) genes (CYP11B). Two of them corresponded to 11 beta-hydroxylase gene (CYP11B1) and aldosterone synthase gene (CYP11B2), respectively. The third one was a novel gene resembling both CYP11B1 and CYP11B2, and was named CYP11B3 gene (CYP11B3). CYP11B2 and CYP11B3 are located tandemly in the genome in the same direction approximately 24 kb apart. These three genes were highly homologous in their amino acid coding regions, with 88% (CYP11B1 to CYP11B2), 89% (CYP11B2 to CYP11B3), 96% (CYP11B1 to CYP11B3) nucleotide identity. The numbers and the locations of the exons of these three genes also exactly corresponded to each other. However, the nucleotide sequences of the 5' upstream regions of CYP11B1 and CYP11B2 were significantly different, suggesting different transcriptional regulations. CYP11B3 had almost the same sequence as CYP11B1 gene in the 5' upstream region. A putative Ad4 site, a cis-acting element present in the promoter regions of all the steroidogenic P-450s so far reported [Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T. (1992) J. Biol. Chem. 267, 17913-17919], was found in the promoter regions of both CYP11B1 and CYP11B2. Gel retardation analysis showed the binding of Ad4BP purified from bovine adrenal cortex to these two Ad4 sites. We analyzed the relative abundance of the mRNAs corresponding to these three genes by the generation of RT-PCR libraries from rat adrenal total RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)
Using pcP-450(11 beta)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a ... more Using pcP-450(11 beta)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a different cDNA clone, pcP-450(11 beta)-3, was isolated from a cDNA library of bovine adrenal cortex. The restriction enzyme map of pcP-450(11 beta)-3 was highly homologous but not identical with that of pcP-450(11 beta)-2. Nucleotide sequence determination revealed the substitutions of 14 nucleotides and 3 amino acids between pcP-450(11 beta)-2 and -3. Blotting analysis involving two different oligonucleotide probes specific to these two cDNAs indicated that at least two kinds of P-450(11 beta) mRNA were expressed in individual animals and that at least two kinds of P-450(11 beta) genes exist in the bovine genome.
Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were iso... more Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.
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