Protease-Activated Receptor 1 and Hematopoietic Cell Tissue Factor Are Required for Hepatic Steatosis in Mice Fed a Western Diet
2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.07.015
ISSN1525-2191
AutoresKaren M. Kassel, A. Phillip Owens, Cheryl E. Rockwell, Bradley P. Sullivan, Ruipeng Wang, Ossama Tawfik, Guodong Li, Grace L. Guo, Nigel Mackman, James P. Luyendyk,
Tópico(s)Liver Disease and Transplantation
ResumoNonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet–induced NAFLD has not been investigated. Using an established mouse model of Western diet–induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell–derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor–deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet–induced NAFLD has not been investigated. Using an established mouse model of Western diet–induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell–derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor–deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet. Obesity is a risk factor for the development of metabolic syndrome (MetS).1Larter C.Z. Chitturi S. Heydet D. Farrell G.C. A fresh look at NASH pathogenesis, part 1: the metabolic movers.J Gastroenterol Hepatol. 2010; 25: 672-690Crossref PubMed Scopus (146) Google Scholar Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of MetS, is estimated to affect at least 25% of the Western population.2Clark J.M. Brancati F.L. Diehl A.M. 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Hepatic steatosis, obesity, and the metabolic syndrome are independently and additively associated with increased systemic inflammation.Arterioscler Thromb Vasc Biol. 2011; 31: 1927-1932Crossref PubMed Scopus (117) Google Scholar, 12Targher G. Bertolini L. Poli F. Rodella S. Scala L. Tessari R. Zenari L. Falezza G. Nonalcoholic fatty liver disease and risk of future cardiovascular events among type 2 diabetic patients.Diabetes. 2005; 54: 3541-3546Crossref PubMed Scopus (482) Google Scholar and diabetes.5Kotronen A. Yki-Jarvinen H. Fatty liver: a novel component of the metabolic syndrome.Arterioscler Thromb Vasc Biol. 2008; 28: 27-38Crossref PubMed Scopus (677) Google Scholar Many studies examining the development of steatosis focus on changes in lipid metabolism. In patients with NAFLD, increased hepatic free fatty acid uptake, de novo synthesis of free fatty acids in the liver, and the conversion of free fatty acids to triglycerides that are stored in hepatocytes8Fabbrini E. Sullivan S. Klein S. Obesity and nonalcoholic fatty liver disease: biochemical, metabolic, and clinical implications.Hepatology. 2010; 51: 679-689Crossref PubMed Scopus (1354) Google Scholar, 13Choi S.S. Diehl A.M. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease.Curr Opin Lipidol. 2008; 19: 295-300Crossref PubMed Scopus (199) Google Scholar yield a liver histologic profile typified by macrovesicular and microvesicular steatosis.14Tiniakos D.G. Vos M.B. Brunt E.M. Nonalcoholic fatty liver disease: pathology and pathogenesis.Annu Rev Pathol. 2010; 5: 145-171Crossref PubMed Scopus (637) Google Scholar, 15Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Yeh M. McCullough A.J. Sanyal A.J. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7112) Google Scholar Although hepatocytes synthesize and store lipids in NAFLD, several studies indicate that proinflammatory cytokines, such as tumor necrosis factor α (TNFα) and monocyte chemoattractant protein-1 (MCP-1), are responsible for mediating changes in gene expression that contribute to the development of steatosis.16Stienstra R. Saudale F. Duval C. Keshtkar S. Groener J.E. van Rooijen N. Staels B. Kersten S. Muller M. Kupffer cells promote hepatic steatosis via interleukin-1β-dependent suppression of peroxisome proliferator-activated receptor α activity.Hepatology. 2010; 51: 511-522Crossref PubMed Scopus (327) Google Scholar, 17Huang W. Metlakunta A. Dedousis N. Zhang P. Sipula I. Dube J.J. Scott D.K. O'Doherty R.M. Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance.Diabetes. 2010; 59: 347-357Crossref PubMed Scopus (374) Google Scholar, 18Tamura Y. Sugimoto M. Murayama T. Minami M. Nishikaze Y. Ariyasu H. Akamizu T. Kita T. Yokode M. Arai H. C-C chemokine receptor 2 inhibitor improves diet-induced development of insulin resistance and hepatic steatosis in mice.J Atheroscler Thromb. 2010; 17: 219-228Crossref PubMed Scopus (71) Google Scholar, 19Rull A. Rodriguez F. Aragones G. Marsillach J. Beltran R. Alonso-Villaverde C. Camps J. Joven J. Hepatic monocyte chemoattractant protein-1 is upregulated by dietary cholesterol and contributes to liver steatosis.Cytokine. 2009; 48: 273-279Crossref PubMed Scopus (41) Google Scholar, 20Wobser H. Dorn C. Weiss T.S. Amann T. Bollheimer C. Buttner R. Scholmerich J. Hellerbrand C. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells.Cell Res. 2009; 19: 996-1005Crossref PubMed Scopus (172) Google Scholar For example, expression of the fatty acid transporter CD36 in livers of obese mice is MCP-1 dependent.21Tamura Y. Sugimoto M. Murayama T. Ueda Y. Kanamori H. Ono K. Ariyasu H. Akamizu T. Kita T. Yokode M. Arai H. Inhibition of CCR2 ameliorates insulin resistance and hepatic steatosis in db/db mice.Arterioscler Thromb Vasc Biol. 2008; 28: 2195-2201Crossref PubMed Scopus (110) Google Scholar These studies indicate that inflammatory mediators are among the necessary stimuli for lipid accumulation in hepatocytes. One potential modifier of the hepatic inflammatory response in NAFLD is the coagulation cascade. The coagulation cascade is initiated by tissue factor (TF), resulting in generation of the serine protease thrombin. Thrombin cleaves circulating fibrinogen to fibrin monomers that are cross-linked into insoluble fibrin clots.22Mackman N. Tissue-specific hemostasis in mice.Arterioscler Thromb Vasc Biol. 2005; 25: 2273-2281Crossref PubMed Scopus (110) Google Scholar In addition to its classic role in hemostasis, thrombin also triggers intracellular signaling by activating protease-activated receptor 1 (PAR-1), a tethered ligand G protein–coupled receptor.23Coughlin S.R. How the protease thrombin talks to cells.Proc Natl Acad Sci U S A. 1999; 96: 11023-11027Crossref PubMed Scopus (522) Google Scholar Obesity and MetS are associated with coagulation cascade activation in patients, as indicated by increased thrombin generation.24Fritsch P. Kleber M. Rosenkranz A. Fritsch M. Muntean W. Mangge H. Reinehr T. Haemostatic alterations in overweight children: associations between metabolic syndrome, thrombin generation, and fibrinogen levels.Atherosclerosis. 2010; 212: 650-655Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Romano M. Guagnano M.T. Pacini G. Vigneri S. Falco A. Marinopiccoli M. Manigrasso M.R. Basili S. Davi G. Association of inflammation markers with impaired insulin sensitivity and coagulative activation in obese healthy women.J Clin Endocrinol Metab. 2003; 88: 5321-5326Crossref PubMed Scopus (104) Google Scholar, 26Sola E. Navarro S. Medina P. Vaya A. Estelles A. Hernandez-Mijares A. Espana F. Activated protein C levels in obesity and weight loss influence.Thromb Res. 2009; 123: 697-700Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Moreover, weight loss is associated with decreased TF expression and a reduction in thrombin generation.27Ay L. Kopp H.P. Brix J.M. Ay C. Quehenberger P. Schernthaner G.H. Pabinger I. Schernthaner G. Thrombin generation in morbid obesity: significant reduction after weight loss.J Thromb Haemost. 2010; 8: 759-765Crossref PubMed Scopus (81) Google Scholar, 28Kopp C.W. Kopp H.P. Steiner S. Kriwanek S. Krzyzanowska K. Bartok A. Roka R. Minar E. Schernthaner G. Weight loss reduces tissue factor in morbidly obese patients.Obes Res. 2003; 11: 950-956Crossref PubMed Scopus (84) Google Scholar An imbalance in hepatic synthesis of several coagulation factors causing a procoagulant state has been observed in patients with NAFLD.9Targher G. Chonchol M. Miele L. Zoppini G. Pichiri I. Muggeo M. Nonalcoholic fatty liver disease as a contributor to hypercoagulation and thrombophilia in the metabolic syndrome.Semin Thromb Hemost. 2009; 35: 277-287Crossref PubMed Scopus (118) Google Scholar, 10Cigolini M. Targher G. Agostino G. Tonoli M. Muggeo M. De Sandre G. Liver steatosis and its relation to plasma haemostatic factors in apparently healthy men: role of the metabolic syndrome.Thromb Haemost. 1996; 76: 69-73PubMed Google Scholar, 29Targher G. Bertolini L. Scala L. Zoppini G. Zenari L. Falezza G. Non-alcoholic hepatic steatosis and its relation to increased plasma biomarkers of inflammation and endothelial dysfunction in non-diabetic men: role of visceral adipose tissue.Diabet Med. 2005; 22: 1354-1358Crossref PubMed Scopus (151) Google Scholar, 30de Larranaga G. Wingeyer S.P. Graffigna M. Belli S. Bendezu K. Alvarez S. Levalle O. Fainboim H. Plasma plasminogen activator inhibitor-1 levels and nonalcoholic fatty liver in individuals with features of metabolic syndrome.Clin Appl Thromb Hemost. 2008; 14: 319-324Crossref PubMed Scopus (14) Google Scholar, 31Kotronen A. Joutsi-Korhonen L. Sevastianova K. Bergholm R. Hakkarainen A. Pietilainen K.H. Lundbom N. Rissanen A. Lassila R. Yki-Jarvinen H. Increased coagulation factor VIII, IX, XI and XII activities in non-alcoholic fatty liver disease.Liver Int. 2011; 31: 176-183Crossref PubMed Scopus (79) Google Scholar Increased coagulation cascade activation in patients with NAFLD is often viewed as potentially increasing the risk of cardiovascular events. In contrast, the possibility that the coagulation cascade acts as a critical determinant of diet-induced hepatic steatosis has not been investigated in detail. One potential mechanism whereby thrombin could contribute to fatty liver disease is by increasing the production of inflammatory mediators responsible for steatosis. PAR-1 contributes to TNFα and MCP-1 expression in mice fed a methionine/choline–deficient (MCD) diet.32Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The MCD diet elicits hepatic changes consistent with nonalcoholic steatohepatitis, but it is not an appropriate model of NAFLD associated with MetS.33Rinella M.E. Green R.M. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.J Hepatol. 2004; 40: 47-51Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar The mechanism of hepatic steatosis triggered by an MCD diet is vastly different from that triggered by a Western diet. For example, MCP-1 contributes to steatosis in mice fed a Western diet18Tamura Y. Sugimoto M. Murayama T. Minami M. Nishikaze Y. Ariyasu H. Akamizu T. Kita T. Yokode M. Arai H. C-C chemokine receptor 2 inhibitor improves diet-induced development of insulin resistance and hepatic steatosis in mice.J Atheroscler Thromb. 2010; 17: 219-228Crossref PubMed Scopus (71) Google Scholar, 19Rull A. Rodriguez F. Aragones G. Marsillach J. Beltran R. Alonso-Villaverde C. Camps J. Joven J. Hepatic monocyte chemoattractant protein-1 is upregulated by dietary cholesterol and contributes to liver steatosis.Cytokine. 2009; 48: 273-279Crossref PubMed Scopus (41) Google Scholar but does not contribute to steatosis in mice fed an MCD diet.34Kassel K.M. Guo G.L. Tawfik O. Luyendyk J.P. Monocyte chemoattractant protein-1 deficiency does not affect steatosis or inflammation in livers of mice fed a methionine-choline-deficient diet.Lab Invest. 2010; 90: 1794-1804Crossref PubMed Scopus (34) Google Scholar In agreement with these studies, we showed previously that PAR-1 deficiency did not affect steatosis in mice fed an MCD diet, despite marked inhibition of MCP-1 expression.32Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The role of the coagulation cascade in regulating inflammation and steatosis in Western diet–induced NAFLD is not known. We tested the hypothesis that coagulation cascade activation is increased in mice fed a Western diet and contributes to the development of steatosis. We evaluated the possibility that thrombin signaling through PAR-1 contributes to liver inflammation and steatosis in mice fed a Western diet using PAR-1–deficient mice. Moreover, using bone marrow transplantation, we tested the hypothesis that TF expressed by hematopoietic cells contributes to hepatic coagulation, inflammation, and steatosis in mice fed a Western diet. Wild-type C57Bl/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME) were fed a control diet [AIN-93M (10% kcal from fat); Dyets Inc., Bethlehem, PA] or a Western diet [Diet #100244 (40% kcal from milk fat); Dyets Inc.] ad libitum for 12 weeks. PAR-1+/+ and PAR-1−/− mice35Connolly A.J. Suh D.Y. Hunt T.K. Coughlin S.R. Mice lacking the thrombin receptor, PAR1, have normal skin wound healing.Am J Pathol. 1997; 151: 1199-1204PubMed Google Scholar backcrossed eight generations onto a C57Bl/6J background were maintained by homozygous breeding. Age-matched PAR-1+/+ and PAR-1−/− mice were fed a control diet or a Western diet ad libitum for 12 weeks. Average food intake was measured weekly for each cage of mice, and mice were weighed weekly. Bone marrow transplantation was performed as described previously.36Pawlinski R. Wang J.G. Owens III, A.P. Williams J. Antoniak S. Tencati M. Luther T. Rowley J.W. Low E.N. Weyrich A.S. Mackman N. Hematopoietic and nonhematopoietic cell tissue factor activates the coagulation cascade in endotoxemic mice.Blood. 2010; 116: 806-814Crossref PubMed Scopus (135) Google Scholar Male 8-week-old low-density lipoprotein receptor–deficient (LDLr−/−) mice were irradiated with 11 Gy (two doses of 550 rad each, 4 hours apart) using a Cs137 irradiator (JL Shepherd, San Fernando, CA). Irradiated mice were repopulated with bone marrow cells isolated from heterozygous control mice (mTF+/−hTF+ mice; n = 10 recipients) or "low TF" mice (mTF−/−hTF+ mice; n = 9 recipients) via retro-orbital injection of 2 × 106 cells. Low-TF mice express human TF at a level approximately 1% of wild-type mice, and this low level of expression rescues the embryonic lethality.37Parry G.C. Erlich J.H. Carmeliet P. Luther T. Mackman N. Low levels of tissue factor are compatible with development and hemostasis in mice.J Clin Invest. 1998; 101: 560-569Crossref PubMed Scopus (181) Google Scholar Four weeks after injection of donor cells, mice were fed a Western diet [Teklad TD.88137 (40% kcal from milk fat); Harlan Laboratories, Madison, WI] ad libitum for 12 weeks. This diet has a formulation identical to Diet #100244 from Dyets Inc. The bone marrow of recipient mice was genotyped to verify successful repopulation of donor cells as described previously.37Parry G.C. Erlich J.H. Carmeliet P. Luther T. Mackman N. Low levels of tissue factor are compatible with development and hemostasis in mice.J Clin Invest. 1998; 101: 560-569Crossref PubMed Scopus (181) Google Scholar All the studies were approved by the Animal Care and Use Committee of the different institutions and complied with National Institutes of Health guidelines. Under isoflurane anesthesia, blood was collected from the caudal vena cava into sodium citrate (final concentration, 0.38%) for the collection of plasma and into an empty syringe for the collection of serum. Citrated whole blood was subjected to centrifugation at 4000 × g for 10 minutes, plasma was collected, and the buffy coat was collected into TRI Reagent (Molecular Research Center Inc., Cincinnati, OH). Sections of liver from the left lateral lobe were fixed in 10% neutral-buffered formalin for 48 hours and embedded in paraffin. The right medial lobe was affixed to a cork using optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA) and immersed for approximately 3 minutes in liquid nitrogen–chilled isopentane. The remaining liver was snap frozen in liquid nitrogen. Paraffin-embedded livers were sectioned at 5 μm and were stained with H&E. The severity of liver steatosis was evaluated by a pathologist (O.T.) in a blinded manner. Oil Red O staining was performed as previously described.38Tanaka Y. Aleksunes L.M. Yeager R.L. Gyamfi M.A. Esterly N. Guo G.L. Klaassen C.D. NF-E2-related factor 2 inhibits lipid accumulation and oxidative stress in mice fed a high-fat diet.J Pharmacol Exp Ther. 2008; 325: 655-664Crossref PubMed Scopus (199) Google Scholar Plasma thrombin-antithrombin (TAT) levels were determined using a commercially available enzyme-linked immunosorbent assay kit (Siemens Healthcare Diagnostics, Deerfield, IL). Lipids were extracted from 100 mg of snap-frozen liver as described,32Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and hepatic and serum triglyceride and cholesterol levels were determined using commercially available reagents (Pointe Scientific Inc., Canton, MI; and Wako, Richmond, VA). Total RNA was isolated from the buffy coat of blood samples (ie, white blood cells) and from approximately 100 mg of snap-frozen liver using TRI Reagent (Molecular Research Center Inc.) per the manufacturer's protocol. One microgram of RNA was used for the synthesis of cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and a C1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA). Levels of stearoyl-CoA desaturase 1 (SCD1), CD36, sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-α (PPARα), fatty acid synthase (FAS), TNFα, MCP-1, TF mRNA, and 18S rRNA were determined using either TaqMan gene expression assays (Applied Biosystems) or TaqMan PrimeTime quantitative PCR assays (IDT, Coralville, IA), iTaq + ROX supermix (Bio-Rad Laboratories), and a StepOnePlus thermal cycler (Applied Biosystems). Mouse CD36 (NM_007643) primer sequences were 5′-GCGACATGATTAATGGCACAG-3′ (forward primer), 5′-GATCCGAACACAGCGTAGATAG-3′ (reverse primer), and 5′-/56-FAM/CAACAAAAG/ZEN/GTGGAAAGGAGGCTGC/3IABkFQ/-3′ (probe). Mouse SCD1 (NM_009127) primer sequences were 5′-CTGACCTGAAAGCCGAGAAG-3′ (forward primer), 5′-AGAAGGTGCTAACGAACAGG-3′ (reverse primer), and 5′-/56-FAM/TGTTTACAA/ZEN/AAGTCTCGCCCCAGCA/3IABkFQ/-3′ (probe). Mouse SREBP-1c (NM_011480) primer sequences were 5′-CCATCGACTACATCCGCTTC-3′ (forward primer), 5′-GCCCTCCATAGACACATCTG-3′ (reverse primer), and 5′-/56-FAM/TCTCCTGCT/ZEN/TGAGCTTCTGGTTGC/3IABkFQ/-3′ (probe). Mouse FAS (NM_007988) primer sequences were 5′-CCCCTCTGTTAATTGGCTCC-3′ (forward primer), 5′-TTGTGGAAGTGCAGGTTAGG-3′ (reverse primer), and 5′-/56-FAM/CAGGCTCAG/ZEN/GGTGTCCCATGTT/3IABkFQ/-3′ (probe). Mouse PPARα (NM_011144) primer sequences were 5′-CATTTCCCTGTTTGTGGCTG-3′ (forward primer), 5′-ATCTGGATGGTTGCTCTGC-3′ (reverse primer), and 5′-/56-FAM/ATAATTTGC/ZEN/TGTGGAGATCGGCCTGG/3IABkFQ/-3′ (probe). Mouse TF (F3) (NM_010171) primer sequences were 5′-CAGTTCATGGAGACGGAGAC-3′ (forward primer), 5′-CAACCACGTTCAGTTTTCTACC-3′ (reverse primer), and 5′-/56-FAM/AGACACAAA/ZEN/CCTCGGACAGCCAG/3IABkFQ/-3′ (probe). 18S (NM_003286) primer sequences were 5′-CTGTAGCCCTGTACTTCATCG-3′ (forward primer), 5′-CTACCACATATTCCTGACCATCC-3′ (reverse primer), and 5′-/56-FAM/CCTTCCTCC/ZEN/TTTTCATTGCCTGCTCT/3IABkFQ/-3′ (probe). CD36, SCD1, FAS, SREBP-1c, PPARα, TF, and 18S primers and probes were purchased from IDT. Mouse TNFα and MCP-1 gene expression assays were purchased from Applied Biosystems (TNFα, Assay ID Mm00443258_m1; MCP-1, Assay ID Mm00441242_m1). The expression of each gene was adjusted relative to 18S expression levels, and the relative expression level was determined using the comparative CT method. Frozen livers were sectioned at 8 μm for staining. Macrophages were identified in liver by CD68 and F4/80 staining, which was performed as previously described.32Luyendyk J.P. Sullivan B.P. Guo G.L. Wang R. Tissue factor-deficiency and protease activated receptor-1-deficiency reduce inflammation elicited by diet-induced steatohepatitis in mice.Am J Pathol. 2010; 176: 177-186Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Immunofluorescent staining for insoluble fibrin was performed as described previously39Copple B.L. Banes A. Ganey P.E. Roth R.A. Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure.Toxicol Sci. 2002; 65: 309-318Crossref PubMed Scopus (68) Google Scholar using a rabbit anti-human fibrinogen antibody (A0080; Dako, Carpinteria, CA). The primary antibody was detected by the addition of goat anti-rabbit IgG conjugated to Alexa 594 (Invitrogen, Carlsbad, CA). Slides were then washed and counterstained with DAPI (Invitrogen). Total protein was isolated from approximately 100 mg of snap-frozen liver using PBS containing 0.1% Triton X-100 and Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA). Samples were rotated for 30 minutes at 4°C and then were subjected to centrifugation at 12,000 × g for 15 minutes at 4°C. Protein concentrations were determined using a DC protein assay (ThermoFisher Scientific). The concentrations of TNFα and MCP-1 in 225 μg of protein were determined using commercially available DuoSet enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN). The concentration of TNFα and MCP-1 protein in serum was determined using a MilliPlex mouse metabolic magnetic bead kit (Millipore, Billerica, MA) and a Bio-Plex 200 System (Bio-Rad Laboratories). Phycoerythrin and xMAP bead fluorescence was detected by a dual-laser detector (532 and 635 nm) and Bio-Plex Manager 5.0 software (Bio-Rad Laboratories). Total protein was isolated from approximately 100 mg of snap-frozen liver using radioimmunoprecipitation assay buffer containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific). Protein concentrations were determined using a DC protein assay (ThermoFisher Scientific). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad Laboratories), and transferred to Immobilon polyvinylidene difluoride membrane (Millipore) by semidry transfer. The membranes were blocked for 1 hour at room temperature in 3% bovine serum albumin in Tris-buffered saline with Tween-20 and incubated overnight at 4°C with either anti-CD36 (1:1000) antibody or anti-PPARα (1:1000) antibody (BioVision, Mountain View, CA) diluted in 1% bovine serum albumin in Tris-buffered saline with Tween-20 or anti-SCD1 (1:1000) antibody (Cell Signaling Technology Inc., Danvers, MA) diluted in 5% bovine serum albumin in Tris-buffered saline with Tween-20. Membranes were then washed with Tris-buffered saline with Tween-20 and incubated for 1 hour with anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:1000) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Membranes were incubated with West Pico electrochemiluminescence reagent (ThermoFisher Scientific) before exposure to Blue Lite autoradiography film (ISC Bioexpress, Kaysville, UT). Before the addition of antibodies, membranes were stained with Ponceau S for 15 minutes and were destained with ddH2O to confirm equal loading of protein, as previously described.40Behari J. Yeh T.H. Krauland L. Otruba W. Cieply B. Hauth B. Apte U. Wu T. Evans R. Monga S.P. Liver-specific β-catenin knockout mice exhibit defective bile acid and cholesterol homeostasis and increased susceptibility to diet-induced steatohepatitis.Am J Pathol. 2010; 176: 744-753Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 41Cieply B. Zeng G. Proverbs-Singh T. Geller D.A. Monga S.P. Unique phenotype of hepatocellular cancers with exon-3 mutations in β-catenin gene.Hepatology. 2009; 49: 821-831Crossref PubMed Scopus (120) Google Scholar Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad Laboratories). Statistics were performed using SigmaPlot 11.0 software (Systat Software, Inc., San Jose, CA). Comparison of two groups was performed using a Student's t-test. Comparison of three or more groups was performed using two-way analysis of variance followed by the Student-Newman-Keuls post hoc test for multiple comparisons. Data are expressed as mean ± SEM. The criterion for s
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