Rat Long Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Uptake and Partitioning to Cellular Triacylglycerol in McArdle-RH7777 Cells

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 μm 1-14Coleic acid. Metabolism of 1-14Coleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of 1-14Coleate and 1,2,3-3Hglycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, 1-14Cacetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage. Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 μm 1-14Coleic acid. Metabolism of 1-14Coleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of 1-14Coleate and 1,2,3-3Hglycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, 1-14Cacetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage. Acyl-CoA synthetase catalyzes the initial step in mammalian fatty acid metabolism. In this reaction, fatty acid, CoA, and ATP are used to form acyl-CoA and AMP. Acyl-CoAs have diverse metabolic fates within the cell and can be used to acylate proteins or be metabolized through catabolic pathways such as β-oxidation or anabolic pathways such as de novo synthesis and reacylation of triacylglycerol (TAG), 2The abbreviations used are: TAGtriacylglycerolDAGdiacylglycerolACSLlong chain acyl-CoA synthetaseACSVLvery long chain acyl-CoA synthetaseASMacid-soluble metabolitesGFPgreen fluorescent proteinGrp7878-kDa glucose regulated proteinSREBP-1csterol regulatory element binding proteinVDACvoltage-dependent anion channelPBSphosphate-buffered salinePIPES1,4-piperazinediethanesulfonic acidTRITCtetramethylrhodamine isothiocyanate. phospholipids, and cholesterol esters. triacylglycerol diacylglycerol long chain acyl-CoA synthetase very long chain acyl-CoA synthetase acid-soluble metabolites green fluorescent protein 78-kDa glucose regulated protein sterol regulatory element binding protein voltage-dependent anion channel phosphate-buffered saline 1,4-piperazinediethanesulfonic acid tetramethylrhodamine isothiocyanate. To date, five isoforms of long chain acyl-CoA synthetase have been cloned and shown to possess activity toward long chain fatty acids. The unique pattern of tissue expression, subcellular localization, and differences in substrate preference among the isoforms suggests that individual isoforms have distinct functions. ACSL1, ACSL4, and ACSL5 appear to be the predominant isoforms in rat liver (1Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. (Tokyo). 1998; 124: 679-685Crossref PubMed Scopus (133) Google Scholar, 2Kang M.J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (217) Google Scholar, 3Fujino T. Kang M.J. Suzuki H. Iijima H. Yamamoto T. J. Biol. Chem. 1996; 271: 16748-16752Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 4Fujino T. Yamamoto T. J. Biochem. (Tokyo). 1992; 111: 197-203Crossref PubMed Scopus (122) Google Scholar, 5Suzuki H. Kawarabayasi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar). ACSL1 and ACSL4 are located in liver endoplasmic reticulum and mitochondrial-associated membrane (6Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), an endoplasmic reticulum fraction possibly involved in lipoprotein synthesis (7Rusinol A.E. Cui Z. Chen M.H. Vance J.E. J. Biol. Chem. 1994; 269: 27494-27502Abstract Full Text PDF PubMed Google Scholar). In addition, ACSL4 is also present on peroxisomes and has a marked preference for C20:4 and C20:5 (2Kang M.J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (217) Google Scholar). ACSL5 is the only isoform found in both mitochondrial membranes and endoplasmic reticulum (6Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) and, similar to ACSL1, has its highest preference for saturated and unsaturated fatty acids of 16-20 carbons (1Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. (Tokyo). 1998; 124: 679-685Crossref PubMed Scopus (133) Google Scholar). Because ACSL5 is the only ACSL isoform known to be located on mitochondria, it is logical to speculate that it has a role in the β-oxidation of fatty acids. In support of this hypothesis, rat liver mitochondrial protein content of ACSL5 increases following a 48-h fast but declines when rats are refed a high sucrose diet for 24 h (6Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). To date, the most direct studies that focus on ACSL used triacsin C, an inhibitor of ACSL1, ACSL3, and ACSL4 but not ACSL5 or ACSL6 (8Kim J.H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 9Van Horn C.G. Caviglia J.M. Li L.O. Wang S. Granger D.A. Coleman R.A. Biochemistry. 2005; 44: 1635-1642Crossref PubMed Scopus (123) Google Scholar). In hepatocytes obtained from fed rats, triacsin C decreases 1-14Coleic acid incorporation into TAG by 70% (10Muoio D.M. Lewin T.M. Wiedmer P. Coleman R.A. Am. J. Physiol. 2000; 279: E1366-E1373Crossref PubMed Google Scholar). In contrast, triacsin C decreases the metabolism of 1-14Coleic acid to phospholipids by 34% and to ASM by 33%. Therefore, inhibiting ACSL1, ACSL3, and ACSL4 decreases fatty acid incorporation into TAG more than into phospholipids or into pathways of fatty acid oxidation. Because hepatocytes express little ACSL6 (4Fujino T. Yamamoto T. J. Biochem. (Tokyo). 1992; 111: 197-203Crossref PubMed Scopus (122) Google Scholar), ACSL5 could account for the triacsin C-resistant activity in hepatocytes and might activate fatty acids destined for β-oxidation. Despite its putative role in fatty acid oxidation, several lines of evidence do not support a catabolic role for ACSL5. Tissue expression of ACSL5 mRNA is highest in intestinal mucosa (1Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. (Tokyo). 1998; 124: 679-685Crossref PubMed Scopus (133) Google Scholar) and brown adipose tissue (11Yu X.X. Lewin D.A. Forrest W. Adams S.H. FASEB J. 2002; 16: 155-168Crossref PubMed Scopus (174) Google Scholar). Although brown adipose tissue is characterized by high levels of fatty acid oxidation and thermogenesis, both intestinal mucosa and brown adipose tissue have a high capacity for TAG synthesis (12Trotter P.J. Storch J. J. Lipid Res. 1991; 32: 293-304Abstract Full Text PDF PubMed Google Scholar, 13Darnley A.C. Carpenter C.A. Saggerson E.D. Biochem. J. 1988; 253: 351-355Crossref PubMed Scopus (18) Google Scholar). Additionally, regulation of ACSL5 mRNA does not support a role in fatty acid β-oxidation. Hepatic ACSL5 message abundance does not change in response to peroxisomal proliferator-activated receptor α agonists (14Lewin T.M. Van Horn C.G. Krisans S.K. Coleman R.A. Arch. Biochem. Biophys. 2002; 404: 263-270Crossref PubMed Scopus (110) Google Scholar) but increases nearly 4-fold in SREBP-1c transgenic mice and is reduced 40% in SREBP cleavage-activating protein knock-out mice (15Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1119) Google Scholar). Administration of leptin to C57BL/6J obese ob/ob mice causes a 1.5-fold decrease in ACSL5 mRNA expression together with down-regulation of lipogenic genes and increased expression of numerous oxidative genes (16Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 49066-49076Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, hepatic ACSL5 message level increases 1.6-fold in zinc-deficient mice with fatty livers (17tom Dieck H. Doring F. Fuchs D. Roth H.P. Daniel H. J. Nutr. 2005; 135: 199-205Crossref PubMed Scopus (87) Google Scholar) and decreases 1.5-fold in estrogen-related receptor knock-out mice, which exhibit a reduction in lipogenic gene expression and reduced total body fat (18Luo J. Sladek R. Carrier J. Bader J.A. Richard D. Giguere V. Mol. Cell. Biol. 2003; 23: 7947-7956Crossref PubMed Scopus (322) Google Scholar). Taken together, there are many discrepancies in the literature between regulation of ACSL5 mRNA and protein abundance and the effects of triacsin C on fatty acid channeling. Therefore, our goal was to use a direct approach to elucidate the function of ACSL5. We ectopically overexpressed ACSL5 in rat hepatoma McArdle-RH7777 cells to determine its role in fatty acid activation and partitioning. Materials—Silica gel G plates were from Whatman. 1-14COleic acid, 1,2,3-3Hglycerol, and 1-14Cacetic acid were from PerkinElmer Life Sciences. Tissue culture dishes were from Corning, and media were obtained from Invitrogen. All chemicals were from Sigma unless otherwise indicated. Construction of Recombinant GFP and ACSL5-FLAG Adenovirus—ACSL5 cDNA was cloned from rat liver total RNA and amplified using primers designed to include the entire open (8Kim J.H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The was into the a and a to form was amplified from the the the entire ACSL5 a at the and the The was with and and into the between the and the a GFP gene by a which direct of the of infection by The was at the of The (Ad-ACSL5) by of the and the S. J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the ACSL5 was used to a for control McArdle-RH7777 and cells were in medium and on were the and cells were at were infected with of infection of or the media were Lipid and of Lipid and media of 1-14Coleic acid to in a The of acid in media was or 500 In μm 1,2,3-3Hglycerol or 1-14Cacetic acid was Additionally, μm triacsin C in was to the media in to ACSL1, ACSL3, and cells were incubated with the of of the media were 24 h after of the media were and used to ASM or J. Biochem. Physiol. PubMed Scopus Google Scholar) to incorporation into media was by the cells with at lipids were J. Biochem. Physiol. PubMed Scopus Google Scholar) and on gel G plates in acid were used to lipids that were by The was used to rates of fatty acid uptake were as Z. P. J. Z. S. J. Biol. Chem. 279: Full Text Full Text PDF PubMed Scopus Google Scholar) that culture dishes were used and the medium of 1-14Coleic acid. for ACSL and cells were with in and on with in a and were at Acyl-CoA synthetase activity was in the of CoA, and μm acid Biophys. PubMed Scopus Google Scholar). initial rates were was by the cells were on and infected with Ad-ACSL5 as 24 cells were in and with for at several at cells were with for on were incubated with a of of of or a of with the for h at several cells were incubated in or for h at in the with was to the cells, and were Confocal microscopy was on an and were used to the and of was used for from rats were with a and RNA was with the and primers for rat ACSL5 were and The to the ACSL5 was The rat was and the was The to the was were on an were using the as in in the were by and was at Ad-ACSL5 and C-resistant determine the function of we infected McArdle-RH7777 cells, a rat hepatoma cell with an ACSL5 and by fluorescent microscopy was to for most and was than Ad-ACSL5 infection of McArdle-RH7777 cells increased total ACSL activity 2-fold when compared with cells ACSL5 is only of five known ACSL isoforms involved in long chain fatty acid we used triacsin C, an inhibitor of ACSL1, ACSL3, and ACSL4, to the increase in activity to ACSL5 In the of μm triacsin C, ACSL activity in Ad-ACSL5-infected cells was higher than the revealed that the only known ACSL not by triacsin C, is not in McArdle-RH7777 cells. we that ACSL5 is for the triacsin C-resistant it for of total ACSL activity in cells and 40% in Ad-ACSL5-infected cells. ACSL5 with and studies that used cell have shown that ACSL5 is present in both and mitochondria in rat hepatocytes (6Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). We microscopy to the subcellular of Ad-ACSL5 in McArdle-RH7777 cells The pattern showed with the an endoplasmic reticulum also colocalized with the a mitochondria did not the on the cell or the Therefore, overexpression of a pattern similar to that of ACSL5. Ad-ACSL5 and to determine the effects of Ad-ACSL5 on fatty acid metabolism, we incubated McArdle-RH7777 cells with or 500 μm acid for h and 1-14Coleic acid incorporation into cellular and medium lipids and oxidation When compared with Ad-ACSL5 did not total acid metabolism, as by incorporation into cell lipids and medium TAG and at or μm but increased metabolism at 500 μm acid a or a Biophys. PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar). To we initial rates of fatty acid uptake In with the increased fatty acid metabolism, we a 30% increase in rates of fatty acid uptake when cells were incubated with 500 μm acid. TAG the predominant form of fatty acids in most cell of 1-14Coleic acid into cellular TAG increased and at and μm acid, in both and Ad-ACSL5-infected cells However, with 500 μm acid in the 42% more 1-14Coleic acid was in TAG in the Ad-ACSL5-infected cells when compared with control cells. to cell medium TAG increased with acid in the but there were differences between and Ad-ACSL5-infected cells at fatty acid Although with 500 μm acid, Ad-ACSL5 increased 1-14Coleic acid incorporation into medium ASM an of body the increase is when compared with the increases in fatty acid uptake and to thus our that ACSL5 fatty acids destined for β-oxidation. were differences in incorporation of 1-14Coleic acid into cellular phospholipids, fatty or cholesterol but Ad-ACSL5 increased 1-14Coleic acid incorporation into cellular 2-fold when compared with cells To the role of ACSL5 in TAG we incubated cells with triacsin C, an inhibitor of ACSL1, ACSL3, and ACSL4, but not ACSL5. in hepatocytes and showed that triacsin TAG synthesis more than ASM (10Muoio D.M. Lewin T.M. Wiedmer P. Coleman R.A. Am. J. Physiol. 2000; 279: E1366-E1373Crossref PubMed Google Scholar, R.A. Wang P. Coleman R.A. Biochem. J. 1997; PubMed Scopus Google Scholar). In cells, triacsin C TAG and ASM synthesis However, Ad-ACSL5 infection triacsin of TAG synthesis and ASM synthesis. Therefore, when ACSL isoforms are overexpressed ACSL5 can for TAG synthesis but can only ASM Ad-ACSL5 TAG through de and the in of fatty we the increase of acid incorporation into cellular TAG in Ad-ACSL5-infected cells was to de novo synthesis or to reacylation pathways. We cells with μm 1,2,3-3Hglycerol 500 μm 1-14Coleic acid to elucidate the of TAG synthesis. Ad-ACSL5-infected cells more acid and more into cellular TAG to The of acid to incorporation was also similar between and Ad-ACSL5 In phospholipids, 1-14Coleic acid incorporation did not differ between but 1,2,3-3Hglycerol incorporation into phospholipids was in the Ad-ACSL5-infected cells. to an increase in the acid to for for that overexpression of ACSL5 de novo synthesis more than The de novo synthesis of fatty acids can a to cellular fatty acid To determine ACSL5 fatty acids derived from de novo we the incorporation of 1-14Cacetic acid into cellular Although the metabolic of acid from acid, we differences between and Ad-ACSL5-infected cells in the metabolism of acid to cellular it that ACSL5 fatty acids from and does not use fatty acids that are ACSL5 mRNA in to and an in obtained from ACSL5 in McArdle-RH7777 cells suggest that ACSL5 partitions fatty acids to TAG synthesis. To into the in regulation of we in ACSL5 mRNA levels in rats that were for or for h and refed a 70% sucrose diet for 24 of ACSL5 mRNA in response to a 48-h but when rats were refed a high sucrose diet after the ACSL5 mRNA increased levels These in in ACSL5 mRNA abundance support the in that ACSL5 in an anabolic ACSL is the for fatty acid activation in known pathways of fatty acid metabolism. that five isoforms have been in the ACSL as as of several of the we suggest that there are distinct roles for the individual isoforms and Coleman R.A. J. D.A. P. W. Hashimoto N. V. A. J.E. A. V. Yamamoto T.T. J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). studies have our of the tissue and, to of the ACSL but have not shown that isoform has a specific role in metabolism. The was designed to the of an individual ACSL on the of fatty acids in the rat hepatoma McArdle-RH7777 cell These cells are fatty acid metabolism is similar to that of hepatocytes and, many hepatoma cell McArdle-RH7777 cells TAG in Biophys. PubMed Scopus Google Scholar). We have shown that overexpression of ACSL5 increases fatty acid metabolism and partitions fatty acids to TAG that is destined for but not increase in TAG synthesis was only at a high fatty acid that be of or a fast Biophys. PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar). suggests that at fatty acid ACSL activity is present to fatty acids to However, when higher of fatty acids are present in the cellular ACSL activity thus fatty acid ACSL5 and total cellular ACSL activity fatty acid can be metabolized more and fatty acid uptake To date, most studies fatty acid uptake have on membrane proteins such as fatty acid or fatty acid Although proteins fatty acid the is not an involved in fatty acids can increase the of fatty acids through a this suggest that uptake might be regulated by of fatty acids. and J.A. Physiol. 2003; PubMed Scopus Google Scholar) that increased of fatty acids to and metabolism fatty acids into the cell by the fatty acid the In support of this hypothesis, overexpression increases acid uptake in both and H. 2001; PubMed Scopus Google Scholar). Although membrane proteins of the metabolism of the fatty acids be more in The 40% increase in fatty acid incorporation into increases in to the that individual ACSL isoforms fatty acids toward distinct pathways. the increase in fatty acid incorporation into cellular TAG did not with increased TAG medium TAG increased with of fatty acid to the we can the that the capacity for very lipoprotein synthesis and was Therefore, fatty acids by ACSL5 be destined for an TAG with a is not known located on mitochondria is more or to or when compared with endoplasmic which include the in TAG diacylglycerol mitochondrial increases cellular TAG very lipoprotein in rat hepatocytes and thus that the TAG in mitochondria the TAG to T.M. Wang S. C.A. Van Horn C.G. Coleman R.A. Am. J. Physiol. 2005; PubMed Scopus Google Scholar). In with the cell rat liver ACSL5 mRNA following a 48-h fast and increased a high sucrose diet for 24 pattern of ACSL5 message abundance suggests that it an involved in a TAG studies have also shown that ACSL5 mRNA is regulated in a with an anabolic Hepatic ACSL5 message abundance increases in SREBP-1c transgenic mice (15Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1119) Google Scholar) and in zinc-deficient mice that have fatty livers (17tom Dieck H. Doring F. Fuchs D. Roth H.P. Daniel H. J. Nutr. 2005; 135: 199-205Crossref PubMed Scopus (87) Google Scholar). In addition, hepatic ACSL5 mRNA levels do not to peroxisomal proliferator-activated receptor α agonists (14Lewin T.M. Van Horn C.G. Krisans S.K. Coleman R.A. Arch. Biochem. Biophys. 2002; 404: 263-270Crossref PubMed Scopus (110) Google Scholar) and decrease in with lipogenic gene expression such as SREBP cleavage-activating protein knock-out mice (15Horton J.D. Shah N.A. Warrington J.A. Anderson N.N. Park S.W. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12027-12032Crossref PubMed Scopus (1119) Google Scholar), estrogen-related receptor knock-out mice (18Luo J. Sladek R. Carrier J. Bader J.A. Richard D. Giguere V. Mol. Cell. Biol. 2003; 23: 7947-7956Crossref PubMed Scopus (322) Google Scholar), and C57BL/6J obese ob/ob mice following leptin (16Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 49066-49076Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In to mRNA rat hepatic mitochondrial ACSL5 protein increases after a 48-h fast and decreases when rats are refed a high sucrose diet (6Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). regulation of ACSL5 mRNA and protein or ACSL5 abundance on mitochondria and using triacsin C, an inhibitor of ACSL1, ACSL3, and ACSL4 but not have shown more decreases in synthesis than in fatty acid β-oxidation (10Muoio D.M. Lewin T.M. Wiedmer P. Coleman R.A. Am. J. Physiol. 2000; 279: E1366-E1373Crossref PubMed Google Scholar, R.A. Wang P. Coleman R.A. Biochem. J. 1997; PubMed Scopus Google Scholar). on ACSL5 was hypothesized to activate fatty acids for β-oxidation. Therefore, it was that Ad-ACSL5-infected cells did not ASM synthesis. is that ACSL5 fatty acids for both anabolic and catabolic pathways and that the of derived from ACSL5 be for TAG synthesis but not for ASM synthesis. could the increase in cellular TAG a change in ASM synthesis. To this hypothesis, we ACSL1, ACSL3, and ACSL4, the known ACSL isoforms in McArdle-RH7777 cells with triacsin overexpression of ACSL5 to the effects of triacsin C on cellular TAG synthesis and the total of fatty acid However, in Ad-ACSL5-infected cells with triacsin C, ASM synthesis be that this decrease was when compared with the decrease in ASM synthesis in cells with triacsin of the derived from ACSL5 were used for ASM synthesis when the ACSL isoforms were Although we that ACSL isoforms have unique roles in fatty it is that an individual isoform fatty acids for a a ACSL isoform activate fatty acids that are metabolized into a specific and be metabolized by pathways. In to the effects of triacsin C in hepatocytes and in McArdle-RH7777 cells, triacsin C had similar effects on both TAG and ASM synthesis. Although the triacsin C a role for ACSL5 in TAG there are many when using triacsin of the ACSL isoforms have most of which have not been for triacsin C long chain acyl-CoA also possess activity toward long chain fatty acids. to the ACSL triacsin C has not been in many of the is to ACSL or subcellular and to triacsin In overexpression of ACSL5 in McArdle-RH7777 cells increased fatty acid uptake into cells that were to fatty acid of a fast or Although high fatty acid were to the increase in fatty acid fatty acid incorporation into cellular TAG was increased at fatty acid Despite the increase in cellular TAG Ad-ACSL5-infected cells did not increase the of TAG into the medium and did not use fatty acids. Taken together with in hepatic mRNA levels in response to and we showed that, in to our hypothesis, ACSL5 is involved in than catabolic pathways. Although it is that the function of overexpressed ACSL5 differ from that of the suggest a unique role for ACSL5 in fatty acid and TAG metabolism in McArdle-RH7777 cells. We from the of and for with the microscopy and the for the with the