The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL
2008; Elsevier BV; Volume: 50; Issue: 1 Linguagem: Inglês
10.1194/jlr.m800204-jlr200
ISSN1539-7262
AutoresMaroun Bou Khalil, Meenakshi Sundaram, Hongyu Zhang, Philip H. Links, Jennifer F. Raven, Boripont Manmontri, Meltem Sarıahmetoğlu, Khai Tran, Karen Reue, David N. Brindley, Zemin Yao,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoPhosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1α and 1β), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1α or -1β increased the synthesis and secretion of [3H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL1 (Sf > 100) and VLDL2 (Sf 20–100). Expression of lipin-1α or -1β increased secretion efficiency and decreased intracellular degradation of [35S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [3H]glycerolipids and [35S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1α not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1α mutant failed to promote [35S]apoB100 synthesis or secretion, and showed compromised stimulation in [3H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion. Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1α and 1β), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1α or -1β increased the synthesis and secretion of [3H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL1 (Sf > 100) and VLDL2 (Sf 20–100). Expression of lipin-1α or -1β increased secretion efficiency and decreased intracellular degradation of [35S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [3H]glycerolipids and [35S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1α not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1α mutant failed to promote [35S]apoB100 synthesis or secretion, and showed compromised stimulation in [3H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion. The major lipoproteins carrying triacylglycerol (TAG) in fasted human plasma are VLDL that are synthesized by the liver. Formation of hepatic VLDL requires the active synthesis of various lipid constituents (e.g., TAG, cholesterol, cholesteryl esters, and phospholipids) that are assembled together with the large, hydrophobic apolipoprotein B100 (apoB100) (1Fisher E.A Ginsberg H.N. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins..J. Biol. Chem. 2002; 277: 17377-17380Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 2Rustaeus S. Lindberg K. Stillemark P. Claesson C. Asp L. Larsson T. Boren J. Olofsson S.O. Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation..J. Nutr. 1999; 129: 463S-466SCrossref PubMed Google Scholar, 3Olofsson S.O Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins..Curr. Opin. Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (188) Google Scholar), and the assembly process is initiated during or immediately after translation and translocation of apoB100 across the endoplasmic reticulum (ER) membranes (4Gibbons G.F Wiggins D. Brown A.M. Hebbachi A.M. Synthesis and function of hepatic very-low-density lipoprotein..Biochem. Soc. Trans. 2004; 32: 59-64Crossref PubMed Scopus (194) Google Scholar). The microsomal triglyceride transfer protein (MTP), encoded by the abetalipoprotenemia gene mttp, is essential for VLDL assembly and secretion (5Gordon D.A Wetterau J.R. Gregg R.E. Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles..Trends Cell Biol. 1995; 5: 317-321Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 6Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.O. The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells..J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 7Olofsson S.O Stillemark-Billton P. Asp L. Intracellular assembly of VLDL: two major steps in separate cell compartments..Trends Cardiovasc. Med. 2000; 10: 338-345Crossref PubMed Scopus (106) Google Scholar), promoting TAG partitioning into the microsomes (8Kulinski A. Rustaeus S. Vance J.E. Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with apoB, as well as for apoB lipidation..J. Biol. Chem. 2002; 277: 31516-31525Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 9Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins..J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 10Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice..J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). The TAG utilized for the assembly process is derived from de novo glycerolipid biosynthesis and also from hydrolysis/re-esterification of preexisting TAG (11Gilham D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates..Mol. Biol. Cell. 2005; 16: 984-996Crossref PubMed Scopus (69) Google Scholar) or phospholipids (12Tran K. Sun F. Cui Z. Thorne-Tjomsland G. St Germain C. Lapierre L.R. McLeod R.S. Jamieson J.C. Yao Z. Attenuated secretion of very low density lipoproteins from McA-RH7777 cells treated with eicosapentaenoic acid is associated with impaired utilization of triacylglycerol synthesized via phospholipid remodeling..Biochim. Biophys. Acta. 2006; 1761: 463-473Crossref PubMed Scopus (29) Google Scholar, 13Wiggins D. Gibbons G.F. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid..Biochem. J. 1996; 320: 673-679Crossref PubMed Scopus (65) Google Scholar). A key enzyme involved in the de novo biosynthesis of TAG and phospholipids is phosphatidate phosphatase-1 (PAP-1), which converts of phosphatidate (PA) to diacylglycerol (DAG) (14Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Inc., Boca Raton1988: 21-77Google Scholar, 15Carman G.M Han G.S. Roles of phosphatidate phosphatase enzymes in lipid metabolism..Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The resulting DAG serves as substrate for the synthesis of TAG as well as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). There are two classes of enzymes that convert PA to DAG in the liver, namely PAP-1 and PAP-2. The PAP-1 activity is specific toward PA and is Mg2+-dependent, and can be inhibited by N-ethylmaleimide (15Carman G.M Han G.S. Roles of phosphatidate phosphatase enzymes in lipid metabolism..Trends Biochem. Sci. 2006; 31: 694-699Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 16Jamal Z. Martin A. Gomez-Munoz A. Brindley D.N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol..J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar, 17Brindley D.N Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer..J. Cell. Biochem. 2004; 92: 900-912Crossref PubMed Scopus (186) Google Scholar, 18Donkor 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 (299) Google Scholar). The PAP-2 enzymes degrade a variety of bioactive lipid phosphates, and their activity is N-ethylmaleimide-insensitive and Mg2+-independent. In hepatic cells, PAP-1 activity is increased by glucocorticoids, an effect that is synergized by glucagon through cAMP formation and antagonized by insulin (19Sturton 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, 20Pittner R.A Fears R. Brindley D.N. Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis..Biochem. J. 1985; 225: 455-462Crossref PubMed Scopus (62) Google Scholar). Thus, increased PAP-1 activity under stress or other aberrant metabolic conditions (such as starvation, diabetes, and post-alcohol consumption) augments the capacity to sequester an increased FA supply to the liver as TAG (14Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Inc., Boca Raton1988: 21-77Google Scholar). Mammalian PAP-1 is encoded by the lipin gene family, which consists of lipin-1, -2, and -3 (18Donkor 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 (299) Google Scholar, 21Peterfy 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 (475) Google Scholar). The Lpin1 gene gives rise to two alternative splicing isoforms, lipin-1α and -1β (22Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis..J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). All of these lipins possess PAP-1 activity specific toward PA (18Donkor 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 (299) Google Scholar). In mice, lipin-1 is expressed at high levels in adipose tissue, heart, and skeletal muscle, where it is the sole PAP-1 enzyme, and at moderate levels in kidney, lung, brain, and liver (18Donkor 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 (299) Google Scholar, 21Peterfy 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 (475) Google Scholar). In mature 3T3-L1 adipocytes, the two lipin-1 splicing isoforms exhibit different subcellular localization, with lipin-1α being primarily nuclear and lipin-1β cytoplasmic (22Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis..J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). This subcellular localization of lipin-1 appears to influence its function; reconstitution of lipin-1-deficient cells with lipin-1α induces adipogenic gene expression, whereas lipin-1β promotes gene expression of enzymes involved in lipid synthesis (22Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis..J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The role of lipin-1 in adipogenic gene expression and TAG biosynthesis in adipose tissue has been documented (22Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis..J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 23Phan J. Peterfy M. Reue K. Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro..J. Biol. Chem. 2004; 279: 29558-29564Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 24Phan J. Peterfy M. Reue K. Biphasic expression of lipin suggests dual roles in adipocyte development..Drug News Perspect. 2005; 18: 5-11Crossref PubMed Scopus (39) Google Scholar). Mutations in the mouse Lpin1 gene prevent normal adipose tissue development, and result in lipodystrophy (21Peterfy 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 (475) Google Scholar, 25Phan J. Reue K. Lipin, a lipodystrophy and obesity gene..Cell Metab. 2005; 1: 73-83Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 26Reue K. Peterfy M. Mouse models of lipodystrophy..Curr. Atheroscler. Rep. 2000; 2: 390-396Crossref PubMed Scopus (29) Google Scholar, 27Reue 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). Transgenic mice expressing lipin-1 specifically in adipocytes have enhanced TAG storage and become obese (23Phan J. Peterfy M. Reue K. Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro..J. Biol. Chem. 2004; 279: 29558-29564Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Lipin-1 also promotes hepatic TAG storage and FA oxidation through transcriptional regulation of the peroxisome proliferator-activated receptor-α (PPARα) and PPAR coactivator protein-1α (PGC-1α) complex (28Finck 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 (436) Google Scholar). Lipin-1 deficiency in the fatty liver dystrophy (fld) mouse strain does not preclude hepatic TAG synthesis or secretion during the neonatal period when the animals consume a high-fat diet (29Langner C.A Birkenmeier E.H. Ben Zeev O. Schotz M.C. Sweet H.O. Davisson M.T. Gordon J.I. The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities..J. Biol. Chem. 1989; 264: 7994-8003Abstract Full Text PDF PubMed Google Scholar). Rather, PAP-1 activity in fld mice is normal, owing to the presence of lipin-2 and probably increased expression of lipin-3 (18Donkor 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 (299) Google Scholar). We have previously shown that TAG synthesis, apoB synthesis and stability, and VLDL secretion in primary rat hepatocytes were all increased by the glucocorticoid dexamethasone, whereas insulin counteracted these effects (30Martin-Sanz P. Vance J.E. Brindley D.N. Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone..Biochem. J. 1990; 271: 575-583Crossref PubMed Scopus (51) Google Scholar, 31Mangiapane E.H Brindley D.N. Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes..Biochem. J. 1986; 233: 151-160Crossref PubMed Scopus (81) Google Scholar, 32Wang C.N McLeod R.S. Yao Z. Brindley D.N. Effects of dexamethasone on the synthesis, degradation, and secretion of apolipoprotein B in cultured rat hepatocytes..Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1481-1491Crossref PubMed Scopus (54) Google Scholar). On the basis of recent discoveries showing that a) the expression of lipin-1, but not lipin-2 or -3, can be upregulated by glucocorticoids and suppressed by insulin in mouse and rat hepatocytes (33Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. 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. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and b) the promoter region of Lpin1 contains a functional glucocorticoid response element (34Zhang P. O'Loughlin L. Brindley D.N. Reue K. Regulation of lipin-1 gene expression by glucocorticoids during adipogenesis..J. Lipid Res. 2008; 49: 1519-1528Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), we postulated that lipin-1 is the link between glucocorticoid treatment and enhanced hepatic VLDL secretion. Upregulation of lipin-1 may provide a mechanism for converting excess FAs, derived from lipolysis in adipose tissue in starvation, diabetes, and other conditions of metabolic stress, into TAGs that are secreted as VLDL or stored as lipid droplets (14Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Inc., Boca Raton1988: 21-77Google Scholar, 35Pittner R.A Fears R. Brindley D.N. Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes..Biochem. J. 1985; 230: 525-534Crossref PubMed Scopus (50) Google Scholar). In the present study, we tested the effect of alterations in hepatic lipin-1 expression on VLDL assembly and secretion using gain- and loss-of-function approaches for lipin-1α and -1β, as well as for a mutant form of lipin-1α in which the nuclear localization signal (NLS) sequences were deleted. Results presented herein provide strong evidence that the level and compartmentalization of lipin-1 exert a major impact on VLDL assembly and secretion. Cell culture media and reagents, mouse anti-V5 monoclonal antibody, goat anti-mouse IgG conjugated with Alexa Fluor 488, goat anti-rabbit IgG conjugated with Alexa Fluor 599, and SlowFade Light AntiFade were purchased from Invitrogen (Burlington, ON). DNA restriction and modification enzymes were obtained from New England Biolabs (Pickering, ON). Protein A-Sepharose™ CL-4B beads, [35S]methionine/cysteine (1,000 Ci/mmol), and HRP-linked anti-mouse IgG antibody were obtained from Amersham Biosciences (Baie d'Urfe, PQ). (2-3H]glycerol (9.6 Ci/mmol) was obtained from Amersham (Piscataway, NJ). Protease inhibitor cocktail (EDTA-free) and chemiluminescent substrates were purchased from Roche Diagnostics (Laval, PQ). Lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL). Antibodies against apoE, lamin A, β-actin, and calnexin were obtained from BioDesign (Saco, ME), Abcam (Hornby, ON), Ambion (Austin, TX), and Stressgen (Ann Arbor, MI), respectively. Polyclonal anti-lipin-1 antiserum against the synthetic polypeptide SKTDSPSRKKDKRSRHLGADG (36Huffman T.A Mothe-Satney I. Lawrence Jr., J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin..Proc. Natl. Acad. Sci. USA. 2002; 99: 1047-1052Crossref PubMed Scopus (188) Google Scholar) and antiserum against rat VLDL were produced in our laboratory. The coding sequences of lipin-1α, -1β (22Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis..J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and ΔNLS (a variant of lipin-1α that lacked the NLS sequence KKRRKRRRK) were originally prepared in the pcDNA3.1/V5-His-TOPO expression system. The coding sequences for lipin-1α and -1β were excised by digestion with KpnI and PmeI and inserted into the pCMV5 vector that had been digested with KpnI and SmaI. The resulting pCMV5-based expression plasmids were transfected into McA-RH7777 cells by the calcium phosphate precipitation method (37Chen C. Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA..Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). All analyses were performed 48 h posttransfection. Double-stranded ON-TARGETplus SMARTpool® short interfering RNAs (siRNAs) (see supplementary Table I) specific for rat lipin-1 were obtained from Dharmacon, Inc. (Lafayette, CO). McA-RH7777 cells (50% confluence) were cultured in DMEM containing 20% FBS, and transfected with 80 pmol lipin-1 siRNA (100 nM final concentration) using different concentrations (0.025, 0.05, or 0.075 μg/μl) of Lipofectamine™ 2000 (Invitrogen), according to the manufacturer's instructions. The highest lipin-1 knockdown was observed at 0.075 μg/μl of Lipofectamine; this concentration was used for all subsequent metabolic labeling experiments and for gene expression analyses by real-time RT-PCR. The transfection medium was replaced with DMEM containing 20% FBS 4.5 h posttransfection. For each experiment, controls for lipin-1 knockdown were performed with functional nontargeting control siRNA (Dharmacon), which can silence firefly luciferase, or with Lipofectamine™ 2000 alone. Cells were harvested 48 h after transfection and subjected to immunoblot analysis (for lipin-1) or used for the metabolic labeling experiments described below. Cell lysates of equal amounts of proteins were resolved by denaturing SDS-PAGE (8% gel), and transferred onto nitrocellulose membranes. Recombinant lipin-1 variants were detected using mouse anti-V5 antibody. In parallel experiments, cells were labeled with [35S]methionine/cysteine (75 μCi/dish) for 4 h in media containing 20% FBS and 0.4 mM oleate. Cell-associated lipin-1 was recovered by immunoprecipitation using polyclonal anti-lipin-1 antiserum, and subjected to denaturing SDS-PAGE (8% gel) and autoradiography. The PAP-1-specific activity in lipin-1-transfected McA-RH7777 cells and primary rat hepatocytes was determined according to protocols described previously (33Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. 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. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cells cultured on fibronectin-coated coverslips were incubated in DMEM ± 0.4 mM oleate for 2 h at 37°C. Cells were then fixed with 4% paraformaldehyde for 30 min, and then permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were blocked with 10% FBS (in PBS) for 1 h, rinsed, and incubated with mouse anti-V5 monoclonal antibody (5 μg/ml) plus rabbit anti-lamin A polyclonal antibody (0.1 μg/ml) for 1 h. After rinsing with PBS, cells were stained with goat anti-mouse IgG conjugated with Alexa Fluor 488 and goat anti-rabbit IgG conjugated with Alexa Fluor 599. The coverslips were mounted onto glass slides using SlowFade Light AntiFade. Confocal images were captured using a 100× NA1.4 oil objective on an Olympus IX81 inverted microscope with appropriate lasers (488 nm argon/krypton laser for Alexa 488, and the 543 nm green helium-neon laser for Alexa 599). Subcellular fractionation was performed as described previously (9Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins..J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Briefly, lipin-1 expressing cells (8–10 100 mm dishes) were homogenized by passing 20 times through a ball-bearing homogenizer. Cell homogenates were centrifuged (12,000 g, 10 min, 4°C) to obtain nuclear membranes (pellet) and the postnuclear supernatant, which was transferred to a quick-seal centrifuge tube and centrifuged using a Beckman TLA-100.4 rotor (174,000 g, 30 min, 4°C) to obtain cytosol (supernatant) and microsomes (pellet). Aliquots of the fractionated samples were subjected to denaturing SDS-PAGE and immunoblot analysis for lipin-1. Antibodies against lamin, calnexin, and actin were used to probe for nucleus, microsomes, and cytosol, respectively. Cells were labeled with [3H]glycerol (4 μCi/ml, 2 ml/dish) in DMEM containing 20% FBS ± oleate (0 to 0.4 mm) for 1, 2, or 4 h. Total lipids were extracted from cells and media, respectively, and separated by TLC as described previously (38Tran K. Wang Y. DeLong C.J. Cui Z. Yao Z. The assembly of very low density lipoproteins in rat hepatoma McA-RH7777 cells is inhibited by phospholipase A2 antagonists..J. Biol. Chem. 2000; 275: 25023-25030Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The radioactivity associated with [3H]TAG, -PC, -PE and -DAG was quantified by scintillation counting. Cells were labeled with [35S]methionine/cysteine (75 μCi/ml) for 4 h in methionine/cysteine-free DMEM containing 20% FBS ± 0.4 mm oleate. At the end of labeling, the media and cells were collected, and the 35S-labeled apoB100, apoE, and lipin-1 were respectively recovered by immunoprecipitation. Proteins were resolved by denaturing SDS-PAGE (5, 8, and 12% gels for apoB100, lipin-1, and apoE, respectively). Radioactivity associated with these proteins was quantified by scintillation counting (9Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins..J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Cells were labeled with [3H]glycerol or [35S]methionine/cysteine as described above. The medium was collected, and the secreted lipoproteins were separated by cumulative rate floatation ultracentrifugation to separate VLDL1 and VLDL2 from the other lipoproteins (9Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins..J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Lipids were extracted from each lipoprotein fraction and resolved by TLC. The [35S]apoB100 was recovered from each lipoprotein fraction by immunoprecipitation and resolved by denaturing SDS-PAGE as described above. Cells were pulse-labeled with [35S]methionine/cysteine (75 μCi/ml) for 30 min. The media were removed and replaced with chase media (DMEM containing 20% FBS and 0.4 mM oleate) for up to 4 h. At the end of chase, [35S]apoB100 and -apoE were recovered from media and cells, respectively, by immunoprecipitation, and subjected to denaturing SDS-PAGE and scintillation counting as described previously (9Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins..J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Lipin-1 expressing cells were suspended in hypotonic buffer (1 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM EGTA) containing protease cocktail inhibitors, and homogenized using a Polytron homogenizer. After centrifugation using a Beckman microcentrifuge at 10,000 rpm, 4°C for 30 min, the supernatants were used for MTP activity assay (with 25 μg protein per assay) as described previously (39Athar H. Iqbal J. Jiang X.C. Hussain M.M. A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride
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