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• W1534724641 abstract "Insulin resistance induces nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). We used a high-fat, high-calorie solid diet (HFD) to create a model of insulin resistance and NASH in nongenetically modified rats and to study the relationship between visceral adipose tissue and liver. Obesity and insulin resistance occurred in HFD rats, accompanied by a progressive increase in visceral adipose tissue tumor necrosis factor (TNF)-α mRNA and in circulating free fatty acids. HFD also decreased adiponectin mRNA and peroxisome proliferator-activated receptor (PPAR)-α expression in the visceral adipose tissue and the liver, respectively, and induced hepatic insulin resistance through TNF-α-mediated c-Jun N-terminal kinase (JNK)-dependent insulin receptor substrate-1Ser307 phosphorylation. These modifications lead to hepatic steatosis accompanied by oxidative stress phenomena, necroinflammation, and hepatocyte apoptosis at 4 weeks and by pericentral fibrosis at 6 months. Supplementation of n-3 polyunsaturated fatty acid, a PPARα ligand, to HFD-treated animals restored hepatic adiponectin and PPARα expression, reduced TNF-α hepatic levels, and ameliorated fatty liver and the degree of liver injury. Thus, our model mimics the most common features of NASH in humans and provides an ideal tool to study the role of individual pathogenetic events (as for PPARα down-regulation) and to define any future experimental therapy, such as n-3 polyunsaturated fatty acid, which ameliorated the degree of liver injury. Insulin resistance induces nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). We used a high-fat, high-calorie solid diet (HFD) to create a model of insulin resistance and NASH in nongenetically modified rats and to study the relationship between visceral adipose tissue and liver. Obesity and insulin resistance occurred in HFD rats, accompanied by a progressive increase in visceral adipose tissue tumor necrosis factor (TNF)-α mRNA and in circulating free fatty acids. HFD also decreased adiponectin mRNA and peroxisome proliferator-activated receptor (PPAR)-α expression in the visceral adipose tissue and the liver, respectively, and induced hepatic insulin resistance through TNF-α-mediated c-Jun N-terminal kinase (JNK)-dependent insulin receptor substrate-1Ser307 phosphorylation. These modifications lead to hepatic steatosis accompanied by oxidative stress phenomena, necroinflammation, and hepatocyte apoptosis at 4 weeks and by pericentral fibrosis at 6 months. Supplementation of n-3 polyunsaturated fatty acid, a PPARα ligand, to HFD-treated animals restored hepatic adiponectin and PPARα expression, reduced TNF-α hepatic levels, and ameliorated fatty liver and the degree of liver injury. Thus, our model mimics the most common features of NASH in humans and provides an ideal tool to study the role of individual pathogenetic events (as for PPARα down-regulation) and to define any future experimental therapy, such as n-3 polyunsaturated fatty acid, which ameliorated the degree of liver injury. Nonalcoholic fatty liver disease (NAFLD) is commonly associated with the clinical features of the metabolic syndrome such as obesity, type II diabetes, and dislipidemia.1Brunt E Nonalcoholic steatohepatitis.Semin Liver Dis. 2004; 24: 3-20Crossref PubMed Scopus (451) Google Scholar The clinical importance of NAFLD is due to its high prevalence (25% of the general population) and its wide spectrum of histological damage ranging from simple steatosis, which is generally nonprogressive, to nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis, hepatocellular carcinoma, and liver failure.1Brunt E Nonalcoholic steatohepatitis.Semin Liver Dis. 2004; 24: 3-20Crossref PubMed Scopus (451) Google Scholar, 2Bellentani S Saccoccio G Masutti F Croce LS Brandi G Sasso F Cristanini G Tiribelli C Prevalence of and risk factors for hepatic steatosis in Northern Italy.Ann Intern Med. 2000; 132: 112-117Crossref PubMed Scopus (1055) Google ScholarInsulin resistance is central to the pathogenesis of the metabolic syndrome, and recent data indicate that NAFLD should be considered the hepatic manifestation of the metabolic syndrome.3Marchesini G Bugianesi E Forlani G Cerrelli F Lenzi M Manini R Natale S Vanni E Villanova N Melchionda N Rizzetto M Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.Hepatology. 2003; 37: 917-923Crossref PubMed Scopus (2165) Google Scholar It has been demonstrated that a series of molecular alterations in insulin signaling occurs in the setting of insulin resistance, finally resulting in triglyceride accumulation in the liver. The insulin receptor belongs to a subfamily of receptor tyrosine kinases that includes insulin-like growth factor-1 and insulin receptor-related receptor. After insulin binding, its receptor undergoes autophosphorylation and catalyzes the tyrosine phosphorylation of cellular proteins such as members of the insulin receptor substrate (IRS) family. In addition to tyrosine phosphorylation, IRS proteins undergo serine phosphorylation, which may attenuate signaling by decreasing insulin-stimulated tyrosine phosphorylation. Several kinases have been implicated in this process, including phosphatidylinositol 3-kinase (PI3K)/Akt, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway.4Saltiel A Kahn CR Insulin signalling and the regulation of glucose and lipid metabolism.Nature. 2001; 414: 799-806Crossref PubMed Scopus (3846) Google ScholarThe study of the pathogenetic or therapeutic factors involved in NASH has been hampered by the absence of a suitable experimental model because most studies use rodents with genetic defects or involve feeding rats a diet lacking in choline and methionine (MCD), creating a nutritional deficiency that is not common in patients with NASH.5Koteish A Diehl A Animal models of steatosis.Semin Liver Dis. 2001; 21: 89-104Crossref PubMed Scopus (408) Google Scholar Recently a high-fat liquid diet, given to normal rats for a short period of time, was shown to cause steatosis, inflammation, oxidative stress, and increased collagen synthesis.6Lieber C Leo M Mak K Xu Y Cao Q Ren C Ponomarenko A DeCarli L Model of nonalcoholic steatohepatitis.Am J Clin Nutr. 2004; 79: 502-509Crossref PubMed Scopus (357) Google Scholar However, the liquid diet represents a time-consuming system to be diffusely used as a model of NASH, and the relationship between the liver and other organs involved in the energy balance, such as adipose tissue, was not investigated.6Lieber C Leo M Mak K Xu Y Cao Q Ren C Ponomarenko A DeCarli L Model of nonalcoholic steatohepatitis.Am J Clin Nutr. 2004; 79: 502-509Crossref PubMed Scopus (357) Google Scholar In this regard, the adipocyte is no longer regarded as a passive depot for storing excess energy in the form of triglyceride but as a cell that actively regulates the energy balance and that secretes proinflammatory cytokines, such as TNF-α, that are able to influence insulin sensitivity and metabolic processes in peripheral tissues, liver included.7Browning J Horton J Molecular mediators of hepatic steatosis and liver injury.J Clin Invest. 2004; 114: 147-152Crossref PubMed Scopus (1721) Google Scholar This occurs via specific transcription factors, such as the nuclear hormone receptor family of peroxisome proliferator-activated receptors (PPARs), which regulate both inflammation and lipid metabolism. The PPARs are nuclear receptors that bind to fatty acid-derived ligands and activate the transcription of genes that regulate lipid metabolism. Three PPAR isoforms have been described: α, δ (or β), and γ. PPARγ is found in adipocytes, macrophages, and muscle cells, where it regulates adipocyte differentiation, fatty acid uptake and storage, and glucose metabolism; PPARγ expression is low in tissues that express predominantly PPARα. The primary sites of action of PPARα are liver, heart, muscle, and kidney. PPARα activates a program of target gene expression involved in fatty acid uptake, β-oxidation, transport into peroxisomes, and ω-oxidation of unsaturated fatty acids. Several experimental studies suggest that PPARα might increase fatty acid catabolism and that, in the pathophysiologic context of the metabolic syndrome or high-fat diet, PPARα-induced fatty acid catabolism might prevent hepatic fat deposition.8Shulman AI Mangelsdorf D Retinoid X receptor heterodimers in the metabolic syndrome.N Engl J Med. 2005; 353: 604-615Crossref PubMed Scopus (330) Google Scholar, 9Yki-Jarvinen H Thiazolidinediones.N Engl J Med. 2004; 351: 1106-1118Crossref PubMed Scopus (1880) Google Scholar, 10Ip E Farrell GC Robertson G Hall P Kirsch R Leclercq I Central role of PPARalpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice.Hepatology. 2003; 38: 123-132Crossref PubMed Scopus (384) Google Scholar, 11Ip E Farrell G Hall P Robertson G Leclercq I Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice.Hepatology. 2004; 39: 1286-1296Crossref PubMed Scopus (301) Google Scholar The hepatic expression and the finding that different compounds such as fibrates and n-3 polyunsaturated fatty acids (PUFA) act as its agonists make PPARα an attractive target in the pathophysiology and treatment of fatty liver.12Clarke S Nonalcoholic steatosis and steatohepatitis: I. Molecular mechanism for polyunsaturated fatty acid regulation of gene transcription.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G865-G869PubMed Google ScholarWe thus took advantage of a high-fat, high-calorie solid diet13Surwit R Feinglos M Rodin J Sutherland A Petro A Opara E Kuhn C Rebuffe-Scrive M Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice.Metabolism. 1995; 44: 645-651Abstract Full Text PDF PubMed Scopus (529) Google Scholar to pursue the aims of our study 1) to create a model of insulin resistance and NASH in nongenetically modified animals, 2) to study the relationship between the visceral adipose tissue and the liver in this model, and 3) to evaluate the possible anti-steatogenic effects of n-3 PUFA as natural ligands of PPARα.Materials and MethodsReagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. The following antibodies were used: anti-diphosphorylated extracellular signal-regulated kinase (ERK)1/2 (pERK) (1:5000 final dilution), anti-ERK2 (1:1000), anti-Akt (1:2000), anti-Ser-473 phosphorylated Akt (pAkt) (1:1000), anti-insulin receptor substrate-1 (IRS-1), anti-JNK2 (1:1000), anti-Thr-183/Tyr185 phosphorylated JNK (pJNK) (1:800), anti-PPARα (1:400) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Ser-307 phosphorylated IRS-1 (IRS-1Ser307) (Cell Signaling Technology, Danvers, MA), and anti-β-actin (Sigma Chemical Co.).Male Sprague-Dawyley CD rats (100 to 120 g body weight) (Charles River Laboratories, Como, Italy) were fed either a control pellet diet (5% of energy derived from fat, 18% from proteins, and 77% from carbohydrates; 3.3 kcal/g) (Harlan Italy, San Pietro al Natisone, UD, Italy) or a high-fat pellet diet (HFD) (58% of energy derived from fat, 18% from protein, and 24% from carbohydrates; 5.6 kcal/g) (Laboratorio Dottori Piccioni, Gessate, Milano, Italy).13Surwit R Feinglos M Rodin J Sutherland A Petro A Opara E Kuhn C Rebuffe-Scrive M Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice.Metabolism. 1995; 44: 645-651Abstract Full Text PDF PubMed Scopus (529) Google Scholar The amount of lipids was provided by saturated (0.9 and 30.4 g/100 g diet) and unsaturated (4.6 and 5.3 g/100 g diet) fat with n-6-to-n-3 PUFA ratios of 11.3 and 85.9 in the control and HFD diet, respectively. Body weight and food intake were measured weekly. Rats were sacrificed after 1, 3, and 6 months of treatment (n = 6 for each time point). Each experiment was performed following the guidelines of the local committee for care and use of laboratory animals.Preparation of Tissue and Histological EvaluationPortal blood was obtained before liver removal. After liver removal, the epididymal fat was removed to measure visceral adipose tissue, and its weight and volume were recorded.14Barzilai N She L Liu B Vuguin P Cohen P Wang J Rossetti L Surgical removal of visceral fat reverses hepatic insulin resistance.Diabetes. 1999; 48: 94-98Crossref PubMed Scopus (332) Google Scholar The degree of necroinflammatory liver injury was quantitated according to Brunt.1Brunt E Nonalcoholic steatohepatitis.Semin Liver Dis. 2004; 24: 3-20Crossref PubMed Scopus (451) Google Scholar The parenchymal extension of fibrotic tissue was determined by morphometry as previously described.15Benedetti A Di Sario A Casini A Ridolfi F Bendia E Pigini P Tonnini C D'Ambrosio L Feliciangeli G Macarri G Svegliati-Baroni G Inhibition of the NA+/H+ exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study.Gastroenterology. 2001; 120: 545-556Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Plasma insulin, glucose, ALT, tumor necrosis factor-α (TNF-α), adiponectin, and free fatty acid (FFA) levels were determined by commercially available kits.Lipid Content of the LiverHepatic lipids were extracted as previously described.16Folch J Lees M Sloane Stanley GH A simple method for the isolation and purification of total lipids from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar The lipid extracts were resuspended in methanol and used for the evaluation of cholesterol and triglycerides levels by commercial kits from Sigma Chemical Co.Measurement of Thiobarbituric Acid Reactive Substances (TBARS) and Lipid Hydroperoxides in the LiverThe levels of hepatic lipid peroxides were spectrophotometrically determined by the thiobarbituric acid method.17Ohkawa H Ohishi N Yagi K Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem. 1979; 95: 351-358Crossref PubMed Scopus (22774) Google Scholar A malondialdehyde solution freshly made by hydrolysis of 1,1,3,3-tetrametoxypropane was used as standard. The results were expressed as nanomoles of malondialdehyde per gram of liver. The levels of hepatic lipid hydroperoxides were evaluated using the xylenol orange assay as previously described.18Hermes-Lima M Willmore W Storey K Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation.Free Radic Biol Med. 1995; 19: 271-280Crossref PubMed Scopus (412) Google Scholar A solution of t-butylhydroperoxide solution (7.3 × 10−4 mol/L) was used as standard. The results were expressed as nanomoles of lipid hydroperoxides per gram of liver. In parallel experiments TBARS and hydroperoxides levels were evaluated either in the absence or in the presence of the antioxidant butyl-hydroxy-toluene (BHT; 1 mmol/L) to block spontaneous and additional generation of reactive oxygen species.Reverse Transcription-Polymerase Chain Reaction (RT-PCR)RNA isolation, reverse transcription, and PCR for TNF-α and adiponectin mRNA were performed as described previously.19Schoemaker MH Gommans WM de la Rosa LC Homan M Klok P Trautwein C van Goor H Poelstra K Haisma HJ Jansen PL Moshage H Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20Ding X Saxena NK Lin S Xu A Srinivasan S Anania FA The roles of leptin and adiponectin: a novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology.Am J Pathol. 2005; 166: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar For every PCR assay, expression of β-actin was used as an internal control. Results were visualized by agarose electrophoresis.Western BlottingWestern blot on total liver proteins or nuclear extracts was performed as previously described.21Svegliati-Baroni G Ridolfi F Caradonna Z Alvaro D Marzioni M Saccomanno S Candelaresi C Trozzi L Macarri G Benedetti A Folli F Regulation and crosstalk of ERK/JNK/P70s6k in two rat models of liver injury and fibrosis: an in vivo and in vitro study.J Hepatol. 2003; 39: 528-537Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Lemberger T Saladin R Vazquez M Assimacopoulos F Staels B Desvergnet B Wahli W Auwerz J Expression of the peroxisome proliferator-activated receptor a gene is stimulated by stress and follows a diurnal rhythm.J Biol Chem. 1996; 271: 1764-1769Crossref PubMed Scopus (281) Google Scholar After electrophoresis on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transfer to a nitrocellulose membrane, immunoblotting was performed according to the manufacturer's instructions for each primary antibody. After electrotransfer, equal loading of the proteins was checked by staining the blots with 0.2% Ponceau-S Red.22Lemberger T Saladin R Vazquez M Assimacopoulos F Staels B Desvergnet B Wahli W Auwerz J Expression of the peroxisome proliferator-activated receptor a gene is stimulated by stress and follows a diurnal rhythm.J Biol Chem. 1996; 271: 1764-1769Crossref PubMed Scopus (281) Google Scholar Immunoreactive proteins were then detected by enhanced chemiluminescence system according to the manufacturer's instructions (ECL; Amersham Biosciences Europe GmbH, Cologno Monzese, Italy). The intensity of the bands was then quantified by scanning video densitometry (Kodak Digital Sciences, Rochester, NY) and expressed as arbitrary units.ImmunoprecipitationTissue samples (5 mg) were lysed in radio-immunoprecipitation assay buffer. Immunoprecipitation was performed for 2 hours at 4°C with 2 μg of an antibody against total IRS-1 (final dilution 1:100) according to manufacturer's instructions. Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and then immunoblotted with an antibody against total IRS-1 or IRS-1Ser307.Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nicked End Labeling (TUNEL) AssayFor in situ detection of apoptosis at the single cell level, we used the TUNEL method (ApopTag kit; Oncor, Gaithersburg, MD). The specimens were examined using a computerized image analysis system (Cue 3; Galai Production Ltd., Migdal Haemek, Israel) connected to an Olympus microscope (Olympus Vanox AHBT3, Olympus Optical Co. Ltd., Tokyo, Japan), and data were expressed as the number of TUNEL-positive cells per field (×100).23Feldstein A Canbay A Angulo P Taniai M Burgart L Lindor K Gores G Hepatocyte apoptosis and Fas expression are prominent features of human nonalcoholic steatohepatitis.Gastroenterology. 2003; 125: 437-443Abstract Full Text Full Text PDF PubMed Scopus (824) Google ScholarMeasurement of n-6-to-n-3 PUFA Ration-6 and n-3 PUFA were measured by means of a gas-chromatographic technique coupled with selected ion monitoring equipment (Agilent Technologies, Cernusco sul Naviglio, Milan, Italy) as elsewhere described, and data were expressed as nanograms per milliliter.24Araya J Rodrigo R Videla LA Thielemann L Orellana M Pettinelli P Poniachik J Increase in long-chain polyunsaturated fatty acid n-6/n-3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease.Clin Sci (Lond). 2004; 106: 635-643Crossref PubMed Scopus (521) Google ScholarJNK Phosphorylation in VitroHepatocytes were isolated from normal CD Sprague-Dawley rats (200 to 250 g body weight) as previously described.25Svegliati Baroni G D'Ambrosio L Ferretti G Casini A Di Sario A Salzano R Ridolfi F Saccomanno S Jezequel A Benedetti A Fibrogenic effect of oxidative stress on rat hepatic stellate cells.Hepatology. 1998; 27: 720-726Crossref PubMed Scopus (261) Google Scholar Hepatocytes were then plated in six-well plates (0.5 × 106 cells/well) coated with rat type I collagen in Waymouth's medium containing 10% fetal bovine serum, 0.1 mmol/L insulin, and 0.1 mmol/L dexamethasone in a humidified atmosphere at 37°C and 5% CO2. After 3 hours, the culture was washed with phosphate-buffered saline, and cells were incubated overnight with hormone-free medium containing 0.2 mmol/L l-glutamine, 5 mg/ml transferrin, 1 nmol/L selenium, and 10 nmol/L free fatty acids in RPMI. Cells were then incubated with TNF-α (30 ng/ml) or insulin (100 nmol/L) for 30 minutes and lysed in radio-immunoprecipitation assay buffer as previously described.26Schwabe R Uchinami H Qian T Bennett B Lemasters J Brenner D Differential requirement for c-Jun NH2-terminal kinase in TNFalpha- and Fas-mediated apoptosis in hepatocytes.FASEB J. 2004; 18: 720-722Crossref PubMed Scopus (135) Google Scholar, 21Svegliati-Baroni G Ridolfi F Caradonna Z Alvaro D Marzioni M Saccomanno S Candelaresi C Trozzi L Macarri G Benedetti A Folli F Regulation and crosstalk of ERK/JNK/P70s6k in two rat models of liver injury and fibrosis: an in vivo and in vitro study.J Hepatol. 2003; 39: 528-537Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Cell extracts (50 μg/lane) were then used for Western blotting as indicated above.Statistical AnalysisResults are expressed as mean ± SD. Group means were compared by analysis of variance followed by the Student-Newman-Keuls test whether the former was significant. A P value of <0.05 was considered statistically significant.ResultsGeneral Effects of Control and High-Fat DietsRats fed HFD consumed significantly more calories on a “per day” basis than controls (Figure 1A), and this was associated with a progressive increase in rat body weight compared with control animals (Figure 1B). The increase in body weight was associated with the development of visceral obesity, as shown by the progressive increase in epididymal fat weight and volume (Figure 1C; data not shown) and by the development of insulin resistance, as shown by higher insulin and glucose values in the portal blood compared with controls (Table 1). Thus, these data indicate that the HFD diet is able to induce obesity and insulin resistance in nongenetically modified animals.Table 1Effect of the HFD on Insulin, Glucose, and ALT Values in the Portal BloodBiochemical parametersControls1 month3 months6 monthsInsulin (μg/L)0.2 ± 0.030.7 ± 0.08*P < 0.05 versus controls.0.7 ± 0.05*P < 0.05 versus controls.0.9 ± 0.01*P < 0.05 versus controls.Glucose (mg/dl)111.8 ± 12.3147.1 ± 18.9*P < 0.05 versus controls.151.0 ± 15.7*P < 0.05 versus controls.203.5 ± 18.2*P < 0.05 versus controls.ALT (IU/ml)39.3 ± 4.297.6 ± 14.7132.1 ± 12.7148.4 ± 19.8* P < 0.05 versus controls. Open table in a new tab Effect of HFD on Visceral Adipose Tissue Synthesis of TNF-α and on Secretion of TNF-α and FFAsIt has been hypothesized that several factors released from the hypertrophic visceral adipose tissue could contribute to the development of hepatic insulin resistance.7Browning J Horton J Molecular mediators of hepatic steatosis and liver injury.J Clin Invest. 2004; 114: 147-152Crossref PubMed Scopus (1721) Google Scholar To study this in our model, we measured TNF-α and adiponectin synthesis in the visceral adipose tissue as well as TNF-α, adiponectin, and FFA levels in the portal blood. By RT-PCR, single bands of 705 and 108 bp for TNF-α and adiponectin mRNA, respectively, were observed in the epididymal adipose tissue of control rats. The level of TNF-α mRNA expression was strikingly elevated in adipose tissue obtained from HFD animals compared with controls. When normalized to the β-actin mRNA bands, a twofold increase was evident at 1 month, followed by a progressive increase up to 6 months (Figure 2, A and B). This increased synthesis was associated with higher TNF-α levels in the portal blood (Figure 2C). In parallel, the intensity of the mRNA adiponectin band decreased as early as 1 month. This reduction peaked at 6 months (60% of control value) and led to a constant decrease in adiponectin serum levels (Figure 2, B and C). Finally, enhanced levels of FFA were observed in the portal blood after 1 month of HFD, again progressively increasing and peaking at 6 months of treatment (Figure 2D).Figure 2Effect of HFD on TNF-α and adiponectin mRNA levels (A and B), levels of TNF-α and adiponectin in serum blood (C), and levels of FFA in serum blood (D). No differences were observed between controls at the different time points, which were thus considered as a single control group. A representative agarose gel of RT-PCR for TNF-α and adiponectin mRNA is shown in A. Gray lines, TNF-α; black lines, adiponectin; white bars, FFA. Data are presented as mean ± SD. *P < 0.05 versus controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of HFD on PPARα ExpressionIn the PPAR family, the α-isoform is involved in hepatic lipid metabolism and is regulated by adipokines such as TNF-α and adiponectin.8Shulman AI Mangelsdorf D Retinoid X receptor heterodimers in the metabolic syndrome.N Engl J Med. 2005; 353: 604-615Crossref PubMed Scopus (330) Google Scholar, 27Beier K Volkl A Fahimi DH TNF-alpha downregulates the peroxisome proliferator activated receptor-alpha and the mRNAs encoding peroxisomal proteins in rat liver.FEBS Lett. 1997; 412: 385-387Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28You M Considine RV Leone TC Kelly DP Crabb DW Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice.Hepatology. 2005; 42: 568-577Crossref PubMed Scopus (220) Google Scholar We thus measured PPARα protein expression in liver nuclear extracts from control and HFD animals. PPARα was readily detected in the liver of control rats, and its level was significantly reduced (by 20%) at 1 month of HFD, reaching 40% of control values at 6 months (Figure 3).Figure 3Effect of HFD on PPARα expression in whole-liver homogenates. No differences were observed between controls at the different time points, which were thus considered as a single control group. A representative Western blot is shown. White bar, controls; black bars, HFD-treated animals. Data are presented as mean ± SD. *P < 0.05 versus controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of HFD on Deposition of Lipid and Production of TBARS and Lipid HydroperoxidesInsulin resistance and decreased PPARα expression were associated with liver steatosis (as shown in Figure 4, A and B). Fatty liver was quantified by measuring triglyceride and cholesterol content in the liver, both of which were increased 1 month after HFD (Figure 4, C and D). This was associated with increased hepatic production of TBARS and lipid hydroperoxides, measured as expression of lipid peroxidation (Figure 4, E and F). Interestingly, hepatic TBARS and hydroperoxide production showed a highly significant linear correlation with both cholesterol and triglyceride liver content (r = 0.78 and 0.63 for triglyceride and r = 0.67 and 0,.84 for cholesterol content versus TBARS and lipid hydroperoxides production, respectively). To evaluate the role of spontaneous and additional generation of reactive oxygen species, TBARS and hydroperoxide levels were evaluated in parallel experiments in both the absence and the presence of the antioxidant BHT (1 mmol/L). The addition of BHT did not modify the differences between controls and HFD-treated animals in TBARS and hydroperoxide levels (data not shown).Figure 4H&E staining in control (A) and HFD-treated (B) rat for 3 months. The effect of HFD on triglyceride and cholesterol content in the liver (C and D) and on TBARS and hydroperoxide production (E and F). No alterations were observed in the liver of the rats fed the control diet (A), whereas HFD induced pronounced hepatic steatosis and inflammatory cells infiltrate surrounding steatotic hepatocytes (lipogranuloma). White bars, controls; black bars, HFD-treated animals. Data are presented as mean ± SD. *P < 0.05 versus controls. Final magnification, ×100.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of HFD on Intracellular Signaling PathwaysIt has been reported that insulin resistance can be associated with specific alterations in the intracellular signaling pathways, such as Ser307 phosphorylation of IRS-1, and that this process can be mediated in vitro by specific kinases such as PI3K/Akt, PKC, and the MAPK system.4Saltiel A Kahn CR Insulin signalling and the regulation of glucose and lipid metabolism.Nature. 2001; 414: 799-806Crossref PubMed Scopus (3846) Google Scholar To elucidate this in the liver in vivo, we took advantage of using antibodies specifically recognizing protein phosphorylation, which parallels enzyme activation21Svegliati-Baroni G Ridolfi F Caradonna Z Alvaro D Marzioni M Saccomanno S Candelaresi C Trozzi L Macarri G Benedetti A Folli F Regulation and crosstalk of ERK/JNK/P70s6k in two rat models of liver injury and fibrosis: an in vivo and in vitro study.J Hepatol. 2003; 39: 528-537Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar (Figure 5, A–D). In rats fed the control diet, no differences were observed in ERK, Akt, and JNK phosphorylation at 1, 3, and 6 months. Animals were thus considered as a single control group. In HFD animals, despite the increase in" @default.
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• W1534724641 title "A Model of Insulin Resistance and Nonalcoholic Steatohepatitis in Rats" @default.
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• W1534724641 doi "https://doi.org/10.2353/ajpath.2006.050953" @default.
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