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Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice

2015; Elsevier BV; Volume: 56; Issue: 4 Linguagem: Inglês

10.1194/jlr.m055962

ISSN

1539-7262

Autores

George G. Schweitzer, Zhouji Chen, Connie Gan, Kyle S. McCommis, Nisreen Soufi, Roman Chrast, Mayurranjan S. Mitra, Kui Yang, Richard W. Gross, Brian N. Finck,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1−/− mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1−/− mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1−/− mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1−/− mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload. Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1−/− mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1−/− mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1−/− mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1−/− mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload. The accumulation of lipid within the liver parenchyma (hepatic steatosis) is common in several hepatic disease states and may be linked to other abnormalities in liver metabolism and function. Indeed, in a subset of individuals, hepatic steatosis can progress to liver injury, dysfunction, and failure. Intrahepatic lipid accumulation is also usually highly correlated with systemic insulin resistance, hyperglycemia, dyslipidemias, and risk of cardiovascular disease. Hepatic steatosis is extremely prevalent in obese individuals, and with the epidemic of obesity, the occurrence of nonalcoholic fatty liver disease has risen dramatically, becoming the most common cause of liver disease in the United States (1Clark J.M. Diehl A.M. Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis.J. 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Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The phosphatidic acid phosphohydrolase (PAP) reaction is not only the penultimate step in TG synthesis, but also a key metabolic branch point. Alterations in PA and DAG concentrations have been linked to regulation of important intracellular signaling cascades including protein kinase C (5Boni L.T. Rando R.R. The nature of protein kinase C activation by physically defined phospholipid vesicles and diacylglycerols.J. Biol. Chem. 1985; 260: 10819-10825Abstract Full Text PDF PubMed Google Scholar, 6Rando R.R. Young N. The stereospecific activation of protein kinase C.Biochem. Biophys. Res. Commun. 1984; 122: 818-823Crossref PubMed Scopus (104) Google Scholar), protein kinase A (7Mitra M.S. Chen Z. Ren H. Harris T.E. Chambers K.T. Hall A.M. Nadra K. Klein S. Chrast R. 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Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 15Kim H.E. Bae E. Jeong D.Y. Kim M.J. Jin W.J. Park S.W. Han G.S. Carman G.M. Koh E. Kim K.S. Lipin1 regulates PPARgamma transcriptional activity.Biochem. J. 2013; 453: 49-60Crossref PubMed Scopus (36) Google Scholar, 16Koh Y.K. Lee M.Y. Kim J.W. Kim M. Moon J.S. Lee Y.J. Ahn Y.H. Kim K.S. Lipin1 is a key factor for the maturation and maintenance of adipocytes in the regulatory network with CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma 2.J. Biol. Chem. 2008; 283: 34896-34906Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and hepatic nuclear receptor 4α (HNF4α) (17Chen Z. Gropler M.C. Mitra M.S. Finck B.N. Complex interplay between the lipin 1 and the hepatocyte nuclear factor 4 alpha (HNF4alpha) pathways to regulate liver lipid metabolism.PLoS ONE. 2012; 7: e51320Crossref PubMed Scopus (31) Google Scholar) nuclear receptors but represses nuclear factor of activated T-cells 4c (18Kim H.B. Kumar A. Wang L. Liu G.H. Keller S.R. Lawrence Jr, J.C. Finck B.N. Harris T.E. Lipin 1 represses NFATc4 transcriptional activity in adipocytes to inhibit secretion of inflammatory factors.Mol. Cell. Biol. 2010; 30: 3126-3139Crossref PubMed Scopus (82) Google Scholar) and the sterol-response element binding protein 1 [SREBP1; (19Peterson T.R. Sengupta S.S. Harris T.E. Carmack A.E. Kang S.A. Balderas E. Guertin D.A. Madden K.L. Carpenter A.E. Finck B.N. et al.mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway.Cell. 2011; 146: 408-420Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar)]. As a general rule, the transcriptional regulatory effects of lipin 1 do not require intact PAP catalytic activity and are mechanistically separable. The outlier to this generalization is repression of SREBP1, which requires both PAP activity and nuclear localization of lipin 1 (19Peterson T.R. Sengupta S.S. Harris T.E. Carmack A.E. Kang S.A. Balderas E. Guertin D.A. Madden K.L. Carpenter A.E. Finck B.N. et al.mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway.Cell. 2011; 146: 408-420Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Thus, lipin 1 is poised to regulate intermediary fat metabolism at multiple regulatory levels. Mice constitutively lacking lipin 1 [fatty liver dystrophic (fld) mice] exhibit very low PAP activity in most tissues, but because lipin 2 is highly expressed in liver, significant hepatic PAP activity can be detected in fld mice (4Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 20Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Indeed, lipin 2 likely encodes a significant proportion of hepatic PAP activity (21Gropler M.C. Harris T.E. Hall A.M. Wolins N.E. Gross R.W. Han X. Chen Z. Finck B.N. Lipin 2 is a liver-enriched phosphatidate phosphohydrolase enzyme that is dynamically regulated by fasting and obesity in mice.J. Biol. Chem. 2009; 284: 6763-6772Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Donkor J. Zhang P. Wong S. O'Loughlin L. Dewald J. Kok B.P. Brindley D.N. Reue K. A conserved serine residue is required for the phosphatidate phosphatase activity but not the transcriptional coactivator functions of lipin-1 and lipin-2.J. Biol. Chem. 2009; 284: 29968-29978Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, hepatic lipin 1 expression is robustly induced by a variety of metabolic stimuli that signal the need for increased PAP activity including fasting (14Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr, J.C. Kelly D.P. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 23Manmontri B. Sariahmetoglu M. Donkor J. Bou Khalil M. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. 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Regulation of hepatic lipin-1 by ethanol: role of AMP-activated protein kinase/sterol regulatory element-binding protein 1 signaling in mice.Hepatology. 2012; 55: 437-446Crossref PubMed Scopus (94) Google Scholar), sterols (25Ishimoto K. Nakamura H. Tachibana K. Yamasaki D. Ota A. Hirano K. Tanaka T. Hamakubo T. Sakai J. Kodama T. et al.Sterol-mediated regulation of human lipin 1 gene expression in hepatoblastoma cells.J. Biol. Chem. 2009; 284: 22195-22205Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and liver regeneration (26Gazit V. Weymann A. Hartman E. Finck B.N. Hruz P.W. Tzekov A. Rudnick D.A. Liver regeneration is impaired in lipodystrophic fatty liver dystrophy mice.Hepatology. 2010; 52: 2109-2117Crossref PubMed Scopus (49) Google Scholar). These findings are congruent with older data, generated before the cloning of lipins, that detected increased PAP activity in response to these stimuli (27Vavrecka M. Mitchell M.P. Hubscher G. The effect of starvation on the incorporation of palmitate into glycerides and phospholipids of rat liver homogenates.Biochem. J. 1969; 115: 139-145Crossref PubMed Scopus (63) Google Scholar, 28Lau D.C. Roncari D.A. Effects of glucocorticoid hormones on lipid-synthetic enzymes from different adipose tissue regions and from liver.Can. J. Biochem. Cell Biol. 1983; 61: 1245-1250Crossref PubMed Scopus (13) Google Scholar, 29Simpson K.J. Venkatesan S. Peters T.J. Martin A. Brindley D.N. Hepatic phosphatidate phosphohydrolase activity in acute and chronic alcohol-fed rats.Biochem. Soc. Trans. 1989; 17: 1115-1116Crossref PubMed Scopus (8) Google Scholar, 30Sturton R.G. Butterwith S.C. Burditt S.L. Brindley D.N. Effects of starvation, corticotropin injection and ethanol feeding on the activity and amount of phosphatidate phosphohydrolase in rat liver.FEBS Lett. 1981; 126: 297-300Crossref PubMed Scopus (14) Google Scholar). Because lipin 1 has the highest intrinsic PAP activity of the lipin family proteins (4Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), the induction of lipin 1 expression by these stimuli fits with this being an adaptive response to augment the hepatic capacity for TG synthesis in response to increased supply of fatty acids. In support of this, the fasting-induced increase in hepatic PAP activity is markedly blunted in fld mice (20Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The caveat to interpreting results from fld mice is the profound lipodystrophic phenotype (31Reue K. Xu P. Wang X.P. Slavin B.G. Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene.J. Lipid Res. 2000; 41: 1067-1076Abstract Full Text Full Text PDF PubMed Google Scholar, 32Péterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (469) Google Scholar) and its effects on lipid availability and metabolism. The marked deficiency in adipose tissue mass makes it difficult to isolate the effects of hepatic lipin 1 on liver TG metabolism. Recently, mice expressing a conditional allele of lipin 1 have been developed (8Nadra K. de Preux Charles A.S. Medard J.J. Hendriks W.T. Han G.S. Gres S. Carman G.M. Saulnier-Blache J.S. Verheijen M.H. Chrast R. Phosphatidic acid mediates demyelination in Lpin1 mutant mice.Genes Dev. 2008; 22: 1647-1661Crossref PubMed Scopus (105) Google Scholar). These mice express, in a tissue-specific manner, lipin 1 protein that is N-terminally truncated and lacks PAP activity but retains its ability to coactivate PPARα (7Mitra M.S. Chen Z. Ren H. Harris T.E. Chambers K.T. Hall A.M. Nadra K. Klein S. Chrast R. Su X. et al.Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.Proc. Natl. Acad. Sci. USA. 2013; 110: 642-647Crossref PubMed Scopus (46) Google Scholar). Herein, we show that mice expressing this allele in a liver-specific manner exhibit significant reductions in hepatic PAP activity but have normal rates of TG synthesis, and steady-state hepatic TG levels are unaffected. The ability of the truncated protein encoded by this allele to activate expression of fatty acid oxidation enzymes was intact. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload. Animal studies were approved by the institutional Animal Use and Care Committees of Washington University School of Medicine and fulfilled National Institutes of Health requirements for humane care. Generation of mice with liver-specific lipin 1 deficiency (Alb-Lpin1−/− mice) has been previously described (7Mitra M.S. Chen Z. Ren H. Harris T.E. Chambers K.T. Hall A.M. Nadra K. Klein S. Chrast R. Su X. et al.Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.Proc. Natl. Acad. Sci. USA. 2013; 110: 642-647Crossref PubMed Scopus (46) Google Scholar, 33Hu M. Yin H. Mitra M.S. Liang X. Ajmo J.M. Nadra K. Chrast R. Finck B.N. You M. Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice.Hepatology. 2013; 58: 1953-1963Crossref PubMed Scopus (56) Google Scholar). Fasting studies were timed so that mice were euthanized at 0800, which was 2 h after the end of the dark cycle. Food removal was timed accordingly to achieve the desired duration of fasting. To induce hepatic steatosis, mice were placed on a diet enriched with fat (40% kcal, mainly trans-fat; trans-oleic and trans-linoleic acids), cholesterol (2% weight [wt.]), and fructose (22% wt.) (HTF-C diet) (D09100301, Research Diets Inc.), which has been demonstrated to cause hepatic injury and inflammation (34Clapper J.R. Hendricks M.D. Gu G. Wittmer C. Dolman C.S. Herich J. Athanacio J. Villescaz C. Ghosh S.S. Heilig J.S. et al.Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 305: G483-G495Crossref PubMed Scopus (176) Google Scholar). Mice were euthanized and tissues were harvested at the end of week 10 of the study after a 4 h fast. Liver, gonadal, and subcutaneous fat tissue samples were frozen in liquid nitrogen and stored at −80°C. The week prior to being euthanized, mice were fasted for 6 h, and tails were snipped to measure baseline blood glucose using a One-Touch Ultra glucometer (Life Scan Inc.). Following an overnight fast, a 10% d-glucose solution (1 g/kg) was administered via intraperitoneal injection and 30, 60, and 120 min after injection, and total area under the curve was calculated. Protein from frozen tissue was homogenized in 0.3–1 ml ice-cold homogenization buffer [25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 8.0, supplemented with 1 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride, 5 mM sodium fluoride, and 1× Complete protease inhibitor cocktail tablet (Roche, Manneheim, Germany; cat. 04693116001)] using high-speed tissue disruption with the TissueLyser II (Qiagen, Valencia, CA). Tissue homogenates were subsequently solubilized by rotating at 4°C at 50 rpm for 1 h before being centrifuged (15,000 g for 15 min at 4°C) and collecting the supernatant. Protein from cultured cells were isolated by scraping adherent cells from culture plates, collecting them in the same homogenization buffer described above, and dispersing them via pipetting. Aliquots of the lysate from tissues and cultured cells were used to determine protein concentration by the bicinchoninic acid assay according to the manufacturer's instructions (Pierce Biotechnology; no. 23227). The remaining lysates were stored at −80°C until further analyzed. Lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were then rinsed with Tris-buffered saline plus Tween (TBST; 0.14 M NaCl, 0.02 M Tris base, pH 7.6, and 0.1% Tween), blocked with 5% BSA in TBST for 1 h at room temperature, washed 3 × 10 min at room temperature, and incubated with the relevant primary antibody 1:1,000 in 5% BSA overnight at 4°C. Blots were then washed 3 × 5 min with TBST, incubated with relevant secondary antibodies for 1 h at room temperature, washed again 3 × 10 min with TBST, and washed 2 × 10 min with TBS. Protein bands were visualized using the Odyssey Imaging System (LiCor Biosciences, Lincoln, NE). Lipin 1 (cat. sc-98450) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). Lipin 2 antibody has been previously described (21Gropler M.C. Harris T.E. Hall A.M. Wolins N.E. Gross R.W. Han X. Chen Z. Finck B.N. Lipin 2 is a liver-enriched phosphatidate phosphohydrolase enzyme that is dynamically regulated by fasting and obesity in mice.J. Biol. Chem. 2009; 284: 6763-6772Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Anti-α-Tubulin Clone B-5-1-2 (cat. T5168) was purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-mouse IRDye 680 (cat. 926-32220), goat anti-rabbit IRDye 680 (cat. 926-68021), and goat anti-rabbit 800 (cat. 926-32211) secondary antibodies were obtained from LiCor Biosciences. Akt (cat. 9272), phospho-Akt Ser473 (cat. 9271), phospho-Akt Thr308 (cat. 9275), glycogen synthase kinase (GSK)-3β (cat. 9315), and phospho- GSK-3β Ser9 (cat. 9323) were obtained from Cell Signaling (Danvers, MA). For all analyses, total RNA from liver or hepatocytes was isolated in RNAzol (Invitrogen) reagent. RNA (500 ng) was subjected to reverse transcription and SYBR GREEN RT-PCR (Applied Biosystems) following the manufacturer's instructions. Results were corrected to the expression of 36B4. Sequence of gene-specific primers is available upon request. The method to assess PAP was modified from Martin et al. (35Martin A. Hales P. Brindley D.N. A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver.Biochem. J. 1987; 245: 347-355Crossref PubMed Scopus (42) Google Scholar). Approximately 50 mg of liver tissue was homogenized in 1 ml of lysis buffer [0.01 M Tris-HCl (pH 7.3), 0.25 M Sucrose, 0.5% Tween-20, 1 mM DTT, 1× EDTA-free protease inhibitor (Roche, Manneheim, Germany; cat. 04693132001)] using a Tissue Master-125. Following homogenization, aliquots of the supernatant were used to determine protein concentration by the bicinchoninic acid assay. The [14C]PA used was prepared as follows: 15 µl 0.1 mCi/ml of the [14C]PAl-α-dioleoyl [oleoyl-1-14C] Na salt ([14C]PA) (American Radiolabeled Chemicals Inc., St. Louis, MO; cat. 1303) was added to 3 mM 3-sn-PA sodium salt (Sigma Aldrich; cat. P9511) and 2 mM l-α-phosphatidylcholine (Sigma Aldrich; cat. P3556) in 1 ml of chloroform, evaporated under a stream of N2, and resuspended in 1 ml of ice cold distilled deionized water. This mixture was sonicated 3 × 10 s and kept on ice in between sonications. To run the assay reaction, 5 µl of each sample was incubated with 40 µl of assay buffer [0.1 M Tris/maleate (pH 6.9), 10 mM MgCl2, 0.2% fatty acid-free BSA, 1 mM DTT, and 1× EDTA-free protease inhibitor with or without 12.5 mM N-ethylmaleimide (Sigma Aldrich; cat. E3876)], and 5 µl of [14C]PA was incubated at 37°C for 20 min. The reaction was stopped by adding 1 ml of a 19:1 (v/v) chloroform-methanol mixture containing 0.8% olive oil to each reaction and vortexing briefly. Then 500 mg of activated aluminum oxide was added to the tubes and capped securely. Three cycles of vortexing for 30 s and being left undisturbed for 10 min followed. Samples were spun down at 10,000 g for 5 min, and 350 µl of sample was added to scintillation fluid to count the 14C radioactivity. For fasting studies, liver TG content was determined from samples prepared in the PAP Activity assay buffer, solubilizing the homogenate 1:1 (v/v) with 1% sodium deoxycholate, vortexing, incubating at 37°C for 5 min, and then using the Infinity TG Reagent (Thermo Fisher Scientific, Waltham, MA; cat: TR22421) as per the manufacturer's instructions. For HTF-C studies, liver lipids were quantified by using ESI/MS analysis as described (36Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Crossref PubMed Scopus (295) Google Scholar, 37Han X. Yang K. Gross R.W. Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses.Mass Spectrom. Rev. 2012; 31: 134-178Crossref PubMed Scopus (417) Google Scholar). In brief, lipids were extracted from mouse liver using a modified Bligh and Dyer technique in the presence of internal standards. ESI/MS analyses were performed utilizing a TSQ Quantum Ultra Plus triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA), or an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA), equipped with an automated nanospray apparatus (Nanomate HD; Advion Bioscience Ltd., Ithaca, NY). TG molecular species were analyzed using TSQ Quantum Ultra Plus triple-quadrupole mass spectrometer in the positive-ion mode in the presence of a small amount of LiOH. Diglyceride molecular species were analyzed by derivatization with dimethylglycine and direct infusion in the presence of 0.1% formic acid, and ionized in the positive ion mode. Due to the low abundance of PA, PA molecular species were analyzed by using LTQ Orbitrap mass spectrometer at resolving powers (at m/z 400 Th) of 60,000 in full-scan mode and with the lock-mass feature engaged. Quantitative analysis of PA molecular species was performed by ratiometric comparisons of the intensities of the high mass accuracy (<5 ppm) PA deprotonated ions with those of internal standard in the negative-ion mode. Hepatocytes from WT and Alb-Lpin1−/− mice were isolated as previously described (38Chen Z. Gropler M.C. Norris J. Lawrence Jr, J.C. Harris T.E. Finck B.N. Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1738-1744Crossref PubMed Scopus (69) Google Scholar) and cultured in DMEM containing 5% FBS for 4 h, followed by a further 1 h incubation period in serum-free DMEM. Triacylglycerol synthesis rates were quantified by using [3H]glycerol in the presence of 0.5 mM BSA-conjugated oleic acid as previously described (39Chen Z. Norris J.Y. Finck B.N. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) stimulates VLDL assembly through activation of cell death-inducing DFFA-like effector B (CideB).J. Biol. Chem. 2010; 285: 25996-26004Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Palmitate oxidation rates were assessed using [3H]palmitate as previously described (38Chen Z. Gropler M.C. Norris J. Lawrence Jr, J.C. Harris T.E. Finck B.N. Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1738-1744Crossref PubMed Scopus (69) Google Scholar, 40Burgess S.C. Leone T.C. Wende A.R. Croce M.A. Chen Z. Sherry A.D. Malloy C.R. Finck B.N. Diminished hepatic gluconeogenesis via defects in tricarboxylic acid cycle flux in peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha)-deficient mice.J. Biol. Chem.

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