malonyl-CoA Decarboxylase Research Articles

AMP‐activated protein kinase control of fat metabolism in skeletal muscle

AMP-activated protein kinase (AMPK) has emerged as a key regulator of skeletal muscle fat metabolism. Because abnormalities in skeletal muscle metabolism contribute to a variety of clinical diseases and disorders, understanding AMPK's role in the muscle is important. It was originally shown to stimulate fatty acid (FA) oxidation decades ago, and since then much research has been accomplished describing this role. In this brief review, we summarize much of these data, particularly in relation to changes in FA oxidation that occur during skeletal muscle exercise. Potential roles for AMPK exist in regulating FA transport into the mitochondria via interactions with acetyl-CoA carboxylase, malonyl-CoA decarboxylase, and perhaps FA transporter/CD36 (FAT/CD36). Likewise, AMPK may regulate transport of FAs into the cell through FAT/CD36. AMPK may also regulate capacity for FA oxidation by phosphorylation of transcription factors such as CREB or coactivators such as PGC-1alpha.

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.

Targeting malonyl CoA inhibition of mitochondrial fatty acid uptake as an approach to treat cardiac ischemia/reperfusion

Cardiovascular disease is the major cause of death and disability in the world, with ischemic heart disease accounting for the vast majority of this health problem. Current treatments for ischemic heart disease are primarily aimed at either increasing blood and oxygen supply to the heart or decreasing the heart's oxygen demand. A novel treatment strategy involves increasing the efficiency of oxygen use by the heart. During and following ischemia, the heart can become inefficient in using oxygen, due in part to an excessive use of fatty acids as a source of fuel. One potential strategy to increase cardiac efficiency is to inhibit this use of fatty acid oxidation as a fuel source, while stimulating the use of glucose oxidation as a fuel source, which allows the heart to produce energy more efficiently and reduces the acidosis associated with ischemia/reperfusion, both of which are beneficial to the heart. Malonyl CoA is a potent endogenous inhibitor of cardiac fatty acid oxidation, secondary to inhibition of carnitine palmitoyl transferase-I, the gatekeeper of mitochondrial fatty acid uptake. Malonyl CoA is synthesized in the heart by acetyl CoA carboxylase and degraded by malonyl CoA decarboxylase (MCD). Strategies aimed at increasing cardiac malonyl CoA levels, such as via inhibition of MCD, are associated with a decrease in fatty acid oxidation rates, and a parallel increase in glucose oxidation rates. This is associated with a decrease in acidosis and an improvement in cardiac function and efficiency during and following ischemia. Therefore, targeting malonyl CoA is a novel exciting approach for the treatment of cardiac ischemia/reperfusion.

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.

Abstract 5381: Insulin-Stimulated Glucose Oxidation is Increased in Hearts from High Fat Diet-Induced Obese Mice Lacking Malonyl CoA Decarboxylase

OBJECTIVE - Diet-induced obesity (DIO) leads to an accumulation of intra-myocardial fatty acid metabolites that have been proposed to cause myocardial insulin resistance and dysfunction. Our goal was to determine the effect of DIO on myocardial fatty acid metabolite accumulation and how this is altered when mitochondrial fatty acid uptake is inhibited. This was achieved by using mice lacking malonyl CoA decarboxylase (MCD−/−), which have higher levels of malonyl CoA, an endogenous inhibitor of mitochondrial fatty acid uptake. METHODS - Wild type (WT) and MCD−/− mice were fed a low (4% kcal from lard) or high (60% kcal from lard) fat diet for 12 weeks to determine the effect of DIO on the intra-myocardial accumulation of long chain acylcarnitines, long chain acyl CoAs, triglycerides (TGs), and ceramides. A parallel feeding study was performed to assess myocardial function and energy metabolism in isolated working hearts in the absence/presence of insulin. RESULTS - We demonstrate that MCD−/− mice do not accumulate intramyocardial long chain acylcarnitines to the same extent as WT mice following DIO (0.56 ± 0.10 vs. 0.28 ± 0.07 pmol myristoylcarnitine/mg protein, P <0.05), but do accumulate similar amounts of long chain acyl CoAs (3.88 ± 0.34 vs. 4.35 ± 1.19 nmol/g wet weight). Interestingly, DIO only lead to an accumulation of TGs in the hearts of MCD−/− mice (3.29 ± 0.62 vs. 10.92 ± 3.72 μmol/g wet weight, P <0.05). Despite this elevation in TGs, MCD−/− mice showed increased insulin-stimulated glucose oxidation (2.46 ± 0.25 vs. 1.74 ± 0.18 fold increase, P <0.05) during aerobic isolated working heart perfusions and did not elicit any dysfunction. CONCLUSIONS - Our data reveal discordance between myocardial TG accumulation and glucose metabolism, suggesting that TG buffers against toxic lipids, and that inhibition of mitochondrial fatty acid oxidation does not cause myocardial dysfunction following DIO.

Topological descriptors in modeling malonyl coenzyme A decarboxylase inhibitory activity: N-Alkyl-N-(1,1,1,3,3,3-hexafluoro-2-hydroxypropylphenyl)amide derivatives

The malonyl-CoA decarboxylase (MCD) inhibition activity of derivatives of N-alkyl-N-(1,1,1,3,3,3-hexafluoro-2-hydroxypropylphenyl)amide has been analyzed through combinatorial protocolin multiple linear regression (CP-MLR) using different topological descriptors obtained from Dragon software for the energy minimized 3D-structures of these molecules. Among the topological descriptor classes considered in the study, the MCD inhibition activity is correlated with simple topological descriptors (TOPO) and 2D-autocorrelation descriptors (2DAUTO). The complementary information contents having neighborhood symmetry of 2-order, CIC2 from the TOPO class, the Geary autocorrelations-lag 8, weighted by atomic Sanderson electronegativities, GATS8e and the Moran autocorrelations-lag 6, weighted by atomic Sanderson electronegativities, MATS6e both from 2DAUTO class have contributed significantly in the development of a statistical significant model.

Regulation of food intake and energy expenditure by hypothalamic malonyl-CoA

Energy balance is monitored by the hypothalamus. Malonyl-CoA, an intermediate in fatty acid synthesis, serves as an indicator of energy status in the hypothalamic neurons. The cellular malonyl-CoA level is determined by its rate of synthesis, catalyzed by acetyl-CoA carboxylase (ACC), and rate of removal, by fatty acid synthase (FAS). Malonyl-CoA functions in the hypothalamic neurons that express orexigenic and anorexigenic neuropeptides. Inhibitors of FAS, administered systemically or intracerebroventricularly to mice, increase hypothalamic malony-CoA and suppress food intake. Recent evidence suggests that the changes of hypothalamic malonyl-CoA during feeding and fasting cycles are caused by changes in the phosphorylation state and activity of ACC mediated via 5'-AMP-activated protein kinase (AMPK). Stereotactic delivery of a viral malonyl-CoA decarboxylase (MCD) vector into the ventral hypothalamus lowers malonyl-CoA and increases food intake. Fasting decreases hypothalamic malonyl-CoA and refeeding increases hypothalamic malonyl-CoA, to alter feeding behavior in the predicted manner. Malonyl-CoA level is under the control of AMP kinase which phosphorylates/inactivates ACC. Malonyl-CoA is an inhibitor of carnitine palmitoyl-CoA transferase-1 (CPT1), an outer mitochondrial membrane enzyme that regulates entry into, and oxidation of fatty acids, by mitochondria. CPT1c, a recently discovered, brain-specific enzyme expressed in the hypothalamus, has high sequence similarity to liver/muscle CPT1a/b and binds malonyl-CoA, but does not catalyze the prototypical reaction. This suggests that CPT1c has a unique function or activation mechanism. CPT1c knockout (KO) mice have lower food intake, weigh less and have less body fat, consistent with the role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c protects against the effects of a high-fat diet. CPT1cKO mice exhibit decreased rates of fatty acid oxidation, consistent with their increased susceptibility to diet-induced obesity. We suggest that CPT1c may be a downstream target of malonyl-CoA that regulates energy homeostasis.

Malonyl-CoA, a Key Signaling Molecule in Mammalian Cells

Malonyl-CoA can be formed within the mitochondria, peroxisomes, and cytosol of mammalian cells. Besides being an intermediate in the pathways of de novo fatty acid biosynthesis and fatty acid elongation, malonyl-CoA has an important signaling function through its allosteric inhibition of carnitine palmitoyltransferase 1, the enzyme that normally exerts flux control over mitochondrial beta-oxidation. Malonyl-CoA is rapidly turned over in mammalian cells, and the activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase are important determinants of its cytosolic concentration. It is now recognized that malonyl-CoA participates in a diverse range of physiological or pathological responses and systems. These include the ketogenic response of the liver to fasting and diabetes, carbohydrate versus fat fuel selection in muscle tissues, metabolic changes in muscle during contracture, alterations in fatty acid metabolism during cardiac ischemia and postischemic reperfusion, stimulation of B cell insulin secretion by glucose, and the hypothalamic control of appetite.

Brain lipogenesis and regulation of energy metabolism

To revise current available information related to the role of brain lipogenic pathways in the regulation of energy homeostasis. The 'classical' hypothalamic neuropeptide view of feeding regulation has been extensively reviewed and revised during the past few years. Accumulating evidence indicates that the modulation of lipogenesis de novo in the hypothalamus, through selective pharmacologic and genetic manipulation of acetyl-CoA carboxylase, AMP-activated protein kinase, carnitine palmitoyltransferase 1, fatty acid synthase and malonyl-CoA decarboxylase enzymes, has a severe impact on food intake and body weight homeostasis. Furthermore, as these manipulations alter the hypothalamic pool of lipids, such as malonyl-CoA or long chain fatty acyl-CoA or both, the concept of lipids as signals of nutrient abundance able to modulate feeding in the hypothalamus has recently re-emerged. In this review, we summarize what is known about brain lipogenesis and energy balance and propose further avenues of research. Defining these novel mechanisms could offer new targets for the treatment of obesity and metabolic syndrome.

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.

Uteroplacental insufficiency down regulates insulin receptor and affects expression of key enzymes of long-chain fatty acid (LCFA) metabolism in skeletal muscle at birth

BackgroundEpidemiological studies have revealed a relationship between early growth restriction and the subsequent development of insulin resistance and type 2 diabetes. Ligation of the uterine arteries in rats mimics uteroplacental insufficiency and serves as a model of intrauterine growth restriction (IUGR) and subsequent developmental programming of impaired glucose tolerance, hyperinsulinemia and adiposity in the offspring. The objective of this study was to investigate the effects of uterine artery ligation on the skeletal muscle expression of insulin receptor and key enzymes of LCFA metabolism.MethodsBilateral uterine artery ligation was performed on day 19 of gestation in Sprague-Dawley pregnant rats. Muscle of the posterior limb was dissected at birth and processed by real-time RT-PCR to analyze the expression of insulin receptor, ACCα, ACCβ (acetyl-CoA carboxylase alpha and beta subunits), ACS (acyl-CoA synthase), AMPK (AMP-activated protein kinase, alpha2 catalytic subunit), CPT1B (carnitine palmitoyltransferase-1 beta subunit), MCD (malonyl-CoA decarboxylase) in 14 sham and 8 IUGR pups.Muscle tissue was treated with lysis buffer and Western immunoblotting was performed to assay the protein content of insulin receptor and ACC.ResultsA significant down regulation of insulin receptor protein (p < 0.05) and reduced expression of ACS and ACCα mRNA (p < 0.05) were observed in skeletal muscle of IUGR newborns. Immunoblotting showed no significant change in ACCα content.ConclusionOur data suggest that uteroplacental insufficiency may affect skeletal muscle metabolism down regulating insulin receptor and reducing the expression of key enzymes involved in LCFA formation and oxidation.

Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced ( approximately 25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased approximately 22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by approximately 83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPARalpha and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size.

QSAR study of malonyl-CoA decarboxylase inhibitors using GA-MLR and a new strategy of consensus modeling

Quantitative structure-activity relationship (QSAR) of a series of structural diverse malonyl-CoA decarboxylase (MCD) inhibitors have been investigated by using the predictive single model as well as the consensus analysis based on a new strategy proposed by us. Self-organizing map (SOM) neural network was employed to divide the whole data set into representative training set and test set. Then a multiple linear regressions (MLR) model population was built based on the theoretical molecular descriptors selected by Genetic Algorithm using the training set. In order to analyze the diversity of these models, multidimensional scaling (MDS) was employed to explore the model space based on the Hamming distance matrix calculated from each two models. In this space, Q(2) (cross-validated R(2)) guided model selection (QGMS) strategy was performed to select submodels. Then consensus modeling was built by two strategies, average consensus model (ACM) and weighted consensus model (WCM), where each submodel had a different weight according to the contribution of model expressed by MLR regression coefficients. The obtained results prove that QGMS is a reliable and practical method to guide the submodel selection in consensus modeling building and our weighted consensus model (WCM) strategy is superior to the simple ACM.

Mitochondrial Medicine

Socrates: … And he who is most skilled in preventing or escaping from a disease is best able to create one? Polemarchus: True Socrates: And is he the best guard of a camp who is best able to steal a march upon the enemy? Polemarchus: Certainly Socrates: Then he who is a good keeper of everything is also a good thief? Polemarchus: That I suppose is to be inferred — —Plato, The Republic Reading Plato one might not be surprised by the apparent paradox in the ability of mitochondria to promote both life and death: On the one hand, they are the major producers of energy in the cell, and on the other hand, they can steal the life of their host “at will.” Mitochondria can sense the supply of fuel (they are important O2 sensors) and via the production of mediators (reactive O2 species [ROS]), communicate with critical effectors in the cell (like redox-sensitive ion channels in the membrane or the nucleus).1 In response to the needs of the cell, mitochondria produce both energy (ie, ATP) and heat, allowing the organism to work and keep a stable core temperature, facilitating optimal adaptation of eukaryotic cells to their environment. If the mitochondria cannot provide optimal adaptation to the metabolic needs and the environment, they have the ability to induce cell death (apoptosis). As strategic regulators of life and death, mitochondria are deeply involved in human disease, but the depth and breadth of mitochondria-based diseases is only now starting to be recognized.2 Article p 2492 Metabolism and apoptosis converge in mitochondria and are both recognized as playing a major role in disease. Primary metabolic abnormalities are now recognized in pulmonary and systemic vascular disease, and the apoptosis resistance seen in proliferative vascular remodeling might have a mitochondrial …

The malonyl CoA axis as a potential target for treating ischaemic heart disease

Cardiovascular disease is the leading cause of death and disability for people living in western societies, with ischaemic heart disease accounting for the majority of this health burden. The primary treatment for ischaemic heart disease consists of either improving blood and oxygen supply to the heart or reducing the heart's oxygen demand. Unfortunately, despite recent advances with these approaches, ischaemic heart disease still remains a major health problem. Therefore, the development of new treatment strategies is still required. One exciting new approach is to optimize cardiac energy metabolism, particularly by decreasing the use of fatty acids as a fuel and by increasing the use of glucose as a fuel. This approach is beneficial in the setting of ischaemic heart disease, as it allows the heart to produce energy more efficiently and it reduces the degree of acidosis associated with ischaemia/reperfusion. Malonyl CoA is a potent endogenous inhibitor of cardiac fatty acid oxidation, secondary to inhibiting carnitine palmitoyl transferase-I, the rate-limiting enzyme in the mitochondrial uptake of fatty acids. Malonyl CoA is synthesized in the heart by acetyl CoA carboxylase, which in turn is phosphorylated and inhibited by 5'AMP-activated protein kinase. The degradation of myocardial malonyl CoA occurs via malonyl CoA decarboxylase (MCD). Previous studies have shown that inhibiting MCD will significantly increase cardiac malonyl CoA levels. This is associated with an increase in glucose oxidation, a decrease in acidosis, and an improvement in cardiac function and efficiency during and following ischaemia. Hence, the malonyl CoA axis represents an exciting new target for the treatment of ischaemic heart disease.

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.

Mitochondrial Overload and Incomplete Fatty Acid Oxidation Contribute to Skeletal Muscle Insulin Resistance

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Abstract 658: Suppressed Hypoxic Pulmonary Vasoconstriction in Malonyl-CoA-Decarboxylase Knock-Out Mice

Background : Hypoxic pulmonary vasoconstriction (HPV) is intrinsic to the pulmonary artery smooth muscle cells (PASMC). A recently proposed mechanism for HPV suggests that hypoxia is sensed within the mitochondria, altering the production of activated oxygen species (AOS) in response to changes in PO 2 . Decreased AOS (like H 2 O 2 ) inhibit Kv channels, depolarize PASMC, increase influx of Ca ++ , causing PASMC contraction. Malonyl-CoA-Decarboxylase (MCD) is a metabolic enzyme, that when inhibited suppresses the mitochondrial-based fatty acid oxidation, promoting glucose oxidation. We have shown that knockout mice lacking MCD (KO-MCD) are resistant to chronic-hypoxia-induced pulmonary hypertension (CH-PHT) but they have a normal phenotype in normoxia. We hypothesized that KO-MCD mice have suppressed HPV. Methods and Results : We compared KO-MCD to wild mice (W-MCD) using PASMC patch clamping, intracellular Ca ++ (fura-2), mitochondrial membrane potential (ΔΨm, using TMRM and confocal microscopy), mitochondrial AOS production (mitosox and confocal microscopy) and isolated intact resistance PA rings; we also studied exercise tolerance (distance covered in a treadmill) during normoxia and acute hypoxia (n =7–10 mice/group for all studies). As expected, in W-MCD mice acute hypoxia inhibited K + current (Ik), increased intracellular Ca ++ and mitochondrial ΔΨm, decreased mitochondrial AOS production, increased PA tone and decreased exercise tolerance (due to the rapid increase in PA pressure and decrease in cardiac output). In contrast, KO-MCD showed an impressive lack of response to acute hypoxia in all the parameters studied (table ). Conclusions : Our data show that acute hypoxia is not sensed in W-MCD mice and might offer at least a partial explanation for the resistance to CH-PHT in these mice. They also offer a novel insight into the metabolic basis of pulmonary vascular reactivity and HPV.

Fatal Malonyl CoA Decarboxylase Deficiency Due to Maternal Uniparental Isodisomy of the Telomeric End of Chromosome 16

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.