Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice
2009; Elsevier BV; Volume: 51; Issue: 3 Linguagem: Inglês
10.1194/jlr.m000976
ISSN1539-7262
AutoresAngela Hall, Elizabeth M. Brunt, Zhouji Chen, Navin Viswakarma, Janardan K. Reddy, Nathan E. Wolins, Brian N. Finck,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoLipid droplet proteins (LDPs) coat the surface of triglyceride-rich lipid droplets and regulate their formation and lipolysis. We profiled hepatic LDP expression in fatty liver dystrophic (fld) mice, a unique model of neonatal hepatic steatosis that predictably resolves between postnatal day 14 (P14) and P17. Western blotting revealed that perilipin-2/ADRP and perilipin-5/OXPAT were markedly increased in steatotic fld liver but returned to normal by P17. However, the changes in perilipin-2 and perilipin-5 protein content in fld mice were exaggerated compared with relatively modest increases in corresponding mRNAs encoding these proteins, a phenomenon likely mediated by increased protein stability. Conversely, cell death-inducing DFFA-like effector (Cide) family genes were strongly induced at the level of mRNA expression in steatotic fld mouse liver. Surprisingly, levels of peroxisome proliferator-activated receptor γ, which is known to regulate Cide expression, were unchanged in fld mice. However, sterol-regulatory element binding protein 1 (SREBP-1) was activated in fld liver and CideA was revealed as a new direct target gene of SREBP-1. In summary, LDP content is markedly increased in liver of fld mice. However, whereas perilipin-2 and perilipin-5 levels are primarily regulated posttranslationally, Cide family mRNA expression is induced, suggesting that these families of LDP are controlled at different regulatory checkpoints. Lipid droplet proteins (LDPs) coat the surface of triglyceride-rich lipid droplets and regulate their formation and lipolysis. We profiled hepatic LDP expression in fatty liver dystrophic (fld) mice, a unique model of neonatal hepatic steatosis that predictably resolves between postnatal day 14 (P14) and P17. Western blotting revealed that perilipin-2/ADRP and perilipin-5/OXPAT were markedly increased in steatotic fld liver but returned to normal by P17. However, the changes in perilipin-2 and perilipin-5 protein content in fld mice were exaggerated compared with relatively modest increases in corresponding mRNAs encoding these proteins, a phenomenon likely mediated by increased protein stability. Conversely, cell death-inducing DFFA-like effector (Cide) family genes were strongly induced at the level of mRNA expression in steatotic fld mouse liver. Surprisingly, levels of peroxisome proliferator-activated receptor γ, which is known to regulate Cide expression, were unchanged in fld mice. However, sterol-regulatory element binding protein 1 (SREBP-1) was activated in fld liver and CideA was revealed as a new direct target gene of SREBP-1. In summary, LDP content is markedly increased in liver of fld mice. However, whereas perilipin-2 and perilipin-5 levels are primarily regulated posttranslationally, Cide family mRNA expression is induced, suggesting that these families of LDP are controlled at different regulatory checkpoints. Lipid droplets (LDs) are metabolically active structures that play important roles in lipid transport, sorting, and signaling cascades (1Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Although adipose tissue is the predominant site of fat storage in higher organisms, most tissues have at least some capacity to store triglyceride (TG) in small LDs that can be used for an immediate energy source. However, several pathologic conditions are associated with marked ectopic fat deposition. For example, fatty liver disease is characterized by striking accumulation of neutral lipid in the cytosol of hepatocytes. The accumulation of LDs in hepatocytes is often, at least overtly, a nonprogressive condition. However, in some individuals, lipotoxicity can result in pathologic changes in hepatic cytoarchitecture, including hepatocyte apoptosis and ballooning and inflammation (steatohepatitis) that subsequently can result in fibrosis and parenchymal remodeling with cirrhosis.The surface layer of LDs is coated by numerous proteins collectively known as lipid droplet proteins (LDPs) (reviewed in ref. 2Ducharme N.A. Bickel P.E. Lipid droplets in lipogenesis and lipolysis.Endocrinology. 2008; 149: 942-949Crossref PubMed Scopus (378) Google Scholar). Although a variety of proteins associate with LDs, the best-studied is the family of PAT proteins (3Brasaemle D.L. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). This family was named after the original three constituents, perilipin, adipocyte differentiation-related protein (ADRP), and tail interacting protein 47 (TIP47). Recently, a standardized nomenclature for PAT family proteins has been adopted (4Kimmel A.R. Brasaemle D.L. McAndrews-Hill M. Sztalryd C. Londos C. Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular, lipid storage droplet proteins.J Lipid Res. 2009; 51: 468-471Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) and this revised naming system for perilipin-1 (perilipin), perilipin-2 (ADRP or adipophilin), and perilipin-3 (TIP47) is used hereafter. Two additional proteins, perilipin-4 (S3-12) and perilipin-5 (also known as OXPAT, MLDP, PAT-1, and LSDP5) (5Wolins N.E. Quaynor B.K. Skinner J.R. Tzekov A. Croce M.A. Gropler M.C. Varma V. Yao-Borengasser A. Rasouli N. Kern P.A. et al.OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.Diabetes. 2006; 55: 3418-3428Crossref PubMed Scopus (244) Google Scholar, 6Wolins N.E. Quaynor B.K. Skinner J.R. Schoenfish M.J. Tzekov A. Bickel P.E. S3–12, Adipophilin, and TIP47 package lipid in adipocytes.J. Biol. Chem. 2005; 280: 19146-19155Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), have now been added to the perilipin family based on sequence and functional similarities. Another family of proteins, known as the cell death-inducing DFFA-like effector (Cide) family of proteins (CideA, CideB, and CideC), has also recently emerged as LDPs that regulate LD metabolism (7Gong J. Sun Z. Li P. CIDE proteins and metabolic disorders.Curr. Opin. Lipidol. 2009; 20: 121-126Crossref PubMed Scopus (136) Google Scholar). The mouse homolog of CideC is known as Fsp27 and will be referenced as such henceforth. Interestingly, work from several groups has convincingly demonstrated that Cide proteins are also physically associated with LDs and modulate LD size and metabolism (8Puri V. Konda S. Ranjit S. Aouadi M. Chawla A. Chouinard M. Chakladar A. Czech M.P. Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage.J. Biol. Chem. 2007; 282: 34213-34218Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 9Puri V. Ranjit S. Konda S. Nicoloro S.M. Straubhaar J. Chawla A. Chouinard M. Lin C. Burkart A. Corvera S. et al.Cidea is associated with lipid droplets and insulin sensitivity in humans.Proc. Natl. Acad. Sci. USA. 2008; 105: 7833-7838Crossref PubMed Scopus (284) Google Scholar).LDPs allow LDs to maintain a dynamic communication with the endoplasmic reticulum and the plasma membrane (1Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Several lines of evidence suggest that perilipin-1 and perilipin-2 are physical barriers to lipolytic enzymes under basal conditions yet can facilitate interactions with lipases and enhance lipolysis in response to lipolytic stimuli (10Subramanian V. Rothenberg A. Gomez C. Cohen A.W. Garcia A. Bhattacharyya S. Shapiro L. Dolios G. Wang R. Lisanti M.P. et al.Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3–L1 adipocytes.J. Biol. Chem. 2004; 279: 42062-42071Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 11Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 12Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation.J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (414) Google Scholar). The significant alterations in lipid metabolism observed in mouse models with targeted deletion of LDPs are strong evidence for important roles for these proteins in regulating metabolism (13Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (488) Google Scholar, 14Nishino N. Tamori Y. Tateya S. Kawaguchi T. Shibakusa T. Mizunoya W. Inoue K. Kitazawa R. Kitazawa S. Matsuki Y. et al.FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets.J. Clin. Invest. 2008; 118: 2808-2821PubMed Google Scholar, 15Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol. Cell. Biol. 2006; 26: 1063-1076Crossref PubMed Scopus (249) Google Scholar).Although LDPs were originally studied from the perspective of their effects in adipose tissue, more recent work has shown critical roles for LDPs in regulating fat metabolism in liver, especially in the context of lipid overload such as occurs in obesity-related fatty liver disease. Specifically, the expression of perilipin-2, CideA, and Fsp27 is induced in liver of ob/ob mice, which exhibit marked hepatic steatosis (16Motomura W. Inoue M. Ohtake T. Takahashi N. Nagamine M. Tanno S. Kohgo Y. Okumura T. Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet.Biochem. Biophys. Res. Commun. 2006; 340: 1111-1118Crossref PubMed Scopus (128) Google Scholar, 17Schadinger S.E. Bucher N.L. Schreiber B.M. Farmer S.R. PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1195-E1205Crossref PubMed Scopus (305) Google Scholar, 18Matsusue K. Kusakabe T. Noguchi T. Takiguchi S. Suzuki T. Yamano S. Gonzalez F.J. Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27.Cell Metab. 2008; 7: 302-311Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Moreover, mice deficient in perilipin-2, CideB, or Fsp27 are protected from developing obesity-related fatty liver disease (15Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol. Cell. Biol. 2006; 26: 1063-1076Crossref PubMed Scopus (249) Google Scholar, 19Li J.Z. Ye J. Xue B. Qi J. Zhang J. Zhou Z. Li Q. Wen Z. Li P. Cideb regulates diet-induced obesity, liver steatosis, and insulin sensitivity by controlling lipogenesis and fatty acid oxidation.Diabetes. 2007; 56: 2523-2532Crossref PubMed Scopus (123) Google Scholar), suggesting that these proteins play a role in driving hepatic lipid accumulation or enhancing the capacity for storing lipid.We sought to evaluate the expression of lipid droplet proteins in liver of fatty liver dystrophic (fld) mice, a lipodystrophic model of fatty liver (20Langner 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). The complex and severe metabolic phenotype of fld mice is caused by a mutation in the gene encoding lipin 1 (Lpin1) (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 (463) Google Scholar). The fld mice appear normal at birth, but rapidly develop an enlarged fatty liver (20Langner 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). Hepatic lipid accumulation in fld mice spontaneously and rapidly resolves prior to weaning (20Langner 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), making this model a unique and interesting system in which to study the expression of the LDP proteins. Herein, we demonstrate that expression of several LDPs is markedly increased in the steatotic liver of fld mice, and decreases as the fatty liver phenotype resolves. Our studies also revealed that the PAT and Cide families of LDPs are controlled by distinct regulatory mechanisms in steatotic hepatocytes.MATERIALS AND METHODSAnimal studiesAll studies were conducted with matched littermate mice. Homozygous fld (fld/fld) mice were compared with littermate heterozygous (fld/+) or wild-type (+/+) control mice. Eighteen-week-old female ob/ob and lean littermate (ob/+) were sacrificed for tissue collection. All animal experiments were approved by the Washington University School of Medicine Animal Studies Committee and conformed to criteria outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Tissue histologyFreshly obtained liver samples were fixed overnight in 10% buffered formalin and embedded in paraffin. Hematoxylin and eosin staining was carried out by the Digestive Disease Research Core Center at Washington University School of Medicine.Determination of liver TG levelsTG concentration was determined by a commercial colorimetric method (Wako Chemicals, Richmond, VA) with solvent extracted lipid, as described (22Schwartz D.M. Wolins N.E. A simple and rapid method to assay triacylglycerol in cells and tissues.J. Lipid Res. 2007; 48: 2514-2520Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Total liver protein was extracted using tissue protein extraction reagent (number 78510; Pierce) and TG concentration is expressed as milligrams of TG/g of liver protein.mRNA isolation and gene expression analysesLiver RNA was extracted with RNAzol Bee (Isotexdiagnostics, Friendswood, TX) according to the manufacturer's instructions. Real time RT-PCR was performed using the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) and the SYBR green kit. Using the standard curve method, the relative amount of specific PCR products for each primer set was generated. For normalization, 36B4 was amplified from each sample, and arbitrary units of target mRNA were corrected to the corresponding level of the 36B4 mRNA. The sequences of the primers used in these studies can be found in supplementary Table I.Protein isolation and cellular fractionation of mouse liversFor Western blot studies in Fig. 2, protein extracts were obtained from liver by using the following lysis buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EDTA, 0.1% sodium deoxycholate, and 1% Triton X-100) containing a protease inhibitor cocktail. For sterol-regulatory element binding protein (SREBP)-1 Western blots, nuclear proteins were isolated using the NXTRACT nuclear protein isolation kit (Sigma Chemical Co., St. Louis, MO). Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Scientific, Rockford, IL).For subcellular fractionation studies, livers from 12-week-old fed fld and wild-type (WT) mice were minced and then homogenized by a Teflon pestle tissue homogenizer in lysis buffer (10 mM HEPES, 1 mM EDTA, pH 7.4). The nuclear fraction was obtained by 2,000 g centrifugation for 5 min. The 2,000 g supernatant was weighted with sucrose to 40% (w/v) with 65% sucrose and was then overlaid with successive layers of 5 ml 35% sucrose and 5 ml 10% sucrose. The tubes were then filled to capacity with lysis buffer. The gradients were centrifuged at 172,000 g for 3 h at 4°. Fractions were harvested as described previously and stored at −80°C until Western blot analyses (23Brasaemle D.L. Wolins N.E. Isolation of lipid droplets from cells by density gradient centrifugation.Curr. Protoc. Cell Biol. 2006; (Chapter 3: Unit 3. 15.)PubMed Google Scholar).Western blot analysisWestern blotting studies were performed with whole-cell lysates (40 µg) or nuclear lysates (20 µg) as indicated. Proteins from sucrose gradient isolation were loaded by volume rather than protein content. Proteins were separated on 4%–12% gradient gels by SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes that were then blocked with 5% (w/v) nonfat dry milk in TBS-Tween (20 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.05% Tween 20) prior to immunoblotting. Generation of rabbit-derived antibodies directed to carboxyl-terminus of perilipin-5, the amino-terminus of perilipin-3, and the amino-terminus of perilipin-2 has been described previously (5Wolins N.E. Quaynor B.K. Skinner J.R. Tzekov A. Croce M.A. Gropler M.C. Varma V. Yao-Borengasser A. Rasouli N. Kern P.A. et al.OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.Diabetes. 2006; 55: 3418-3428Crossref PubMed Scopus (244) Google Scholar, 6Wolins N.E. Quaynor B.K. Skinner J.R. Schoenfish M.J. Tzekov A. Bickel P.E. S3–12, Adipophilin, and TIP47 package lipid in adipocytes.J. Biol. Chem. 2005; 280: 19146-19155Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Antibodies to glycogen synthase (Proteintech Group, Chicago, IL), peroxisome proliferator-activated receptor γ (PPARγ) (Santa Cruz, San Diego, CA), SREBP-1 (Santa Cruz), CideA (Genway, San Diego, CA), Fsp27 (generous gift of Dr. Vishwajeet Puri), and actin (Sigma) were used according to the manufacturer's instructions.Primary mouse hepatocyte isolationPrimary mouse hepatocytes were isolated from mice as previously described (24Chen Z. Fitzgerald R.L. Averna M.R. Schonfeld G. A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.J. Biol. Chem. 2000; 275: 32807-32815Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, mice were anesthetized and then perfused through the portal vein with warmed HBSS containing collagenase. Livers were mechanically disrupted with forceps. Released cells were washed extensively and plated onto collagen coated dishes and grown in culture in DMEM supplemented with 5% FBS.Pulse-chase experimentAfter plating and adherence, hepatocytes from adult WT mice were washed three times with PBS and incubated in Met- and Cys-free DMEM for 1 h to deplete the cellular pool of Met and Cys. Thereafter, the medium was replaced with 1 ml of Met- and Cys-free DMEM containing 200 µCi of [35S]Promix with or without oleic acid (OA) (0.4 mM; to give an OA/BSA ratio of 6) for 1 h. After the 1 h pulse, the cells were washed twice with PBS and incubated in 1 ml of DMEM containing 10 mM Met and 3 mM Cys for 12 h with or without the specified OA.Immunofluorescence microscopyHepatocytes from P14 WT or fld mice were isolated, plated, and then fixed with formaldehyde after adhering for 1 h. Fixed hepatocytes were then stained as described previously (25Wolins N.E. Skinner J.R. Schoenfish M.J. Tzekov A. Bensch K.G. Bickel P.E. Adipocyte protein S3–12 coats nascent lipid droplets.J. Biol. Chem. 2003; 278: 37713-37721Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) with antibodies below. The cells were stained with anti- perilipin-2 (Fitzgerald Industries International, Flanders NJ, CAT# 20R-AP002), perilipin-3 (6Wolins N.E. Quaynor B.K. Skinner J.R. Schoenfish M.J. Tzekov A. Bickel P.E. S3–12, Adipophilin, and TIP47 package lipid in adipocytes.J. Biol. Chem. 2005; 280: 19146-19155Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), or perilipin-5 (5Wolins N.E. Quaynor B.K. Skinner J.R. Tzekov A. Croce M.A. Gropler M.C. Varma V. Yao-Borengasser A. Rasouli N. Kern P.A. et al.OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.Diabetes. 2006; 55: 3418-3428Crossref PubMed Scopus (244) Google Scholar) antibodies. The secondary antibodies were goat Anti-Guinea Pig Alexa 488 and Donkey Anti-Rabbit Alexa 594 (Invitrogen, CAT# A-11073, CAT #A-21207), respectively. Images were captured on a Nikon Eclipse TE2000U by using a 60× oil objective lens on a photometric cool-snap camera (Nikon Instruments) driven by Metamorph software (UIC, Downingtown, PA). Appropriate filters were used to image the signals from the two secondary antibodies separately.Adenovirus studiesTo produce an adenovirus expressing a constitutively active form of human SREBP-1a (26Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar), a cDNA fragment (∼1.5 kb) encoding the amino acids 1–460 of human SREBP-1a was generated from poly-A RNA isolated from HepG2 cells by RT-PCR using the following primers: 5′ primer, 5′-GGGAAGCTTGCTCCCTAGGAAGGGCCGTACGAGGCG-3′; and 3′ primer, 5′-GTCTAGACTACTAGTCAGGCTCCGAGTCACTGCCACTGCCAC-3′. The resultant PCR product was subcloned into a TA-cloning vector and subcloned into the Ad-track shuttle vector. For overexpression of PPARγ, a full-length, FLAG-tagged, mouse PPARγ1 cDNA was cloned into the Adtrack vector. The final adenovirus constructs were produced using the AdEasy system as described (27Finck 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 (425) Google Scholar). Mouse hepatocytes were infected with the specified adenovirus at an MOI of 8 as described previously (28Chen 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 (67) Google Scholar). RNA was isolated 48 h after adenoviral infection.Promoter-luciferase reporter studiesHEK-293 cells were maintained in DMEM-10% fetal calf serum. Cells were transfected using calcium-phosphate coprecipitation and luciferase activity in cell lysates determined by Dual-Glo (Promega) assays 60 h after transfection. The Cidea promoter constructs have been previously described (29Viswakarma N. Yu S. Naik S. Kashireddy P. Matsumoto K. Sarkar J. Surapureddi S. Jia Y. Rao M.S. Reddy J.K. Transcriptional regulation of Cidea, mitochondrial cell death-inducing DNA fragmentation factor alpha-like effector A, in mouse liver by peroxisome proliferator-activated receptor alpha and gamma.J. Biol. Chem. 2007; 282: 18613-18624Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and contain -1385 (pCID2), -888 (pCID3), or -578 (pCID4) of the 5′-flanking sequence of the Cidea gene (all notations are relative to the transcriptional start site). Cidea promoter constructs were cotransfected with expression constructs driving expression of the caSREBP-1 ((26), generous gift of Jay Horton) or empty vector control and SV40-driven renilla luciferase expression construct. All values are normalized to 1.0 and firefly luciferase values were corrected to renilla luciferase activity.ChIP analysesChromatin immunoprecipitation (ChIP) experiments were performed as previously described (27Finck 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 (425) Google Scholar). Hepatocytes from WT mice were dissociated with collagenase, plated, and infected with adenovirus to express caSREBP-1 and/or GFP. After 24 h of infection, hepatocytes were cross-linked in 1% formaldehyde for 15 min. Chromatin-bound proteins were immunoprecipitated using antibodies directed against SREBP-1 or IgG control. PCR primers (supplementary Table I) were designed to amplify a region of the Cidea gene promoter identified in the promoter deletion series as being responsive to SREBP-1 or an exon of the Acadm gene (27Finck 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 (425) Google Scholar, 30Dongol B. Shah Y. Kim I. Gonzalez F.J. Hunt M.C. The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter.J. Lipid Res. 2007; 48: 1781-1791Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).Statistical analysesStatistical comparisons were made using ANOVA or t-test. All data are presented as means ± SEM, with a statistically significant difference defined as a P value < 0.05.RESULTSTime-course of hepatic steatosis in neonatal fld miceWe sought to evaluate the hepatic histological and biochemical phenotype of young fld mice at 3 day intervals. Histological examination of hepatic sections from WT and fld mice at P8 and P11 revealed virtual replacement of the parenchyma by hepatocytes distended with tiny fat droplets. Hepatocytes had a "foamy" appearance, which is a morphologic appearance consistent with microvesicular steatosis (Fig. 1). Hepatic triglyceride content was strikingly elevated 30-fold and 40-fold at P8 and P11, respectively, in fld mice compared with WT mice (Table 1). By P14, microvesicular LD were greatly reduced in fld mice. However, occasional large lipid droplets remained present and hepatic TG content remained elevated 8-fold compared with WT. At P17, fld and WT liver sections were indistinguishable in biochemical TG and histological analysesFig. 1Lipid droplets in fld mouse liver exhibit a microvesicular distribution. Representative hematoxylin and eosin -stained liver sections from WT and fld mice at postnatal day 8 (P8), P11, P14, and P17 are shown. As can be noted, the marked microvesicular steatosis in P8 and 11 is gone in P14 and P17. In p14 mice, only scattered hepatocytes with macrovesicular fat droplets were noted, whereas P17 mice livers were indistinguishable from WT. (Hematoxylin and eosin; 20×).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Hepatic TG content in WT and fld mice at indicated postnatal dayTG contentWTflddaymg liver TG/gmg liver TG/gP84.97 ± 3.22159.62 ± 71.54aP < 0.05.P115.42 ± 4.35195.63 ± 100.83aP < 0.05.P144.94 ± 0.6737.50 ± 16.43aP < 0.05.P173.71 ± 1.075.31 ± 3.27a P < 0.05. Open table in a new tab PAT protein expression is increased in steatotic fld mouse liverLDP expression is induced in many models of hepatic steatosis secondary to obesity or lipodystrophy (16Motomura W. Inoue M. Ohtake T. Takahashi N. Nagamine M. Tanno S. Kohgo Y. Okumura T. Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet.Biochem. Biophys. Res. Commun. 2006; 340: 1111-1118Crossref PubMed Scopus (128) Google Scholar, 18Matsusue K. Kusakabe T. Noguchi T. Takiguchi S. Suzuki T. Yamano S. Gonzalez F.J. Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27.Cell Metab. 2008; 7: 302-311Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 31Bell M. Wang H. Chen H. McLenithan J.C. Gong D.W. Yang R.Z. Yu D. Fried S.K. Quon M.J. Londos C. et al.Consequences of lipid droplet coat protein downregulation in liver cells: abnormal lipid droplet metabolism and induction of insulin resistance.Diabetes. 2008; 57: 2037-2045Crossref PubMed Scopus (147) Google Scholar, 32Imai Y. Varela G.M. Jackson M.B. Graham M.J. Crooke R.M. Ahima R.S. Reduction of hepatosteatosis and lipid levels by an adipose differentiation-related protein antisense oligonucleotide.Gastroenterology. 2007; 132: 1947-1954Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 33Straub B.K. Stoeffel P. Heid H. Zimbelmann R. Schirmacher P. Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis.Hepatology. 2008; 47: 1936-1946Crossref PubMed Scopus (186) Google Scholar), but whether these genes are regulated in fld mice is unknown. Of the five PAT genes, only the hepatic expression of perilipin-2 mRNA
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