Original Article Rosiglitazone Inhibits Acyl-CoA Synthetase Activity and Fatty Acid Partitioning to Diacylglycerol and Triacylglycerol via a Peroxisome Proliferator–Activated Receptor-␥–Independent Mechanism in Human Arterial Smooth Muscle Cells and Macrophages Bardia Askari,1 Jenny E. Kanter,1 Ashley M. Sherrid,1 Deidre L. Golej,1 Andrew T. Bender,2 Joey Liu,3 Willa A. Hsueh,3 Joseph A. Beavo,2 Rosalind A. Coleman,4 and Karin E. Bornfeldt1 Rosiglitazone is an insulin-sensitizing agent that has re- cently been shown to exert beneficial effects on atheroscle- rosis. In addition to peroxisome proliferator–activated ong-chain acyl-CoA synthetases (ACSLs) receptor (PPAR)-␥, rosiglitazone can affect other targets, (E.C.6.2.1.3) catalyze esterification of long-chain such as directly inhibiting recombinant long-chain acyl- fatty acids, mediating the partitioning of fatty CoA synthetase (ACSL)-4 activity. Because it is unknown if acids in mammalian cells. ACSL isoforms ACSL4 is expressed in vascular cells involved in athero- L sclerosis, we investigated the ability of rosiglitazone to (ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6) generate inhibit ACSL activity and fatty acid partitioning in human bioactive fatty acyl-CoAs from CoA, ATP, and long-chain and murine arterial smooth muscle cells (SMCs) and mac- (C12–C20) fatty acids (1). ACSLs exhibit different tissue rophages. Human and murine SMCs and human macro- distribution, subcellular localization, fatty acid preference, phages expressed Acsl4, and rosiglitazone inhibited Acsl responsiveness to pharmacological inhibitors, and tran- activity in these cells. Furthermore, rosiglitazone acutely scriptional regulation. Previous studies have characterized inhibited partitioning of fatty acids into phospholipids in these enzymes in different tissues (2); however, expression human SMCs and inhibited fatty acid partitioning into levels and function in vascular cells are unknown. Until diacylglycerol and triacylglycerol in human SMCs and mac- rophages through a PPAR-␥–independent mechanism. Con- recently, inconsistencies in the ACSL nomenclature led to versely, murine macrophages did not express ACSL4, and confusion in the literature. The standard nomenclature rosiglitazone did not inhibit ACSL activity in these cells, now uses ACSL1 and ACSL3–6 for human and Acsl1 and nor did it affect acute fatty acid partitioning into cellular Acsl3–6 for murine members of this family (3). lipids. Thus, rosiglitazone inhibits ACSL activity and fatty Thiazolidinediones (TZDs) are a class of oral insulin- acid partitioning in human and murine SMCs and in human sensitizing agents, extensively used in the treatment of macrophages through a PPAR-␥–independent mechanism type 2 diabetes. Whereas members of this class of agents, likely to be mediated by ACSL4 inhibition. Therefore, such as rosiglitazone and pioglitazone, have been demon- rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis. Diabetes 56: strated to provide effective glycemic control in patients 1143–1152, 2007 with diabetes (4), their effect on lipid metabolism is less clear (5). Perhaps more interesting are the recent findings that rosiglitazone treatment results in fewer inflammatory cells and increased collagen content in advanced athero- From the 1Department of Pathology, University of Washington School of sclerotic plaques (6,7; rev. in 8), suggesting that rosiglita- Medicine, Seattle, Washington; the 2Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington; the 3Departments of zone might exert a stabilizing effect on atherosclerotic Nutrition and Pediatrics, University of North Carolina, Chapel Hill, North plaques. Carolina; and the 4Division of Endocrinology, Diabetes, and Hypertension, The mechanism of TZD action is mediated largely David E. Geffen School of Medicine, University of California, Los Angeles, ␥ California. through the activation of PPAR- , a member of the super Address correspondence and reprint requests to Dr. Karin E. Bornfeldt, family of ligand-activated nuclear transcription factors. In Department of Pathology, 1959 NE Pacific St., University of Washington, vascular tissue, peroxisome proliferator–activated recep- Seattle, WA 98195-7470. E-mail: [email protected]. Received for publication 24 February 2006 and accepted in revised form 16 tor (PPAR)-␥ is expressed in smooth muscle cells, macro- January 2007. phages, and endothelial cells. TZDs may also exert Published ahead of print at http://diabetes.diabetesjournals.org on 26 Jan- nongenomic effects. For example, rosiglitazone has been uary 2007. DOI: 10.2337/db06-0267. ⌬12,14 shown to activate 5Ј-AMP protein kinase (AMPK) through 15-dPGJ2, 15-deoxy- prostaglandin J2; AA, arachidonic acid; ACSL, long-chain acyl-CoA synthetase; AICAR, 5-aminoimidazole-4-carboxaide 1-␤- a PPAR-␥–independent mechanism (9). With regard to D-ribo-furanoside; DAG, diacylglycerol; hSMC, human aortic smooth muscle Acsl isoforms, in vitro studies of rat recombinant proteins cell; IC50, half-maximal inhibition; OA, oleic acid; SMC, smooth muscle cell; TAG, triacylglycerol; TZD, thiazolidinedione. have demonstrated that TZDs can directly inhibit the © 2007 by the American Diabetes Association. activity of one of the gene products, Acsl4 (10). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance Because of the strong association among increased fatty with 18 U.S.C. Section 1734 solely to indicate this fact. acid load, atherosclerosis, and cardiovascular disease, DIABETES, VOL. 56, APRIL 2007 1143 ROSIGLITAZONE INHIBITS ACSL4 ACTIVITY particularly in the setting of type 2 diabetes, we investi- separated via gel electrophoresis in 2% agarose gels and visualized with gated the ability of rosiglitazone to inhibit ACSL activity ethidium bromide. Murine brain and liver were used as positive controls for the Acsl3, Acsl5, and Acsl6 primers. A human neuroblastoma cell line and acute fatty acid partitioning in smooth muscle cells (SH-SY5Y) was used as a positive control for human ACSL6. (SMCs) and macrophages. We show that human and Western blot analysis of ACSL1 and ACSL4. Lysates were prepared as murine SMCs and human macrophages express a number previously described (12,14,16), separated using 7.5% Tris-glycine SDS-PAGE, of ACSL isoforms and that ACSL4, the rosiglitazone- and transferred to polyvinylidene difluoride membranes. The membranes sensitive isoform, is expressed in these cells. We also were then incubated overnight at 4°C with rabbit polyclonal anti-rat Acsl1 or show that rosiglitazone inhibits ACSL activity and fatty Acsl4 antibodies (1:10,000) generated against the amino acid sequences acid partitioning in human and murine SMCs and in human MEVHELFRYFRMPELIDIR and EIHSMQSVEELGSKPENSSI, respectively ␥ (17). Horseradish peroxidase–linked anti-rabbit secondary antibodies were macrophages through a PPAR- –independent mechanism used at a final dilution of 1:5,000 for1hatroom temperature. Signal detection likely to be mediated by direct inhibition of ACSL4. was performed by enhanced chemiluminescence. Recombinant rat Acsl4 (3 Furthermore, rosiglitazone might exert effects on fatty ␮g) was used as a positive control. Rat liver high-speed supernatant was used acid partitioning in human macrophages that are not as a positive control for ACSL1 expression. Briefly, rat liver was homogenized mimicked by mouse models, because murine macro- (1:3; weight:volume) using a Potter-Elvehjem homogenizer in a 320 mmol/l sucrose buffer containing Tris-HCl, pH 7.4 (20 mmol/l), EDTA (1 mmol/l), phages are devoid of Acsl4. EGTA (1 mmol/l), leupeptin (20 ␮g/ml), benzamidine (1 mmol/l), aprotinin (10 ␮g/ml), pepstatin (5 ␮g/ml), dithiothreitol (1 mmol/l), and phenylmethylsulfo- nyl fluoride (0.1 mmol/l). The homogenate was centrifuged at 7,700g for 2 min. RESEARCH DESIGN AND METHODS The supernatant was then centrifuged at 12,000g for 12 min and then at Oleic acid (OA) (sodium salt) was purchased from Nucheck Prep (Elysian, 177,000g for 1 h. The resulting high-speed supernatant was used for analyses. MN). RPMI-1640, fetal bovine serum, and penicillin/streptomycin were ob- Analysis of ACSL activity. Total acyl-CoA synthetase activity was measured tained from Gibco/Invitrogen (Grand Island, NY). CoA, ATP, essentially fatty via a modified protocol by Kim et al. (10), involving the formation of OA-CoA acid–free BSA, sodium thioglycollate, dithiothreitol, EDTA, EGTA, leupeptin, from OA. Briefly, cells were scraped into ice-cold cell sonication buffer aprotinin, pepstatin, phenylmethylsulfonyl fluoride, and benzamidine were containing 50 mmol/l potassium phosphate (pH 7.4), 10% glycerol, 1 mmol/l obtained from Sigma-Aldrich (St. Louis, MO). Triascin C, T0070907, 5-amino- EDTA, 20 ␮g/ml leupeptin, 5 ␮g/ml pepstatin, 10 ␮g/ml aprotinin, and 5 mmol/l imidazole-4-carboxaide 1-␤-D-ribo-furanoside (AICAR), 15-deoxy-⌬12,14 prosta- benzamidine and sonicated with two bursts of 10-s duration at 50 W. Protein glandin J2 (15-dPGJ2), and Wy14643 were from Biomol Research Laboratories concentration was determined via BCA protein assays (Pierce, Rockford, IL). (Plymouth Meeting, PA). Rosiglitazone was obtained from Alexis Biochemical The lysate (200 ␮g/sample) was then incubated in the presence of increasing (San Diego, CA). Anti-rabbit IgG horseradish peroxidase–linked antibodies, concentrations of rosiglitazone, triacsin C, 15-dPGJ2, AICAR, or Wy16463