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Research Notes: 3-hydroxy-3-methylglutaryl-CoA Synthase DeficiencySee also 3-Hydroxy-3-Methylglutaryl-CoA Lyase Deficiency. J Inherit Metab Dis. 2006 Feb. Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed. J Pediatr. 2002 Jun. 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. Biochem Pharmacol. 1999 May 1. 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 beta-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. Biochem J. 1999 Mar 15. 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. Introduction The two 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthases The enzyme HMG-CoA synthase (EC 4.1.3.5) catalyses the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA plus free CoA. HMG-CoA synthase activity is located in two different compartments: the cytosol and the mitochondria. The HMG-CoA produced by the cytosolic HMG-CoA synthase is transformed into mevalonate by the action of HMG-CoA reductase. This starts the isoprenoid pathway which, in addition to cholesterol as the main end-product, produces several important products, such as ubiquinone, dolichol, isopentenyl adenosine and farnesyl groups, which covalently modify proteins. The HMG-CoA produced inside the mitochondria by the mitochondrial HMG-CoA synthase is transformed into acetoacetate by the action of HMG-CoA lyase. Acetoacetate is transformed into hydroxybutyrate and acetone; all of these are known as ketone bodies. In 1975 the two HMG-CoA synthase isoforms were characterized by Lane's group as different chemical entities, but some uncertainty remained as to whether only one gene produced these two proteins that catalysed the same reaction. In 1986, the group of Goldstein and Brown reported the cloning and sequencing, first of the cDNA and then of the gene, of the hamster cytosolic HMG-CoA synthase. The existence of two genes was firmly established in 1990, when we cloned the cDNA, and then the gene, for the rat mitochondrial HMG-CoA synthase. Although the percentage identity in amino acid residues between hamster cytosolic and rat mitochondrial HMG-CoA synthases was high (65%), it became clear that they were the products of two different genes, and that they were differently regulated. The cytosolic synthase was repressed by fasting and cholesterol feeding. In contrast, the mitochondrial HMG-CoA synthase was increased by fasting. Structural and functional comparisons of the promoter regions of the two synthases has indeed shown that the two promoters are very different. Cytosolic HMG-CoA synthase contains sterol regulatory elements that modulate transcriptional activity by sterols, mediated by sterol regulatory element binding proteins (SREBP)-1 and -2, which have not been observed in the promoter of the mitochondrial HMG-CoA synthase. Conversely, the peroxisome proliferator regulatory element (PPRE) is present in the mitochondrial HMG-CoA synthase promoter, but has not been detected in the promoter of the cytosolic synthase. These lines of evidence emphasize that the promoter of each gene is responsible for the control of one of the two different pathways: the cytosolic HMG-CoA synthase is a control site of the isoprenoid biosynthetic pathway, and the mitochondrial HMG-CoA synthase is an important control site of the ketogenic pathway. Ketogenesis Ketogenesis is a mitochondrial process by which acetyl-CoA, mostly derived from the b-oxidation of fatty acids, is converted through four reactions, usually known as the HMG-CoA pathway, into acetoacetate, b-hydroxybutyrate and acetone, all of which are commonly called ketone bodies (Scheme 1).
Acetone is formed by the non-enzymic breakdown of acetoacetate and is unlikely to be important in the metabolism of the intact animal. The other two products are used by different tissues as fuels, thus saving glucose. Ketogenesis is mainly hepatic, but it also occurs in the intestines of suckling mammals, and to a lesser extent in kidney and in neonatal cortical astrocytes. Factors that induce ketogenesis are (in addition to diabetes) fasting and intense lipolysis. In the transition from the fed to the fasted condition, carbohydrate utilization and fatty acid synthesis in the liver cease and are replaced by the oxidation of fatty acids and the induction of ketogenesis. The influence of fatty acids on ketogenesis depends, in turn, on the metabolic state of the organism. The production of acetoacetate and -hydroxybutyrate in perfused rat liver incubated with fatty acids shows marked differences depending on whether the hepatocytes are taken from a fed, fasted or diabetic animal. The control of ketogenesis by substrate availability in mammals after the entry of fatty acids into mitochondria, regulated by carnitine palmitoyltransferase I (CPT I), has been extensively reviewed. A second factor that has aroused vigorous interest as a participant in the control of ketogenesis is the expression of the genes specifically involved in ketogenesis: those for CPT I and mitochondrial HMG-CoA synthase. Mitochondrial HMG-CoA synthase as a control site of ketogenesis Williamson et al. were the first to propose that mitochondrial HMG-CoA synthase is the rate-limiting enzyme of the ketogenic pathway, in studies of acetoacetate production in sonicated liver particles. Later, the work of Chapman et al. and Clinkenbeard et al. using mitochondrial subfractions supported this view. Dashti and Ontko unequivocally showed that the activity of mitochondrial HMG-CoA synthase was responsible for the control of synthesis of acetoacetate in the HMG-CoA pathway. In aged mitochondria, in which ketogenesis had decreased, only mitochondrial HMG-CoA synthase was decreased in the same proportion, whereas thiolase and HMG-CoA lyase retained 100% activity. The question of whether CPT I regulated by malonyl-CoA is the main ketogenic control site, or whether it merely provides the mitochondria with a ketogenic substrate, led to many studies. Grantham and Zammit concluded that, although CPT I is an important locus for the control of hepatic fatty acid oxidation, and hence ketogenesis, during the onset of diabetic ketosis, it does not appear to play such a role during the acute reversal of ketosis. Moreover, acute depression of the ketogenic capacity of the liver on refeeding is not accompanied during the first few hours of refeeding by any reversal of the changes in CPT I induced by starvation. Analogous results were found during liver regeneration. It is now increasingly evident that, during certain transition periods in rats (e.g. foetal–neonatal, suckling–weaning, starved–fed and diabetic–insulin-treated), significant control over ketogenesis may be exerted at another intramitochondrial site distal to CPT I, more specifically HMG-CoA synthase. Top-down control analysis in isolated rat liver mitochondria showed that, under conditions of non-regulation of CPT I by malonyl-CoA, the absence of NADH and the inhibition of the Krebs cycle by malonate, 28% of the control over ketogenesis is invested in the group of enzymes responsible for the production of acetyl-CoA and 72% resides with the enzymes of the HMG-CoA pathway that convert acetyl-CoA into acetoacetate. Studies on short-term inactivation by succinylation and desuccinylation and on the transcriptional regulation of the HMG-CoA synthase gene by nutritional and hormonal effectors confirmed HMG-CoA synthase as the main control point over ketogenesis, providing that enough substrate is available in mitochondria. Pediatr Res. 1998 Sep. There are at least two isoenzymes of 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (EC 4.1.3.5) located in the mitochondrial matrix and the cytoplasm of hepatocytes, respectively. The mitochondrial enzyme is necessary for the synthesis of ketone bodies, which are important fuels during fasting. We report a child with a deficiency of this isoenzyme. He presented at 16 mo with hypoglycemia. There was no rise in ketone bodies during fasting or after a long chain fat load but there was a small rise after a leucine load. Measurement of beta-oxidation flux in fibroblasts was normal. Using antibodies specific for mitochondrial HMG-CoA synthase, no immunoreactive material could be detected on Western blotting. Total HMG-CoA synthase activity in liver homogenate was only slightly lower than in control samples. Presumably, as there was no mitochondrial HMG-CoA synthase enzyme protein, this activity arose from the cytoplasmic or other (e.g. peroxisomal) isoenzymes. With avoidance of fasting, our patient has had no problems since presentation and is developing normally at 4 y of age. Biochem Biophys Res Commun. 1998 Jan 26. 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 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. N Engl J Med. 1997 Oct 23. Fasting is accompanied by a decrease in the availability of glucose for energy use in peripheral tissues and, consequently, an increased reliance of these tissues on the availability of ketone bodies and fatty acids for energy. The availability of ketone bodies depends almost exclusively on hepatic ketogenesis. Failure of ketogenesis may occur in patients with any defect of the enzymes associated with the mitochondrial oxidation of fatty acids. These defects are typically manifested by hypoglycemia, which results from the inadequate supply of alternative substrate (ketones). Other clinical features are more variable and may include myopathy, cardiomyopathy, hepatocellular damage, and neuropathies. Studies in rats have indicated a pivotal role for mitochondrial 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) synthase in the control of ketogenesis. HMG-CoA synthase has cytosolic and mitochondrial forms that, although structurally similar, are controlled by different genes. Both forms catalyze the combination of acetoacetyl-CoA and acetyl-CoA to form beta-hydroxy-beta-methylglutaryl-CoA, which in the cytosol is a precursor of sterols and in the mitochondria is converted to acetoacetate (Figure 1). During fasting, acetyl-CoA produced by mitochondrial beta-oxidation of fatty acids in the liver is largely directed toward the production of ketones, with minimal use of the tricarboxylic acid cycle, so that the vast majority of the two-carbon units produced by fatty-acid oxidation are directed through HMG-CoA synthase to the production of ketone bodies.
In this report, we describe an 11-year-old boy with deficiency of mitochondrial HMG-CoA synthase. This case report underlines the importance of the enzyme as a control point in ketogenesis and confirms the functional difference between the cytosolic and mitochondrial forms of HMG-CoA synthase. Case Report A boy of Chinese descent first presented at six years of age after mild gastroenteritis with poor oral intake for two to three days that culminated in a brief generalized seizure, which left him semicomatose. His blood glucose concentration was 9 mg per deciliter (0.5 mmol per liter), and a urine dipstick test was negative for ketones. He had previously been well and tolerated minor illnesses without difficulty. Physical examination was normal. The child responded within five minutes to intravenous dextrose, with further improvement over the next hour. Blood lactate and plasma ammonia, aminotransferase, and carnitine concentrations were normal, as was urinary excretion of organic acids when measured two days later. The patient was given a normal diet, and the parents were advised not to allow the boy to go without food for prolonged periods. Subsequently, he continued to tolerate minor illnesses with no difficulty, and on one occasion strenuous exercise for one hour did not provoke increases in serum creatine kinase or cholesterol concentrations. His physical and mental development during the next five years were normal, as were plasma carnitine, creatine kinase, creatinine, cholesterol, and aminotransferase concentrations on several occasions. Plasma alanine and lactate concentrations were normal even during periods of stress, suggesting that gluconeogenesis was normal. There have been no further seizures; the seizure on presentation was attributed to hypoglycemia. The boy's parents are not related, and he has one sister, who is healthy and has never had any symptoms such as his. Methods Provocative Tests At the age of seven the patient fasted for 22 hours. Blood was collected every two to four hours from hour 14 to hour 22 for measurements of blood glucose and plasma free fatty acid and beta-hydroxybutyrate concentrations. Urine was collected for organic-acid analysis at the end of the fast. After a 15-hour fast, the boy was given 1.5 g of medium-chain triglycerides per kilogram of body weight orally, and then plasma beta-hydroxybutyrate was measured at hourly intervals for 3 hours, blood glucose was measured every 15 to 30 minutes for 3 hours, and urine and plasma were collected for organic-acid analysis at 3 hours. A similar protocol was followed after the child had fasted for 12 hours and was then given long-chain triglycerides in the form of safflower oil (1.5 g per kilogram). The boy's parents gave informed consent for all the diagnostic investigations. Analyses of Enzymes and Metabolites [...] Liver Biopsy [...] HMG-CoA Synthase Activity HMG-CoA synthase activity was measured in whole homogenates of liver tissue and in cultured skin fibroblasts by radioisotopic modification of a coupled enzyme assay. The assay measures the conversion of acetyl-CoA to acetoacetate by means of HMG-CoA and therefore depends on the activities of acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA lyase. In the liver, 90 percent of acetoacetate is produced in mitochondria. Citrate synthase activity was measured in the liver homogenates as a control mitochondrial enzyme to assess sample preservation. Results After fasting for 22 hours, the patient had a blood glucose concentration of 41 mg per deciliter (2.3 mmol per liter) and a plasma beta-hydroxybutyrate concentration of 0.2 mmol per liter (normal, >2.0). He rapidly became comatose, waking about 30 minutes after receiving a bolus intravenous injection of dextrose (500 mg per kilogram) followed by a continuous infusion at a rate of 10 mg per kilogram per minute. This response is slower than that expected in children with brief episodes of hypoglycemia, in whom coma usually resolves within minutes. After the ingestion of long-chain triglycerides, the patient's plasma free fatty-acid concentrations increased appropriately, but plasma beta-hydroxybutyrate concentrations remained below 0.15 mmol per liter (normal response, >0.5). The patient's plasma beta-hydroxybutyrate concentrations (Table 1) also remained low after the ingestion of medium-chain triglycerides, indicating that enzyme function after the oxidation of long-chain fatty acids, the first step in ketone-body formation, was abnormal. Four hours after the ingestion of medium-chain triglycerides the boy became comatose, with a blood glucose concentration of 50 mg per deciliter (2.8 mmol per liter). He was then given intravenous dextrose (in the same dose as before) and responded in about one hour. Urinary excretion of organic acids was normal on several occasions during minor illnesses and after fasting and the ingestion of long-chain triglycerides. After the ingestion of medium-chain triglycerides plasma concentrations of hydroxy fatty acids, including 3-hydroxyhexanoate and 3-hydroxyoctanoate, increased markedly (Table 1). Urinary excretion of ethyl malonate was slightly elevated (38 µmol per millimole of creatinine), and traces of 3-ketohexanoate were also present. Hydroxy fatty acids were not detected in plasma during fasting, and their presence after the ingestion of medium-chain triglycerides therefore most likely reflects defective metabolism of medium-chain triglycerides, raising the possibility that fatty-acid toxicity contributed to the patient's coma. After the ingestion of long-chain triglycerides, plasma 3-hydroxyhexanoate and 3-hydroxyoctanoate concentrations were 6 and 2 µmol per liter, respectively, as compared with respective base-line values of 4 and less than 2 µmol per liter. Histologic examination of the liver showed mild fatty infiltration, mainly in the periportal regions, but no other abnormalities. Electron microscopy revealed moderate variation in the size of the mitochondria, and many contained nonspecific crystalline inclusions. The total carnitine content of the liver was 1278 nmol per gram of wet weight (normal, 900 to 1800), and the free carnitine content was 698 nmol per gram of wet weight (normal, approximately 70 percent of the total content). Analysis of blood spots on filter paper by tandem mass spectrometry revealed a normal acylcarnitine profile. The HMG-CoA synthase activity in the patient's liver was 5 to 20 percent of that in samples of normal liver (Table 2). In mixing experiments, HMG-CoA synthase activity in combined samples was equal to the sum of the values in samples from the patient and the normal subjects. This finding rules out the possibility that an inhibitor was responsible for the reduced activity in the patient's liver and confirms that the defect was in the rate-limiting step (i.e., at the level of HMG-CoA synthase). Hepatic citrate synthase activity was similar in the patient and the normal subjects. The activities of other enzymes involved in fatty-acid oxidation in fibroblasts were normal. Discussion The occurrence of episodes of hypoketotic, hypoglycemic coma during fasting in our patient is typical of deficiencies of enzymes involved in mitochondrial fatty-acid oxidation. The studies performed while the patient was fasting confirmed that ketogenesis was defective, and the fat-loading studies indicated that the defect was not in the metabolism of long-chain fatty acids. Plasma carnitine concentrations are low in many patients with defects of fatty-acid beta-oxidation, with normal values previously having been associated with carnitine palmitoyltransferase I deficiency and multiple mild defects in dehydrogenation. The normal plasma and liver values in our patient, together with the results of the fat-loading tests and the finding of normal urinary excretion of organic acids, raised the suspicion of a fault in the final stages of ketogenesis. Our patient had no evidence of HMG-CoA lyase deficiency, and acetoacetyl-CoA thiolase deficiency typically presents with hyperketosis. Thus, a deficiency of HMG-CoA synthase appeared likely. The patient's hepatic HMG-CoA synthase activity was approximately 10 percent of that in normal liver. This residual activity almost certainly reflected cytosolic HMG-CoA synthase activity, which contributes about 10 percent of the overall HMG-CoA synthase activity in normal liver. The results in our patient therefore support the diagnosis of a deficiency of hepatic mitochondrial HMG-CoA synthase activity. Control of hepatic ketogenesis is exerted by two major regulatory enzymes, carnitine palmitoyltransferase I and mitochondrial HMG-CoA synthase. Carnitine palmitoyltransferase I has a clear role in controlling the initiation of beta-oxidation and, hence, ketogenesis, but appears to have little influence on later control (down-regulation) of the process. HMG-CoA synthase is active in both the initiation and the down-regulation of ketogenesis, as well as having a specific role in the control of beta-oxidation of medium-chain triglycerides. This specific role would explain the sensitivity of our patient to medium-chain triglycerides and the appearance of abnormal metabolites after the ingestion of medium-chain triglycerides. Although the mitochondrial HMG-CoA synthase gene has been sequenced in rats and birds, the location of the gene in humans is not known. The diagnosis of mitochondrial HMG-CoA synthase deficiency is not straightforward. Like other defects of fatty-acid oxidation, this condition should be considered in any patient with coma induced by fasting or a life-threatening event from the newborn period through infancy to middle childhood. In particular, this deficiency may possibly contribute to the sudden infant death syndrome and Reye's syndrome. Low levels of HMG-CoA synthase have been identified in lymphocytes, intestine, kidney, testis, and ovary, but our patient had no symptoms to suggest the involvement of these tissues. HMG-CoA synthase therefore appears to cause symptoms only in relation to its hepatic location, and these symptoms are similar to those that occur in patients with most other defects in fatty-acid oxidation. However, some of these defects involve multiple organs, and the absence of such involvement in the presence of normal plasma carnitine concentrations and normal urinary excretion of organic acids should raise the suspicion of HMG-CoA synthase deficiency. However, the results of diagnostic tests of value in other disorders of fatty-acid oxidation were normal in our patient, and specific diagnosis currently relies on liver-enzyme assay. The clinical course in our patient confirms the importance of hepatic synthesis of ketones to the maintenance of the energy supply during fasting. The absence of any demonstrable disturbance of cholesterol metabolism underlines the functional distinction between the cytosolic and mitochondrial forms of HMG-CoA synthase. Our patient was asymptomatic except during prolonged fasting. In contrast, patients with deficiency of HMG-CoA lyase, the final enzyme of the ketogenic pathway, typically have more prominent symptoms. This difference could be due to the fact that metabolic stress on pathways of protein metabolism, as well as on those of fatty-acid metabolism, can induce symptoms in the latter condition or, possibly, to the variation in acyl-CoA accumulation between the two disorders, the latter being reflected in the relative derangements in carnitine metabolism. Although ketones are thought to be important for the developing brain, the normal neurologic development in both our patient and many with HMG-CoA lyase deficiency indicates that ketone synthesis is not essential to brain development as long as prolonged fasting is avoided. Arch Biochem Biophys. 1997 Apr 15. 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 1 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. J Biol Chem. 1996 Mar 29. We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice. J Biol Chem. 1994 Jul 22. Fatty acids induce an increase in the transcription of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which encodes an enzyme that has been proposed as a control site of ketogenesis. We studied whether the peroxisome proliferator-activated receptor (PPAR) is involved in the mechanism of this transcriptional induction. We found that cotransfection of a rat mitochondrial HMG-CoA synthase promoter-chloramphenicol acetyltransferase reporter plasmid and a PPAR expression plasmid in the presence of the peroxisome proliferator clofibrate led to a more than 30-fold increase in chloramphenicol acetyltransferase activity, relative to the activity in the absence of both PPAR and inducer. Linoleic acid, a polyunsaturated fatty acid, increased this activity as potently as does clofibrate and more effectively than does monounsaturated oleic acid. We have identified, by deletional analysis, an element located 104 base pairs upstream of the mitochondrial HMG-CoA synthase gene, which confers PPAR responsiveness to homologous and heterologous promoters. This is the first example of a peroxisome proliferator-responsive element (PPRE) in a gene encoding a mitochondrial protein. This element contains an imperfect direct repeat that is similar to those described in the PPREs of other genes. Furthermore, gel retardation and cotransfection assays revealed that, as for other genes, PPAR heterodimerizes with retinoid X receptor and that both receptors cooperate for binding to the mitochondrial HMG-CoA synthase PPRE and subsequent activation of the gene. In conclusion, our data demonstrate that regulation of mitochondrial HMG-CoA synthase gene expression by fatty acids is mediated by PPAR, supporting the hypothesis that PPAR has an important role at the transcriptional level in the regulation of lipid metabolism. Arch Biochem Biophys. 1993 Nov 15. We have determined the levels of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase under different metabolic situations to examine its potential role as a regulatory protein in the ketogenic pathway. We used specific antibodies directed against a peptide of the amino acid sequence of the protein as deduced from the cDNA sequence. The amount of mitochondrial HMG-CoA synthase protein rapidly increased in response to cyclic AMP, dexamethasone, starvation, fat feeding, and diabetes, whereas it was decreased by insulin and refeeding. Insulin was also able to counteract the increase in mitochondrial HMG-CoA synthase levels observed under the diabetic condition. Furthermore, the finding that quantitative changes in HMG-CoA synthase protein were less marked than those in the corresponding mRNA in starved and diabetic rats suggests either translational control or increased degradation of either mRNA or protein. All these results indicate that mitochondrial HMG-CoA synthase is a regulatory element in the ketogenic process. Biochem J. 1993 Jun 1. The tissue-specific expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene was studied in 15-day-old suckling rats. The mRNA and protein were present in liver, intestine and kidney, but were absent from brain, heart, skeletal muscles, brown and white adipose tissues. Kidney-cortex mitochondria from suckling rats were able to produce low amounts of ketone bodies from oleate. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels increased slowly during foetal life and markedly after birth. The postnatal increase in liver HMG-CoA synthase mRNA could be due to the increase in plasma glucagon levels, since it rapidly induced the accumulation of HMG-CoA synthase mRNA in cultured foetal hepatocytes. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels remained elevated throughout the suckling period or in rats weaned on to a high-fat carbohydrate-free diet (HF), but decreased by 50% in the liver and totally disappeared from the intestine and the kidney of rats weaned on to a high-carbohydrate low-fat diet (HC). When HC-weaned rats were fed on a HF-diet for a week, HMG-CoA synthase mRNA was re-induced in the intestine and the kidney. The role of hormones and nutrients in the regulation of HMG-CoA synthase gene expression is discussed. J Lipid Res. 1993 Jun. Ketogenesis has been thought to occur exclusively in the mitochondrial compartment of liver cells. After analysis of five different rat tissues, it was shown that the gene for mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, one of the major control points in the pathway (1992. Casals et al. Biochem. J. 283: 261-264) was expressed only in liver (1990. Ayte et al. Proc. Natl. Acad. Sci. USA. 87: 3874-3878). However, exhaustive analysis of organs and tissues has shown that, in addition to liver cells, testis and ovary express this committed gene in levels similar to those of liver, not only as mRNAs but also as immunodetectable mitochondrial HMG-CoA synthase protein. Immunocytochemical studies locate the mitochondrial HMG-CoA synthase protein in Leydig cells, theca interna cells of ovarian follicle, corpus luteum cells of ruptured ovarian follicle, and epidermal cells of the oviduct. The development of gonadal function appears to be accompanied by mitochondrial HMG-CoA synthase gene expression, as hypophysectomy reduces the expression pattern in gonads. Changes induced in mitochondrial HMG-CoA synthase levels after the depletion of lipoprotein levels in blood closely mimic those of the cholesterogenic cytosolic HMG-CoA synthase and HMG-CoA reductase. These results suggest that mitochondrial HMG-CoA synthase could perform a function similar to that of cytosolic HMG-CoA synthase in de novo cholesterogenesis in gonads, at variance with its ketogenic role in liver. Biochem J. 1992 Apr 1. We have explored the role of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in regulating ketogenesis. We had previously cloned the cDNA for mitochondrial HMG-CoA synthase and have now studied the regulation in vivo of the expression of this gene in rat liver. The amount of processed mitochondrial HMG-CoA synthase mRNA is rapidly changed in response to cyclic AMP, insulin, dexamethasone and refeeding, and is greatly increased by starvation, fat feeding and diabetes. We conclude that one point of ketogenic control is exercised at the level of genetic expression of mitochondrial HMG-CoA synthase. Eur J Biochem. 1991 Jan 30. (1) We assayed active and total (i.e. active plus succinylated) 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in mitochondria isolated from foetal, neonatal, suckling or weaned rats. (2) HMG-CoA synthase was substantially succinylated and inactivated in mitochondria isolated from term-foetal, (1-h-old, 6-h-old, 1-day-old) neonatal, suckling and high carbohydrate/low-fat (hc)-weaned rats. Succinylation of HMG-CoA synthase was very low in mitochondria isolated from the livers of foetal, 30-min-old neonatal and high-fat/carbohydrate-free (hf)-weaned rats. (3) There was a negative correlation between active HMG-CoA synthase and succinyl-CoA content in mitochondria isolated from term-foetal, suckling and hc-weaned rats. (4) Differences in active enzyme could not be entirely accounted for by differences in succinylation and inactivation of the synthase. Immunoassay confirmed that the absolute amounts of mitochondrial HMG-CoA synthase increased during the foetal/neonatal transition and decreased with hc weaning. The levels remained elevated with hf weaning. (5) From these data we propose that mitochondrial HMG-CoA synthase is controlled by two different mechanisms in young rats. Regulation by succinylation provides a mechanism for rapid modification of existing enzyme in response to changing metabolic states. Changes in the absolute amounts of HMG-CoA synthase provide a more long-term control in response to nutritional changes. Eur J Biochem. 1990 Jan 12. 1. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rat liver mitochondria can be inactivated by succinyl-CoA and activated by incubation in a medium designed to cause desuccinylation ('desuccinylation medium'). 2. The enzyme is less active in extracts of whole liver from control rats than from rats treated with glucagon or mannoheptulose. Incubation in desuccinylation medium raises the activity in extracts from control rats to the same value as treated rats, suggesting that the extent of succinylation in vivo is greater in controls than in hormone-treated animals. 3. This result is also obtained in liver homogenates and in isolated mitochondria. 4. Increasing the succinyl-CoA content of mitochondria to the same high level lowers the enzyme activity to the same value in mitochondria isolated from control or treated rats. In each case subsequent incubation of the lysates in desuccinylation medium raises the enzyme activity by the same extent. 5. Measurement of the incorporation of radiolabel from 2-oxo[5-14C]glutarate into protein is consistent with the proposal that all these changes in activity in isolated mitochondria may be explained by changes in the extent of succinylation of the enzyme. 6. From these data and our earlier work we conclude that, in vivo, mitochondrial HMG-CoA synthase in fed rats is normally substantially succinylated (about 40%) and inactivated, and that glucagon increases the activity of HMG-CoA synthase by lowering the concentration of succinyl-CoA and thus decreasing the extent of succinylation of the enzyme (to less than 10%). This may be an important control mechanism in ketogenesis. Biochem J. 1989 Aug 15. 1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase. Biochem J. 1985 Nov 15. Succinyl-CoA (3-carboxypropionyl-CoA) inactivates ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) in a time-dependent manner, which is partially prevented by the presence of substrates of the enzyme. The inactivation is due to the enzyme catalysing its own succinylation. Complete inactivation corresponds to about 0.5 mol of succinyl group bound/mol of enzyme dimer. The succinyl-enzyme linkage appears to be a thioester bond and is probably formed with the active-site cysteine residue that is normally acetylated by acetyl-CoA. Succinyl-CoA binds to 3-hydroxy-3-methylglutaryl-CoA synthase with a binding constant of 340 microM and succinylation occurs with a rate constant of 0.57 min-1. Succinyl-enzyme breaks down with a half-life of about 40 min (k = 0.017 min-1) at 30 degrees C and pH 7 and is destabilized by the presence of acetyl-CoA and succinyl-CoA. A control mechanism is postulated in which flux through the 3-hydroxy-3-methylglutaryl-CoA cycle of ketogenesis is regulated according to the extent of succinylation of 3-hydroxy-3-methylglutaryl-CoA synthase. Biochemistry. 1985 Jun 18. 3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue. Biol Neonate. 1983. Rat hepatic mitochondrial hydroxymethylglutaryl-CoA (HMG-CoA) synthase activity increases throughout the perinatal period, indicating that the factors necessary for the induction of the enzyme are present in utero. Reciprocal changes occur in 3-ketoacid transferase activity during late fetal life, suggesting that ketone body formation and utilization may be subject to coordinate regulation. Cytosolic HMG-CoA synthase activity is low during fetal life and rises rapidly after birth. The activities of both cytosolic and mitochondrial enzymes in fetal liver are dependent upon maternal diet and different dietary effects are observed at different fetal ages. Neurochem Res. 1982 Nov. The properties and developmental change in the activity of cytosolic 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) synthase in brain was examined and whether or not HMG-CoA lyase is present in cytosol and mitochondria from brain was determined. Although mitochondrial fractions contained significant HMG-CoA lyase activity, the enzyme activity was not detected in brain cytosol. The synthase activity was present in both mitochondrial and cytosolic fraction. The HMG-CoA synthesis by brain cytosol was optimal at pH 8.0 and did not require Mg2+ or exogenous acetoacetyl CoA. This indicates that brain cytosol can synthesize sufficient quantity of acetoacetyl CoA from acetyl CoA to be utilized for HMG-CoA synthesis. Our results also showed that the specific activity (nmol acetyl CoA incorporated/mg protein) of HMG-CoA synthase in brain cytosol was high (between 2-11 days of postnatal age) when the cholesterol content of brain is increasing rapidly, and the activity declined slowly thereafter. This suggests that in brain, cytosolic enzyme HMG-CoA synthase plays a role in the regulation of cholesterol synthesis. Clin Sci (Lond). 1979 Mar. 1. Succinyl-CoA inhibits 3-hydroxy-3-methyl-glutaryl-CoA lyase (EC 4.1.3.4) when added to purified preparations of the enzyme. 2. The apparent Ki value is 2.1 x 10(-4) mol/l and the inhibition has the features of a partially competitive inhibition. 3. The effect of succinyl-CoA both added and enzymically produced on the lyase activity of sonically disrupted rat liver mitochondria results in decreased acetoacetate formation. 4. This occurs with mitochondria obtained from normal, starved and streptozotocin-diabetic rats. Biochem J. 1978 Dec 15. 1. Data are provided that indicate that the rat brain acetoacetyl-CoA deacylase is almost exclusively mitochondrial. Developmental studies show that this enzyme more than doubles its activity during suckling (0--21 days) and then maintains this activity in adults (approx. 1.1 units/g wet wt.). 2. Kinetic studies (on the acetoacetyl-CoA deacylase) in a purified brain mitochondrial preparation give a Vmax. of 47 nmol/min per mg of protein, and a Km for acetoacetyl-CoA of 5.2 micron and are compatible with substrate inhibition by acetoacetyl-CoA above concentrations of 47 micron. 3. The total brain 3-hydroxy-3-methyl-glutaryl-CoA synthase remains constant in the developing and adult rat brain (approx. 1.2 units/g wet wt.). This enzyme is located in both the mitochondrial and cytosolic fractions. During suckling (0--21 days) the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase represents approx. one-third of the total, but this increases markedly to about 60% of the total in the adult. The cytosolic enzyme correspondingly falls to approx. 40% of the total. 4. The role of the acetoacetyl-CoA deacylase in providing cytosolic acetoacetate for biosynthetic activities in the developing brain is discussed. Enzyme. 1977. The activities of hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA lyase have been determined in the mitochondria and cytosol of the liver during development of the rat. Both mitochondrial enzymes exhibit similar developmental patterns with rapid rises in activity after birth, peaks of activity during early and late suckling and a trough during the mid-suckling period and a slight fall in activity (to the adult values) after weaning. Both cytosolic enzymes have low activities at all ages studied and exhibit no major developmental changes. J Biol Chem. 1975 Apr 25. Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase. |
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