Mitochondrial HMG-CoA Synthase Research Articles

Histone deacetylase inhibitors stimulate mitochondrial HMG-CoA synthase gene expression via a promoter proximal Sp1 site

The expression of mitochondrial HMG-CoA synthase in the colon has been correlated with the levels of butyrate present in this tissue. We report here that the effect of butyrate on mitochondrial HMG-CoA synthase gene expression is exerted in vivo at the transcriptional level, and that trichostatin A (TSA), a specific histone deacetylase inhibitor, also induces transcriptional activity and mRNA expression of the gene in human cell lines derived from colon carcinoma. Using chromatin immunoprecipitation assays, we show that histone deacetylase 1 (HDAC1) is associated with the endogenous mitochondrial HMG-CoA synthase promoter and that TSA induction correlates with hyperacetylation of H4 histone associated with the 5' flanking region of the gene. Overexpression of HDAC1 activity leads consistently to mitochondrial HMG-CoA synthase promoter hypoacetylation and reduces its transcriptional activity. The effect of butyrate and TSA maps to a single Sp1 site present in the proximal promoter of the gene, which is able to bind Sp1 and Sp3 proteins. Interestingly, the binding affinity of Sp1 and Sp3 proteins to the Sp1 site correlates with the TSA responsiveness of the promoter. Using a one-hybrid system (GAL4-Sp1 and GAL4-Sp3), we show that both proteins can mediate responsiveness to TSA in CaCo-2 cells employing distinct mechanisms.

The diagnosis of mitochondrial HMG-CoA synthase deficiency

Deficiency of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, the only disorder exclusively affecting hepatic ketogenesis, is a cause of hypoglycemic coma. We report that the diagnosis can be made by typical laboratory findings (hypoketosis, elevated free fatty acids, normal acylcarnitines, specific urinary organic acids) during acute episodes. (J Pediatr 2002;140:778-80)

Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α: PPARα FAILS TO INDUCE PEROXISOME PROLIFERATION-ASSOCIATED GENES IN HUMAN CELLS INDEPENDENTLY OF THE LEVEL OF RECEPTOR EXPRESSION

We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) alpha agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPAR alpha agonists in human hepatocytes. Moreover, other genes that are regulated by PPAR alpha ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPAR alpha agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPAR alpha to levels between 6- and 26-fold over normal human hepatocytes. These PPAR alpha-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPAR alpha agonist treatment in the PPAR alpha-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPAR alpha levels determine the species-specific response of human and rat hepatocytes to the induction of PP.

Identification of Peroxisome Proliferator-responsive Human Genes by Elevated Expression of the Peroxisome Proliferator-activated Receptor α in HepG2 Cells

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.

Low Activity of Mitochondrial HMG-CoA Synthase in Liver of Starved Piglets Is Due to Low Levels of Protein Despite High mRNA Levels

The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65–73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.

Colonic delivery of sodium butyrate via oral route: acrylic coating design of pellets and in vivo evaluation in rats.

Few pharmaceutical studies, with the exception of those on rectal solutions, are described on short chain fatty acid (SCFA) formulations-especially for sodium butyrate, which is a colonocyte preferential substrate. Highly dosed butyrate pellets (90%) were prepared and their coating was designed for colonic delivery. In vivo determination (pH and transit time of pellets in rats) allowed to respectively choose the grade and thickness (resistance of 6 h) of the pH-dependent coating (Eudragi L+S, 1:1). The coated pellets were administered to naturally butyrate-deprived rats. The rats' colonic mucosa had the particularity to weakly express mitochondrial HMG-CoA synthase, an enzyme that responds to luminal butyrate. The results did not show early absorption of butyrate, but a probable cecal loss in the rat cecum as cecal residence time of the pellets was important and as pH was propitious for the coating hydrolysis. It seemed that butyrate, given daily for 7 days without the other main SCFA. was unable to induce the enzyme and/or that the dose (0.32 mmol/day) was insufficient.

Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats.

Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.

Two-dimensional electrophoresis of liver proteins: Characterization of a drug-induced hepatomegaly in rats

Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.

Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase and carnitine palmitoyltransferase II as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation

3-Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. Administration of 3-thia fatty acids to rats was followed by significantly increased levels of plasma ketone bodies, whereas the levels of plasma non-esterified fatty acids decreased. The hepatic mRNA levels of fatty acid binding protein and formation of acid-soluble products, using both palmitoyl-CoA and palmitoyl- l-carnitine as substrates, were increased. Hepatic mitochondrial carnitine palmitoyltransferase (CPT) -II and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activities, immunodetectable proteins, and mRNA levels increased in parallel. In contrast, the mitochondrial CPT-I mRNA levels were unchanged and CPT-I enzyme activity was slightly reduced in the liver. The CoA ester of the monocarboxylic 3-thia fatty acid, tetradecylthioacetic acid, which accumulates in the liver after administration, inhibited the CPT-I activity in vitro, but not that of CPT-II. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased, whereas the citrate synthase activity was decreased. The present data suggest that 3-thia fatty acids increase both the transport of fatty acids into the mitochondria and the capacity of the β-oxidation process. Under these conditions, the regulation of ketogenesis may be shifted to step(s) beyond CPT-I. This opens the possibility that mitochondrial HMG-CoA synthase and CPT-II retain some control of ketone body formation.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis

Cytosolic and mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases were first recognized as different chemical entities in 1975, when they were purified and characterized by Lane's group. Since then, the two enzymes have been studied extensively, one as a control site of the cholesterol biosynthetic pathway and the other as an important control site of ketogenesis. This review describes some key developments over the last 25 years that have led to our current understanding of the physiology of mitochondrial HMG-CoA synthase in the HMG-CoA pathway and in ketogenesis in the liver and small intestine of suckling animals. The enzyme is regulated by two systems: succinylation and desuccinylation in the short term, and transcriptional regulation in the long term. Both control mechanisms are influenced by nutritional and hormonal factors, which explains the incidence of ketogenesis in diabetes and starvation, during intense lipolysis, and in the foetal-neonatal and suckling-weaning transitions. The DNA-binding properties of the peroxisome-proliferator-activated receptor and other transcription factors on the nuclear-receptor-responsive element of the mitochondrial HMG-CoA synthase promoter have revealed how ketogenesis can be regulated by fatty acids. Finally, the expression of mitochondrial HMG-CoA synthase in the gonads and the correction of auxotrophy for mevalonate in cells deficient in cytosolic HMG-CoA synthase suggest that the mitochondrial enzyme may play a role in cholesterogenesis in gonadal and other tissues.

Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element

Low expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene during development correlates with an unusually low hepatic ketogenic capacity and lack of hyperketonaemia in piglets. Here we report the isolation and characterization of the 5' end of the pig mitochondrial HMG-CoA synthase gene. The 581 bp region proximal to the transcription start site permits transcription of a reporter gene, confirming the function of the promoter. The pig mitochondrial HMG-CoA synthase promoter is trans-activated by the peroxisomal proliferator-activated receptor (PPAR), and a functional response element for PPAR (PPRE) has been localized in the promoter region. Pig PPRE is constituted by an imperfect direct repeat (DR-1) and a downstream sequence, both of which are needed to confer PPAR-sensitivity to a thymidine kinase promoter and to form complexes with PPAR.retinoid X receptor heterodimers. A role of PPAR trans-activation in starvation-associated induction of gene expression is suggested.

Transcriptional regulation of mitochondrial HMG-CoA synthase in the control of ketogenesis

Mitochondrial and cytosolic HMG-CoA synthases are encoded by two different genes. Control of ketogenesis is exerted by transcriptional regulation of mitochondrial HMG-CoA synthase. Fasting, cAMP, and fatty acids increase its transcriptional rate, while refeeding and insulin repress it. Fatty acids increase transcription through peroxisomal proliferator regulatory element (PPRE), to which peroxisome proliferator activated receptor (PPAR) can bind. Other transcription factors such as chicken ovalbumin upstream promoter transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF-4) compete for the PPRE site, modulating the response of PPAR.

The Hepatocyte Nuclear Factor 4 (HNF-4) Represses the Mitochondrial HMG-CoA Synthase Gene

We have recently shown that the gene for the mitochondrial HMG-CoA synthase is a target for PPAR and that this receptor mediates the induction of this gene by fatty acids. With the aim of gaining further insight into the function and regulation of this gene we examined the effect of other members of the nuclear hormone receptor superfamily on its expression. We previously identified a regulatory element in the mitochondrial HMG-CoA synthase gene promoter that confers transcriptional regulation by PPAR, RXR and the orphan nuclear receptor COUP-TF. In this study we demonstrate a trans-repressing regulatory function for HNF-4 at this same nuclear receptor response element (NRRE). HNF-4 binds to the mitochondrial HMG-CoA synthase NRRE, and, in cotransfection assays in HepG2 cells, it represses PPAR-dependent activation of a reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter.These results suggest that the mitochondrial HMG-CoA synthase gene is subject to differential regulation by the interplay of multiple members of the nuclear hormone receptor superfamily.

The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats.

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mit. HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mit. HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mit. HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mit. HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.

Chicken ovalbumin upstream-promoter transcription factor (COUP-TF) could act as a transcriptional activator or repressor of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene.

The chicken ovalbumin upstream-promoter transcription factor (COUP-TF) has a dual effect on the regulation of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene. COUP-TF could act as a transcriptional activator or repressor of this gene through different DNA sequences. COUP-TF induces expression of a reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter in human hepatoma HepG2 cells, but represses it in a Leydig tumour cell line (R2C); in both these cell lines the expression of the mitochondrial HMG-CoA synthase gene mimics that of liver and testis. The activation is promoted by a fragment of the gene from coordinates -62 to +28, which contains a GC box and a TATA box, and where no COUP-TF binding site was observed by in vitro DNA binding studies. On the other hand, the COUP-TF inhibitory effect is mainly due to repression of peroxisome-proliferator-activated receptor-dependent activation of the gene, interacting with the region from -104 to -92. To our knowledge this work represents the second example of a target gene for COUP-TF I that could be either activated or repressed by the action of this receptor through different DNA sequences of the same gene.

Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.

The Effect of Fasting/Refeeding and Insulin Treatment on the Expression of the Regulatory Genes of Ketogenesis in Intestine and Liver of Suckling Rats

The influence of fasting/refeeding and insulin treatment on ketogenesis in 12-day-old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT I) and mitochondrial HMG-CoA synthase. Fasting produced hardly any change in mRNA or activity of CPT I in intestine, but led to a decrease in mitochondrial (mit.) HMG-CoA synthase. In liver, while mRNA levels and activity for CPT I increased, neither parameter was changed in HMG-CoA synthase. The comparison of these values with the ketogenic rate of both tissues under the fasting/refeeding treatment shows that HMG-CoA synthase could be the main gene responsible for regulation of ketogenesis in suckling rats. The small changes produced in serum ketone bodies in fasting/refeeding, with a profile similar to the ketogenic rate of the liver, indicate that liver contributes most to ketone body synthesis in suckling rats under these experimental conditions. Short-term insulin treatment produced increases in mRNA levels and activity in CPT I in intestine, but it also decreased both parameters in mit. HMG-CoA synthase. In liver, graphs of mRNA and activity were nearly identical in both genes. There was a marked decrease in mRNA levels and activity, resembling those values observed in adult rats. As in fasting/refeeding, the ketogenic rate correlated better to mit. HMG-CoA synthase than CPT I, and liver was the main organ regulating ketogenesis after insulin treatment. Serum ketone body concentrations were decreased by insulin but recovered after the second hour. Long-term insulin treatment had little effect on the mRNA levels for CPT I or mit. HMG-CoA synthase, but both the expressed and total activities of mit. HMG-CoA synthase were reduced by half in both intestine and liver. The ketogenic rate of both organs was decreased to 40% by long-term insulin treatment. The different effects of refeeding and insulin treatment on the expression of both genes, on the ketogenic rate, and on ketone body concentrations are discussed.

Bacterial status is a key-factor controlling expression of the mitochondrial HMG-CoA synthase gene in the rat intestinal mucosa

Bacterial status is a key-factor controlling expression of the mitochondrial HMG-CoA synthase gene in the rat intestinal mucosa

Transfection of the ketogenic mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase cDNA into Mev-1 cells corrects their auxotrophy for mevalonate.

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 (called Mev-1), auxotrophic for mevalonate by virtue of a complete lack of detectable cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase activity, was transfected with a plasmid containing the cDNA for ketogenic mitochondrial HMG-CoA synthase under the control of SV40 early promoter. The resulting stable cell line (Mev-SM) was able to grow in the absence of mevalonate. Analysis of Western blot showed that the new cell line strongly expressed mitochondrial HMG-CoA synthase protein. Immunocytochemical studies using specific antibodies against mitochondrial HMG-CoA synthase showed that the protein was located exclusively inside the mitochondria. The prototroph cell line Mev-SM can incorporate labeled acetate into cholesterol in the absence of mevalonate. These results show that the new cell line may circumvent the lack of cytosolic HMG-CoA synthase activity by producing cholesterol-convertible HMG-CoA inside the mitochondria.

Human Mitochondrial HMG CoA Synthase: Liver cDNA and Partial Genomic Cloning, Chromosome Mapping to 1p12-p13, and Possible Role in Vertebrate Evolution

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholesterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of a HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. The nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistent with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain.