ATGL and DGAT1 are involved in the turnover of newly synthesized triacylglycerols in hepatic stellate cells
2016; Elsevier BV; Volume: 57; Issue: 7 Linguagem: Inglês
10.1194/jlr.m066415
ISSN1539-7262
AutoresMaidina Tuohetahuntila, Martijn R. Molenaar, Bart Spee, Jos F. Brouwers, Martin Houweling, Arie B. Vaandrager, J. Bernd Helms,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoHepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation. Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation. Hepatic stellate cells (HSCs) are the main vitamin A (retinol)-storing cells of the body (1Blaner W.S. O'Byrne S.M. Wongsiriroj N. Kluwe J. D'Ambrosio D.M. Jiang H. Schwabe R.F. Hillman E.M. Piantedosi R. Libien J. Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.Biochim. Biophys. Acta. 2009; 1791: 467-473Crossref PubMed Scopus (309) Google Scholar, 2Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol. Rev. 2008; 88: 125-172Crossref PubMed Scopus (2071) Google Scholar). In a healthy liver, HSCs store vitamin A in the form of retinyl esters (REs) in large lipid droplets (LDs), together with triacylglycerols (TAGs) and cholesteryl esters (CEs). HSCs are located in the space of Disse, between the sinusoidal endothelial cells and the hepatocytes. Upon liver injury, quiescent HSCs can transdifferentiate into an activated myofibroblastic phenotype (1Blaner W.S. O'Byrne S.M. Wongsiriroj N. Kluwe J. D'Ambrosio D.M. Jiang H. Schwabe R.F. Hillman E.M. Piantedosi R. Libien J. Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.Biochim. Biophys. Acta. 2009; 1791: 467-473Crossref PubMed Scopus (309) Google Scholar). Activated macrophages, in concert with the HSCs, may initiate this transition by secreting cytokines, such as transforming growth factor β (TGF-β), which stimulate the synthesis of matrix proteins and the release of retinoids by HSCs (1Blaner W.S. O'Byrne S.M. Wongsiriroj N. Kluwe J. D'Ambrosio D.M. Jiang H. Schwabe R.F. Hillman E.M. Piantedosi R. Libien J. Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.Biochim. Biophys. Acta. 2009; 1791: 467-473Crossref PubMed Scopus (309) Google Scholar, 3Pellicoro A. Ramachandran P. Iredale J.P. Fallowfield J.A. Liver fibrosis and repair: immune regulation of wound healing in a solid organ.Nat. Rev. Immunol. 2014; 14: 181-194Crossref PubMed Scopus (856) Google Scholar). The loss of retinoids is associated with a gradual disappearance of the LDs inside the HSCs. We previously reported that LD degradation in activated rat HSCs occurs in two phases (4Testerink N. Ajat M. Houweling M. Brouwers J.F. Pully V.V. van Manen H.J. Otto C. Helms J.B. Vaandrager A.B. Replacement of retinyl esters by polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during activation.PLoS One. 2012; 7: e34945Crossref PubMed Scopus (66) Google Scholar). Upon activation of the HSCs, the LDs reduce in size, but increase in number, during the first 7 days in culture before they disappear in a later phase. Raman and lipidomic studies showed that in the initial phase of HSC activation, the REs disappear rapidly, whereas the TAG content is transiently increased (4Testerink N. Ajat M. Houweling M. Brouwers J.F. Pully V.V. van Manen H.J. Otto C. Helms J.B. Vaandrager A.B. Replacement of retinyl esters by polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during activation.PLoS One. 2012; 7: e34945Crossref PubMed Scopus (66) Google Scholar). Interestingly, this increase in TAGs in rat HSCs is predominantly caused by a large and specific increase in PUFA-containing TAG species during the first 7 days in culture, mediated by an increase in expression of the PUFA-specific FA, long-chain acyl-CoA synthase (ACSL)4, and a decrease in expression of the other ASCLs, especially ASCL1 (5Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar). So far, the molecular mechanisms and identity of the enzymes involved in the observed increase in LD number and their subsequent breakdown during HSC activation are not well understood. An increase in number can be accomplished by the de novo synthesis of new LDs (6Wilfling F. Haas J.T. Walther T.C. Farese Jr, R.V. Lipid droplet biogenesis.Curr. Opin. Cell Biol. 2014; 29: 39-45Crossref PubMed Scopus (270) Google Scholar) or fission of existing large LDs (7Long A.P. Manneschmidt A.K. VerBrugge B. Dortch M.R. Minkin S.C. Prater K.E. Biggerstaff J.P. Dunlap J.R. Dalhaimer P. Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.Traffic. 2012; 13: 705-714Crossref PubMed Scopus (57) Google Scholar). Breakdown of LDs is best characterized in adipose cells, in which key roles are assigned to adipose triglyceride lipase [ATGL, also known as patatin-like phospholipase domain containing (PNPLA)2], its coactivator, CGI-58, and hormone-sensitive lipase (HSL) (8Zechner R. Kienesberger P.C. Haemmerle G. Zimmermann R. Lass A. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores.J. Lipid Res. 2009; 50: 3-21Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The first two proteins are known to have a more general function, as deficiencies in either one lead to neutral lipid storage diseases (9Schweiger M. Lass A. Zimmermann R. Eichmann T.O. Zechner R. Neutral lipid storage disease: genetic disorders caused by mutations in adipose triglyceride lipase/PNPLA2 or CGI-58/ABHD5.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E289-E296Crossref PubMed Scopus (216) Google Scholar). Both ATGL and CGI-58 were present on LDs in the HSC line, HSC-T6 (10Eichmann T.O. Grumet L. Taschler U. Hartler J. Heier C. Woblistin A. Pajed L. Kollroser M. Rechberger G. Thallinger G.G. et al.ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.J. Lipid Res. 2015; 56: 1972-1984Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and rat HSCs were shown to express ATGL, although it was downregulated upon activation (11Mello T. Nakatsuka A. Fears S. Davis W. Tsukamoto H. Bosron W.F. Sanghani S.P. Expression of carboxylesterase and lipase genes in rat liver cell-types.Biochem. Biophys. Res. Commun. 2008; 374: 460-464Crossref PubMed Scopus (36) Google Scholar). In mouse HSCs, lipid breakdown was shown to be partially mediated by a lipophagic pathway, as inhibition of autophagy increased the amount of LDs (12Thoen L.F. Guimaraes E.L. Dolle L. Mannaerts I. Najimi M. Sokal E. van Grunsven L.A. A role for autophagy during hepatic stellate cell activation.J. Hepatol. 2011; 55: 1353-1360Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Hernández-Gea V. Ghiassi-Nejad Z. Rozenfeld R. Gordon R. Fiel M.I. Yue Z. Czaja M.J. Friedman S.L. Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.Gastroenterology. 2012; 142: 938-946Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Because inhibition of autophagy was shown to impair HSC activation in mice and this effect could be partially reversed by addition of exogenous FAs, it was suggested that LD breakdown is required to fulfill the energy demands of HSCs during activation (13Hernández-Gea V. Ghiassi-Nejad Z. Rozenfeld R. Gordon R. Fiel M.I. Yue Z. Czaja M.J. Friedman S.L. Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.Gastroenterology. 2012; 142: 938-946Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). On the other hand, HSC activation was shown to be relatively undisturbed in the absence of REs and LDs in lecithin:retinol acyltransferase (LRAT) knockout mice (14Kluwe J. Wongsiriroj N. Troeger J.S. Gwak G.Y. Dapito D.H. Pradere J.P. Jiang H. Siddiqi M. Piantedosi R. O'Byrne S.M. et al.Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis.Gut. 2011; 60: 1260-1268Crossref PubMed Scopus (98) Google Scholar). In this study, we addressed the question of whether a change in lipid metabolism is causally related to the activation process in rat and mouse HSCs. We identified enzymes involved in LD formation and breakdown in HSCs in vitro and studied the effect of inhibition of these enzymes on HSC activation. D4-palmitate, D8-arachidonate, and Atglistatin were purchased from Cayman Chemical (Ann Arbor, MI). DMEM, FBS, and penicillin/streptomycin were obtained from Gibco (Paisley, UK). BSA fraction V was obtained from PAA (Pasching, Austria). T863 and collagenase (Clostridium histolyticum type I) was obtained from Sigma-Aldrich (St. Louis, MO), and saponin from Riedel-de Haën (Seelze, Germany). The mouse monoclonal antibody against α-smooth muscle actin (α-SMA) was from Thermo Scientific (Waltham, MA). LD staining dye, LD540, was kindly donated by Dr. C. Thiele, Bonn, Germany. Hoechst 33342 was obtained from Molecular Probes (Paisley, UK), paraformaldehyde (PF) (8%) was obtained from Electron Microscopy Sciences (Hatfield, PA). FluorSave was obtained from Calbiochem (Billerica, MA), all HPLC-MS solvents were from Biosolve (Valkenswaard, The Netherlands) with the exception of chloroform (Carl Roth, Karlsruhe, Germany) and were of HPLC grade. Silica-G (0.063–0.200 mm) was purchased from Merck (Darmstadt, Germany). We used 10- to 12-week-old male and female Atgl+/+ (WT) and Atgl−/− mice, generated by crosses of Atgl+/− C57BL/6J mice (15Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1015) Google Scholar) and paired on sex and age from the same nest and adult male Wistar rats (300–400 g). Procedures of mouse and rat care and handling were in accordance with governmental and international guidelines on animal experimentation, and were approved by the Animal Experimentation Committee (Dierexperimentencommissie) of Utrecht University (Dierexperimentencommissie numbers: 2010.III.09.110, 2012.III.10.100, and 2013.III.09.065). Stellate cells were isolated from livers of mice and rats by collagenase digestion followed by differential centrifugation (16Riccalton-Banks L. Bhandari R. Fry J. Shakesheff K.M. A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue.Mol. Cell. Biochem. 2003; 248: 97-102Crossref PubMed Scopus (73) Google Scholar) and cultured, as described previously (5Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar), in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 4 μl/ml Fungizone and cells were maintained in a humidified 5% CO2 incubator at 37°C. Medium was changed every 3 days. Gene-silencing experiments were performed using siRNA for target genes or nontargeting siRNA as a control (ON-TARGETplus SMARTpool of four siRNAs; Thermo-Scientific, Rochester, NY) according to the manufacturer's instructions. Briefly, 2 days after plating, the cells were treated with 40 nM siRNA using 5 μl/ml RNAiMAX (Invitrogen, Breda, The Netherlands) in antibiotic-free complete medium (with FBS). After 6 h of transfection, medium was changed to standard culturing conditions up to day 7. Total RNA was isolated from HSCs grown in a 24-well plate using RNeasy Mini kit (Qiagen, Venlo, The Netherlands) including the optional on-column DNase digestion (Qiagen RNase-free DNase kit). RNA was dissolved in 30 μl of RNase-free water and was quantified spectrophotometrically using a Nanodrop ND-1000 (Isogen Life Science, IJsselstein, The Netherlands). An iScript cDNA synthesis kit (Bio-Rad, Veenendaal, The Netherlands) was used to synthesize cDNA. Primer design and quantitative (q)PCR conditions were as described previously (17van Steenbeek F.G. Van den Bossche L. Grinwis G.C. Kummeling A. van Gils I.H. Koerkamp M.J. van Leenen D. Holstege F.C. Penning L.C. Rothuizen J. et al.Aberrant gene expression in dogs with portosystemic shunts.PLoS One. 2013; 8: e57662Crossref PubMed Scopus (22) Google Scholar). Briefly, qPCR reactions were performed in duplicate using the Bio-Rad detection system. Amplifications were carried out in a volume of 25 μl containing 12.5 μl of 2× SYBR Green Supermix (Bio-Rad), 1 ul of forward and reverse primer, and 1 μl cDNA. Cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 45 cycles of denaturation (95°C for 10 s), annealing temperature (see supplementary Tables 1, 2) for 30 s, and elongation (72°C for 30 s). To determine the relative expression of a gene, a 4-fold dilution series from a pool of all samples of all genes tested was used. The amplification efficiency was between 95 and 105%, amplicon sequencing confirmed the specificity and for each sample a melt-curve analysis was performed. IQ5 real-time PCR detection system software (Bio-Rad) was used for data analysis. Expression levels were normalized by using the average relative amount of the reference genes. Reference genes used for normalization are based on their stable expression in stellate cells, namely, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz), hypoxanthine phosphoribosyl transferase (Hprt), and hydroxylmethylbilane synthase (Hmbs). Primers of reference and target genes are listed in supplementary Tables 1, 2. Freshly isolated HSCs were grown on glass coverslips in 24-well plates at 37°C for 7 days. Staining of cells was performed as follows: cells were fixed in 4% (v/v) PF at room temperature for 30 min and stored in 1% (v/v) PF at 4°C for a maximum of 1 week. HSCs were washed twice in PBS, permeabilized [0.1% (w/v) saponin] and blocked with 2% BSA for 1 h at room temperature. After blocking, slides were incubated 1 h with the primary antibody against α-SMA (50–75 ug/ml), washed again, and incubated for 1 h with anti-mouse antibody (15 ug/ml) supplemented with Hoechst (4 ug/ml) for nuclear counterstaining and LD dye, LD540 (0.05 ug/ml). Thereafter, coverslips were mounted with FluorSave on microscopic slides. Image acquisition was performed on a Leica TCS SPE-II confocal microscope at the Center of Cellular Imaging, Faculty of Veterinary Medicine, Utrecht University. To quantify LD size and numbers per cell, confocal images of LD540 (LDs) and Hoechst33342 (nuclei) were analyzed with CellProfiler v2.1.1. Recognized LDs and nuclei were overlayed on the original image to confirm the identity. The error rate was <5% for LDs and 1,000) and the observation that the various TAG species fragment to a different degree depending on the saturation of their acyl chains (5Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar). Nevertheless, quantitation of a number of representative intact and fragmented TAG ions allowed us to estimate that WT HSCs at day 1 have 10% TAGs with one or more PUFAs, whereas this was approximately 5% in Atgl−/− HSCs. At day 14, the fraction of PUFA-TAGs in WT HSCs was similar as on day 1, but in ATGL−/− cells it was increased to more than 30% (supplementary Figs. 1D, 2). This allowed us to estimate that the PUFA-TAG levels in Atgl−/− HSCs at days 1, 7, and 14 are 0.13, 0.33, and 0.29 nmol/mmol cholesterol, respectively. We previously reported that the increase in PUFA-TAGs in WT HSCs upon culturing in vitro was dependent on a preferential incorporation of exogenous FAs into TAG (4Testerink N. Ajat M. Houweling M. Brouwers J.F. Pully V.V. van Manen H.J. Otto C. Helms J.B. Vaandrager A.B. Replacement of retinyl esters by polyunsaturated triacylglycerol species in lipid droplets of hepatic stellate cells during activation.PLoS One. 2012; 7: e34945Crossref PubMed Scopus (66) Google Scholar, 5Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar). We therefore determined the incorporation and subsequent chase of deuterium-labeled FAs (D4-palmitate and D8-arachidonate) in mouse HSCs. As shown in Fig. 2A, approximately 5% of all the TAG species were labeled with D4-palmitate in either TAG (16:0, 16:0, x) or TAG (16:0, 18:1, x) (sum of labeled species in first and fifth bar). The predominant D4-palmitate-labeled species were the TAGs with two palmitoyl groups, from which more than 50% were labeled (Fig. 2A, first bar). ATGL is involved in the breakdown of this newly formed TAG pool, as the labeled TAG pool was lost at a significantly lower relative rate in Atgl−/− HSCs (half time >2 days) compared with WT cells (half time <1 day; Fig. 2C, gray and dark gray bars). The breakdown of nonlabeled, mainly preexisting, TAGs was not different in the ATGL-deficient cells (Fig. 2C, white bars), assuming an equal resynthesis of unlabeled TAG species during the chase in both mouse types from FAs from the medium. Similar results were observed for the predominant D8-arachidonate-labeled TAG species (Fig. 2B, C), indicating that ATGL is specific for newly formed TAGs, irrespective of the degree of unsaturation of the FA moieties. We previously reported that the enrichment of PUFAs into newly synthesized TAGs is caused by a downregulation of the general ASCL1 isoform concomitant with an upregulation of the PUFA-specific ACSL4 isoform upon rat HSC activation (5Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar). To see whether a similar shift occurred in mouse HSCs to explain the increase in PUFA-TAGs observed in the ATGL-deficient HSCs, we determined the relative mRNA expression of ASCL1 and ASCL4 in mouse HSCs at days 1 and 7. We found that the ratio of the ACSL4 mRNA to ASCL1 mRNA levels increased from 0.8 ± 0.2 at day 1 to 4.6 ± 0.7 at day 7 (n = 6). No difference in increase in the ASCL4/ASCL1 ratio was observed between WT and ATGL−/− HSCs. This demonstrates a clear enrichment of the PUFA-specific ASCL4 isoform upon activation of mouse HSCs. Furthermore, we observed that D8-arachidonate was incorporated at a higher rate into TAG in activated HSCs at day 6 than in relatively quiescent cells at day 1 (Fig. 3B). To investigate which of the two acyl-CoA:DAG acyltransferase (DGAT) enzymes was involved in the formation of the new TAG species in mouse HSCs during activation, we determined the DGAT1 and DGAT2 mRNA expression in mouse HSCs at day 1 and day 7. The mRNA levels of DGAT1 were higher compared with those of DGAT2 at day 1 (relative mRNA levels of 118 ± 26 and 35 ± 36, respectively; n = 6), and the difference between DGAT1 and DGAT2 mRNA expression increased during activation at day 7 (relative mRNA levels of 136 ± 20 and 14 ± 16, respectively; n = 6). To test the role of DGAT1 on the synthesis of new TAGs, we determined the incorporation of deuterium-labeled FAs into TAG in the presence of the DGAT1-specific inhibitor, T863 (21Cao J. Zhou Y. Peng H. Huang X. Stahler S. Suri V. Qadri A. Gareski T. Jones J. Hahm S. et al.Targeting acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases.J. Biol. Chem. 2011; 286: 41838-41851Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). As shown in Fig. 3A, B, the DGAT1 inhibitor inhibited the incorporation of both D4-palmitate and D8-arachidonate into TAG. The DGAT1-specific inhibitor, T863, also caused a clear decrease in the levels of PUFA-TAGs in both WT and Atgl−/− HSCs during activation, but had almost no effect on
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