Liver-specific Loss of Long Chain Acyl-CoA Synthetase-1 Decreases Triacylglycerol Synthesis and β-Oxidation and Alters Phospholipid Fatty Acid Composition
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.022467
ISSN1083-351X
AutoresLei O. Li, Jessica M. Ellis, Heather A. Paich, Shuli Wang, Nan Gong, George Altshuller, Randy Thresher, Timothy R. Koves, Steven M. Watkins, Deborah M. Muoio, Gary W. Cline, Gerald I. Shulman, Rosalind Coleman,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoIn mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25–35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1L−/− liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [14C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [14C]oleate suggested that hepatic ACSL1 is important for mitochondrial β-oxidation of long chain fatty acids. Because the Acsl1L−/− mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance. In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25–35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1L−/− liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [14C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [14C]oleate suggested that hepatic ACSL1 is important for mitochondrial β-oxidation of long chain fatty acids. Because the Acsl1L−/− mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance. Acyl-CoA synthetase (ACSL) 3The abbreviations used are: ACSLlong chain acyl-CoA synthetaseAGPATacylglycerol-3-phosphate acyltransferaseANOVAanalysis of varianceASMacid-soluble metaboliteCEcholesteryl esterDAGdiacylglycerolFAfatty acidFATPfatty acid transport proteinGPATglycerol-3-phosphate acyltransferaseITTinsulin tolerance testLPAlysophosphatidic acidLPClysophosphatidylcholineneoneomycinOGTToral glucose tolerance testPCphosphatidylcholinePEphosphatidylethanolaminePSphosphatidylserinePPARperoxisome proliferator-activated factorTAGtriacylglycerol. 3The abbreviations used are: ACSLlong chain acyl-CoA synthetaseAGPATacylglycerol-3-phosphate acyltransferaseANOVAanalysis of varianceASMacid-soluble metaboliteCEcholesteryl esterDAGdiacylglycerolFAfatty acidFATPfatty acid transport proteinGPATglycerol-3-phosphate acyltransferaseITTinsulin tolerance testLPAlysophosphatidic acidLPClysophosphatidylcholineneoneomycinOGTToral glucose tolerance testPCphosphatidylcholinePEphosphatidylethanolaminePSphosphatidylserinePPARperoxisome proliferator-activated factorTAGtriacylglycerol. activates long chain fatty acid (FA) to acyl-CoA, thereby enhancing vectorial FA transport across the plasma membrane (1Mashek D.G. Coleman R.A. Curr. Opin. Lipidol. 2006; 17: 274-278Crossref PubMed Scopus (115) Google Scholar) and providing substrates for most downstream pathways that metabolize FA. ACSL1 is one of five ACSL isoforms, each encoded by a separate gene. Its mRNA expression is highest in adipose tissue, liver, and heart (2Mashek D.G. Li L.O. Coleman R.A. J. Lipid Res. 2006; 47: 2004-2010Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar); and because Acsl1 mRNA and total ACSL1 activity increase 160-fold (3Marszalek J.R. Kitidis C. Dararutana A. Lodish H.F. J. Biol. Chem. 2004; 279: 23882-23891Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and 100-fold (4Coleman R.A. Reed B.C. Mackall J.C. Student A.K. Lane M.D. Bell R.M. J. Biol. Chem. 1978; 253: 7256-7261Abstract Full Text PDF PubMed Google Scholar), respectively, in differentiating 3T3-L1 adipocytes, ACSL1 has been thought to be important in activating FA destined for triacylglycerol (TAG) synthesis. In support of this idea, overexpressing ACSL1 in mouse heart increases cardiac myocyte TAG accumulation 12-fold and induces apoptotic pathways, cardiac hypertrophy, left ventricular dysfunction, and heart failure (5Chiu H.C. Kovacs A. Ford D.A. Hsu F.F. Garcia R. Herrero P. Saffitz J.E. Schaffer J.E. J. Clin. Invest. 2001; 107: 813-822Crossref PubMed Scopus (606) Google Scholar). However, Acsl1 mRNA expression is up-regulated in liver and adipose tissue by activators of peroxisome proliferator-activated factor α (PPARα) (6Schoonjans K. Staels B. Grimaldi P. Auwerx J. Eur. J. Biochem. 1993; 216: 615-622Crossref PubMed Scopus (105) Google Scholar, 7Frederiksen K.S. Wulff E.M. Sauerberg P. Mogensen J.P. Jeppesen L. Fleckner J. J. Lipid Res. 2004; 45: 592-601Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) via a PPAR response element in the promoter region of Acsl1 (8Schoonjans K. Watanabe M. Suzuki H. Mahfoudi A. Krey G. Wahli W. Grimaldi P. Staels B. Yamamoto T. Auwerx J. J. Biol. Chem. 1995; 270: 19269-19276Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar), suggesting a possible function related to the β-oxidation of fatty acids. Moreover, overexpression of ACSL1 in rat primary hepatocytes increases oleate incorporation into diacylglycerol (DAG) but does not increase TAG mass (9Li 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 (81) Google Scholar). Thus, the exact role of ACSL1 in providing acyl-CoA for lipogenesis versus β-oxidation has remained uncertain. long chain acyl-CoA synthetase acylglycerol-3-phosphate acyltransferase analysis of variance acid-soluble metabolite cholesteryl ester diacylglycerol fatty acid fatty acid transport protein glycerol-3-phosphate acyltransferase insulin tolerance test lysophosphatidic acid lysophosphatidylcholine neomycin oral glucose tolerance test phosphatidylcholine phosphatidylethanolamine phosphatidylserine peroxisome proliferator-activated factor triacylglycerol. long chain acyl-CoA synthetase acylglycerol-3-phosphate acyltransferase analysis of variance acid-soluble metabolite cholesteryl ester diacylglycerol fatty acid fatty acid transport protein glycerol-3-phosphate acyltransferase insulin tolerance test lysophosphatidic acid lysophosphatidylcholine neomycin oral glucose tolerance test phosphatidylcholine phosphatidylethanolamine phosphatidylserine peroxisome proliferator-activated factor triacylglycerol. It has been suggested that lipid intermediates, including FAs, long chain acyl-CoAs, DAG, ceramide, and phosphatidic acid, rather than TAG accumulation per se, might underlie the development of insulin resistance (10Savage D.B. Petersen K.F. Shulman G.I. Physiol. Rev. 2007; 87: 507-520Crossref PubMed Scopus (743) Google Scholar, 11Postic C. Girard J. J. Clin. Invest. 2008; 118: 829-838Crossref PubMed Scopus (878) Google Scholar, 12Wymann M.P. Schneiter R. Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (921) Google Scholar, 13Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2144) Google Scholar). For example, long chain acyl-CoAs directly affect glucose metabolism by allosterically inhibiting glycogen synthase, pyruvate dehydrogenase, glucose-6-phosphatase, and glucokinase (14Faergeman N.J. Knudsen J. Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (576) Google Scholar, 15Thompson A.L. Cooney G.J. Diabetes. 2000; 49: 1761-1765Crossref PubMed Scopus (109) Google Scholar); and FAs or acyl-CoAs may also be ligands for hepatocyte nuclear factor 4α, a transcription factor that regulates aspects of lipoprotein and glucose metabolism (16Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Further, DAG activates protein kinase C isoforms that can phosphorylate insulin receptor substrates on serine and threonine residues and thereby impair insulin signaling (10Savage D.B. Petersen K.F. Shulman G.I. Physiol. Rev. 2007; 87: 507-520Crossref PubMed Scopus (743) Google Scholar). However, alterations of acyl-CoAs do not always correlate with changes in insulin sensitivity (17Neschen S. Morino K. Hammond L.E. Zhang D. Liu Z.X. Romanelli A.J. Cline G.W. Pongratz R.L. Zhang X.M. Choi C.S. Coleman R.A. Shulman G.I. Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Although acyl-CoA content in muscle has been associated with diminished insulin sensitivity (18Timmers S. Schrauwen P. de Vogel J. Physiol. Behav. 2008; 94: 242-251Crossref PubMed Scopus (108) Google Scholar), in liver the relationship between acyl-CoAs and insulin sensitivity is less clear. For example, when lipoprotein lipase is overexpressed and FA flux into the liver from lipoprotein particles is increased, hepatic insulin resistance is associated with an increased hepatic content of TAG and acyl-CoA (19Kim J.K. Fillmore J.J. Chen Y. Yu C. Moore I.K. Pypaert M. Lutz E.P. Kako Y. Velez-Carrasco W. Goldberg I.J. Breslow J.L. Shulman G.I. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7522-7527Crossref PubMed Scopus (579) Google Scholar). However, when DAG acyltransferase-2 is overexpressed in liver, the mice retain normal hepatic and whole body insulin sensitivity despite hepatic steatosis and an elevated acyl-CoA content (20Monetti M. Levin M.C. Watt M.J. Sajan M.P. Marmor S. Hubbard B.K. Stevens R.D. Bain J.R. Newgard C.B. Farese Sr., R.V. Hevener A.L. Farese Jr., R.V. Cell Metab. 2007; 6: 69-78Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Similarly, the absence of glycerol-3-phosphate acyltransferase 1 (Gpat1−/−) led to protection against hepatic insulin resistance despite a 64% increase in hepatic acyl-CoA content, arguing against the importance of long chain acyl-CoAs in promoting insulin resistance (17Neschen S. Morino K. Hammond L.E. Zhang D. Liu Z.X. Romanelli A.J. Cline G.W. Pongratz R.L. Zhang X.M. Choi C.S. Coleman R.A. Shulman G.I. Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Although a higher content of acyl-CoAs might contribute to insulin resistance in some models, it is not known whether protection against insulin resistance might be achieved by decreasing hepatic acyl-CoA content. To understand the function of ACSL1 in liver, we constructed a mouse model with a liver-specific knock-out of ACSL1 (Acsl1L−/−). Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25–35% decrease in long chain acyl-CoA content. This model has allowed us to determine whether ACSL1 deficiency protects liver from hepatic steatosis and alters the incorporation of FAs into specific glycerolipids and to learn whether a decrease in long chain acyl-CoA content is sufficient to protect the liver from high fat diet-induced insulin resistance. Liver-specific ACSL1 knock-out mice (Acsl1L−/−) were created by LoxP-Cre strategy (21Kos C.H. Nutr. Rev. 2004; 62: 243-246PubMed Google Scholar). The gene-targeting vector was designed to produce a floxed Acsl1 exon 2 (Fig. 1A) and was constructed in a standard plasmid backbone containing neomycin phosphotransferase (neo) and thymidine kinase cassettes for positive and negative selection, respectively. The 5′ arm of homology was 4.5 kb in length and was derived from intron 1, whereas the 3′ arm was 3.8 kb in length and derived from a portion of intron 2, exon 3, and a portion of intron 3. Between the arms, a floxed exon 2 was cloned. The targeting vector was electroporated into E14Tg2A (E14) embryonic stem cells, and the cells were grown in medium supplemented with G418 and gancyclovir. Targeted cells were identified by PCR across both the 3′ and 5′ arms of homology using primers specific to neo and primers flanking the arms of homology. Neo was then excised from the targeted allele via transient expression of flpE recombinase (Fig. 1B). Targeted, neo-cells were microinjected into blastocysts derived from mouse strain C57BL/6 to produce transmitting chimeras. Twenty-one high quality chimeras were produced that transmitted the targeted allele. Duplex PCR was performed to distinguish wild-type and Flox alleles with wild-type specific primers (forward, 5′-AGCAAGCCACATGAAGGCATGTGTG-3′ and reverse, 5′-AAGTGGGGGACATAGGTGCCACT-3′) and LoxP-specific primers (forward, 5′-TAGAAAGTATAGGAACTTCGGCGCG-3′ and reverse, 5′-GCCCCTATATCACTTTTGGCGACA-3′). Mice heterozygous for the targeted allele (Acsl1Flox/+) were identified and back-crossed six times to C57BL/6 mice. The established Acsl1Flox/+ mice were then crossed with rat albumin promoter-Cre transgenic mice (Alb-CreTg/0), which express Cre recombinase exclusively in postpartum liver (22Postic C. Magnuson M.A. Genesis. 2000; 26: 149-150Crossref PubMed Scopus (306) Google Scholar). The resulting double heterozygous mice carrying one floxed allele and Alb-Cre recombinase (Acsl1Flox/+-Alb-CreTg/0, i.e. ACSL1 heterozygote) were interbred to yield Acsl1L−/− mice (Acsl1Flox/Flox-Alb-CreTg/0), as well as five different groups of littermate controls (Fig. 1C). The presence or absence of the Alb-Cre transgene was determined by duplex PCR using Cre-specific primers (forward, 5′-GCGGTCTGGCAGTAAAAACTATC-3′ and reverse, 5′-GTGAAACAGCATTGCTGTCACTT-3′) and IL-2 as internal control for amplification (forward, 5′-CATGGCCACAGAATTGAAAGATCT-3′ and reverse, 5′-GTAGGTGGAAATTCTAGCATCATCC-3′) (Fig. 1C). To confirm the knock-out of exon 2 in Acsl1L−/− mice, total RNA was extracted (Qiagen RNeasy Mini kit) from Acsl1L−/− mice and control mice, and cDNA was synthesized by reverse transcription (Applied Biosystems). PCR was conducted with forward primer 5′-GCGGAGGAGAATTCTGCATAGAGAA-3′ and reverse primer 5′-ATATCAGCACATCATCTGTGGAAG-3′. PCR amplification of the region between coding exon 1 and exon 10 yielded a band of ∼1 kb in control mice and a smaller band of ∼900 bp in Acsl1L−/− mice (Fig. 1D). DNA sequencing (University of North Carolina Genome Analysis Facility) of the purified bands (QIAquick gel extraction kit) showed that in Acsl1L−/− mice, exon 1 was followed immediately by exon 3, proving that the shorter PCR product in Acsl1L−/− mice was due to the missing exon 2 (118 bp). The absence of exon 2 causes a frameshift and introduces a stop codon (TAG) immediately after exon 1, resulting a truncated peptide of 70 amino acids in the Acsl1L−/− mice compared with a 699-amino acid protein in the control mice (sequence not shown). Male mice (age 12–17 weeks) were fed a standard diet (Prolab Isopro® RMH 3000, LabDiet) and killed after either a 4-h or a 24-h fast unless otherwise indicated. Plasma was collected from the retroorbital sinus. Liver, heart, and adipose tissues were snap frozen in liquid nitrogen and stored at −80 °C. For high fat diet studies, 8-week-old Acsl1L−/− and control (Acsl1Flox/Flox) mice were fed a high fat Western diet (45% calories from fat (mostly lard), 35% from carbohydrate (sucrose and corn starch) Research Diets D12451) or an isocaloric standard control diet (10% calories from fat (lard and soybean oil), 70% from carbohydrate (sucrose and corn starch) Research Diets D12450B) for 14–18 weeks. Mice were weighed weekly. Body composition was determined by magnetic resonance imaging. At ∼25 weeks, mice were killed, and tissues were snap frozen in liquid nitrogen and analyzed for enzyme activity, quantitative real time-PCR, Western blotting, and lipid content. Primary hepatocytes were obtained from fed 12-week-old male Acsl1Flox/Flox and Acsl1L−/− mice, by in situ liver perfusion using a modification of the collagenase method (23Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-520Crossref PubMed Scopus (3601) Google Scholar). Briefly, the livers were first perfused with a Ca2+-free perfusion buffer (10 mm HEPES, 132 mm NaCl, 20 mm dextrose, 6.7 mm KCl, 1 mm adenosine, 1.5 mm NaOH, 0.1 mm EGTA, 0.02 unit/ml insulin) and then with Ca2+-containing collagenase buffer (0.3 mg/ml; type CLS I, 230 units/ml; Worthington, Lakewood, NJ). Hepatocytes were collected after low speed centrifugation and seeded (1.0 × 106 cells/60-mm dishes) onto collagen-coated plates as described previously (9Li 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 (81) Google Scholar). The next day, hepatocytes were labeled with 2 ml of medium containing 0.5 μCi of [1-14C]oleate (final concentration, 500 μm oleate) for 3 h and then collected for cellular lipid extraction (24Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar) and medium acid-soluble metabolites (ASMs) as a measure of incomplete FA oxidation (9Li 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 (81) Google Scholar). Lipid extracts were separated by thin layer chromatography in hexane:ethyl ether:acetic acid (80:20:1, v/v). The 14C-labeled lipids were detected and quantified with a Bioscan 200 imaging system. Oral glucose tolerance tests (OGTTs) and insulin tolerance tests (ITTs) were performed in 5-month-old mice (after 3 months of high fat or control diet feeding). Mice were fasted for 4 h after the start of the light cycle and gavaged with glucose (2.5 g/kg) for OGTTs or injected intraperitoneally with insulin (0.75 unit/kg) for ITTs. Blood glucose was determined before and 15, 30, 60, 90, and 120 min after the glucose or insulin load with a glucometer (LifeScan, Inc.) (25Buhl E.S. Jessen N. Pold R. Ledet T. Flyvbjerg A. Pedersen S.B. Pedersen O. Schmitz O. Lund S. Diabetes. 2002; 51: 2199-2206Crossref PubMed Scopus (205) Google Scholar). In 11–13-week-old mice that had been fed a high fat (safflower oil) diet for the preceding 3 weeks, clamp studies were performed as described previously (17Neschen S. Morino K. Hammond L.E. Zhang D. Liu Z.X. Romanelli A.J. Cline G.W. Pongratz R.L. Zhang X.M. Choi C.S. Coleman R.A. Shulman G.I. Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) with a prime-continuous [3-3H]glucose infusion 10-μCi bolus, 0.1 μCi/min) to determine rates of whole body glucose turnover followed by a primed-continuous insulin infusion (2.5 milliunits/kg/min; Humulin, Eli Lilly, Indianapolis, IN) to raise insulin levels within a physiologic range. A single 2-deoxy-d-[1-14C]glucose injection was administered at 75 min. Male mice (∼5 months old) were fasted overnight (16 h) and anesthetized with Avertin. Insulin (1 unit/kg of body weight) or phosphate-buffered saline was injected via the portal vein. Two min later, the left liver lobe was snap frozen in liquid nitrogen. Liver tissue was pulverized and homogenized on ice in a HEPES buffer (pH 7.4) containing 1% Nonidet P-40, 100 mm NaCl, 2% glycerol, 5 mm NaF, 1 mm EDTA, and proteinase inhibitor mixture and phosphatase inhibitor cocktails I and II (Sigma). Homogenates were centrifuged at 16,000 × g for 30 min at 4 °C, and supernatants were used for Western blotting with antibody against phosphorylated AKT. ACSL1 expression was determined in liver, gonadal adipose tissue, and heart using a polyclonal peptide antibody (a gift from Cell Signaling, 4047). Homogenates from liver and heart or total particulate from gonadal adipose tissue was separated by electrophoresis on an 10% polyacrylamide gel. Phosphorylation of AKT (Ser-473) was determined in the liver supernatant after injection of insulin through the portal vein. Anti-phospho-AKT (Ser-473) and anti-AKT were from Cell Signaling. Adobe Photoshop software was used for densitometry analysis. Initial rates of total ACSL activity in liver homogenates were measured with 2–4 μg of liver homogenate at 37 °C in the presence of 175 mm Tris (pH 7.4), 8 mm MgCl2, 5 mm dithiothreitol, 10 mm ATP, 250 μm CoA, 50 μm [1-14C]palmitic acid in 500 μm Triton X-100, and 10 μm EDTA in a total volume of 200 μl (9Li 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 (81) Google Scholar). GPAT specific activity was assayed with 20–40 μg of liver homogenate at room temperature in a 200-μl reaction mixture containing 75 mm Tris-HCl (pH 7.5), 4 mm MgCl2, 1 mg/ml bovine serum albumin (essentially FA-free), 1 mm dithiothreitol, 8 mm NaF, 800 μm [3H]glycerol 3-phosphate, and 80 μm palmitoyl-CoA (26Coleman R.A. Haynes E.B. J. Biol. Chem. 1983; 258: 450-456Abstract Full Text PDF PubMed Google Scholar). AGPAT activity was determined by measuring the conversion of [3H]lysophosphatidic acid (LPA) to [3H]phosphatidic acid in a 200-μl reaction mixture containing 100 mm Tris-HCl (pH 7.4), 10 μm oleoyl-LPA, 50 μm oleoyl-CoA, 0.25 μCi of [3H]oleoyl-LPA, and 1 mg/ml bovine serum albumin (essentially FA-free) (27Agarwal A.K. Barnes R.I. Garg A. Arch. Biochem. Biophys. 2006; 449: 64-76Crossref PubMed Scopus (51) Google Scholar). The reaction was started by adding 0.2–0.8 μg of liver total particulate followed by incubation for 6 min at 37 °C. Lipids were extracted by a modified Folch method (28Samuel V.T. Liu Z.X. Qu X. Elder B.D. Bilz S. Befroy D. Romanelli A.J. Shulman G.I. J. Biol. Chem. 2004; 279: 32345-32353Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar, 29Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), and liver TAG (Stanbio) and total cholesterol (Wako) mass were determined enzymatically. To determine hepatic lipid metabolite profiles, liver (100 mg) was homogenized with appropriate internal standards in specific organic reagents (17Neschen S. Morino K. Hammond L.E. Zhang D. Liu Z.X. Romanelli A.J. Cline G.W. Pongratz R.L. Zhang X.M. Choi C.S. Coleman R.A. Shulman G.I. Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). After separation, purification, and elution, lipid metabolite extracts were separated by high performance liquid chromatography, and individual and total lipid species were analyzed by liquid chromatography/tandem mass spectrometry (17Neschen S. Morino K. Hammond L.E. Zhang D. Liu Z.X. Romanelli A.J. Cline G.W. Pongratz R.L. Zhang X.M. Choi C.S. Coleman R.A. Shulman G.I. Cell Metab. 2005; 2: 55-65Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Liver glycogen was extracted and digested with amyloglucosidase for 30 min at 37 °C (30Suzuki Y. Lanner C. Kim J.H. Vilardo P.G. Zhang H. Yang J. Cooper L.D. Steele M. Kennedy A. Bock C.B. Scrimgeour A. Lawrence Jr., J.C. DePaoli-Roach A.A. Mol. Cell Biol. 2001; 21: 2683-2694Crossref PubMed Scopus (129) Google Scholar). Glucose concentration was determined using the Glucose Autokit CII assay (Wako). For acylcarnitine species, male mice fed a standard diet were fasted 48 h before livers were collected. Specimens of powdered liver were homogenized in deionized water and extracted, and acylcarnitine species were measured by direct injection electrospray tandem mass spectrometry (31An J. Muoio D.M. Shiota M. Fujimoto Y. Cline G.W. Shulman G.I. Koves T.R. Stevens R. Millington D. Newgard C.B. Nat. Med. 2004; 10: 268-274Crossref PubMed Scopus (357) Google Scholar). For plasma chemistries and lipids, after Avertin anesthesia, blood was obtained from the tail vein or the retroorbital venous plexus. Plasma glucose, TAG, total cholesterol, free FA, β-hydroxybutyrate (Wako, Stanbio) and insulin (Roche Applied Science) were determined with commercially available kits (32Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Crossref PubMed Scopus (159) Google Scholar). Total liver RNA was isolated using the RNeasy Plus Mini kit (Qiagen), and 20 μg of total RNA was reverse transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative PCR was performed with the iCycler Thermal Cycler instrument (Bio-Rad) in 25 μl using SYBR Green fluorescein reagent mix and 10 nm fluorescein as the calibration dye (Applied Biosystems). Each reaction contained cDNA derived from 20 ng of total RNA. Oligonucleotide primers were designed using the Primer-BLAST program (National Center for Biotechnology Information). Sequences of primers are listed in supplemental Table 1. The fold change in expression of the target genes was normalized to the endogenous control (β-actin) and was calculated relative to the control group using the 2−ΔΔCT method (33Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar). Values are expressed as means ± S.E. Statistical comparisons between control and ACSL1 knock-out mice were determined using an unpaired, two-tailed Student's t test. Statistically significant effects of diet and genotype were identified using a two-way analysis of variance (ANOVA) with interaction. A p value < 0.05 was considered significant unless otherwise indicated. The generation of targeting vector, embryonic stem cells containing floxed Acsl1 allele, and chimeras was described under "Experimental Procedures." Acsl1L−/− mice (Acsl1Flox/Flox-Alb-CreTg/0) were produced by interbreeding double heterozygotes (Fig. 1C). For the experiments described, Acsl1L−/− were crossed with Acsl1Flox/Flox littermates lacking the Alb-Cre transgene (Acsl1Flox/Flox-Alb-Cre0/0) to yield Acsl1L−/− mice and Acsl1Flox/Flox mice. These two genotypes were born at the expected frequency (1:1), indicating that the combination of the floxed allele and the Cre transgene did not affect reproduction. Acsl1Flox/Flox mice were used as controls because pilot studies showed that neither the loxP sites nor the presence of albumin-Cre altered ACSL1 expression or phenotype (data not shown). Acsl1 mRNA was virtually absent in Acsl1L−/− liver, confirming the ACSL1 deletion (Fig. 2A). No difference was observed for the mRNA from the other two major ACSL isoforms in liver, Acsl4 and Acsl5. The increase in the mRNA amount of the less abundant ACSL isoform, Acsl3, was not significant. The mRNA abundance for fatty acid transport proteins FATP2, 4, and 5, which have very long chain acyl-CoA synthetase activity, also remained unchanged in Acsl1L−/− liver. Using a rabbit peptide antibody against human ACSL1, we detected a ∼75 kDa band in liver from control liver, but not in liver from Acsl1L−/− mice, whereas the band was present in gonadal adipose tissue and heart from both control and knock-out mice, indicating that the deletion of ACSL1 was specific to liver (Fig. 2B). Consistent with the quantitative real time-PCR and Western blotting results, total ACSL activity in liver homogenates was 56 and 47% lower than in control male and female mice, respectively (Fig. 2C). This decrease in ACSL activity indicates that ACSL1 contributes approximately 50% of total ACSL activity in liver and that other ACSL isoforms did not compensate for the loss. The remaining ACSL activity probably represents activity from other ACSL and FATP isoforms present in liver. At 12–14 weeks, Acsl1L−/− mice showed no significant difference from control mice for body weight, organ weights, adiposity, or plasma metabolites (supplemental Table 2). Because it has been reported that ACSL1 is up-regulated by PPARα, a transcription factor that increases FA β-oxidation (6Schoonjans K. Staels B. Grimaldi P. Auwerx J. Eur. J. Biochem. 1993; 216: 615-622Crossref PubMed Scopus (105) Google Scholar, 7Frederiksen K.S. Wulff E.M. Sauerberg P. Mogensen J.P. Jeppesen L. Fleckner J. J. Lipid Res. 2004; 45: 592-601Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), we compared Acsl1L−/− and control mice after a 24-h fast. After a 24-h fast, both knock-out and control mice lost approximately 3 g of body weight and had similar changes in blood glucose, insulin, TAG, total cholesterol, ketone bodies, and hepatic glycogen (supplemental Table 3). No difference was observed when the 12–14-week-old mice were subjected to OGTTs and ITTs (data not shown), sugges
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