OBJECTIVEPeripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function.RESEARCH DESIGN AND METHODSGSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis.RESULTSGSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-α treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion.CONCLUSIONSThese results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.

Abstract-AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPK␣2 protects the heart against systolic overload-induced ventricular hypertrophy and dysfunction, we studied the response of AMPK␣2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPK␣2 KO had no effect on ventricular structure or function under control conditions, AMPK␣2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPK␣2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPK␣2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinase Thr389 , p-S6 Ser235 , and p-eukaryotic initiation factor 4E Ser209 . AMPK␣2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-Akt Ser473 . These data indicate that AMPK␣2 exerts a cardiac protective effect against pressure-overloadinduced ventricular hypertrophy and dysfunction. Key Words: hypertrophy Ⅲ congestive heart failure Ⅲ mTOR I ncreases of cardiac work resulting from systolic overload necessitate an increase of ATP use in proportion to the increase in left ventricular (LV) systolic wall stress. 1,2 In response to chronic systolic overload, cardiac myocyte hypertrophy occurs, characterized by increased protein synthesis, whereas myocardial oxygen consumption and carbon substrate use are increased to accommodate the need for increased energy availability. This initially occurs with no change in high energy phosphate levels, but with the development of pathological hypertrophy and congestive heart failure, ATP levels fall and cytosolic free ADP levels increase (as indicated by a decrease of the myocardial phosphocreatine:ATP ratio). 2,3 In this situation, the adenylate kinase reaction can catalyze the reaction of 2 molecules of ADP to produce 1 molecule of ATP and 1 molecule of AMP. An increased AMP:ATP ratio results in activation of the energy stress sensor known as AMP activated protein kinase (AMPK).AMPK is composed of 1 catalytic ␣ subunit (either ␣1 or ␣2) and 2 regulatory subunits (␤ and ␥). AMPK␣2 is the dominant catalytic subunit in the heart, 3,4 where it is predominantly expressed in cardiac myocytes. AMPK is activated by metabolic stres...

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor g and CCAAT/enhancer binding protein a. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an ?25% reduction in basal 3 H-labeled fatty acid uptake and a complete loss of insulin-stimulated 3 H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using ␣-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V max /K m (C16:0)/ V max /K m (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.

Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD ؉ / NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades.

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a ϳ15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.Fatty acid influx and efflux mechanisms and their regulation affect lipid storage and metabolism in adipocytes. Imbalances in adipose lipid metabolism have been shown to significantly contribute to the development of obesity and associated metabolic diseases, such as type 2 diabetes, hypertension, and cardiovascular disease (1-3). Although the molecular mechanisms involved in fatty acid efflux are still undefined, several proteins implicated in fatty acid influx have been proposed: CD36 (fatty acid translocase), acyl-CoA synthetases (fatty acid transport protein (FATP) 2 and acyl-CoA synthetase (ACSL) family members), plasma membrane fatty acid-binding protein, and caveolin-1 (4 -9).FATPs and long-chain ACSLs are membrane-bound enzymes that catalyze the ATP-dependent esterification of long chain (ACSL) and very long-chain (FATP) fatty acids to their acyl-CoA derivatives (10, 11). Both types of CoA synthetases have common ATP/AMP binding and fatty acid binding signature motifs. In mammals, six different isoforms of FATP (FATP1-FATP6) and five different isoforms of ACSL (ACSL1, -3, -4, -5, and -6) have been identified with tissue-specific expression patterns (12). White adipose tissue predominantly express FATP1, FATP4, and ACSL1, whereas brown adipose tissue in addition expresses ACSL5. Our recent results have confirmed a major role of FATP1 and CD36, but not FATP4, in insulin-stimulated LCFA uptake in 3T3-L1 adipocytes (6).ACSL1 is a ϳ78-kDa intrinsic membrane protein localized to multiple sites in a variety of different cells. In liver, ACSL1 has been shown to be localized to the endoplasmic reticulum and mitochond...

Human pluripotent stem cells derived cardiomyocytes (PSC-CMs) have been widely used for disease modeling, drug safety screening, and preclinical cell therapy to regenerate myocardium. Most studies have utilized PSC-CM grown in vitro for a relatively short period after differentiation. These PSC-CMs demonstrated structural, electrophysiological, and mechanical features of primitive cardiomyocytes. A few studies have extended in vitro PSC-CM culture time and reported improved maturation of structural and electromechanical properties. The degree of mitochondrial maturation, however, remains unclear. This study characterized the development of mitochondria during prolonged in vitro culture. PSC-CM demonstrated an improved mitochondrial maturation with prolonged culture, in terms of increased mitochondrial relative abundance, enhanced membrane potential, and increased activity of several mitochondrial respiratory complexes. These are in parallel with the maturation of other cellular components. However, the maturation of mitochondria in PSC-CMs grown for extended in vitro culture exhibits suboptimal maturation when compared with the maturation of mitochondria observed in the human fetal heart during similar time interval.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) plays a central role in translating nutrient abundance into cell growth and proliferation. Although specific proteins have been described as mediators of this nutrient input, their mechanistic linkage remains incomplete. Two studies have added phospholipase D (PLD) as a mediator of nutrients to mTORC1. Furthermore, these studies link PLD and its product phosphatidic acid to previously identified activators of mTORC1 signaling, including the class III phosphoinositide-3 kinase, and provide evidence of the existence of two parallel nutrient-regulated pathways that converge on mTORC1 at late endosomes and/or lysosomes.