malonyl-CoA Decarboxylase Activity Research Articles

Cardiovascular involvement in later-onset malonyl-CoA decarboxylase deficiency: Case studies and literature review

BackgroundMalonyl-CoA decarboxylase deficiency (MLYCDD) is an ultra-rare inherited metabolic disorder, characterized by multi-organ involvement manifesting during the first few months of life. Our aim was to describe the clinical, biochemical, and genetic characteristics of patients with later-onset MLYCDD. MethodsClinical and biochemical characteristics of two patients aged 48 and 29 years with a confirmed molecular diagnosis of MLYCDD were examined. A systematic review of published studies describing the characteristics of cardiovascular involvement of patients with MLYCDD was performed. ResultsTwo patients diagnosed with MLYCDD during adulthood were identified. The first presented with hypertrophic cardiomyopathy and ventricular pre-excitation and the second with dilated cardiomyopathy (DCM) and mild-to-moderate left ventricular (LV) systolic dysfunction. No other clinical manifestation typical of MLYCDD was observed. Both patients showed slight increase in malonylcarnitine in their plasma acylcarnitine profile, and a reduction in malonyl-CoA decarboxylase activity. During follow-up, no deterioration of LV systolic function was observed.The systematic review identified 33 individuals with a genetic diagnosis of MLYCDD (median age 6 months [IQR 1–12], 22 males [67%]). Cardiovascular involvement was observed in 64% of cases, with DCM the most common phenotype. A modified diet combined with levocarnitine supplementation resulted in the improvement of LV systolic function in most cases. After a median follow-up of 8 months, 3 patients died (two heart failure-related and one arrhythmic death). ConclusionsFor the first time this study describes a later-onset phenotype of MLYCDD patients, characterized by single-organ involvement, mildly reduced enzyme activity, and a benign clinical course.

The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism.

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075-1096. © 2016 Wiley Periodicals, Inc.

P84 – 2801: Two novel, disease-causing mutations in the ACSF3 gene in a patient with combined malonic and methylmalonic aciduria

Background Combined malonic and methylmalonic aciduria (CMAMMA) is a rare inborn error of metabolism with elevated excretion of malonic acid (MA) and methylmalonic acid (MMA) in urine commonly caused by mutations in the MLYCD gene leading to malonyl-CoA decarboxylase (MCD) deficiency. Clinical features are metabolic acidosis, seizures, axial hypotonia, developmental delay, failure to thrive, microcephaly, cardiomyopathy, elevated transaminases and gastrointestinal symptoms. Benign clinical courses and asymptomatic patients summarized as non-classical phenotype with mostly normal MCD activity are described. In these patients mutations in the ACSF3 gene encoding a methylmalonyl-CoA and malonyl-CoA synthetase are frequently detected. Case report We describe a 3-year-old boy diagnosed with CMAMMA at the age of 26 months. After an uneventful pregnancy a boy of unrelated Afghan parents was born at term with a birth weight of 2955 g. At three weeks he presented with two generalized seizures and metabolic acidosis. Plasma lactat level was normal. Initially moderately elevated levels of MMA were found in urine. Plasma amino acids, carnitine levels, acylcarnitine profile, homocysteine, folate, vitamin B12, liver and renal function parameters were normal. Initial electroencephalogram (EEG) was pathologic and anticonvulsive therapy with phenobarbital was initiated. No further seizures occurred, EEG normalized at the age of three months and antiepileptic medication was stopped. Besides microcephaly the development is age-appropriate up to now. Asymptomatic cholelithiasis persisted despite therapy with ursodeoxycholic acid. Echocardiography was normal. Genetic analysis revealed two novel, disease-causing mutations in the ACSF3 gene and led to the diagnosis auf CMAMMA. Excretion of MMA in urine decreased spontaneously. A mild protein restricted diet was started. A younger sister also presented generalized seizure and elevated MMA in urine at the age of four weeks. Her genetic analysis is in progress. Conclusion We found two novel mutations in the ACSF3 gene which we assume to cause a non-classic CMAMMA phenotype.

Chemical-genetic induction of Malonyl-CoA decarboxylase in skeletal muscle.

BackgroundDefects in skeletal muscle fatty acid oxidation have been implicated in the etiology of insulin resistance. Malonyl-CoA decarboxylase (MCD) has been a target of investigation because it reduces the concentration of malonyl-CoA, a metabolite that inhibits fatty acid oxidation. The in vivo role of muscle MCD expression in the development of insulin resistance remains unclear.ResultsTo determine the role of MCD in skeletal muscle of diet induced obese and insulin resistant mouse models we generated mice expressing a muscle specific transgene for MCD (Tg-fMCDSkel) stabilized posttranslationally by the small molecule, Shield-1. Tg-fMCDSkel and control mice were placed on either a high fat or low fat diet for 3.5 months. Obese and glucose intolerant as well as lean control Tg-fMCDSkel and nontransgenic control mice were treated with Shield-1 and changes in their body weight and insulin sensitivity were determined upon induction of MCD. Inducing MCD activity >5-fold in skeletal muscle over two weeks did not alter body weight or glucose intolerance of obese mice. MCD induction further potentiated the defects in insulin signaling of obese mice. In addition, key enzymes in fatty acid oxidation were suppressed following MCD induction.ConclusionAcute induction of MCD in the skeletal muscle of obese and glucose intolerant mice did not improve body weight and decreased insulin sensitivity compared to obese nontransgenic controls. Induction of MCD in skeletal muscle resulted in a suppression of mitochondrial oxidative genes suggesting a redundant and metabolite driven regulation of gene expression.

Effects of Teucrium polium aerial parts extracts on malonyl-CoA decarboxylase level

A B S T R A C T Malonyl-CoA decarboxylase (MCD) is an enzyme involved in the decarboxylation of malonyl-CoA to acetyl-CoA. In order to explore the hypothesis that the changing plant materials’ MCD activity level can serve as therapy to diabetics, the effect of Teucrium polium compounds was studied in a diabetic rat model. In this experimental study, two groups of rats, a control and a diabetic group, each including six rats, were used. At the end of the experiment, all rats were exterminated by ether anesthesia, their pancreases removed and dissected. Isolated rat pancreas was cultured in buffers with or without 100-500µg/l T. polium aerial parts extracts containing arginine and leucine. MCD and insulin levels were measured after culture at 37°C and 5% CO2, for 1, 3 and 5 days. Results showed that T. polium aqueous and the alcoholic extract decreased MCD activity. Present data also indicate that incubation of pancreatic tissue at a concentration of 2.8 and 16.7 mmol/L glucose stimulated insulin release. For the first time it seems that aqueous and alcoholic extracts of this plant decreased MCD activity.

SIRT4 Coordinates the Balance between Lipid Synthesis and Catabolism by Repressing Malonyl CoA Decarboxylase

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.

Structural Asymmetry and Disulfide Bridges among Subunits Modulate the Activity of Human Malonyl-CoA Decarboxylase

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif.

Ethylmalonyl-CoA Decarboxylase, a New Enzyme Involved in Metabolite Proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.

Malonyl-CoA decarboxylase inhibition is selectively cytotoxic to human breast cancer cells

Fatty acid synthase (FAS) inhibition initiates selective apoptosis of cancer cells both in vivo and in vitro, which may involve malonyl-CoA metabolism. These findings have led to the exploration of malonyl-CoA decarboxylase (MCD) as a potential novel target for cancer treatment. MCD regulates the levels of cellular malonyl-CoA through the decarboxylation of malonyl-CoA to acetyl-CoA. Malonyl-CoA is both a substrate for FAS and an inhibitor of fatty acid oxidation acting as a metabolic switch between anabolic fatty acid synthesis and catabolic fatty acid oxidation. We now report that the treatment of human breast cancer (MCF7) cells with MCD small interference RNA (siRNA) reduces MCD expression and activity, reduces adenosine triphosphate levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we synthesized a small-molecule inhibitor of MCD, 5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-pentanoic acid methyl ester (MPA). Similar to MCD siRNA, MPA inhibits MCD activity in MCF7 cells, increases cellular malonyl-CoA levels and is cytotoxic to a number of human breast cancer cell lines in vitro. Taken together, these data indicate that MCD-induced cytotoxicity is likely mediated through malonyl-CoA metabolism. These findings support the hypothesis that MCD is a potential therapeutic target for cancer therapy.

Rosiglitazone and fenofibrate improve insulin sensitivity of pre-diabetic OLETF rats by reducing malonyl-CoA levels in the liver and skeletal muscle

Aims Rosiglitazone and fenofibrate, specific agonists of the peroxisome proliferator activated receptors-γ (PPARγ) and -α (PPARα), respectively, improve insulin sensitivity in diabetic animals and in patients with type 2 diabetes. Here we investigated how pre-diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats fed with normal and high-fat diets respond to these PPAR agonists. Main methods Pre-diabetic OLETF rats were subjected to high-fat or standard diets with or without rosiglitazone or fenofibrate for 2 weeks. The metabolism of the rats and the levels of malonyl-CoA and activities of malonyl-CoA decarboxylase (MCD), acetyl-CoA carboxylase (ACC), and AMP-activated protein kinase (AMPK) in metabolic tissues were assessed. Key findings Rosiglitazone and fenofibrate significantly improved insulin sensitivity and reduced the levels of plasma triglycerides and free fatty acids in OLETF rats fed with a high-fat diet. Fenofibrate particularly reduced the body weight, fat, and total cholesterol in high fat diet OLETF rats. The highly elevated malonyl-CoA levels in the skeletal muscle and liver of OLETF rat were significantly reduced by rosiglitazone or fenofibrate due to, in part, the increased MCD activities and expression. On the other hand, ACC activities were unchanged in skeletal muscle and decreased in liver in high fat diet group. AMPK activities were dramatically decreased in OLETF rats and not affected by these agonists. Significance These results demonstrate that treatment of pre-diabetic OLETF rats–particularly those fed a high-fat diet–with rosiglitazone and fenofibrate significantly improves insulin sensitivity and fatty acid metabolism by increasing the activity of MCD and reducing malonyl-CoA levels in the liver and skeletal muscle.

Myocardial Hypertrophy and the Maturation of Fatty Acid Oxidation in the Newborn Human Heart

After birth dramatic decreases in cardiac malonyl CoA levels result in the rapid maturation of fatty acid oxidation. We have previously demonstrated that the decrease in malonyl CoA is due to increased activity of malonyl CoA decarboxylase (MCD), and decreased activity of acetyl CoA carboxylase (ACC), enzymes which degrade and synthesize malonyl CoA, respectively. Decreased ACC activity corresponds to an increase in the activity of 5'-AMP activated protein kinase (AMPK), which phosphorylates and inhibits ACC. These alterations are delayed by myocardial hypertrophy. As rates of fatty acid oxidation can influence the ability of the heart to withstand an ischemic insult, we examined the expression of MCD, ACC, and AMPK in the newborn human heart. Ventricular biopsies were obtained from infants undergoing cardiac surgery. Immunoblot analysis showed a positive correlation between MCD expression and age. In contrast, a negative correlation in both ACC and AMPK expression and age was observed. All ventricular samples displayed some degree of hypertrophy, however, no differences in enzyme expression were found between moderate and severe hypertrophy. This indicates that increased expression of MCD, and the decreased expression of ACC and AMPK are important regulators of the maturation of fatty acid oxidation in the newborn human heart.

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP‐activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 microM of palmitate did not affect cell viability but significantly reduced FA oxidation by approximately 26.5%, approximately 43.5%, approximately 50%, and approximately 47%, respectively. Interestingly, this occurred despite significant increases in AMPK ( approximately 2.5-fold) and ACC ( approximately 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity ( approximately 38-60%). Low concentrations of palmitate (50-100 microM) caused an increase ( approximately 30%) in CPT-1 activity. However, as the concentration of palmitate increased, CPT-1 activity decreased by approximately 32% after exposure for 8 h to 800 microM of palmitate. Although FA uptake was reduced ( approximately 35%) in cells exposed to increasing palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values approximately 2.3-, approximately 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 microM palmitate, respectively. Interestingly, myotubes exposed to 400 microM of palmitate for 1 h increased basal glucose uptake and glycogen synthesis by approximately 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8 h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake ( approximately 65%) and glycogen synthesis ( approximately 30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs, such as in obesity and Type 2 Diabetes.

A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20 nM acetyl-CoA, compared to 3 μM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening and yields excellent assay statistics (Z′ > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that generate acetyl-CoA.

Clinical, enzymatic and molecular characterization of nine new patients with malonyl‐coenzyme A decarboxylase deficiency

We report nine new patients with malonic aciduria associated with enzyme-confirmed malonyl-CoA decarboxylase (MCD) deficiency in eight. Clinical details were available on eight, and molecular genetic characterization was obtained for nine. As for 15 previously described patients, cardinal clinical manifestations included developmental delay and cardiomyopathy; metabolic perturbations (e.g. acidosis) and seizures, however, were infrequent or not observed in our patients. For all, detection of elevated malonic acid in urine (+/- increased C3DC acylcarnitine by analysis employing tandem mass spectrometry) led to pursuit of enzyme studies. MCD activities (nmol/h PER mg protein) revealed: control (n = 22), 16.2 +/- 1.8 (SEM; range 5.7-46.2); patients (n = 8, assayed in duplicate), 1.7 +/- 0.3 (10% of parallel control; range 0.6-2.8). Molecular characterization by DNA sequence analysis and multiplex ligation-dependent probe amplification revealed nine novel mutations (c.796C>T; p.Gln266X, c.481delC; p.Leu161CysfsX18, c.1367A>C; p.Tyr456Ser, c.1319G>T; p.Ser440Ile, c.1430C>T; p.Ser477Phe, c.899G>T; p.Gly300Val, c.799-1683_949-1293del3128, and two other large genomic deletions comprising exons 1 or the complete gene) and two known mutations in the MLYCD gene. Our findings increase the number of enzyme-confirmed MCD-deficient patients by >50%, and expand our understanding of the phenotypic and molecular heterogeneity of this rare disorder.

Malonyl-CoA Decarboxylase Inhibition as a Novel Approach to Treat Ischemic Heart Disease

During and following cardiac ischemia the levels of circulating fatty acids are elevated, resulting in fatty acid oxidation dominating as a source of oxidative metabolism at the expense of pyruvate oxidation. A decrease in the levels of myocardial malonyl-CoA (an endogenous inhibitor of mitochondrial fatty acid uptake) contributes to these high fatty acid oxidation rates. Low pyruvate oxidation rates during and following ischemia results in the accumulation of metabolic byproducts (lactate and protons) that leads to impaired cardiac function, decreased cardiac efficiency, and increased myocardial tissue injury. One approach to increasing pyruvate oxidation during and following ischemia is to inhibit fatty acid oxidation, which results in an improvement of both cardiac function and cardiac efficiency. A novel approach to decreasing fatty acid oxidation and increasing pyruvate oxidation is to increase myocardial levels of malonyl-CoA. This can be achieved by pharmacologically inhibiting malonyl-CoA decarboxylase (MCD), the principal enzyme involved in the degradation of cardiac malonyl-CoA. Studies with either genetic deletion of MCD in the mouse or with novel MCD inhibitors show that decreased MCD activity increases cardiac malonyl-CoA, resulting in an inhibition of fatty acid oxidation and a stimulation of pyruvate oxidation. The beneficial effects of MCD inhibition on cardiac function and cardiac efficiency suggest that this approach could be an effective means to treat ischemic heart disease.

Role of malonyl-CoA in heart disease and the hypothalamic control of obesity

Obesity is an important contributor to the risk of developing insulin resistance, diabetes, and heart disease. Alterations in tissue levels of malonyl-CoA have the potential to impact on the severity of a number of these disorders. This review will focus on the emerging role of malonyl-CoA as a key "metabolic effector" of both obesity and cardiac fatty acid oxidation. In addition to being a substrate for fatty acid biosynthesis, malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase (CPT) 1, a key enzyme involved in mitochondrial fatty acid uptake. A decrease in myocardial malonyl-CoA levels and an increase in CPT1 activity contribute to an increase in cardiac fatty acid oxidation. An increase in malonyl-CoA degradation due to increased malonyl-CoA decarboxylase (MCD) activity may be one mechanism responsible for this decrease in malonyl-CoA. Another mechanism involves the inhibition of acetyl-CoA carboxylase (ACC) synthesis of malonyl-CoA, due to AMP-activated protein kinase (AMPK) phosphorylation of ACC. Recent studies have demonstrated a role of malonyl-CoA in the hypothalamus as a regulator of food intake. Increases in hypothalamic malonyl-CoA and inhibition of CPT1 are associated with a decrease in food intake in mice and rats, while a decrease in hypothalamic malonyl-CoA increases food intake and weight gain. The exact mechanism(s) responsible for these effects of malonyl-CoA are not clear, but have been proposed to be due to an increase in the levels of long chain acyl CoA, which occurs as a result of malonyl-CoA inhibition of CPT1. Both hypothalamic and cardiac studies have demonstrated that control of malonyl-CoA levels has an important impact on obesity and heart disease. Targeting enzymes that control malonyl-CoA levels may be an important therapeutic approach to treating heart disease and obesity.

Assay of the activity of malonyl‐CoA decarboxylase (MCD) by gas chromatography‐mass spectrometry (GC‐MS)

We developed a GC-MS assay for the activity of MCD in tissue homogenates. Extracts of rat liver (0.25 mg tissue equivalent/assay) are incubated at 37°C with 0.4 mM [U-13C3]malonyl-CoA to form [1,2-13C2]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-13C2]acetyl-CoA by acetyl-CoA hydrolase present in the tissue extracts. After stopping the reaction with methanol, an internal standard of [2H3, 1-13C]acetyl-CoA is added before reacting the two forms of acetyl-CoA with thiophenol. The product acetylthiophenol is analyzed by ammonia positive chemical ionization GC-MS, monitoring 170 (M + NH4+) to 174 (M + NH4+ + 4). The assay was applied to a study of the kinetics of MCD in rat liver, yielding Km and Vmax of 171 μM and 2.5 μmol·min 1·(g liver) 1, respectively. Also, 10 μM CBM-301940, a potent MCD inhibitor (Chugai Pharma), decreased MCD activity 7-fold. Lastly, liver MCD activities (μmol·min 1·g 1 ± S.D.) in three groups of rats that were fed, one-day fasted or two-day fasted were 1.66 ± 0.53, 2.35 ± 0.38 (p < 0.05), and 2.72 ± 0.62 (p < 0.05) respectively. Supported by NIH Roadmap grant R33DK070291.

Exercise training decreases the concentration of malonyl-CoA and increases the expression and activity of malonyl-CoA decarboxylase in human muscle

The study was designed to evaluate whether changes in malonyl-CoA and the enzymes that govern its concentration occur in human muscle as a result of physical training. Healthy, middle-aged subjects were studied before and after a 12-wk training program that significantly increased VO2 max by 13% and decreased intra-abdominal fat by 17%. Significant decreases (25-30%) in the concentration of malonyl-CoA were observed after training, 24-36 h after the last bout of exercise. They were accompanied by increases in both the activity (88%) and mRNA (51%) of malonyl-CoA decarboxylase (MCD) in muscle but no changes in the phosphorylation of AMP kinase (AMPK, Thr172) or of acetyl-CoA carboxylase. The abundance of peroxisome proliferator-activated receptor (PPAR)gamma coactivator-1alpha (PGC-1alpha), a regulator of transcription that has been linked to the mediation of MCD expression by PPARalpha, was also increased (3-fold). In studies also conducted 24-36 h after the last bout of exercise, no evidence of increased whole body insulin sensitivity or fatty acid oxidation was observed during an euglycemic hyperinsulinemic clamp. In conclusion, the concentration of malonyl-CoA is diminished in muscle after physical training, most likely because of PGC-1alpha-mediated increases in MCD expression and activity. These changes persist after the increases in AMPK activity and whole body insulin sensitivity and fatty acid oxidation, typically caused by an acute bout of exercise in healthy individuals, have dissipated.

Effects of PPAR-α and -γ Agonists on Fatty Acid Metabolism of Muscle Cells in Hyperlipidemic and Hyperglycemic Conditions

Background: Studies for the regulation of fatty acid metabolism are deficient relatively in skeletal muscle and heart. The investigations in pathological conditions for malonyl-CoA decarboxylase (MCD) and for the relation of MCD and PPAR-․-γ agonists are insufficient in particular. Methods: In the current study, fully differentiated H9c2 muscle cells were exposed to pathological conditions such as hyperlipidemic (0.1 mM Palmitate) and hyperglycemic (16.5 mM Glucose) condition with 5 uM PPAR-γ agonist (rosiglitazone) and 10 uM PPAR- agonist (WY14,643) and then experiments such as MCD activity assay, MCD real-time RT-PCR, MCD reporter gene assay, MCD Western blotting, PPAR- Western blotting, and palmitate oxidation test were carried out. Results: Only PPAR- agonist increased MCD activity. In the result of real-time RT-PCR, both PPAR- and PPAR-γ agonists elevated MCD mRNA expression in hyperlipidemic condition. MCD protein expression was decreased in hyperlipidemic condition, however, increased in rosiglitazone, or WY14,643 treated conditions. Rosiglitazone, and WY14,643 treated groups showed incresed MCD protein expression in hyperglycemic condition. Hyperlipidemic control group and PPAR-․ﾇ-γ agonists treated groups presented about 3.8 times more increased palmitate oxidation level than normolipidemic control group in hyperlipidemic condition. PPAR- agonist treated group showed 49% more increased palmitate oxidation rate than hyperlipidemic control group in primary cultured rat skeletal muscle cells. The amount of palmitate oxidation from differentiated H9c2 muscle cells that had overexpressed PPAR- structural genes was more increased than control group. Conclusion: This study suggests that PPAR- agonist ameliorates the defects induced by hyperlipidemic condition through the regulation of MCD. In summary, a closely reciprocal relation among PPAR- agonist, MCD, and fatty acid oxidation existed distinctly in hyperlipidemic condition, but not in hyperglycemic condition.

Malonyl-CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes

A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.