PPARα activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes
2005; Elsevier BV; Volume: 47; Issue: 2 Linguagem: Inglês
10.1194/jlr.m500203-jlr200
ISSN1539-7262
AutoresUlrika Edvardsson, Anna Ljungberg, Daniel Lindén, Lena William‐Olsson, Helena Peilot-Sjögren, Andrea Ahnmark, Jan Oscarsson,
Tópico(s)Lipid metabolism and biosynthesis
ResumoAdipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor α (PPARα) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARα activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARα activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor α (PPARα) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARα activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARα activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP. Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that plays a key role in the regulation of genes involved in carbohydrate, lipid, and lipoprotein metabolism (for review, see 1Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr. Rev. 1999; 20: 649-688Google Scholar). PPARα is highly expressed in tissues with high mitochondrial and peroxisomal β-oxidation activities, such as liver, heart, kidney, and skeletal muscle (2Braissant O. Foufelle F. Scotto C. Dauça M. Wahli W. 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Li L. Gustafsson M. Borén J. Olofsson S.O. Influence of peroxisome proliferator-activated receptor alpha agonists on the intracellular turnover and secretion of apolipoprotein (Apo) B-100 and ApoB-48.J. Biol. Chem. 2002; 277: 23044-23053Google Scholar, 17Lamb R.G. Koch J.C. Bush S.R. An enzymatic explanation of the differential effects of oleate and gemfibrozil on cultured hepatocyte triacylglycerol and phosphatidylcholine biosynthesis and secretion.Biochim. Biophys. Acta. 1993; 1165: 299-305Google Scholar). However, in vivo in rats, incorporation of palmitate into liver triglycerides was unchanged, whereas triglyceride secretion decreased (15Petit D. Bonnefis M.T. Rey C. Infante R. Effects of ciprofibrate and fenofibrate on liver lipids and lipoprotein synthesis in normo- and hyperlipidemic rats.Atherosclerosis. 1988; 74: 215-225Google Scholar), indicating that other effects of PPARα activation than decreased triglyceride biosynthesis gave rise to the decreased triglyceride secretion. One suggested possibility is that PPARα activation increases diacylglycerol acyltransferase (DGAT) activity in the cytoplasm while inhibiting DGAT activity in the microsomal compartment, thereby diverting triglycerides away from the secretory pathway without influencing total cellular triglyceride synthesis (18Waterman I.J. Zammit V.A. Differential effects of fenofibrate or simvastatin treatment of rats on hepatic microsomal overt and latent diacylglycerol acyltransferase activities.Diabetes. 2002; 51: 1708-1713Google Scholar). Adipose differentiation-related protein (ADRP) is a lipid storage droplet-associated protein belonging to the PAT (Perilipin, ADRP, and TIP 47) family (19Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells.Semin. Cell Dev. Biol. 1999; 10: 51-58Google Scholar). ADRP was first identified as an early marker of adipocyte differentiation (20Jiang H.P. Serrero G. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.Proc. Natl. Acad. Sci. USA. 1992; 89: 7856-7860Google Scholar). However, studies have shown that ADRP is expressed in a variety of tissues and cultured cells (21Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J. Lipid Res. 1997; 38: 2249-2263Google Scholar, 22Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.Cell Tissue Res. 1998; 294: 309-321Google Scholar). It has been suggested to be a marker of lipid accumulation, as the cellular protein level of ADRP is related to the total mass of neutral lipids within the cell (21Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J. Lipid Res. 1997; 38: 2249-2263Google Scholar, 23Steiner S. Wahl D. Mangold B.L.K. Robison R. Raymackers J. Meheus L. Anderson N.L. Cordier A. Induction of the adipose differentiation-related protein in liver of etomoxir-treated rats.Biochem. Biophys. Res. Commun. 1996; 218: 777-782Google Scholar). A few studies have explored the function of ADRP. Overexpression of ADRP in fibroblasts resulted in lipid accumulation and lipid droplet formation without induction of adipogenic genes (24Imamura M. Inoguchi T. Ikuyama S. Taniguchi S. Kobayashi K. Nakashima N. Nawata H. ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts.Am. J. Physiol. Endocrinol. Metab. 2002; 283: E775-E783Google Scholar). Also in macrophages, ADRP overexpression gave rise to lipid accumulation without changed expression of the genes involved in lipid efflux (25Larigauderie G. Furman C. Jaye M. Lasselin C. Copin C. Fruchart J.C. Castro G. Rouis M. Adipophilin enhances lipid accumulation and prevents lipid efflux from THP-1 macrophages: potential role in atherogenesis.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 504-510Google Scholar). Furthermore, transfection of COS-7 cells with ADRP was shown to promote the uptake of long-chain fatty acids (26Gao J. Serrero G. Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake.J. Biol. Chem. 1999; 274: 16825-16830Google Scholar). Together, these results indicate that ADRP increases intracellular lipids without changing the expression of genes involved in lipid metabolism, but the mechanisms of this effect are still unclear. ADRP expression has been reported to be regulated by long-chain fatty acids at the transcriptional level (27Gao J. Ye H. Serrero G. Stimulation of adipose differentiation related protein (ADRP) expression in adipocyte precursors by long-chain fatty acids.J. Cell. Physiol. 2000; 182: 297-302Google Scholar). In macrophages and in colorectal cancer cells, ADRP expression was induced by agonists of PPAR subtypes α, γ, and δ (28Vosper H. Patel L. Graham T.L. Khoudoli G.A. Hill A. Macphee C.H. Pinto I. Smith S.A. Suckling K.E. Wolf C.R. et al.The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages.J. Biol. Chem. 2001; 276: 44258-44265Google Scholar, 29Hodgkinson C.P. Ye S. Microarray analysis of peroxisome proliferator-activated receptor-gamma induced changes in gene expression in macrophages.Biochem. Biophys. Res. Commun. 2003; 308: 505-510Google Scholar, 30Gupta R.A. Brockman J.A. Sarraf P. Willson T.M. DuBois R.N. Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells.J. Biol. Chem. 2001; 276: 29681-29687Google Scholar). Peroxisome proliferator-activated receptor response elements (PPREs) were recently identified both in the murine and human ADRP promoters (31Chawla A. Lee C.H. Barak Y. He W. Rosenfeld J. Liao D. Han J. Kang H. Evans R.M. PPARdelta is a very low-density lipoprotein sensor in macrophages.Proc. Natl. Acad. Sci. USA. 2003; 100: 1268-1273Google Scholar, 32Targett-Adams P. McElwee M.J. Ehrenborg E. Gustafsson M.C. Palmer C.N. McLauchlan J. A PPAR response element regulates transcription of the gene for human adipose differentiation-related protein.Biochim. Biophys. Acta. 2005; 1728: 95-104Google Scholar), showing that PPAR agonists regulate ADRP expression by increasing transcription of the gene. However, the regulation of ADRP by PPARα in liver and hepatocytes has not been investigated. The aims of this study were to investigate the effects of the specific PPARα agonist Wy14,643 (Wy) (33Ljung B. Bamberg K. Dahllöf B. Kjellstedt A. Oakes N.D. Östling J. Svensson L. Camejo G. AZ 242, a novel PPARalpha/gamma agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats.J. Lipid Res. 2002; 43: 1855-1863Google Scholar) on the expression of ADRP in mouse liver in vivo and in primary mouse hepatocytes in vitro and to determine the importance of changed ADRP expression for the effects of PPARα activation on triglyceride secretion and intracellular triglyceride accumulation. C57BL/6 mice were from Taconic Europe (Ry, Denmark). Homozygous PPARα null mice and corresponding wild-type control mice, on a pure Sv/129 genetic background, were used for in vitro experiments (kindly provided by Dr. F. J. Gonzalez, National Institutes of Health, Bethesda, MD) (34Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.Mol. Cell. Biol. 1995; 15: 3012-3022Google Scholar). PPARα null mice and littermate controls, backcrossed for two generations with C57BL/6, were used for in vivo experiments. The animals were housed individually and maintained under standardized conditions of temperature (21–22°C) and humidity (40–60%), with light from 6:00 AM to 6:00 PM for at least 1 week before the experiments. The mice were given either standard laboratory chow containing (energy %) 12% fat, 62% carbohydrates, and 26% protein, with a total energy content of 12.6 kJ/g (R3; Lactamin AB, Kimstad, Sweden), or a high-fat diet containing 48% fat (mainly saturated), 15% protein, and 37% carbohydrates, with a total energy content of 21.4 kJ/g (Lactamin). The mice were fed laboratory chow or high-fat diet for 3 weeks and treated with Wy (30 μmol/kg/day; Chemsyn Science Laboratories, Lenaxa, KS) in 0.5% (w/v) methyl cellulose by gavage once daily for the last 2 weeks. Age-matched control mice received only vehicle. Food intake was estimated by weighing the food twice weekly. The mice were anesthetized with isoflurane (Forene; Abbot Scandinavia AB), and the livers were removed, immediately frozen in liquid nitrogen, and stored at − 70°C. The study protocol was approved by the Ethics Committee of Göteborg University. All experiments were conducted in accordance with accepted standards of humane animal care. Frozen livers were homogenized in isopropanol (1 ml/50 mg tissue) and incubated at 4°C for 1 h. The samples were centrifuged at 4°C for 5 min at 2,500 rpm, and triglyceride concentrations in the supernatants were measured with an enzymatic colorimetric assay (Roche, Mannheim, Germany). A pBluescript SK(+) vector containing full-length ADRP was kindly provided by Björn Magnusson (Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, Göteborg, Sweden). The ADRP construct was then transferred to a pENTR vector (Invitrogen, Carlsbad, CA) and recombined into pAd/CMV/V5-DEST (Invitrogen) according to the manufacturer's manual. The packaging cell line Ad-293 (Stratagene, La Jolla, CA) was grown in Dulbecco's modified Eagle's medium with 10% FBS supplemented with 100,000 IU/l penicillin and 100 mg/l streptomycin. Cells were seeded in 25 cm2 culture flasks, cultured to 90–95% confluence, and transfected with adenoviral constructs for ADRP or the control zsGreen (35Lindén D. William-Olsson L. Rhedin M. Asztely A.K. Clapham J.C. Schreyer S. Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis.J. Lipid Res. 2004; 45: 1279-1288Google Scholar) digested with Pac-1 using Lipofectamine 2000 in Opti-MEM according to the manufacturer's manual. After 4 h of incubation, DMEM containing 20% FBS was added, resulting in a concentration of 10% FBS. Cells were cultured for 10–14 days until cytopathic effects were ∼80%. Viruses were harvested by repeated freeze/thaw cycles in 10 mM Tris-HCl, pH 8.0. After large-scale amplification using Cell Factories (Nunc, Rochester, NY), recombinant adenoviruses were purified by two rounds of CsCl density gradient ultracentrifugation. The purified virus stocks were desalted over 10DG columns (Bio-Rad, Hercules, CA) and eluted in sterile PBS. Glycerol (65%) was added (1:5 dilution with virus suspension) before the virus stocks were divided into aliquots and stored at −80°C until use. Infectious viral titers were determined using the Adeno-X Rapid Titer Kit (Clontech, Palo Alto, CA). All purified virus stocks were screened for possible wild-type virus contamination according to Zhang, Koch, and Roth (36Zhang W.W. Koch P.E. Roth J.A. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.Biotechniques. 1995; 18: 444-447Google Scholar) before use. Mouse hepatocytes were obtained by nonrecirculating collagenase perfusion through the portal vein of the mice (10–16 weeks of age) as described (16Lindén D. Lindberg K. Oscarsson J. Claesson C. Asp L. Li L. Gustafsson M. Borén J. Olofsson S.O. Influence of peroxisome proliferator-activated receptor alpha agonists on the intracellular turnover and secretion of apolipoprotein (Apo) B-100 and ApoB-48.J. Biol. Chem. 2002; 277: 23044-23053Google Scholar, 37Carlsson L. Nilsson I. Oscarsson J. Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin.Endocrinology. 1998; 139: 2699-2709Google Scholar, 38Lindén D. Alsterholm M. Wennbo H. Oscarsson J. PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.J. Lipid Res. 2001; 42: 1831-1840Google Scholar). In brief, the cells were seeded at 100,000 cells/cm2 in dishes (Falcon, Plymouth, UK) coated with laminin-rich matrigel (BD Biosciences, Bedford, MA). The cells were cultured during the first 16–18 h in Williams' E medium with Glutamax (Invitrogen) supplemented as described (37Carlsson L. Nilsson I. Oscarsson J. Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin.Endocrinology. 1998; 139: 2699-2709Google Scholar). The cells were then treated for up to 3 days with 1 or 10 μM Wy (Chemsyn Science Laboratories) dissolved in DMSO [final concentration, 0.15% (v/v)] in medium supplemented as described above plus 1 nM dexamethasone (Sigma, St. Louis, MO) and 3 nM insulin (Actrapid; Novo Nordisk A/S). In experiments with adenoviral overexpression of ADRP or the control zsGreen, cells were infected with virus (500 infectious units/cell) in 0.75 ml of culture medium (10 cm2 dish) starting 4 h after seeding. Two hours later, medium was added to a final volume of 2 ml and infection was continued overnight. After 17 h of infection with virus, the medium was replaced with virus-free medium. Analyses were performed 3 days after infection. Total RNA of cultured primary hepatocytes and mouse liver was isolated with TriReagent™ (Sigma) according to the manufacturer's protocol, and the concentration of RNA was determined spectrophotometrically at 260 nm. To remove contaminating DNA, total RNA was treated with DNA free (Ambion, Austin, TX) before being retrotranscribed using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Quantitative real-time PCR analysis was performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) using SYBR Green detection chemistry. All samples were analyzed in triplicate. To exclude that the amplification-associated fluorescence was associated with residual genomic DNA and/or from the formation of primer dimers, controls without reverse transcriptase or DNA template were analyzed. RT-PCR products were also analyzed by electrophoresis in ethidium bromide-stained agarose gels to check that a single amplicon of the expected size was obtained. The expression data were normalized to the endogenous control acidic ribosomal phosphoprotein P0 (36B4). The expression of 36B4 was not influenced by the various treatments in this study. The relative expression levels were calculated according to the formula 2−ΔCt, where ΔCt is the difference in threshold cycle (Ct) values between the target and the 36B4 endogenous control. Specific primers for each gene (Table 1) were designed using Primer Express software (Applied Biosystems).TABLE 1.Primers used for quantitative real-time PCRGeneForward Primer (5′–3′)Reverse Primer (5′–3′)ADRPTGGCAGCAGCAGTAGTGGATCAGGTTGGCCACTCTCATCACPT-ITGAGTGGCGTCCTCTTTGGCAGCGAGTAGCGCATAGTCATGLCADGCGAAATACTGGGCATCTGAATCCGTGGAGTTGCACACATTACOCAGCAGGAGAAATGGATGCAGGGCGTAGGTGCCAATTATCT36B4GAGGAATCAGATGAGGATATGGGAAAGCAGGCTGACTTGGTTGCACO, acyl-coenzyme A oxidase; ADRP, adipose differentiation-related protein; CPT-I, carnitine palmitoyltransferase I; LCAD, long-chain acyl-coenzyme A dehydrogenase; 36B4, acidic ribosomal phosphoprotein P0. Open table in a new tab ACO, acyl-coenzyme A oxidase; ADRP, adipose differentiation-related protein; CPT-I, carnitine palmitoyltransferase I; LCAD, long-chain acyl-coenzyme A dehydrogenase; 36B4, acidic ribosomal phosphoprotein P0. Matrigel was removed from cultured primary hepatocytes by incubation on ice for 60 min in PBS containing 5 mM EDTA followed by washings in PBS. Total protein extracts from frozen livers and cultured hepatocytes were prepared as described previously (39Améen C. Oscarsson J. Sex difference in hepatic microsomal triglyceride transfer protein expression is determined by the growth hormone secretory pattern in the rat.Endocrinology. 2003; 144: 3914-3921Google Scholar), and protein concentrations were determined with the RC/DC Protein Assay Kit II (Bio-Rad). Proteins were separated on 10–20% Tris-glycine gels (Invitrogen) and transferred to Hybond-P polyvinylidene difluoride transfer membrane (Amersham Biosciences, Bucks, UK). Equal loading was confirmed by staining the membranes with 0.2% Ponceau S (Serva, Heidelberg, Germany). Immunoblotting was performed using a guinea pig polyclonal anti-ADRP antibody at 1:2,000 (Research Diagnostics, Inc., Flanders, NJ) and a horseradish peroxidase-conjugated anti-guinea pig antibody at 1:20,000 (Dako, Glostrup, Denmark), followed by detection using the enhanced chemiluminescence plus detection system (Amersham Biosciences). Band intensity was quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Williams' E medium with Glutamax, supplemented as described above, containing [9,10(n)-3H]palmitic acid was prepared as described by Leung and Ho (40Leung K.C. Ho K.K. Stimulation of mitochondrial fatty acid oxidation by growth hormone in human fibroblasts.J. Clin. Endocrinol. Metab. 1997; 82: 4208-4213Google Scholar). To each culture dish (10 cm2), 1 ml of medium containing 110 μM unlabeled palmitic acid and 8.3 μCi of [9,10(n)-3H]palmitic acid (specific activity, 54 Ci/mmol) was added. The fatty acid oxidation was shown to be linear between 30 and 120 min of incubation at 37°C (data not shown). Fatty acid oxidation was thereafter determined after 60 or 90 min of incubation with labeled palmitic acid. Labeled water-soluble products were isolated and analyzed as described previously (35Lindén D. William-Olsson L. Rhedin M. Asztely A.K. Clapham J.C. Schreyer S. Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis.J. Lipid Res. 2004; 45: 1279-1288Google Scholar) but with one additional precipitation step. Background radioactivity was determined by precipitation of fatty acids in medium that had not been in contact with cells. The fatty acid oxidation was related to the DNA content in each culture dish, which was determined according to Labarca and Paigen (41Labarca C. Paigen K. A simple, rapid, and sensitive DNA assay procedure.Anal. Biochem. 1980; 102: 344-352Google Scholar). Triglyceride biosynthesis in cultured hepatocytes was estimated by measurement of incorporated [9,10(n)-3H]palmitic acid (concentration as described above) in cellular triglycerides after 60–90 min of incubation at 37°C. Accumulation of newly synthesized triglycerides in the medium was determined after 2–6 h of incubation with [9,10(n)-3H]palmitic acid at 37°C. Cells and medium were then collected and lipid extraction was performed according to Bligh and Dyer (42Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Med. Sci. 1959; 37: 911-917Google Scholar). Lipids were separated by thin-layer chromatography (silica gel 60 on plastic sheets; Merck, Darmstadt, Germany) with chloroform-acetic acid (96:4). The bands corresponding to triglycerides were recovered and extracted from the silica gel with 1 ml of cyclohexane followed by the addition of 10 ml of scintillation solution (Ready Safe™; Beckman Coulter, Fullerton, CA) before the radioactivity was measured. Triglyceride synthesis and the accumulation of newly synthesized triglycerides in the cell culture medium were related to the DNA content in each culture dish as described above. The secretion of apoB-48 and apoB-100 from primary mouse hepatocyte cultures was estimated by labeling the cells with a [35S]methionine-cysteine mix (Amersham Biosciences) for 2 h followed by a 4 h chase in culture medium supplemented with 10 mM methionine, as described (38Lindén D. Alsterholm M. Wennbo H. Oscarsson J. PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.J. Lipid Res. 2001; 42: 1831-1840Google Scholar, 43Sjöberg A. Oscarsson J. Boström K. Innerarity T.L. Edén S. Olofsson S.O. Effects of growth hormone on apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48 and apoB 100 synthesis and secretion in the rat liver.Endocrinology. 1992; 130: 3356-3364Google Scholar, 44Lindén D. Sjöberg A. Asp L. Carlsson L. Oscarsson J. Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes.Am. J. Physiol. Endocrinol. Metab. 2000; 279: E1335-E1346Google Scholar). Labeled apoB-48 and apoB-100 were isolated by immunoprecipitation with 10 μl of polyclonal rabbit anti-human apoB antibodies (DakoCytomation, Glostrup, Denmark), followed by 5% polyacrylamide gel electrophoresis containing SDS. The bands corresponding to apoB-48 and apoB-100 were quantified using a FLA-3000 phosphorimager (Fujifilm). The densities were related to the total amount of DNA in each culture dish, as described above. Intracellular triglyceride content in hepatocytes was determined by
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