Enzymatic and transcriptional regulation of the cytoplasmic acetyl-CoA hydrolase ACOT12.

Acyl-CoA thioesterase 12 (ACOT12) is the major enzyme known to hydrolyze the thioester bond of acetyl-CoA in the cytosol in the liver. ACOT12 contains a catalytic thioesterase domain at the N terminus and a steroidogenic acute regulatory protein-related lipid transfer (START) domain at the C terminus. We investigated the effects of lipids (phospholipids, sphingolipids, fatty acids, and sterols) on ACOT12 thioesterase activity and found that the activity was inhibited by phosphatidic acid (PA) in a noncompetitive manner. In contrast, the enzymatic activity of a mutant form of ACOT12 lacking the START domain was not inhibited by the lipids. These results suggest that the START domain is important for regulation of ACOT12 activity by PA. We also found that PA could bind to thioesterase domain, but not to the START domain, and had no effect on ACOT12 dissociation. ACOT12 is detectable in the liver but not in hepatic cell lines such as HepG2, Hepa-1, and Fa2N-4. ACOT12 mRNA and protein levels in rat primary hepatocytes decreased following treatment with insulin. These results suggest that cytosolic acetyl-CoA levels in the liver are controlled by lipid metabolites and hormones, which result in allosteric enzymatic and transcriptional regulation of ACOT12.

In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17ß-estradiol and suppressed by insulin deprivation.

BACKGROUND: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. METHODS: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. RESULTS: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. GENERAL SIGNIFICANCE: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.

ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma.

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)ß subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.

Re-characterisation of Saccharomyces cerevisiae Ach1p: fungal CoA-transferases are involved in acetic acid detoxification.

Saccharomyces cerevisiae and Neurospora crassa mutants defective in the so-called acetyl-CoA hydrolases Ach1p and Acu-8, respectively, display a severe growth defect on acetate, which is most strongly pronounced under acidic conditions. Acetyl-CoA hydrolysis is an energy wasting process and therefore denoted as a biochemical conundrum. Acetyl-CoA hydrolases show high sequence identity to the CoA-transferase CoaT from Aspergillus nidulans. Therefore, we extensively re-characterised the yeast enzyme. Ach1p showed highest specific activity for the CoASH transfer from succinyl-CoA to acetate and only a minor acetyl-CoA-hydrolase activity. Complementation of an ach1 mutant with the coaT gene reversed the growth defect on acetate confirming the in vivo function of Ach1p as a CoA-transferase. Our results imply that Ach1p is involved in mitochondrial acetate detoxification by a CoASH transfer from succinyl-CoA to acetate. Thereby, Ach1p does not perform the energy wasting hydrolysis of acetyl-CoA but conserves energy by the detoxification of mitochondrial acetate.

Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase.

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).

Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Physiological difference between dietary obesity-susceptible and obesity-resistant Sprague Dawley rats in response to moderate high fat diet.

The primary aim of the present study was to define central and peripheral physiological differences between dietary obesity-susceptible (DOS) and obesity-resistant (DOR) outbred Sprague Dawley (SD) rats when given a moderate high fat diet containing 32.34% of energy as a fat. After a 9-week feeding period, the DOS-SD rats consumed significantly more feed (11.1%) and had higher abdominal (39.9%) and epididymal (27.5%) fat pads than the DOR-SD rats. In addition, serum leptin and insulin levels were significantly increased in the DOS-SD rats compared with those in the DOR-SD rats. However, we did not observe significant differences in serum triglyceride, cholesterol and glucose. No differences in hypothalamic OB-Ra and Rb mRNA expressions were found between the two groups. In contrast, arcuate NPY immunohistochemical expression was much higher in the DOS-SD rats than in the DOR-SD rats, though NPY expression in the supraoptic and paraventricular nuclei was not different between the two phenotypes. In peripheral tissues, the DOS-SD rats showed noticeably increased acetyl CoA carboxylase (ACC) mRNA expression in the liver, not epididymal fat. However, Western blot of peroxisomal proliferator activated factor gamma (PPAR gamma) in the liver and epididymal fat was not different between the two phenotypes of SD rats. It was concluded that different body weight phenotypes within outbred SD population responded differently to the development of dietary induced obesity via altered anabolic features in the hypothalamus and liver.

Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae.

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

Mouse cytosolic acetyl-CoA hydrolase, a novel candidate for a key enzyme involved in fat metabolism: cDNA cloning, sequencing and functional expression.

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.

Acetyl-CoA hydrolase involved in acetate utilization in Saccharomyces cerevisiae.

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA or CoASH pools. The yeast enzyme is encoded by ACHl (acetyl-CoA hydrolase) and the expression of ACH1 is repressed by glucose (Lee, F.-J.S., Lin, L.-W. and Smith, J.A. (1990) J. Biol. Chem. 265, 7413-7418). In order to study the biological function of the acetyl-CoA hydrolase, a null mutation (achl-1) was created by gene replacement. The mutation, while not lethal, slows down acetate utilization. In comparison to wild-type, homozygote achl-l diploids, the onset of sporulation was delayed. When measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity, we demonstrated that ACHl was highly expressed during sporulation process. These results indicated that acetyl-CoA hydrolase in yeast cells involved in acetate utilization and subsequently affected the sporulation process.

An acetate-sensitive mutant of Neurospora crassa deficient in acetyl-CoA hydrolase.

The predicted amino acid sequence of the product of the acetate-inducible acu-8 gene of Neurospora crassa, previously of unknown function, has close homology to the recently published sequence of Saccharomyces cerevisiae acetyl-CoA hydrolase. An acu-8 mutant strain, previously characterized as acetate non-utilizing, shows strong growth-inhibition by acetate, but will use it as carbon source at low concentrations. The mutant was shown to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate. As in Saccharomyces, the Neurospora enzyme is acetate-inducible.

Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO, ACY, NP, CKBB, ADH2, and ME1 loci in mink (Mustela vison).

To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.

A glucose-repressible gene encodes acetyl-CoA hydrolase from Saccharomyces cerevisiae.

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA pool. Recently, a yeast acetyl-CoA hydrolase was purified to homogeneity from Saccharomyces cerevisiae and partially characterized (Lee, F.-J. S., Lin, L.-W., and Smith, J. A. (1989) Eur. J. Biochem. 184, 21-28). In order to study the biological function and regulation of the acetyl-CoA hydrolase, we cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase. RNA blot analysis indicates that acetyl-CoA hydrolase is encoded by a 2.5-kilobase mRNA. DNA blot analyses of genomic and chromosomal DNA reveal that the gene (so-called ACH1, acetyl-CoA hydrolase) is present as a single copy located on chromosome II. Acetyl-CoA hydrolase is established to be a mannose-containing glycoprotein, which binds concanavalin A. By measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity in different growth phases and by examining the effects of various carbon sources, we have demonstrated that ACH1 expression is repressed by glucose.