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• W2000692520 abstract "Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-γ, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-α and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-14C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis. Long chain acyl-CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids to acyl-CoAs in the initial step of fatty acid metabolism. Numerous isoforms of ACSL and FATP exist with different tissue distribution patterns, intracellular locations, and substrate preferences, suggesting that each isoform has distinct functions in channeling fatty acids into different metabolic pathways. Because fatty acids, acyl-CoAs, and downstream lipid metabolites regulate various transcription factors that control hepatic energy metabolism, we hypothesized that ACSL or FATP isoforms differentially regulate hepatic gene expression. Using small interference RNA (siRNA), we knocked down each liver-specific ACSL and FATP isoform in rat primary hepatocyte cultures and subsequently analyzed reporter gene activity of numerous transcription factors and performed quantitative mRNA analysis of their target genes. Compared with control cells, which were transfected with control siRNA, knockdown of acyl-CoA synthetase 3 (ACSL3) significantly decreased reporter gene activity of several lipogenic transcription factors such as peroxisome proliferator activation receptor-γ, carbohydrate-responsive element-binding protein, sterol regulatory element-binding protein-1c, and liver X receptor-α and the expression of their target genes. These findings were further supported by metabolic labeling studies that showed [1-14C]acetate incorporation into lipid extracts was decreased in cells treated with ACSL3 siRNAs and that ACSL3 expression is up-regulated in ob/ob mice and mice fed a high sucrose diet. ACSL3 knockdown decreased total acyl-CoA synthetase activity without substantially altering the expression of other ACSL isoforms. In summary, these results identify a novel role for ACSL3 in mediating transcriptional control of hepatic lipogenesis. Intracellular fatty acids and downstream metabolites affect a host of physiological processes, including transcriptional control of energy metabolism (1Mashek D.G. Li L.O. Coleman R.A. Future Lipidol. 2007; 2: 465-476Crossref PubMed Scopus (174) Google Scholar, 2Jump D.B. Botolin D. Wang Y. Xu J. Christian B. Demeure O. J. Nutr. 2005; 135: 2503-2506Crossref PubMed Scopus (376) Google Scholar). Fatty acids and/or their metabolites regulate hepatic transcription factors such as peroxisome proliferator activation receptors (PPARs), 2The abbreviations used are: PPARperoxisome proliferator-activated receptorACCacetyl-CoA carboxylaseACSLlong chain acyl-CoA synthetaseAMPKAMP kinaseChREBPcarbohydrate-responsive element-binding proteinFASfatty acid synthaseFATPfatty acid transport proteinsLXRliver X receptorPGC-1βPPAR-γ co-activator-1βSREBPsterol regulatory element-binding proteinsiRNAsmall interference RNARTreverse transcriptionTAGtriacylglycerol. carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element-binding protein (SREBP)-1c, and liver X receptor (LXR)-α (3Zhang Y.L. Hernandez-Ono A. Siri P. Weisberg S. Conlon D. Graham M.J. Crooke R.M. Huang L.S. Ginsberg H.N. J. Biol. Chem. 2006; 281: 37603-37615Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 4Pawar A. Xu J. Jerks E. Mangelsdorf D.J. Jump D.B. J. Biol. Chem. 2002; 277: 39243-39250Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 5Xu J. Teran-Garcia M. Park J.H. Nakamura M.T. Clarke S.D. J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 6Stoeckman A.K. Ma L. Towle H.C. J. Biol. Chem. 2004; 279: 15662-15669Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 7Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1423) Google Scholar). The effects of fatty acids are partially determined by their chemical structure and intracellular source. For instance, EPA (C20:5) and DHA (C22:6) bind and activate PPAR-α and up-regulate genes involving fatty acid oxidation and gluconeogenesis (8Pawar A. Jump D.B. J. Biol. Chem. 2003; 278: 35931-35939Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). These polyunsaturated fatty acids also suppress activity of ChREBP, SREBP-1c, and LXR-α through multiple mechanisms (2Jump D.B. Botolin D. Wang Y. Xu J. Christian B. Demeure O. J. Nutr. 2005; 135: 2503-2506Crossref PubMed Scopus (376) Google Scholar, 4Pawar A. Xu J. Jerks E. Mangelsdorf D.J. Jump D.B. J. Biol. Chem. 2002; 277: 39243-39250Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 5Xu J. Teran-Garcia M. Park J.H. Nakamura M.T. Clarke S.D. J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 9Dentin R. Benhamed F. Pégorier J.P. Foufelle F. Viollet B. Vaulont S. Girard J. Postic C. J. Clin. Investig. 2005; 115: 2843-2854Crossref PubMed Scopus (241) Google Scholar, 10Lorente-Cebrián S. Pérez-Matute P. Martínez J.A. Marti A. Moreno-Aliaga M.J. J. Physiol. Biochem. 2006; 62: 61-69Crossref PubMed Google Scholar), whereas saturated fatty acids activate SREBP-1c by recruiting the SREBP-1c co-activator, PPAR-γ co-activator-1β (PGC-1β) (2Jump D.B. Botolin D. Wang Y. Xu J. Christian B. Demeure O. J. Nutr. 2005; 135: 2503-2506Crossref PubMed Scopus (376) Google Scholar,11Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. Newgard C.B. Spiegelman B.M. Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Also intracellular fatty acids derived from de novo lipogenesis or hydrolysis of triacylglycerol (TAG) or phospholipid can activate these transcription factors. For example, recent evidence suggests that modulating specific pathways, such as de novo fatty acid synthesis or TAG hydrolysis, which supply intracellular fatty acids, regulates gene expression (12Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 13Sapiro J.M. Mashek M.T. Greenberg A.S. Mashek D.G. J. Lipid Res. 2009; 50: 1621-1629Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Such evidence that different regulation of these transcription factors by fatty acid type (unsaturated versus saturated) or source (intracellular versus exogenous) implicates that certain proteins or enzymes that control cellular uptake or trafficking of fatty acids and their downstream metabolites could mediate their effects on gene expression. peroxisome proliferator-activated receptor acetyl-CoA carboxylase long chain acyl-CoA synthetase AMP kinase carbohydrate-responsive element-binding protein fatty acid synthase fatty acid transport proteins liver X receptor PPAR-γ co-activator-1β sterol regulatory element-binding protein small interference RNA reverse transcription triacylglycerol. Acyl-CoA synthetase (ACSL) and fatty acid transport protein (FATP) activate fatty acids to acyl-CoAs in the presence of ATP and CoA. After this initial step, acyl-CoAs enter multiple metabolic pathways for lipid synthesis or β-oxidation (1Mashek D.G. Li L.O. Coleman R.A. Future Lipidol. 2007; 2: 465-476Crossref PubMed Scopus (174) Google Scholar, 14Mashek D.G. Coleman R.A. Curr. Opin. Lipidol. 2006; 17: 274-278Crossref PubMed Scopus (118) Google Scholar). FATP, also termed very long chain acyl-CoA synthetase, share 20–40% of sequence similarity with ACSL and have substrate preferences toward very long chain fatty acids (C22–26) but also show activity toward long chain fatty acids (1Mashek D.G. Li L.O. Coleman R.A. Future Lipidol. 2007; 2: 465-476Crossref PubMed Scopus (174) Google Scholar, 15Watkins P.A. J. Biol. Chem. 2008; 283: 1773-1777Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Each family of these enzymes has several isoforms that have unique cellular localization patterns, substrate preferences, and enzyme kinetics (1Mashek D.G. Li L.O. Coleman R.A. Future Lipidol. 2007; 2: 465-476Crossref PubMed Scopus (174) Google Scholar, 14Mashek D.G. Coleman R.A. Curr. Opin. Lipidol. 2006; 17: 274-278Crossref PubMed Scopus (118) Google Scholar, 15Watkins P.A. J. Biol. Chem. 2008; 283: 1773-1777Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 16Mashek D.G. Li L.O. Coleman R.A. J. Lipid Res. 2006; 47: 2004-2010Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Gain- or loss-of-function studies also suggest unique roles for the individual ACSL and FATP isoforms in fatty acid channeling. Adenovirus-mediated overexpression of ACSL1 in rat primary hepatocytes results in channeling of [1-14C]oleic acid toward diacylglycerol and phospholipid synthesis and away from cholesterol esterification (17Li L.O. Mashek D.G. An J. Doughman S.D. Newgard C.B. Coleman R.A. J. Biol. Chem. 2006; 281: 37246-37255Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Knockdown of ACSL3 in human hepatocytes decreases [1-14C]oleic acid incorporation to phospholipids for very low density lipoprotein synthesis (18Yao H. Ye J. J. Biol. Chem. 2008; 283: 849-854Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) thus indicating an anabolic role in energy metabolism. Overexpression of ACSL5 in rat hepatoma cell lines increases fatty acid incorporation into TAG with substrate selectivity toward exogenous fatty acids, but not endogenous fatty acids, and without changes in β-oxidation or phospholipid synthesis (19Mashek D.G. McKenzie M.A. Van Horn C.G. Coleman R.A. J. Biol. Chem. 2006; 281: 945-950Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Differential channeling of fatty acids into diverse metabolic pathways suggest that ACSL and FATP isoforms regulate distinct pools of intracellular lipids. Based on the differential regulation of ACSL or FATP enzymes on fatty acid channeling and the importance of fatty acids or their downstream metabolites on regulating transcription factors involving energy metabolism, we hypothesized that ASCL or FATP isoforms would differentially regulate hepatic gene expression. Therefore, the aim of this study was to determine which ACSL or FATP isoforms are responsible for modulating the activity of transcription factors in hepatic energy metabolism by utilizing siRNA specifically targeting the predominant ACSL or FATP isoforms expressed in the liver. We found that ACSL3 siRNA-transfected cells uniquely down-regulated PPAR-γ activity. Further characterization revealed that knockdown of ACSL3 decreased the activity of several lipogenic transcription factors, their target gene expression, and rates of de novo lipogenesis. Thus we conclude that ACSL3 mediates hepatic lipogenesis through transcriptional regulation of gene expression. Tissue culture plates were from Nunc, and media were obtained from Invitrogen. Rat-tail collagen I was obtained from BD Biosciences. [1-14C]Acetic acid was from PerkinElmer Life Sciences. pSG5-GAL4-hPPAR-γ or pSG5-GAL4-hPPAR-α expression plasmid and a TKMH-UAS-LUC reporter plasmid were provided by Philippe Thuillier (Oregon Health and Science University, Portland, OR). pCMX-hLXR-α expression plasmid and TK-hcyp7a-LXRE(X3)-Luc reporter plasmid were provided by Dr. David Mangelsdorf (University of Texas-Southwestern). For ChREBP measurements, the ACC carbohydrate response element-containing reporter plasmid and pCMVS4-ChREBP expression vector were provided by Dr. Howard Towle (University of Minnesota), and for SREBP1-c analysis, the SRE sequence on the FAS gene and the pCMV-SREBP-1c expression vector, which contains a constitutively activated form of SREBP-1c, were provided by Dr. Timothy Osborne (University of California, Irvine, CA). Rosiglitazone and T0901317 were obtained from Cayman Chemical. All other chemicals were obtained from Sigma unless otherwise indicated. Male Sprague-Dawley rats (250–300 g) were maintained on a 12:12-h light:dark cycle and were allowed free access to food before hepatocyte isolation. Hepatocytes were isolated by using the collagenase perfusion method (20Stoeckman A.K. Towle H.C. J. Biol. Chem. 2002; 277: 27029-27035Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and cell viability, as measured by trypan blue exclusion, was over 90%. Hepatocytes were plated at a density of 0.5 × 106 cells/22-mm well in M199 medium (23 mm HEPES, 26 mm sodium bicarbonate, 10% fetal bovine serum, 50 IU/ml penicillin, 50 μg/ml streptomycin, 100 nm dexamethasone, 100 nm insulin, and 11 mm glucose). Animal protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. Duplexes of siRNA targeting ACSL or FATP isoforms were synthesized by Qiagen and are listed in Table 1. Nonspecific sequence targeted siRNA, which was designed by Qiagen, served as a control. Recombinant siRNA was transfected into primary hepatocytes with 1 μg of siRNA per 0.5 × 106 cells using Effectene reagent (Qiagen) when cells were plated. Transfection media were removed and replaced by M199 media supplemented with 23 mm HEPES, 26 mm sodium bicarbonate, 50 IU/ml penicillin, 50 μg/ml streptomycin, 10 nm insulin, 10 nm dexamethasone, and 5.5 mm glucose unless noted otherwise.TABLE 1Target sequences of siRNA duplexes for ACSL or FATP isoformsTarget geneNCBI accession no.Nucleotide positionsiRNA sequences (5′-3′)bpACSL1NM_012820253–273CAA GCT CTT GCT GTA CTA CTAACSL3-aNM_0571071368–1389CTG GGT GGA AAG AGG CGC GTTACSL3-bNM_0571072137–2157GCC TTC AAG TTG AAA CGT AAAACSL4NM_0536231758–1778CAG ATT ATC GAT CGT AAG AAAACSL5NM_053607423–443GCC CTA CAA GTG GAT ATC CTAFATP2NM_0317361970–1990AAG GCA CGA GCT GAT CAA GTAFATP4XM_0010794091740–1760AAC AAG AAG AAT GCT AGT GATFATP5NM_024143884–904CCA AGC TTC GTG CTA ATA TAA Open table in a new tab Hepatocytes were co-transfected with the pSG5-GAL4-hPPAR-γ or pSG5-GAL4-hPPAR-α expression plasmid (50 ng) and a TKMH-UAS-LUC reporter plasmid (250 ng) per 0.5 × 106 cells for measuring PPAR-γ and PPAR-α activity. pCMX-hLXR-α expression plasmid (20 ng) and TK-hCYP7a-LXRE(X3)-Luc reporter plasmid (180 ng per 0.5 × 106 cells) was used for measurement of LXR-α. For ChREBP and SREBP1-c, firefly luciferase reporter driven by ACC carbohydrate response element-containing promoter region (200 ng) and the SRE sequence on the FAS gene (200 ng) were transfected in 0.5 × 106 cells for reporter gene assay. For overexpression of ChREBP and SREBP1-c, pCMVS4-ChREBP and pCMV-SREBP1-c expression vectors were used (20 ng per 0.5 × 106 cells). A Renilla luciferase vector (pRL-SV40, Promega, Madison, WI) was used at a concentration of 20 ng per 0.5 × 106 cells as an internal control for adjusting transfection efficiency of each reporter gene plasmid. Cells were then harvested for luciferase reporter gene assay at 50 h after transfection (Promega), and activity of each transcription factor was expressed as relative luciferase units. Total RNA was isolated with TRIzol (Invitrogen) and stored at −80 °C until use. First-strand cDNA was synthesized with the SuperScript III reverse transcriptase and random hexamer primers (Invitrogen). Synthesized cDNAs were mixed with 2× SYBR Green PCR Master Mix (Invitrogen) and subjected to the real-time PCR quantification on an ABI Prism 7700 sequence detection system (Applied Biosystems). Fluorescence data were acquired for 40 cycles with an annealing temperature of 60 °C. Primers for each gene are listed in Table 2. Data were analyzed using the ΔΔCt method, and the mRNA abundance of each gene was normalized to RPL-32.TABLE 2Primer sequences for quantitative reverse transcription-PCR analysisGeneForward primerReverse primerACSL1AAC GAT GTA CGA TGG CTT CCCAT ATG GCT GGT TTG GCT TTACSL3GGG ACT ACA ATA CCG GCA GAATA GCC ACC TTC CTC CCA GTACSL4AAA TGC AGC CAA ATG GAA AGCAC TCG GCA GTT CAC TTC AAACSL5ATC TGC CTC CTG ACA TTT GGGCT CCT CCC TCA ATC CCT ACFATP2CTG CAT GTC TTC TTG GAG CAGCG TAG GTA AGC GTC TCG TCFATP4CAC TGC CTT GAC ACC TCA AAACC AGA GCA GAA GAG GGT GAFATP5GGA ACT CTA CGG CTC CAC AGGGC TCT GCC GTC TCT ATG TCFASAGG ATG TCA ACA AGC CCA AGACA GAG GAG AAG GCC ACA AASCD-1TGT TCG TCA GCA CCT TCT TGGGA TGT TCT CCC GAG ATT GAACC-αATT GTG GCT CAA ACT GCA GGTGCC AAT CCA CTC GAA GAC CAACC-βCAA AGC CTC TGA AGG TGG AGGGA CAC TGC GTT CCC ATA CTCD36GGC TGT GTT TGG AGG CAT TCTCAA AAA CTG GGT GAA AAC GGGL-PKGTA CAG AAA ATC GGC CCA GAAGG TCC ACC TCA GTG TTT GGSREBP-1CGGA GCC ATG GAT TGC ACA TTAGA AGA GAA GCT CTC AGG AGChREBPCGG GAC ATG TTT GAT GAC TATAAT AAA GGT CGG ATG AGG ATGPPAR-γ1/2CGA GAA GGA GAA GCT GTT GGTCA GCG GGA AGG ACT TTA TGLXR-αTAC AAC CGG GAA GAC TTT GCTGC AGA GAA GAT GCT GAT GGPGC-1βCAA GAA GCG GCG GGG AAGCT CAT GTG ACC GGA GAG ATT TRPL32AAA CTG GCG GAA ACC CAG AGGCA GCA CTT CCA GCT CCT TGRPL32 (mouse)AAC CCA GAG GCA TTG ACA ACATT GTG GAC CAG GAA CTT GC Open table in a new tab For measuring protein expression of ACSL3 and AMP kinase (AMPK) phosphorylation, cell monolayers were harvested and lysed in 10 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 0.1% Triton X-100, and 1% protease inhibitor mixture (Roche Applied Science). Aliquots of total proteins (15–150 μg) were denatured at 100 °C for 10 min in SDS sample loading buffer (50 mm Tris, pH 6.8, 2% SDS, 10% glycerol, 1% bromphenol blue, and 15% β-mercaptoethanol). Samples were then separated by SDS-PAGE using a 7.5% resolving gel and electroblotted to a polyvinylidene difluoride membrane (Millipore). Equal transfer of proteins was confirmed by Ponceau S staining. After transfer, the membrane was blocked in 5% nonfat dry milk in phosphate-buffered saline (pH 7.4) with 1% Tween 20 and then incubated with ACSL3 (Santa Cruz Biotechnology, Santa Cruz, CA), ChREBP (Novus Biologicals), AMPK, phosphorylated AMPK at Thr-172 (Cell Signaling Technology), or β-actin (Sigma) antibodies. The antigens were detected by ECL chemiluminescent assay following incubation with a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology). Hepatocytes transfected with control or ACSL siRNAs were washed twice with cold phosphate-buffered saline and collected in cold Med I buffer (10 mm Tris, pH 7.4, 250 mm sucrose, 1 mm EDTA, 1 mm dithiothreitol, and protease inhibitor mixture) and homogenized on ice with 10 strokes of a tissue homogenizer (Biospec). Homogenate aliquots were stored at −80 °C until use. Protein concentrations were determined by using the BCA method (Pierce). Acyl-CoA synthetase specific activity was determined by measuring the production of [1-14C]acyl-CoAs in the presence of 175 mm Tris-HCl, pH 7.4, 8 mm MgCl2, 5 mm dithiothreitol, 10 mm ATP, 0.25 mm CoA, and 500 μm [1-14C]palmitate in 0.5 mm Triton X-100, 0.01 mm EDTA. The assay was performed in a total volume of 200 μl at 37 °C for 5 min. The reaction was started by adding 1–2 μg of homogenate protein, terminated with 1 ml of Dole reagent (isopropanol, heptane, 1 m H2SO4, 80:20:2, v/v), and fatty acids were extracted with sequential hexane washes prior to scintillation counting of the aqueous phase containing the acyl-CoAs. Seventy-two hours after plating or siRNA transfection, hepatocytes were labeled with 1 ml of M199 containing 4.0 and 1.0 μCi of [1-14C]acetic acid for either 15 min or 3 h as indicated in the figure legends. Hepatocytes were washed twice with 1% bovine serum albumin in phosphate-buffered saline at 37 °C, and cellular lipids were extracted (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). Aliquots of the lipid extracts from the cells were separated by TLC on 0.25-mm Silica Gel G plates in hexane:ethyl ether:acetic acid (80:20:1, v/v) together with synthetic lipid standards (Sigma and BioChemika) in parallel. The 14C-labeled lipids in aliquots of the total lipid extracts and lipids scraped from iodine vapor-stained TLC plates were quantified using liquid scintillation counting (LS6000IC, Beckman). Fifty hours after plating or siRNA transfection, lipids were extracted (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar) and total intracellular free fatty acids were separated by TLC on 0.25-mm silica gel G plates in hexane:ethyl ether:acetic acid (80:20:1, v/v). Isolated free fatty acids were methylated in 3 n methanolic HCl at 100 °C for 90 min. The fatty acids methyl esters were extracted with hexane and subjected to gas chromatography analysis (22Forsythe C.E. Phinney S.D. Fernandez M.L. Quann E.E. Wood R.J. Bibus D.M. Kraemer W.J. Feinman R.D. Volek J.S. Lipids. 2008; 43: 65-77Crossref PubMed Scopus (241) Google Scholar). Data were expressed as means ± S.E. Significance of data were declared at p < 0.05 by Student t test. Initial studies were designed to test a minimum of three siRNA sequences to each ACSL or FATP gene expressed in liver for knockdown of at least 70%. Expression of each ACSL and FATP mRNA was successfully suppressed after 24 h compared with control siRNA-transfected cells by at least one of the siRNAs tested (Fig. 1A). Once we verified the efficacy of isoform-specific siRNA, we co-transfected these siRNAs in rat primary hepatocytes with reporter gene plasmids for PPAR-γ or PPAR-α. Of all the siRNA targeting different ACSL or FATP isoforms, ACSL3 siRNA significantly and uniquely suppressed PPAR-γ activity compared with control cells (Fig. 1B). None of the ACSL or FATP siRNAs affected reporter gene activity of PPAR-α (Fig. 1C). Based upon these findings, we sought to further characterize the role of ACSL3 in modulating hepatic gene expression to control energy metabolism. To confirm that the effect of the ACSL3 siRNA was not due to off-target effects, we developed an additional ACSL3 siRNA targeting a different region of ACSL3 mRNA and confirmed its efficacy. Quantitative RT-PCR revealed that the level of ACSL3 mRNA was reduced by >70% in cells transfected with ACSL3 siRNA targeting different regions of the ACSL3 gene (Fig. 2A). Both ACSL3 siRNAs also decreased the level of ACSL3 protein expression at 50 h following transfection (Fig. 2B). Subsequently, we tested whether other ACSL or FATP isoforms had compensational expression in response to knockdown of ACSL3. To test this possibility, mRNA of each ACSL or FATP isoform was analyzed in ACSL3 siRNA-transfected cells after 50 h of transfection, which is the time point that the protein level of ACSL3 is suppressed. Although the expression of ACSL5 mRNA was modestly up-regulated in cells transfected with one of the ACSL3 siRNA (ACSL3-b siRNA), the expressions of the other ACSL or FATP isoforms were not significantly affected by the low expression of ACSL3 (Fig. 2C). These data indicate that the expression of the other isoforms of ACSL or FATP do not compensate for a deficiency of ACSL3. Transfection of ACSL3 siRNAs decreased total acyl-CoA synthetase activity 25 and 60%, respectively (Fig. 2D), when compared with control siRNA-transfected cells. As we observed for the first ACSL3 siRNA, the second ACSL3 siRNA also suppressed PPAR-γ activity by 55% (Fig. 3E).FIGURE 3Knockdown of ACSL3 suppresses the activity of lipogenic transcription factors in rat primary hepatocytes. ACSL3-a or ACSL3-b siRNA were transfected in rat primary hepatocytes, and reporter gene activities were quantified after 50 h. A and B, for SREBP, firefly luciferase reporter of SRE sequence on FAS gene were transfected for reporter gene assay. Cells in B were also transfected with pCMV-SREBP1-c, a constitutively active form of SREBP. C and D, firefly luciferase reporter driven by the ACC carbohydrate response element-containing promoter region was transfected for measurement of ChREBP in cells treated with 5 mm (C) and 25 mm glucose (D). E and F, pSG5-GAL4-hPPAR-γ expression plasmids were co-transfected with TK-MH-UAS-Luc reporter plasmids in cells treated with DMSO (E) or 10 μm rosiglitazone (F) for 18 h to determine whether synthetic ligands normalize PPAR-γ. G and H, pCMX-hLXR-α expression plasmid and TK-hcyp7a-LXRE(X3)-Luc reporter plasmid were used for measurement of LXR-α activity in cells treated with DMSO (G) or 15 μm T0901317 (H) for 18 h. Values shown are mean ± S.E. from a representative experiment performed in triplicate that was repeated two or three times. *, p < 0.05 when compared with controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although most characterization of PPAR-γ has been reported in adipocytes (2Jump D.B. Botolin D. Wang Y. Xu J. Christian B. Demeure O. J. Nutr. 2005; 135: 2503-2506Crossref PubMed Scopus (376) Google Scholar, 23He Z. Jiang T. Wang Z. Levi M. Li J. Am. J. Physiol. Endocrinol. Metab. 2004; 287: E424-E430Crossref PubMed Scopus (70) Google Scholar), this transcription factor has also been linked to lipogenesis and lipid accumulation in liver (3Zhang Y.L. Hernandez-Ono A. Siri P. Weisberg S. Conlon D. Graham M.J. Crooke R.M. Huang L.S. Ginsberg H.N. J. Biol. Chem. 2006; 281: 37603-37615Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 24Schadinger S.E. Bucher N.L. Schreiber B.M. Farmer S.R. Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1195-E1205Crossref PubMed Scopus (319) Google Scholar, 25Westerbacka J. Kolak M. Kiviluoto T. Arkkila P. Sirén J. Hamsten A. Fisher R.M. Yki-Järvinen H. Diabetes. 2007; 56: 2759-2765Crossref PubMed Scopus (263) Google Scholar). Thus, based on our initial findings that PPAR-γ was suppressed by ACSL3 knockdown, we wanted to further characterize how ACSL3 influenced other transcription factors such as SREBP-1c, LXR-α, and ChREBP, all of which contribute to the regulation of lipogenic gene expression. ACSL3 siRNAs decreased reporter gene activity of SREBP1-c (25 and 30%) and LXR-α (50 and 55%) (Fig. 3, A and G). The reporter gene activity assays for SREBP were performed with reporter plasmids containing sterol regulatory elements, but SREBP itself was not overexpressed. To determine if endogenous SREBP or its subsequent processing were limiting, we overexpressed the constitutively active nuclear form of SREBP-1c in hepatocytes in conjunction with reporter plasmids. However, ACSL3 siRNA-mediated suppression of SREBP reporter gene activity was not overcome by overexpression of SREBP-1c (Fig. 3B). ChREBP is thought to be the primary glucose-responsive transcription factor that governs the expression of glycolytic and lipogenic genes (6Stoeckman A.K. Ma L. Towle H.C. J. Biol. Chem. 2004; 279: 15662-15669Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 9Dentin R. Benhamed F. Pégorier J.P. Foufelle F. Viollet B. Vaulont S. Girard J. Postic C. J. Clin. Investig. 2005; 115: 2843-2854Crossref PubMed Scopus (241) Google Scholar). Similar to the other lipogenic transcription factors, ACSL3 knockdown suppressed ChREBP reporter gene activity (Fig. 3C). Additionally, we challenged the cells with high (25 mm) glucose concentrations to stimulate ChREBP activity. ACSL3 siRNAs suppressed ChREBP under high glucose conditions and largely prevented the increase in ChREBP activity following exposure to high glucose. Specifically, control cells exhibited a 25-fold increase in ChREBP activity in response to high glucose, whereas cells with suppressed ACSL3 expression only responded with a 3- to 3.5-fold increase (Fig. 3D). Therefore, ACSL3 appears to inhibit basal ChREBP activity and its glucose-mediated induction. To determine if ligands for these transcription factors can rescue PPAR-γ or LXR-α activity in cells transfected with ACSL3 siRNA, we incubated cells with 10 μm rosiglitazone or 15 μm T0901317, synthetic ligands for PPAR-γ and LXR-α, respectively. Exposure to these ligands increased PPAR-γ (∼8-fold) or LXR-α (∼2.5-fold) activity (Fig. 3, F and H). However, these ligands were unab" @default.
• W2000692520 created "2016-06-24" @default.
• W2000692520 creator A5006953866 @default.
• W2000692520 creator A5070099135 @default.
• W2000692520 creator A5080799718 @default.
• W2000692520 date "2009-10-01" @default.
• W2000692520 modified "2023-10-15" @default.
• W2000692520 title "Suppression of Long Chain Acyl-CoA Synthetase 3 Decreases Hepatic de Novo Fatty Acid Synthesis through Decreased Transcriptional Activity" @default.
• W2000692520 cites W1495212988 @default.
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