PNPLA3 mediates hepatocyte triacylglycerol remodeling
2014; Elsevier BV; Volume: 55; Issue: 4 Linguagem: Inglês
10.1194/jlr.m046607
ISSN1539-7262
AutoresHanna Ruhanen, Julia Perttilä, Maarit Hölttä‐Vuori, You Zhou, Hannele Yki‐Järvinen, Elina Ikonen, Reijo Käkelä, Vesa M. Olkkonen,
Tópico(s)Diet, Metabolism, and Disease
ResumoThe I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3WT) and PNPLA3I148M on HuH7 cell lipidome after [13C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [13C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3WT and PNPLA3I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3WT associated in PCA with newly synthesized [13C]TAGs, particularly 52:1 and 50:1, while PNPLA3I148M associated with similar preexisting TAGs. PNPLA3WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3I148M. PNPLA3I148M localized more extensively to lipid droplets (LDs) than PNPLA3WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3I148M. The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3WT) and PNPLA3I148M on HuH7 cell lipidome after [13C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [13C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3WT and PNPLA3I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3WT associated in PCA with newly synthesized [13C]TAGs, particularly 52:1 and 50:1, while PNPLA3I148M associated with similar preexisting TAGs. PNPLA3WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3I148M. PNPLA3I148M localized more extensively to lipid droplets (LDs) than PNPLA3WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3I148M. Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem closely associated with all features of the metabolic syndrome (1–3) Romeo et al. (4Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2212) Google Scholar) first described a single-nucleotide polymorphism (rs738409; C>G/I148M) in the patatin-like phospholipase domain containing 3 (PNPLA3, adiponutrin) gene, to be strongly associated with NAFLD. A meta-analysis of 16 studies demonstrated that homozygous carriers of PNPLA3I148M have on the average a 73% higher liver fat content than weight-matched homozygous carriers of the major allele (5Sookoian S. Pirola C.J. Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.Hepatology. 2011; 53: 1883-1894Crossref PubMed Scopus (668) Google Scholar). However, NAFLD associated with PNPLA3I148M is distinct from obesity-associated common NAFLD, as it is not characterized by features of the metabolic syndrome such as hyperinsulinemia or dyslipidemia (1Cohen J.C. Horton J.D. Hobbs H.H. Human fatty liver disease: old questions and new insights.Science. 2011; 332: 1519-1523Crossref PubMed Scopus (1566) Google Scholar, 5Sookoian S. Pirola C.J. Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.Hepatology. 2011; 53: 1883-1894Crossref PubMed Scopus (668) Google Scholar). In vitro assays using recombinant PNPLA3 have suggested that the WT PNPLA3 (PNPLA3WT) hydrolyzes emulsified triacylglycerol (TAG) and that the I148M substitution in PNPLA3 (PNPLA3I148M) abolishes this activity (6He S. McPhaul C. Li J.Z. Garuti R. Kinch L. Grishin N.V. Cohen J.C. Hobbs H.H. A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.J. Biol. Chem. 2010; 285: 6706-6715Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 7Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. 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Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar), and to prefer oleic acid (18:1n-9) as the fatty acyl moiety (9Huang Y. Cohen J.C. Hobbs H.H. Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.J. Biol. Chem. 2011; 286: 37085-37093Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Opposing a putative role as a lipase, PNPLA3 is induced by glucose and insulin (10Moldes M. Beauregard G. Faraj M. Peretti N. Ducluzeau P.H. Laville M. Rabasa-Lhoret R. Vidal H. Clement K. Adiponutrin gene is regulated by insulin and glucose in human adipose tissue.Eur. J. Endocrinol. 2006; 155: 461-468Crossref PubMed Scopus (48) Google Scholar, 11Rae-Whitcombe S.M. Kennedy D. Voyles M. Thompson M.P. Regulation of the promoter region of the human adiponutrin/PNPLA3 gene by glucose and insulin.Biochem. Biophys. Res. Commun. 2010; 402: 767-772Crossref PubMed Scopus (32) Google Scholar, 12Soronen J. Laurila P.P. Naukkarinen J. Surakka I. Ripatti S. Jauhiainen M. Olkkonen V.M. Yki-Järvinen H. Adipose tissue gene expression analysis reveals changes in inflammatory, mitochondrial respiratory and lipid metabolic pathways in obese insulin-resistant subjects.BMC Med. Genomics. 2012; 5: 9Crossref PubMed Scopus (61) Google Scholar) and is a target gene of the lipogenic transcription factors SREBP-1c and the carbohydrate responsive element binding protein, ChREBP (13Huang Y. He S. Li J.Z. Seo Y.K. Osborne T.F. Cohen J.C. Hobbs H.H. A feed-forward loop amplifies nutritional regulation of PNPLA3.Proc. Natl. Acad. Sci. USA. 2010; 107: 7892-7897Crossref PubMed Scopus (276) Google Scholar, 14Dubuquoy C. Robichon C. Lasnier F. Langlois C. Dugail I. Foufelle F. Girard J. Burnol A.F. Postic C. Moldes M. Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes.J. Hepatol. 2011; 55: 145-153Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Perttilä J. Huaman-Samanez C. Caron S. Tanhuanpää K. Staels B. Yki-Järvinen H. Olkkonen V.M. PNPLA3 is regulated by glucose in human hepatocytes, and its I148M mutant slows down triglyceride hydrolysis.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E1063-E1069Crossref PubMed Scopus (70) Google Scholar, 16Qiao A. Liang J. Ke Y. Li C. Cui Y. Shen L. Zhang H. Cui A. Liu X. Liu C. et al.Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis.Hepatology. 2011; 54: 509-521Crossref PubMed Scopus (62) Google Scholar). Kumari et al. (17Kumari M. Schoiswohl G. Chitraju C. Paar M. Cornaciu I. Rangrez A.Y. Wongsiriroj N. Nagy H.M. Ivanova P.T. Scott S.A. et al.Adiponutrin functions as a nutritionally regulated lysophosphatidic acid acyltransferase.Cell Metab. 2012; 15: 691-702Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) suggested that the protein acts as lipogenic lysophosphatidic acid (LPA) acyltransferase, converting LPA to phosphatidic acid (PA), and that the I148M substitution increases this activity. Because PA acts as a precursor for both phospholipids and TAGs, this provided an alternative explanation for the hepatic fat accumulation in the PNPLA3I148M allele carriers. Even though PNPLA3 knockout mice have no metabolic phenotype (18Basantani M.K. Sitnick M.T. Cai L. Brenner D.S. Gardner N.P. Li J.Z. Schoiswohl G. Yang K. Kumari M. Gross R.W. et al.Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome.J. Lipid Res. 2011; 52: 318-329Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 19Chen W. Chang B. Li L. Chan L. Patatin-like phospholipase domain-containing 3/adiponutrin deficiency in mice is not associated with fatty liver disease.Hepatology. 2010; 52: 1134-1142Crossref PubMed Scopus (180) Google Scholar), Kumashiro et al. (20Kumashiro N. Yoshimura T. Cantley J.L. Majumdar S.K. Guebre-Egziabher F. Kursawe R. Vatner D.F. Fat I. Kahn M. Erion D.M. et al.Role of patatin-like phospholipase domain-containing 3 on lipid-induced hepatic steatosis and insulin resistance in rats.Hepatology. 2013; 57: 1763-1772Crossref PubMed Scopus (69) Google Scholar) reported that reducing PNPLA3 in rat liver via RNA interference prevented hepatic steatosis, an effect attributed to decreased FA esterification. In apparent contradiction with these findings, the study by Pirazzi et al. (21Pirazzi C. Adiels M. Burza M.A. Mancina R.M. Levin M. Stahlman M. Taskinen M.R. Orho-Melander M. Perman J. Pujia A. et al.Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) affects hepatic VLDL secretion in humans and in vitro.J. Hepatol. 2012; 57: 1276-1282Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) in human subjects showed a reduced rate of large TAG-rich VLDL secretion in I148M carriers versus 148II homozygotes for any amount of liver fat, suggesting that PNPLA3I148M represents a loss-of-function mutant that promotes hepatocyte TAG accumulation via reduction of VLDL assembly, putatively by inhibiting the mobilization of TAG FAs. Li et al. (22Li J.Z. Huang Y. Karaman R. Ivanova P.T. Brown H.A. Roddy T. Castro-Perez J. Cohen J.C. Hobbs H.H. Chronic overexpression of PNPLA3I148M in mouse liver causes hepatic steatosis.J. Clin. Invest. 2012; 122: 4130-4144Crossref PubMed Scopus (193) Google Scholar) recently developed transgenic mice overexpressing WT human PNPLA3 or the I148M variant either in liver or adipose tissue. Expression of PNPLA3I148M, but not the WT protein, in the liver recapitulated the fatty liver phenotype. The above-mentioned metabolic studies suggested that the increase in hepatic TAGs associated with the I148M allele results from multiple changes in hepatic TAG metabolism. Both PNPLA3WT and PNPLA3I148M localize prominently on cytoplasmic lipid droplets (LDs) (6He S. McPhaul C. Li J.Z. Garuti R. Kinch L. Grishin N.V. Cohen J.C. Hobbs H.H. A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.J. Biol. Chem. 2010; 285: 6706-6715Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 23Chamoun Z. Vacca F. Parton R.G. Gruenberg J. PNPLA3/adiponutrin functions in lipid droplet formation.Biol. Cell. 2013; 105: 219-233Crossref PubMed Scopus (72) Google Scholar). Thus, the mechanism by which PNPLA3I148M causes NAFLD is still controversial. In the present study, we examined the mechanisms by which PNPLA3 and its I148M variant modify the lipid content and composition of human hepatic cells in vitro. We acutely overexpressed PNPLA3WT or PNPLA3I148M in the HuH7 hepatoma cell line, which expresses no detectable endogenous PNPLA3, cultured the cells under stable isotope labeling of TAGs with [13C]glycerol, and performed turnover experiments with oleic acid (18:1n-9) labeled with 17 deuterium atoms ([D17]18:1n-9). Cellular TAGs, diacylglycerols (DAGs), and phospholipids were examined by MS. Further evidence of the functional difference between PNPLA3WT and PNPLA3I148M was obtained by comparing their subcellular distributions in the presence or absence of FA supplementation. HuH7 cells (24Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar) were grown in MEM (GIBCO/Life Technologies, Carlsbad, CA), containing 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The PNPLA3 cDNA open-reading frame corresponding to NM_025225 was isolated by PCR from human subcutaneous adipose tissue and inserted into the EcoRI site of pcDNA4HisMaxC (Invitrogen/Life Technologies) and pEGFP-C2 (Clontech/Takara Bio, Mountain View, CA). The I148M substitution corresponding to the rs738409 G allele was introduced in the PNPLA3 cDNA with the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Rabbit anti-PNPLA3, anti-β-actin (Sigma-Aldrich, St. Louis, MO), and anti-green fluorescent protein (GFP) (Life Technologies, Grand Island, NY) were employed for detection of Western blots. For analysis of preexisting and newly synthesized glycerolipids, HuH7 cells cultured on 6-well plates were transfected with an empty vector, PNPLA3WT, or PNPLA3I148M-pcDNAHisMaxC using Lipofectamine 2000TM (Invitrogen). One day after transfection medium was changed to a labeling medium containing 500 μg/ml of [13C]glycerol (Cambridge Isotope Laboratories, Andover, MA), incubated for 24 h, washed with PBS, and scraped into 1 ml of ice-cold 0.25 M sucrose. An aliquot of 100 μl was withdrawn for total cell protein analysis using the BCA assay (Thermo Fisher Scientific, Waltham, MA). The data for glycerolipid synthesis represents seven parallel cell cultures originating from two independent labeling experiments [the TAG and phosphatidylcholine (PC) species profiles in both experiments were in general the same and are shown in detail for the first experiment, supplementary Tables I, II]. For analysis of TAG hydrolysis, the cells were cultured on 6 cm dishes and transfected as described above, washed, and changed into growth medium supplemented with 5% delipidated FBS and 5 μM triacsin C (Enzo Life Sciences, Farmingdale, NY). After 0, 6, or 24 h chase times in this medium, the cells were washed with PBS and scraped into 1 ml of ice-cold 0.25 M sucrose. For analysis of TAG turnover, HuH7 cells in complete growth medium without antibiotics were transfected for 24 h as above and then labeled for 24, 48, or 72 h with 15 μg/ml [D17]18:1n-9 (71-1851; Larodan Fine Chemicals, Malmö, Sweden) using fat-free BSA as a vehicle. The lipids were extracted, and the unlabeled and labeled TAG species detected by MS as detailed below. Cellular lipids were extracted according to Folch et al. (25Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and dissolved in chloroform:methanol 1:2. Immediately before MS, 1% NH4OH was added along with an internal standard mixture containing polar and neutral lipid species. The samples were infused into an ion trap ESI mass spectrometer (Esquire-LC, Bruker-Franzen Analytik, Bremen, Germany) and spectra recorded by employing both positive and negative ionization mode in the range of m/z 500–1,000 (26Käkelä R. Somerharju P. Tyynelä J. Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry.J. Neurochem. 2003; 84: 1051-1065Crossref PubMed Scopus (91) Google Scholar). The unlabeled [M] and [13C]glycerol-labeled [M+3] ions were resolved by comparing the spectra of cell samples with or without labeling, and deconvolution into different peaks was performed by the best profile fitting available in LIMSA software (27Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (166) Google Scholar). In addition, FA composition of total lipids was determined by GC as detailed in Käkelä et al. (28Käkelä R. Käkelä A. Kahle S. Becker P.H. Kelly A. Furness R. Fatty acid signatures in plasma of captive herring gulls as indicators of demersal or pelagic fish diet.Mar. Ecol. Prog. Ser. 2005; 293: 191-200Crossref Scopus (58) Google Scholar). In order to confirm glycerolipid species structures, the precursor scans for the main acyl chains detected by GC were later recorded in ESI-MS/MS experiments for TAGs (positive mode neutral loss scans for the FAs) and PCs (negative mode precursor scans for the FAs of PC formate adducts) using a triple quadrupole mass spectrometer (Agilent 6490 Triple Quad LC/MS with iFunnel Technology; Agilent Technologies, Santa Clara, CA). The [D17]18:1n-9 incorporation into TAG and the following turnover were studied using the triple quadrupole equipment by scanning for the positive mode neutral loss of the unlabeled and deuterated 18:1n-9 moiety. For analysis of DAGs and PAs by ion trap ESI-MS, cellular lipids were extracted using a modified Bligh/Dyer procedure (29Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42681) Google Scholar). First, 800 μl of ice-cold 0.1 N HCl:methanol (1:1) was added to each cell pellet and DAG 24:0 was inserted as an internal standard. Then 400 μl of ice-cold chloroform was added and the separated lower phase was collected, evaporated to dryness under nitrogen flow, and finally the original volume was restored by adding chloroform:methanol (1:9). The mass spectra for the DAG species were recorded in the positive ionization mode over the range of m/z 400–750. The spectra for PA species were recorded over the range of m/z 600–800 by using samples brought into chloroform:methanol (1:2) and using PA 34:0 as internal standard. The ion trap ESI-MS spectra were processed by Bruker Daltonics (Billerica, MA) data analysis software and the triple quadrupole ESI-MS/MS spectra by Agilent Mass Hunter software. Individual lipid species were quantified by using the internal standards and the LIMSA software (27Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (166) Google Scholar). To analyze the subcellular distribution of PNPLA3, HuH7 cells were transfected for 48 h with plasmids encoding GFP- PNPLA3WT or GFP-PNPLA3I148M by using Lipofectamine 2000. Prior to fixation with 4% paraformaldehyde, cells were treated for indicated times with 200 μM oleic acid-BSA as described (30Hölttä-Vuori M. Salo V.T. Ohsaki Y. Suster M.L. Ikonen E. Alleviation of seipinopathy-related ER stress by triglyceride storage.Hum. Mol. Genet. 2013; 22: 1157-1166Crossref PubMed Scopus (31) Google Scholar). For LD visualization, cells were stained immediately after fixation with LipidTox Red (Invitrogen/Life Technologies) according to the manufacturer's instructions and imaged with a TCS SP2 confocal microscope (Leica, Wetzlar, Germany). The percentage of cells exhibiting predominantly reticular/cytosolic PNPLA3 distribution was determined by visual inspection of cells from 10 randomly selected fields under a wide-field AX70 microscope (Olympus, Hamburg, Germany). For univariate comparisons of the cellular lipid levels, statistical significance for the differences between control, PNPLA3WT, and PNPLA3I148M overexpressing samples were assessed by using one-way ANOVA followed by Newman-Keuls test of means (SPSS Statistics, IBM, North Castle, NY). For multivariate comparisons of detailed lipid profiles, principal component analysis (PCA) (Sirius, PRS, Bergen, Norway) was used. The PCA describes compositional differences between the samples, and highlights the lipid species mainly responsible for the variation in the data. PCA was computed using arcsine transformed data and the relative positions of the samples and variables were plotted using the first two principal components. In addition, quantitative multivariate measures of the compositional differences among the sample groups were determined by soft independent modeling of class analogy (SIMCA; Sirius) (31Wold, S., Sjöström, M. 1977. SIMCA: a method for analyzing chemical data in terms of similarity and analogy. In Chemometrics: Theory and Application. B. Kowalski, editor. American Chemical Society, Washington, DC. 243–282.Google Scholar). To analyze the rate of TAG remodeling, regression lines of groups were compared by measuring the effect of a categorical factor on the responding variable, by using the ‘aov’ function in the ‘stats’ package of R. The statistical differences in the proportion of cells where PNPLA3 was present in cytosol or endoplasmic reticulum (ER) were tested by Student's t-test. We acutely overexpressed PNPLA3WT or PNPLA3I148M in the hepatoma cell line HuH7, which contains no endogenous PNPLA3 protein (with an apparent molecular mass of 53 kDa) detectable by Western blot analysis (Fig. 1A). After 24 h of transfection, the cells were subjected to 24 h metabolic labeling of glycerolipids with [13C]glycerol, and the cellular lipids were thereafter analyzed by MS. After the [13C]glycerol labeling (total transfection time of 48 h), the cells expressing PNPLA3I148M displayed a marked net increase of unlabeled TAGs compared with mock-transfected controls, while expression of PNPLA3WT had no such effect (Fig. 1B). While the unlabeled TAGs represent lipids that were present in the cells already before the labeling, the [13C]TAGs represent the newly synthesized species. After 48 h of transfection, the concentrations of [13C]TAGs were similar in controls and cells expressing either form of PNPLA3 (Fig. 1C), suggesting that PNPLA3 does not primarily enhance de novo TAG synthesis. Consistent with earlier findings (6He S. McPhaul C. Li J.Z. Garuti R. Kinch L. Grishin N.V. Cohen J.C. Hobbs H.H. A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.J. Biol. Chem. 2010; 285: 6706-6715Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 15Perttilä J. Huaman-Samanez C. Caron S. Tanhuanpää K. Staels B. Yki-Järvinen H. Olkkonen V.M. PNPLA3 is regulated by glucose in human hepatocytes, and its I148M mutant slows down triglyceride hydrolysis.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E1063-E1069Crossref PubMed Scopus (70) Google Scholar), when the cells were incubated for 6 or 24 h in a medium supplemented with 5% delipidated serum and the long chain FA-CoA synthase inhibitor Triacsin C, PNPLA3I148M caused a kinetic delay in TAG hydrolysis (data not shown). Thus, PNPLA3I148M expression results in net TAG accumulation in the absence of altered synthesis of TAGs, but with a delay in their hydrolysis. To analyze in detail the effect of PNPLA3 on the hepatocellular glycerolipid dynamics, we studied the lipid species profiles of the [13C]glycerol-labeled cells (values for the glycerolipid species from the experiment described in Fig. 2 are listed in supplementary Tables I–IV). According to PCA, the TAG profiles of both PNPLA3WT and PNPLA3I148M cells were found to differ significantly from those of the controls (Fig. 2A). The most important principal component extracted (PC1) explained 35% of the TAG variation between the constructs and reflected the degree of FA unsaturation. Cells expressing either form of PNPLA3 were characterized by increased relative amounts of TAGs with saturated FA (SFA) and MUFA moieties. The WT and I148M cells differed with respect to principal component 2 (PC2) (explaining 23% of the variation), a component influenced by the presence/absence of [13C]glycerol in certain TAG species. PNPLA3WT cell TAGs (high position on PC2 axis) contained, e.g., more newly synthesized [13C]52:1 and [13C]50:1 species (52:1H and 50:1H in Fig. 2A) and less of their unlabeled counterparts than the I148M cells (low on PC2 axis). This observation is consistent with a more active remodeling of these TAGs in cells expressing the PNPLA3WT as compared with the PNPLA3I148M cells. The differences between the cells (control, WT, and I148M) seen on the PCA biplot were studied quantitatively by the SIMCA method, which confirmed that the TAG profiles of the three transfected cell pools significantly differed from each other (P < 0.05, test graphics not shown). When applied for DAG species, the PCA followed by SIMCA (P < 0.05) showed that the profile of PNPLA3I148M-expressing cells was significantly different from those of the controls and the PNPLA3WT cells: the I148M cells were enriched with species containing SFAs or MUFAs. PNPLA3WT cells localized in the PCA between the PNPLA3I148M and control cells, and did not differ in SIMCA from the latter (Fig. 2B). The total cellular DAG content was unaffected by PNPLA3WT/PNPLA3I148M expression. Analysis of the species profiles of cellular PCs, the major membrane phospholipid class of mammalian hepatocytes, showed a significant shift (SIMCA, P < 0.05) toward species carrying PUFAs in both PNPLA3WT- and PNPLA3I148M-transfected cells, especially arachidonic acid (20:4n-6) (e.g., 36:4, 38:5, and 38:4), while the total cellular PC content did not differ significantly between the control and PNPLA3WT/PNPLA3I148M-transfected cells (Fig. 2C). Regarding the composition of PC species, cells transfected with either of the PNPLA3 constructs differed significantly (SIMCA, P < 0.05) from the controls, and the PNPLA3WT induced a more pronounced shift in the PC species composition than PNPLA3I148M (Fig. 2C). This result, in the face of TAGs enriched with SFAs and MUFAs, suggests net transfer of PUFAs from TAGs to membrane phospholipids by the protein. The same phenomenon was seen in two independent experiments (data of the repeat experiment not shown). In extended cultures beyond 72 h, the FA composition of the PNPLA3I148M-expressing cells was also slightly altered. The relative amounts of MUFAs 18:1n-7 and 16:1n-7 (vaccenic and palmitoleic acids, respectively) were significantly increased as compared with the controls or PNPLA3WT-transfected cells (ANOVA, P < 0.05; supplementary Table V). Prompted by the report of Kumari et al. (17Kumari M. Schoiswohl G. Chitraju C. Paar M. Cornaciu I. Rangrez A.Y. Wongsiriroj N. Nagy H.M. Ivanova P.T. Scott S.A. et al.Adiponutrin functions as a nutritionally regulated lysophosphatidic acid acyltransferase.Cell Metab. 2012; 15: 691-702Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) suggesting that PNPLA3 displays LPA acyltransferase activity, we analyzed the PA contents and species profiles in the transfected HuH7 cells. The PA patterns of PNPLA3WT/PNPLA3I148M-expressing cells or the total cellular amount of PAs did not differ significantly from the controls (Fig. 2D). To directly assess the possibility that PNPLA3 facilitates the remodeling of hepatocyte TAGs, we carried out labeling of transfected HuH7 cells in serum containing growth medium with stable isotope [D17]18:1n-9, the preferred substrate of PNPLA3 (9Huang Y. Cohen J.C. Hobbs H.H. Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.J. Biol. Chem. 2011; 286: 37085-37093Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), for 24, 48, or 72 h, followed by lipid extraction and mass spectrometric analysis of the TAGs. At the 24 h time point, analysis of the relative amount of [D17]-labeled versus unlabeled 18:1-containing TAGs revealed a significant increase of [D17]18:1n-9 incorporation into TAGs in cells expressing PNPLA3WT, an effect not observed with PNPLA3I148M (Fig. 3). After this time point, the relative amount of [D17]TAGs reduced in PNPLA3WT-expressing cells more rapidly as compared with PNPLA3I148 or the mock-transfected control (P < 0.01), all three groups of transfected cells ending up at the same level at the 72 h time point (Fig. 3). These observations suggest that PNPLA3WT enhances both 18:1n-9 incorporation into TAGs and its removal from these lipids, a remodeling activity defective in PNPLA3I148M. To assess the subcellular distribution and the putative impact of PNPLA3 on LD morphology, we ex
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