Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A 2 (iPLA 2 ) dual signature nucleotide ((G/A)XGXXG) and lipase (GX-SXG) consensus sequence motifs identified a novel subfamily of three putative iPLA 2 /lipase family members designated iPLA 2 ⑀, iPLA 2 , and iPLA 2 (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA 2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA 2 ⑀, iPLA 2 , and iPLA 2 also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA 2 s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC 50 ‫؍‬ 0.1-0.5 M). Quantitative PCR revealed dramatically increased expression of iPLA 2 ⑀ and iPLA 2 transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA 2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.Obesity and its associated clinical sequelae (e.g. type 2 diabetes, atherosclerosis, and hypertension) represent the major and most rapidly expanding health epidemic in industrialized nations (1-4). Obesity results from an abnormal increase in white adipose tissue mass, primarily in the form of triglycerides, and in humans is thought to be caused by a complex array of genetic, environmental, and hormonal factors (1, 4). Under conditions of obesity, serum nonesterified fatty acids are elevated, contributing to the accumulation of triglycerides in nonadipose tissues (e.g. hepatic, myocardial, and pancreatic) and to the development of the type 2 metabolic syndrome (5). The combined effects of excess cellular triglycerides, fatty acylCoAs, and free fatty acids are believed to be primary mediators of the lipotoxic effects of obesity that include decreased insulin sensitivity, increased oxidative stress, reduced metabolic capacity, and increased rates of cellular apoptosis in multiple organ systems (5-8).Triacylglycerol/fatty acid recycling is an important mechanism by which adipocytes modulate fatty acyl flux in response to changing metab...

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Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A 2 ␥ (iPLA 2 ␥), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA 2 ␥ in cellular bioenergetics, we generated mice null for the iPLA 2 ␥ gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA 2 ␥ display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IVmediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA 2 ␥ is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.Mitochondria transduce chemical energy from multiple dietary substrates into ATP and heat and are important participants in cellular lipid metabolism and signaling (1, 2). Through the precisely regulated partitioning of energy derived from substrates between ATP synthesis and heat production, a finely tuned dynamic balance is maintained to promote organism survival. In addition, excess energy derived from substrates, not immediately needed to fulfill chemical or thermodynamic demands, is directed into the production of lipid synthetic intermediates for energy storage (e.g. mitochondrial synthesis of lysophosphatidic acid, a precursor of triglycerides). Moreover, mitochondria generate diverse chemical signals that reflect the bioenergetic status of the cell (e.g. NO and cytochrome c) and thus integrate multiple energetic, metabolic, and signaling cascades (1-5).Mitochondrial phospholipases are important participants in the regulation of mitochondrial function and signaling (6 -11). Mitochondrial phospholipases catalyze the production of nonesterified fatty acids that regulate UCP function, release lipid second messengers, such as 2-arachidonoyl lysophosphatidylcholine (LPC), 2 and modulate membrane molecular dynamics (12). Maladaptive activation of phospholipases has deleterious metabolic effects in many systems, and previous studies have implicated calcium-independent phospholipases A 2 (iPLA 2 s) as the enzymic mediators of membrane dysfunction in diabetic cardiomyopathy (13-16). Metabolic flexibility is dependent on the efficient coordinated transitions of substrate flux that occur during alterations in energy sources (e.g. glucose versus fatty acid) or energy demand (e.g. exercise). Obesity and type II diabetes are thought to result from disruption of these transitions (17). Diabetic cardiomyopathy is characterized by altered lipid metabolism (i.e. greatly increased utilization of fatty acids) that results in changes in the chemical composition of cardiac membran...

Women with acute liver disease during pregnancy may have a Glu474Gln mutation in long-chain hydroxyacyl-CoA dehydrogenase. Their infants are at risk for hypoketotic hypoglycemia and fatty liver.

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Mitochondrial long chain fatty acid 13-oxidation provides the major source of energy in the heart. Deficiencies of human 13-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.211] catalyzes the third step in 13-oxidation, and this activity is present on the C-terminal portion of the a subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (et subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated severe complications during pregnancies with the affected infants. The analysis in two affected children revealed a G to C mutation at position 1528 (G1528C) of the a subunit of the trifunctional protein on both alleles. This is in the LCHAD domain and substitutes glutamine for glutamic acid at position 474 of mature a subunit. The third child had this G1528C mutation on one allele and a different mutation (C1132T) creating a premature termination codon (residue 342) on the second allele. Our results demonstrate that mutations in the LCHAD domain of the trifunctional protein a subunit in affected offspring are associated with maternal acute fatty liver of pregnancy. This is the initial delineation of the molecular basis of isolated LCHAD deficiency.Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NADI oxidoreductase, EC 1.1.1.211] activity catalyzes the third step in the mitochondrial (3-oxidation spiral. (3-Oxidation provides the major source of energy in heart and skeletal muscle and is essential for intermediary metabolism in the liver. The (3-oxidation of long chain fatty acids requires four sequential enzyme activities: the initial fatty acyl-CoA dehydrogenase, a 2,3-enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenation step, and a 3-ketoacyl-CoA thiolase. With each cycle through the spiral, the long chain fatty acid substrate is reduced by two carbons, producing acetyl CoA. Because of the range of fatty acid substrate lengths, each of these reactions is catalyzed by two to four separate enzymes encoded by individual nuclear genes and with differing substrate specificities.

Abstract-Fatty acid oxidation (FAO) defects are inborn errors of metabolism clinically associated with cardiomyopathy and sudden infant death syndrome (SIDS). FAO disorders often present in infancy with myocardial dysfunction and arrhythmias after exposure to stresses such as fasting, exercise, or intercurrent viral illness. It is uncertain whether the heart, in the absence of stress, is normal. We generated very-long-chain acyl-coenzyme A dehydrogenase (VLCAD)-deficient mice by homologous recombination to define the onset and molecular mechanism of myocardial disease. We found that VLCAD-deficient hearts have microvesicular lipid accumulation, marked mitochondrial proliferation, and demonstrated facilitated induction of polymorphic ventricular tachycardia, without antecedent stress. The expression of acyl-CoA synthase (ACS1), adipophilin, activator protein 2, cytochrome c, and the peroxisome proliferator activated receptor ␥ coactivator-1 were increased immediately after birth, preceding overt histological lipidosis, whereas ACS1 expression was markedly downregulated in the adult heart. We conclude that mice with VLCAD deficiency have altered expression of a variety of genes in the fatty acid metabolic pathway from birth, reflecting metabolic feedback circuits, with progression to ultrastructural and physiological correlates of the associated human disease in the absence of stress.

Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.