Amelioration of lipid-induced insulin resistance in rat skeletal muscle by overexpression of Pgc-1ß involves reductions in long-chain acyl-CoA levels and oxidative stress.

AIMS/HYPOTHESIS: To determine if acute overexpression of peroxisome proliferator-activated receptor, gamma, coactivator 1 beta (Pgc-1ß [also known as Ppargc1b]) in skeletal muscle improves insulin action in a rodent model of diet-induced insulin resistance. METHODS: Rats were fed either a low-fat or high-fat diet (HFD) for 4 weeks. In vivo electroporation was used to overexpress Pgc-1ß in the tibialis cranialis (TC) and extensor digitorum longus (EDL) muscles. Downstream effects of Pgc-1ß on markers of mitochondrial oxidative capacity, oxidative stress and muscle lipid levels were characterised. Insulin action was examined ex vivo using intact muscle strips and in vivo via a hyperinsulinaemic-euglycaemic clamp. RESULTS: Pgc-1ß gene expression was increased >100% over basal levels. The levels of proteins involved in mitochondrial function, lipid metabolism and antioxidant defences, the activity of oxidative enzymes, and substrate oxidative capacity were all increased in muscles overexpressing Pgc-1ß. In rats fed a HFD, increasing the levels of Pgc-1ß partially ameliorated muscle insulin resistance, in association with decreased levels of long-chain acyl-CoAs (LCACoAs) and increased antioxidant defences. CONCLUSIONS: Our data show that an increase in Pgc-1ß expression in vivo activates a coordinated subset of genes that increase mitochondrial substrate oxidation, defend against oxidative stress and improve lipid-induced insulin resistance in skeletal muscle.

A full-length cDNA encoding mouse adrenodoxin.

A full-length cDNA encoding mouse adrenodoxin was isolated from a Y1 adrenocortical tumor cell lambda ZAP cDNA library. The 883 bp cDNA contains a 567 bp open reading frame encoding a protein containing 188 amino acids. Mouse adrenodoxin shows high amino acid sequence identity with other mammalian adrenodoxins and the four cysteines involved in the formation of the iron-sulfur cluster are present.

Distribution and regulation of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase in the rat central nervous system.

5 alpha-Androstane-3 beta,17 beta-diol hydroxylase (3 beta-diol OHase), a form of cytochrome P-450 whose main function is thought to be the elimination of dihydrotestosterone (DHT) from its target tissues, was found in the central nervous system (CNS) of both male and female rats at a level which is at least 300-fold higher than any other P-450 catalyzed steroid hydroxylase in the brain. In order to gain some insight into the physiological function of this enzyme we have studied its distribution throughout the CNS as well as the effects of age and hormonal manipulations of rats. We have also partially purified the enzyme from the brain and compared it with the 3 beta-diol OHase in the prostate. 3 beta-Diol OHase activity was distributed throughout the CNS of adult male and female rats with levels between 70-153 nmol products formed/g tissue.h. The products were identified as 5 alpha-androstane-3 beta,7 beta,17 beta-triol,5 alpha-androstane-3 beta,6 alpha,17 beta-triol and 5 alpha-androstane-3 beta,7 alpha,17 beta-triol. The ratio of these three triols was 1:9:3. Catalytic activity was not affected by castration or adrenalectomy of male rats, or during late pregnancy or lactation in females. In the brains of 14-day-old fetuses and 2-day-old rats, the level of 3 beta-diol OHase was 30% of that of adults and it increased to adult levels by day 28. Cytochrome P-450 was partially purified from microsomes of whole brain, hypothalamus, and prostate and 3 beta-diol OHase activity reconstituted. On the basis of the ratios of the three triols formed from 3 beta-diol, it can be concluded that the 3 beta-diol OHase in the brain is similar to that in the prostate. From a comparison of the turnover numbers in reconstituted systems with P-450 prepared from the prostate, brain, and hypothalamus, it can be estimated that up to 50% of the P-450 in the hypothalamus and 10% in whole brain is 3 beta-diol OHase. Supportive evidence for this relative distribution of 3 beta-diol OHase in the hypothalamus was obtained by comparing the sodium dodecyl sulfate electrophoretic profiles of P-450 obtained from the hypothalamus and prostate microsomes.

Elevated expression of lanosterol 14alpha-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats.

Mammalian CYP51 encodes lanosterol 14alpha-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14alpha-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII-XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observed. The rat male germ cell-specific CYP51 transcript is translated in vitro to two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher in protein extracts of testes and germ cells of sexually mature rats than in prepubertal animals, in which postmeiotic germ cells are not yet present. This shows increased capacity for the production of MAS sterols by male germ cells that have already completed meiosis, suggesting that they serve a role different from meiosis activation.

Endocrine regulation of cytochrome P-450 in the rat brain and pituitary gland.

In an effort to understand the physiological functions of cytochrome P-450 in the central nervous system and pituitary gland, we evaluated changes in the level of the enzyme as a function of the endocrine status of rats and the ability of these tissues to synthesize or degrade steroids. The P-450 content of microsomes prepared from the hypothalamic preoptic area (HPOA), the olfactory lobes and the cerebrum was 0.040 +/- 0.009 and in the pituitary gland 2.2 +/- 0.6 (S.D.) nmol/g tissue. The P-450 content of the HPOA and olfactory lobes, but not of the rest of the cerebrum, was influenced by the endocrine status of rats. In microsomes it increased five- to tenfold over control levels during late pregnancy in the olfactory lobes and during lactation in the HPOA, and in both brain regions treatment of rats with 5 alpha-dihydrotestosterone (DHT) caused an eight- to tenfold increase in the P-450 content. Androstenedione was not a good substrate for brain P-450. The level of androstenedione 19-hydroxylase in the olfactory lobe microsomal fraction was 0.50 +/- 0.06 nmol 19-hydroxyandrostenedione formed/g tissue per h. This activity was tenfold lower in other brain areas and was not detectable in the pituitary gland. The rate of aromatization of androstenedione to oestradiol in the HPOA and olfactory lobe of lactating rats was 0.46 +/- 0.14 and 0.38 +/- 0.05 pmol/oestradiol formed/g tissue per h respectively. 5 alpha-Androstane-3 beta,17 beta-diol (A-5 alpha-3 beta,17 beta-diol) was a much better substrate for P-450 throughout the brain and pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain.

Using the reverse transcription polymerase chain reaction, mRNAs encoding steroidogenic P450s as well as NADPH-cytochrome P450 reductase (P450 reductase), adrenodoxin and the transcription factor steroidogenic factor 1 (SF-1) were all detected in rodent brain, but their distribution between brain regions varied. Adrenodoxin and P450 reductase were detected in all regions, suggesting the presence of both mitochondrial and microsomal P450s throughout the brain. Messenger RNAs encoding P450scc (CYP11A1) and P45017 alpha (CYP17) were also detected in all brain regions, this being the first report of CYP17 in the brain. P450c21 (CYP21) was detected only in the brain stem. P45011 beta (CYP11B1) and P450aldo (CYP11B2) are expressed in rat brain, but not in mouse brain; CYP11B1 primarily in the cerebrum, whereas CYP11B2 was detected in all brain regions. In both species, highest levels of aromatase P450 (CYP19) mRNA were detected in the cerebrum. SF-1 expression was restricted to the cerebrum minus cortex. Thus, although SF-1 is required for high level expression of the steroidogenic enzymes in adrenals and gonads, other factors may influence the expression of these genes in the brain. If the mRNAs detected by RT-PCR are indeed translated into functional enzymes, these studies suggest that different brain regions have different capacities for local steroid hormone production and metabolism. This raises the technical challenge of locating the specific sites of synthesis as well as the function of such locally produced ligands.

Regulation of cytochrome P450 in the central nervous system.

The role of brain P450 in the physiology, pharmacology and toxicology of the brain is the subject of this study. Cytochrome P450 was isolated from the brains of rats and quantitated spectrally. The contribution of the known hepatic forms of the enzyme to the forms constitutive in the brain as well as those which are induced by hormones are xenobiotics were characterized on Western blots. We have found that the level of P450 in the brain is increased during pregnancy and lactation, by partial hepatectomy and by ethanol. In each case the profile of P450s induced is different. In pregnancy and lactation the P450 content of the hypothalamic preoptic area and olfactory lobes were increased up to 10-fold and the only subfamily identified on Western blots was 4A. There was no detectable 1A, 2A, 2B, 2C, or 2E1. Ethanol increases the level of brain P450 3- to 5-fold and P450 2C, 2E1 and 4A are induced. Upon partial hepatectomy P450 1A, 2C and 4A were detected on Western blots but there was no 2E1. The inducibility of these forms of P450 in the brain suggests that there is in situ metabolism of steroids, fatty acids, prostaglandins, ethanol and other xenobiotics in the brain and raises questions about the role of brain P450 in the development of tolerance to drugs and the neurotoxicity of xenobiotics. More importantly, the action of neurotransmitters such as dopamine which utilize fatty acids metabolites as intracellular mediators, could be influenced by the levels of 2C and 4A P450s.

Cloning and characterization of a novel member of the cytochrome P450 subfamily IVA in rat prostate.

To isolate cDNAs for forms of cytochrome P450 from rat prostate, a lambda gt11 cDNA library from this tissue was screened with a mixture of oligonucleotide probes directed against the conserved heme binding region of different P450 isozymes. A cDNA clone (PP1) encoding a part of a novel form of cytochrome P450 was isolated and the deduced amino acid sequence showed 76% identity with cytochrome P450 IVA1, indicating that PP1 is a member of the same subfamily. Northern blot analysis of total RNA from prostates of untreated rats revealed that two mRNAs of approximately 2.8 and 2.2 kb hybridize to PP1. The level of mRNA was induced fivefold above the level in intact animals by androgen treatment of castrated rats. Analysis of poly(A)+RNA levels in different tissues on Northern blots showed high constitutive expression of PP1 in the kidney, but no signal was detectable with RNA from liver; a weak signal was detected in the retina. Subsequent screening of a rat kidney cDNA library led to the isolation of the full-length clone KP1, which differs from Pp1 only in three nucleotide positions. KP1 is 1,957 bp long and contains a 1,527-bp-long open reading frame encoding a protein of 508 amino acids. In situ hybridization of rat kidney sections with PP1 showed that this P450 form is expressed in the outer stripe of the outer medulla, indicating its localization in the proximal tubules.

The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols.

Sterol biosynthesis requires the removal of the 14 alpha-methyl group from lanosterol in animals and fungi and from obtusifoliol in plants. This reaction is catalyzed by a microsomal cytochrome P450, the sterol 14 alpha-demethylase (P450(14DM), which is the only P450 described so far to be expressed in different phyla. A cDNA encoding human P450(14DM) was isolated from a liver cDNA library using a partial rat lanosterol 14 alpha-demethylase cDNA probe. The deduced amino acid sequence is 93% and 38--42% identical to rat and fungal P450(14DM), respectively. Expression of the human CYP51 cDNA in Escherichia coli showed that the cDNA encodes an enzyme having lanosterol 14 alpha-demethylase activity. Northern blot analysis showed that CYP51 mRNA is ubiquitously expressed with highest levels in testis, ovary, adrenal, prostate, liver, kidney, and lung. Many genes involved in cholesterol homeostasis are regulated by cholesterol or its metabolites. In the case of CYP51, cholesterol deprivation led to a 2.6- to 3.8-fold induction of mRNA levels in human adrenocortical H295R cells and this effect was suppressed by the addition of 25-hydroxycholesterol. In human hepatoma HepG2 cells, no effect of cholesterol deprivation was observed; however, the levels of CYP51 mRNA were reduced 4- to 6-fold by the addition of 25-hydroxycholesterol. Thus, like several other genes in the cholesterol biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, HMG CoA reductase, squalene synthase, and farnesyl diphosphate synthase, the expression of the human CYP51 is suppressed by oxysterols.

Cytochrome P450s of the 4A subfamily in the brain.

Members of the P450 4A subfamily are key enzymes in the synthesis and degradation of metabolites of arachidonic acid, which are of physiological importance in the brain. In the rat, four members of this subfamily, 4A1, 4A2, 4A3, and 4A8, have been described. In this study, the expression of members of the 4A subfamily in the rat brain has been examined by PCR amplification, by western and northern blotting, and by protein N-terminal sequencing. With PCR all four members of the subfamily were detectable in the liver and kidney. P450 4A1 was found exclusively in the liver and kidney, whereas P450 4A2 was detectable in all the tissues tested, including the lung, seminal vesicles, prostate, cerebral cortex, hypothalamic preoptic area, cerebellum, and brainstem. The tissue distribution of P450 4A3 was similar to that of 4A2 except that it was not detectable in seminal vesicles. A P450 4A8-specific fragment was amplified from the kidney, liver, and prostate and weakly from the cerebral cortex but not from other brain regions. Despite the evidence of their presence by PCR, no members of the 4A family were detectable on northern blots with mRNA from the brain. On western blots a P450 4A-specific antiserum recognized a band in P450 fractions prepared from the brain. The intensity of the signal with 30 pmol of P450 from the brain was similar to that with 10 pmol of liver microsomal P450. The brain P450 was extracted from 1 g of brain, whereas the 10 pmol of liver P450 is the equivalent of 1 mg of liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450 forms in the rodent lung involved in the metabolic activation of food-derived heterocyclic amines.

The metabolic activation of the promutagens 2-amino-3,8- dimethylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by rat and mouse lung microsomes was studied using Salmonella mutagenicity (strain TA98). Lungs from uninduced animals were found to activate all three compounds. A 4-6 fold higher mutagenic activity was obtained with IQ compared to MeIQx and the mutagenic response of PhIP was 1-2 orders of magnitude lower than that of IQ. In order to characterize the forms of P450 in the lung responsible for the metabolic activation of these food mutagens Western blots were performed with microsomes and partially purified P450 fractions from the lung. Western blots revealed the presence of cytochrome P450 2A, 2B and 4A forms in untreated rats. In the lung CYP 1A1 was only detectable after BNF treatment of rats. The CYP 4A isozymes, which have not previously been described in the rat lung, were further identified after PCR amplification from lung mRNA as 4A2 and 4A8. Antibody inhibition studies showed that CYP 2A3 catalyzed a major part (70%) of the metabolic activation of IQ by uninduced rat lung microsomes. The metabolic activation of MeIQx was not influenced by this antibody. An antibody against CYP 2B isozymes also partially inhibited the activation of IQ by uninduced rat lung microsomes. However, since induction of CYP 2B isozymes in the liver by phenobarbital treatment did not increase the metabolic activation of the heterocyclic amines over controls it is unlikely that the rat lung CYP 2B1 is participating in the activation of heterocyclic amines. The inhibition of the IQ-dependent mutagenicity by the CYP 2B antibody is probably due to cross-reaction with CYP 2A3. Alfa-naphthoflavone (ANF), considered to be a specific inhibitor of CYP 1A isozymes at 10 microM, partly inhibited the activation of IQ (30-40%) and MeIQx (60-80%) by uninduced rat and mouse lung microsomes. Upon pretreatment of rats with BNF, lung microsomes activated MeIQx at a rate that was 2-10-fold higher than control lung microsomes, whereas the increase in EROD activity was approximately 100-fold in the same lung preparations. These results suggest that CYP 1A1 may not be the enzyme responsible for the activation of MeIQx in the control rat despite the inhibition with ANF. It is likely that ANF can inhibit other P450 enzymes in the lung, including CYP 2A3.(ABSTRACT TRUNCATED AT 400 WORDS)

The three human cytochrome P450 lanosterol 14 alpha-demethylase (CYP51) genes reside on chromosomes 3, 7, and 13: structure of the two retrotransposed pseudogenes, association with a line-1 element, and evolution of the human CYP51 family.

The three human lanosterol 14 alpha-demethylase (CYP51) genes have been mapped to human chromosomes 3, 7, and 13 using a polychromosomal somatic cell hybrid panel. Two of the genes have been cloned from human chromosome 3-specific (CYP51P1) or from human chromosome 13-containing (CYP51P2) cell hybrids. Both were found to be processed pseudogenes, the first reported in the cytochrome P450 (CYP) gene superfamily. The functional CYP51 gene resides on human chromosome 7. CYP51P1 is 96.5% identical to the human CYP51 coding sequence and is not interrupted with introns but has six in-frame stop codons resulting from point mutations. The intronless CYP51P2 gene is 97.2% identical to the CYP51 cDNA coding region. It has a 1-bp insertion leading to a change of reading frame after codon 9 and a stop codon after amino acid 81. In addition, the CYP51P2 sequence is interrupted with a 5' truncated 131-bp LINE-1 element after nucleotide 606. The element belongs to the youngest LINE subfamily Sb and is 98.2% identical to the LINE-1 element expressed in human teratocarcinoma cells. CYP51 processed pseudogenes are the only known examples of the reverse flow of genetic information during evolution of the large (more than 480 genes) CYP superfamily, suggesting expression in the germ line and a housekeeping function of the lanosterol 14 alpha-demethylase gene. CYP51 pseudogenes evolved by two independent reverse transcription events of the human CYP51 mRNA approximately 9.5 MYR (CYP51P2) and approximately 11.7 MYR (CYP51P1) ago and were inactivated soon after the insertion. The truncated L1 element was inserted into CYP51P2 approximately 6 MYR ago.

Role of brain cytochrome P450 in regulation of the level of anesthetic steroids in the brain.

The role of brain cytochrome P450 (P450) in regulating the levels of the potent anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) has been investigated. By analogy with the elimination of androgen from its target tissues, we present evidence that it is 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP) and not 3 alpha-OH-DHP that represents the major pathway for the formation of more polar metabolites and thus the elimination of the 5 alpha-reduced metabolites of progesterone from target tissues. No polar metabolites were formed when 3 alpha-OH-DHP was incubated with microsomal fractions prepared from rat brain, but 3 beta-OH-DHP was hydroxylated at the 6 alpha- and 7 alpha-positions. These 3 beta-diols were not formed to any detectable extent in the liver or kidney but were formed in prostate, pituitary, brain, and breast. The highest catalytic activity, 512 nmol of products formed/g of tissue/hr, was found in the prostate. The corresponding rates in the pituitary, brain, and breast were 71.9, 28.1, and 6.7 nmol/g/hr, respectively. These hydroxylations were confirmed to be P450-catalyzed reactions by solubilization of the P450 from prostate, brain, and breast microsomes and reconstitution of the catalytic activity with NADPH-P450 reductase (EC 1.6.2.4) and lipid. Because 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol) has been shown to be a good substrate for prostate and brain P450, competition experiments were performed to determine whether the same form of P450 is involved in the elimination of 3 beta-Adiol and 3 beta-OH-DHP in the brain. These two substrates competed with each other for metabolism in microsomal fractions and in reconstitution experiments with P450 extracted from the brain or prostate. To test the hypothesis that the hydroxylation of 3 beta-OH-DHP represents a pathway for regulation of the level of 3 alpha-OH-DHP in the brain, the effect of inhibition of the hydroxylation of 3 beta-OH-DHP on the duration of 3 alpha-OH-DHP-induced anesthesia was examined. The nonanesthetic steroid 3 beta-Adiol was used as a competitive inhibitor of the metabolism of 3 beta-OH-DHP. The duration of anesthesia upon intravenous administration of 3 alpha-OH-DHP was increased by 33% when 3 beta-Adiol was coadministered.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure and mapping of the human lanosterol 14alpha-demethylase gene (CYP51) encoding the cytochrome P450 involved in cholesterol biosynthesis; comparison of exon/intron organization with other mammalian and fungal CYP genes.

Sterol 14alpha-demethylase (P45014DM) encoded by CYP51 is a member of the cytochrome P450 (CYP) gene superfamily involved in sterol biosynthesis in fungi, plants, and animals. Constraints imposed by the specific function of CYP51 have severely limited sequence divergence in this family. Consequently, CYP51 is the only P450 family recognizable across all eukaryotic phyla. We have determined the structure of the functional human CYP51 gene, which spans 22 kb, is divided into 10 exons, and maps to 7q21.2-q21.3. The 5' portion of intron 1 is GC-rich and contains potential binding sites for several transcription factors. Primer extension studies reveal predominant transcription initiation sites in liver, kidney, lung, and placenta 250 and 249 bp upstream from the translation start site and a second major site at -100 bp. Ubiquitous expression of human CYP51 (Strömstedt et al., Arch. Biochem. Biophys. 329: 73-81, 1996), the absence of TATA and CAAT patterns, a GC-rich sequence in the promoter region, and initiation of CYP51 transcription at more than one site indicate that CYP51 is a housekeeping gene. The 5'-flanking region, exon 1, and a portion of intron 1 show the characteristics of a CpG island, with the observed/expected CpG ratio of 0.79. Sterol responsive element-like motifs were present in this region, suggesting regulation by oxysterols via a mechanism similar to that associated with other genes involved in cholesterol homeostasis. Comparison of the human CYP51 gene structure with structures of other mammalian and fungal CYP gene families shows that 7 of the 9 CYP51 introns are located at unique positions. More than 80 intron locations exist in mammalian and fungal CYP gene families, and it seems very unlikely that all these introns could have been present in the primordial CYP gene.

Pretranslational regulation of cytochrome P4504A1 by free fatty acids in primary cultures of rat hepatocytes.

The effect of different fatty acids on cytochrome P4504A1 mRNA levels was studied in primary cultures of rat hepatocytes, using a solution hybridization assay. All fatty acids tested induced P4504A1 mRNA levels in a dose- and time-dependent manner. Most potent were docosahexaenoic acid (22:6) and arachidonic acid (20:4), both of which gave a 6-fold increase in mRNA levels at 300 microM, followed by linolenic acid (18:3) and lauric acid (12:0). The effect of three different sulfur-substituted fatty acids was investigated. Tetradecylthioacetic acid, which is blocked for beta-oxidation by sulfur substitution of the beta-carbon, induced P4504A1 mRNA levels 8-fold at 300 microM concentration, whereas tetradecylthiopropionic acid, which can undergo one round of beta-oxidation, only gave a 2-fold increase at the same concentration. The most pronounced effect was seen with 3,14-dithiahexadecanedioic acid, a dicarboxylic acid with both beta-carbons blocked for beta-oxidation, which gave a 31-fold induction of mRNA levels at 300 microM. In time-course studies the effect of docosahexaenoic acid, 3,14-dithiahexadecanedioic acid and the potent peroxisomal proliferator Wy 14,643 on P4504A1 mRNA levels was already detectable after 2 h and maximal after 48 h of treatment, which was reflected in increased levels of P4504A1 protein. Taken together, these results show that endogenous fatty acids, such as docosahexaenoic acid and arachidonic acid, act as pretranslational regulators of P4504A1 when added to primary cultures of rat hepatocytes. Blocking their metabolism (beta-oxidation) leads to significant enhancement of their activity.

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.

Characteristics of the heterologously expressed human lanosterol 14alpha-demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the purified human and Candida albicans CYP51 with azole antifungal agents.

Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.

Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis.

Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH-cytochrome P450 reductase (P450-reductase), adrenodoxin, lanosterol 14-demethylase P450 (CYP51), 17 alpha-hydroxylase P450 (CYP17), cholesterol side-chain cleavage P450 (CYP11A1), sterol 27-hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription-polymerase chain reaction (RT-PCR). In mouse blastocysts, mRNAs of P450-reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.

Fatty acid activation of the peroxisome proliferator activated receptor, a member of the nuclear receptor gene superfamily.

The peroxisome proliferator activated receptor is a member of the steroid receptor gene superfamily, sharing amino acid sequence homology with other receptors and also showing similarities at the level of gene structure. This receptor is activated both by xenobiotic compounds that induce peroxisome proliferation and by fatty acids at physiological concentrations. Upon activation the receptor mediates transcription of responsive genes through binding to peroxisome proliferator response elements. These genes include those encoding peroxisomal enzymes and members of the cytochrome P450 family of drug metabolizing enzymes. It is therefore possible that the peroxisome proliferator activated receptor may play a crucial role in regulating lipid homeostasis.