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...

The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine-and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine-and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity. Diabetes 54:3190 -3197, 2005 O besity, which is characterized by an excess of fat stores, is the most important risk factor for type 2 diabetes. Adipose tissue lipolysis leads to the hydrolysis of triglycerides and release of free fatty acids (FFAs). Because of the link between elevated circulating FFA levels and the development of insulin resistance and the metabolic syndrome (1,2), adipose tissue lipolysis constitutes a target for the drug industry. Nicotinic acid, which acts by inhibiting adipose tissue lipolysis, was the first extensively used lipid-lowering agent (3). Catecholamines and natriuretic peptides are the major hormones stimulating this catabolic pathway in humans (4). Resistance to catecholamine-induced lipolysis in subcutaneous adipose tissue has been demonstrated in obese adults and children (5,6) and is attributed to decreased expression of lipolytic ␤ 2 -adrenoceptors (7), increased antilipolytic properties of ␣ 2 -adrenoceptors (8), and decreased expression of hormone-sensitive lipase (HSL) (9). It is possible that the HSL defect is the most important factor because it is also observed in nonobese first-degree relatives to obese subjects (10) and because there is a positive relationship between lipolytic capacity and HSL expression in human subcutaneous fat cells (11).The rate-limiting role of HSL in adipose tissue lipolysis has been challenged by the data from HSL knockout mice (12-15). Catecholamine-induced lipolysis is abrogated, but residual basal lipolysis is observed in adipocytes from HSL-null mice. These data suggest the existence of non-HSL lipases in adipos...

During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two ϳ10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium-independent phospholipases A 2 . Detailed inspection of the identified sequence (residues 79,671-109,912 GenBank TM accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A 2 through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0.3 nmol/mg⅐min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC 50 ‫؍‬ 3 M), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, and sn-2-lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A 2 .Phospholipases A 2 catalyze the esterolytic cleavage of fatty acids from the sn-2-position of phospholipids, thereby regulating the release of lipid second messengers (e.g. eicosanoids and lysophospholipids), growth factors (lysophosphatidic acid), and membrane physical properties (1-5). In most cell types, the availability of nonesterified arachidonic acid is the rate-limiting step in the production of biologically active eicosanoid metabolites (1, 2, 6, 7). Thus, phospholipase A 2 activity modulates cellular growth programs (e.g. peroxisome proliferation (by prostaglandin J 2 )), inflammation (e.g. prostaglandins and leukotrienes), vascular tone (e.g. 20-hydroxyeicosatetraenoic acid), and ion channel function (e.g. P450 products and arachidonic acid) (1, 2, 6, 7-11). Accordingly, substantial attention has focused on the molecular identification of the polypeptides that catalyze phospholipase A 2 activity, regulate its kinetic properties, and facilitate its topologic and topographic distribution in normal and stimulated ce...

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, ␤-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A 2 (iPLA 2 s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA 2 s has been extended to include iPLA 2 ␥, which previously could not be pharmacologically distinguished from iPLA 2 ␤. To determine whether iPLA 2 ␤ or iPLA 2 ␥ (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA 2 ␤ and iPLA 2 ␥ in intact cells. We hypothesized that the R-and S-enantiomers of BEL would possess different inhibitory potencies for iPLA 2 ␤ and iPLA 2 ␥. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA 2

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...