Serotonin Mediates Oxidative Stress and Mitochondrial Toxicity in a Murine Model of Nonalcoholic Steatohepatitis
2007; Elsevier BV; Volume: 133; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2007.05.019
ISSN1528-0012
AutoresAntonio Nocito, Felix Dahm, Wolfram Jochum, Jae Hwi Jang, Panco Georgiev, Michael Bäder, Eberhard L. Renner, Pierre‐Alain Clavien,
Tópico(s)Alcohol Consumption and Health Effects
ResumoBackground & Aims: Nonalcoholic steatohepatitis (NASH) is one of the most common causes of liver enzyme elevation in the West. Its prevalence is likely to increase further, paralleling the epidemic increase of the metabolic syndrome. Serotonin degradation by monoamine oxidase A (MAO-A) was recently implicated as an important source of reactive oxygen species. We therefore tested the pathogenetic role of serotonin in a murine model of diet-induced steatohepatitis. Methods: Wild-type and serotonin-deficient mice, tryptophan hydroxylase 1 (Tph1−/−) were fed a choline-methionine–deficient diet for 2 and 6 weeks. MAO-A was inhibited with clorgyline. Steatosis, hepatocyte injury, and hepatic inflammation were assessed by histology, immunohistochemistry, and biochemical analysis. Expression levels of MAO-A and serotonin transporter were analyzed by reverse-transcription polymerase chain reaction and Western blot. Oxidative stress was detected by measuring lipid peroxidation. Mitochondrial damage was determined by electron microscopy and quantification of cytochrome c release. Results: After choline-methionine–deficient diet, Tph1−/− mice displayed an equal degree of steatosis, yet reduced hepatocellular injury and less severe inflammation. The difference in these NASH-defining features could be attributed to an increased uptake and catabolism of serotonin, yielding enhanced levels of reactive oxygen species and lipid peroxides, which mediated hepatocellular injury by mitochondrial damage and inflammation. Inhibition of MAO-A reduced hepatocellular damage in wild-type mice. Correspondingly, MAO-A expression was up-regulated significantly in human NASH. Conclusions: This study provides evidence that serotonin plays a role in the pathogenesis of steatohepatitis, and therefore might represent a novel target for the prevention and treatment of NASH. Background & Aims: Nonalcoholic steatohepatitis (NASH) is one of the most common causes of liver enzyme elevation in the West. Its prevalence is likely to increase further, paralleling the epidemic increase of the metabolic syndrome. Serotonin degradation by monoamine oxidase A (MAO-A) was recently implicated as an important source of reactive oxygen species. We therefore tested the pathogenetic role of serotonin in a murine model of diet-induced steatohepatitis. Methods: Wild-type and serotonin-deficient mice, tryptophan hydroxylase 1 (Tph1−/−) were fed a choline-methionine–deficient diet for 2 and 6 weeks. MAO-A was inhibited with clorgyline. Steatosis, hepatocyte injury, and hepatic inflammation were assessed by histology, immunohistochemistry, and biochemical analysis. Expression levels of MAO-A and serotonin transporter were analyzed by reverse-transcription polymerase chain reaction and Western blot. Oxidative stress was detected by measuring lipid peroxidation. Mitochondrial damage was determined by electron microscopy and quantification of cytochrome c release. Results: After choline-methionine–deficient diet, Tph1−/− mice displayed an equal degree of steatosis, yet reduced hepatocellular injury and less severe inflammation. The difference in these NASH-defining features could be attributed to an increased uptake and catabolism of serotonin, yielding enhanced levels of reactive oxygen species and lipid peroxides, which mediated hepatocellular injury by mitochondrial damage and inflammation. Inhibition of MAO-A reduced hepatocellular damage in wild-type mice. Correspondingly, MAO-A expression was up-regulated significantly in human NASH. Conclusions: This study provides evidence that serotonin plays a role in the pathogenesis of steatohepatitis, and therefore might represent a novel target for the prevention and treatment of NASH. Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver enzyme level increases in the West.1Ioannou G.N. Boyko E.J. Lee S.P. The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999-2002.Am J Gastroenterol. 2006; 101: 76-82Crossref PubMed Scopus (252) Google Scholar Its prevalence within the normal population has been estimated at 3%–20%, depending on the diagnostic criteria.2Bedogni G. Miglioli L. Masutti F. Tiribelli C. Marchesini G. Bellentani S. Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos nutrition and liver study.Hepatology. 2005; 42: 44-52Crossref PubMed Scopus (1005) Google Scholar, 3Ruhl C.E. Everhart J.E. Relation of elevated serum alanine aminotransferase activity with iron and antioxidant levels in the United States.Gastroenterology. 2003; 124: 1821-1829Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Furthermore, the prevalence of NAFLD is likely to increase further, paralleling the epidemic increase of obesity.4Marchesini 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, 5Mokdad A.H. Ford E.S. Bowman B.A. Dietz W.H. Vinicor F. Bales V.S. Marks J.S. Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001.JAMA. 2003; 289: 76-79Crossref PubMed Scopus (4513) Google Scholar Of note, in a series of obese patients (body mass index >35) undergoing bariatric surgery, more than 90% had NAFLD on liver histology.6Dixon J.B. Bhathal P.S. O’Brien P.E. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese.Gastroenterology. 2001; 121: 91-100Abstract Full Text PDF PubMed Scopus (1149) Google ScholarNAFLD typically is divided into steatosis, thought to be a benign condition, and nonalcoholic steatohepatitis (NASH), which is a progressive disease leading to cirrhosis in 20%–30% of patients within 20 years.7Angulo P. Keach J.C. Batts K.P. Lindor K.D. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis.Hepatology. 1999; 30: 1356-1362Crossref PubMed Scopus (1398) Google Scholar, 8Matteoni C.A. Younossi Z.M. Gramlich T. Boparai N. Liu Y.C. McCullough A.J. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.Gastroenterology. 1999; 116: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar Indeed, NASH seems to be the main cause for cryptogenic cirrhosis,9Bugianesi E. Leone N. Vanni E. Marchesini G. Brunello F. Carucci P. Musso A. De Paolis P. Capussotti L. Salizzoni M. Rizzetto M. Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma.Gastroenterology. 2002; 123: 134-140Abstract Full Text Full Text PDF PubMed Scopus (1248) Google Scholar which carries a grim prognosis.10Ratziu V. Bonyhay L. Di Martino V. Charlotte F. Cavallaro L. Sayegh-Tainturier M.H. Giral P. Grimaldi A. Opolon P. Poynard T. Survival, liver failure, and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis.Hepatology. 2002; 35: 1485-1493Crossref PubMed Scopus (435) Google Scholar Biopsy examination findings mimic the histopathologic picture of alcoholic steatohepatitis in patients lacking a history of significant alcohol consumption.11Ludwig J. Viggiano T.R. McGill D.B. Oh B.J. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.Mayo Clin Proc. 1980; 55: 434-438PubMed Google Scholar Histologic features include the NASH-defining components of hepatocellular injury, steatosis, and mild lobular neutrophilic inflammation, as well as facultative features such as Mallory’s hyaline and megamitochondria.12Brunt E.M. Nonalcoholic steatohepatitis: definition and pathology.Semin Liver Dis. 2001; 21: 3-16Crossref PubMed Scopus (762) Google ScholarConceptually, NASH is thought to result from a 2-hit process.13Day C.P. James O.F. Steatohepatitis: a tale of two “hits”?.Gastroenterology. 1998; 114: 842-845Abstract Full Text Full Text PDF PubMed Scopus (3404) Google Scholar The first hit is the hepatocellular accumulation of fatty acids, which sensitizes the liver to further injury. Oxidative stress acts as a second hit, leading to lipid peroxidation, mitochondrial damage (megamitochondria), hepatocellular injury (ballooning, Mallory bodies), and, finally, to chronic inflammation and fibrosis. In a mouse model of nutritional steatohepatitis, induction of the cytochrome P450 isoenzyme (CYP2E1) was shown to be a major source of reactive oxygen species (ROS), leading to lipid peroxidation and cellular injury.14Leclercq I.A. Farrell G.C. Field J. Bell D.R. Gonzalez F.J. Robertson G.R. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.J Clin Invest. 2000; 105: 1067-1075Crossref PubMed Scopus (660) Google Scholar The pathophysiologic relevance of ROS is underscored further by the protective effects of experimental treatment with antioxidants in a murine model of NASH,15Laurent A. Nicco C. Tran Van Nhieu J. Borderie D. Chereau C. Conti F. Jaffray P. Soubrane O. Calmus Y. Weill B. Batteux F. Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis.Hepatology. 2004; 39: 1277-1285Crossref PubMed Scopus (109) Google Scholar as well as in human disease.16Harrison S.A. Torgerson S. Hayashi P. Ward J. Schenker S. Vitamin E and vitamin C treatment improves fibrosis in patients with nonalcoholic steatohepatitis.Am J Gastroenterol. 2003; 98: 2485-2490Crossref PubMed Scopus (596) Google ScholarSerotonin, named for its vasoconstrictor properties, is a biogenic amine widely appreciated as a neurotransmitter with numerous functions in the central nervous system. Outside the central nervous system, serotonin is produced in intestinal enterochromaffin cells involving the rate-limiting enzyme tryptophan hydroxylase 1, and is stored and distributed by platelets. After its release, serotonin is taken up rapidly by various cell types via the specific membrane-bound serotonin transporter (SERT). Besides its functions in the central nervous system, serotonin also has emerged as a key mediator of various biological processes in peripheral tissues, such as regulation of bowel motility,17Bulbring E. Crema A. Observations concerning the action of 5-hydroxytryptamine on the peristaltic reflex.Br J Pharmacol Chemother. 1958; 13: 444-457Crossref PubMed Scopus (20) Google Scholar cell proliferation,18Balasubramanian S. Paulose C.S. Induction of DNA synthesis in primary cultures of rat hepatocytes by serotonin: possible involvement of serotonin S2 receptor.Hepatology. 1998; 27: 62-66Crossref PubMed Scopus (88) Google Scholar and differentiation.19Battaglino R. Fu J. Spate U. Ersoy U. Joe M. Sedaghat L. Stashenko P. Serotonin regulates osteoclast differentiation through its transporter.J Bone Miner Res. 2004; 19: 1420-1431Crossref PubMed Scopus (178) Google Scholar Our group recently showed serotonin to be a key mediator of liver regeneration.20Lesurtel M. Graf R. Aleil B. Walther D.J. Tian Y. Jochum W. Gachet C. Bader M. Clavien P.A. Platelet-derived serotonin mediates liver regeneration.Science. 2006; 312: 104-107Crossref PubMed Scopus (601) Google ScholarDegradation of serotonin is catalyzed by the mitochondrial enzyme monoamine oxidase A (MAO-A), generating 5-hydroxyindolic acid and ROS such as hydrogen peroxide. ROS generated by MAO-A–mediated catabolism of serotonin were reported recently to play a pivotal role in cardiomyocyte death.21Bianchi P. Kunduzova O. Masini E. Cambon C. Bani D. Raimondi L. Seguelas M.H. Nistri S. Colucci W. Leducq N. Parini A. Oxidative stress by monoamine oxidase mediates receptor-independent cardiomyocyte apoptosis by serotonin and postischemic myocardial injury.Circulation. 2005; 112: 3297-3305Crossref PubMed Scopus (211) Google Scholar Because serotonin is degraded substantially in the liver, we tested whether serotonin-derived ROS play a central role in the pathogenesis of NASH by initiating lipid peroxidation, mitochondrial damage, cellular injury, and inflammation.Materials and MethodsAnimal ExperimentsAll animal experiments were performed in accordance with Swiss federal regulations on animal experimentation and approved by the appropriate local regulatory body (Cantonal Veterinary Office, Zurich, Switzerland). Female C57Bl/6 (Harlan, Horst, The Netherlands) and tryptophan hydroxylase 1 deficient (Tph1−/−) mice (on a C57BL/6 background; own breeding), 8–12 weeks of age, were kept on a 12-hour day/night cycle with free access to food and water. As previously described,22Walther D.J. Peter J.U. Bashammakh S. Hortnagl H. Voits M. Fink H. Bader M. Synthesis of serotonin by a second tryptophan hydroxylase isoform.Science. 2003; 299: 76Crossref PubMed Scopus (1187) Google Scholar Tph1−/− mice have a disrupted gene for tryptophan hydroxylase 1 and therefore lack serotonin outside of the central nervous system. Mice were fed a choline-methionine–deficient (CMD) diet or the corresponding control chow ad libitum (MP Biomedicals, Heidelberg, Germany) for 2 or 6 weeks. Inhibition of MAO-A was achieved by daily intraperitoneal injections of 10 mg/kg clorgyline, which has been reported to cause rapid hepatic MAO-A inhibition of 85%.23Moron J.A. Perez V. Pasto M. Lizcano J.M. Unzeta M. FA-70, a novel selective and irreversible monoamine oxidase-A inhibitor: effect on monoamine metabolism in mouse cerebral cortex.J Pharmacol Exp Ther. 2000; 292: 788-794PubMed Google Scholar Serotonin receptor antagonists were administered twice daily by subcutaneous injections at doses of 3 mg/kg (ketanserine) or 1 mg/kg (SB206553).20Lesurtel M. Graf R. Aleil B. Walther D.J. Tian Y. Jochum W. Gachet C. Bader M. Clavien P.A. Platelet-derived serotonin mediates liver regeneration.Science. 2006; 312: 104-107Crossref PubMed Scopus (601) Google Scholar All substances were from Sigma Aldrich (Buchs, Switzerland) and were dissolved in 0.9% saline. Control groups received corresponding injections of saline. There were 6 mice per group for each condition. Food intake and weight were assessed twice weekly.Serum Levels of TransaminasesBlood samples were obtained before euthanasia under isoflurane anesthesia from the inferior vena cava and immediately centrifuged at 6000 rounds per minute for 6 minutes. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using a serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson Inc., Rochester, NY).Histologic ExaminationThe middle and the left hepatic lobes were immersion-fixed in 4% PBS-buffered formalin, embedded in paraffin, sectioned, and stained with H&E or Sirius red or chromotrope-anilin-blue using standard histologic techniques. In addition, slides were immunostained for myeloperoxidase (MPO) (polyclonal rabbit antibody; NeoMarkers, Fremont, CA), CD3 (monoclonal rabbit antibody; Neomarkers), B220 (monoclonal rat antibody; BD Biosciences Pharmingen, San Diego, CA), and F4/80 (monoclonal rat antibody; BMA Biomedicals, Augst, Switzerland) using the Ventana Discovery automated staining system with the 3,3′-diaminobenzidine tetrahydrochloride Map kit (Ventana, Tucson, AZ). All immunostains were counterstained with hematoxylin. Quantifications were performed as follows: MPO-positive cell nests (accumulation of ≥3 MPO-positive cells) were counted on 2 entire transections of the middle and left hepatic lobe; CD3-, B220-, and F4/80-positive cells were counted on 10 randomly selected high-power fields (400×). All counts were performed by 2 investigators blinded with respect to the experimental group. Lipid vacuoles, chromotrope-anilin-blue–positive inclusions, and Sirius red collagen fibers were quantified on 10 randomly chosen images with the analySIS^D imaging software using a semiautomatic thresholding algorithm (Olympus, Volketswil, Switzerland).Biochemical Analysis of Total Hepatic Lipid ContentTotal liver lipids were extracted from 10–20 mg of liver homogenate using the method of Folch et al.24Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar Total lipids were determined in aliquots of lipid extracts by the sulphophospho-vanillin colorimetric method.25Drevon B. Schmit J.M. [The Sulfo-Phospho-Vanillic reaction in the evaluation of the serum lipids and lipoproteins of the monkey (Cynocephalus babuin); comparison with human serum.].C R Seances Soc Biol Fil. 1964; 158: 778-780PubMed Google ScholarQuantitative Real-Time Polymerase Chain ReactionTotal RNA was extracted from 50 mg of liver tissue using TRIzol reagent (Invitrogen, Basel, Switzerland) following the manufacturer’s instructions. Five micrograms of RNA were reverse transcribed using the ThermoScript reverse-transcription polymerase chain reaction System (Invitrogen), yielding the cDNA template. Quantitative real-time PCR amplification and data analysis were performed using an ABI Prism 7000 Sequence Detector System (PE Applied Biosystems, Rotkreuz, Switzerland). TaqMan gene expression assays (PE Applied Biosystems) for interleukin-1β (IL-1β) (Mm00434228_m1), IL-6 (Mm 00446190_m1), tumor necrosis factor-α (Mm 00443258_m1), KC (Mm00433859_m1), CXCL2 (Mm00436450_m1), SERT (Mm00439391_m1), MAO-A (Mm00558004_m1), transforming growth factor-β1 (Mm00441724_m1), and procollagen I α1 (Mm00801666_g1) were used to quantify the messenger RNA (mRNA) expression of the respective genes. The mRNA expression levels of each sample were normalized to 18S RNA (TaqMan ribosomal RNA control reagents; PE Applied Biosystems). The results shown represent fold induction of mRNA expression in CMD diet–fed animals (C57Bl/6 or Tph1−/−) compared with that in wild-type mice (C57Bl/6) fed control diet.Isolation of Hepatic MitochondriaMitochondria isolation was performed as described.26Manfredi G. Yang L. Gajewski C.D. Mattiazzi M. Measurements of ATP in mammalian cells.Methods. 2002; 26: 317-326Crossref PubMed Scopus (193) Google Scholar Briefly, the whole mouse liver was homogenized with 8 mL of buffer containing 0.22 mol/L D-mannitol, 0.07 mol/L sucrose, 20 mmol/L HEPES, 1 mmol/L ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, and 1% bovine serum albumin, pH 7.2. The homogenate was centrifuged at 1500g for 5 minutes. The supernatant was centrifuged at 10,000g for 10 minutes. The mitochondrial pellet was resuspended in a buffer containing 150 mmol/L KCl, 25 mmol/L Tris-HCl, 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% bovine serum albumin, 10 nmol/L potassium phosphate, and 0.1 mmol/L MgCl.Western BlottingMitochondrial (for MAO-A) or cytosolic fractions (for cytochrome c) were diluted in sample buffer (187.5 mmol/L Tris-HCL [pH 6.8], 6% sodium dodecyl sulfate, 30% glycerol, 150 mmol/L dithiothreitol, and 0.3% bromophenol blue), and boiled for 10 minutes at 90°C. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed and samples were blotted onto a polyvinylidene difluoride membrane. Primary antibodies were mouse anti–cytochrome c monoclonal (BD Biosciences PharMingen) and rabbit anti–MAO-A polyclonal antibody, as well as rabbit anti–glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), used to ascertain equal loading. Secondary staining and detection was performed according to standard protocols with the enhanced chemiluminescence detection reagent (GE Healthcare Ltd., Buckinghamshire, UK).Electron MicroscopyImmediately after harvesting, liver tissue was prefixed with 1.5% glutaraldehyde and 0.8% paraformaldehyde (0.1 mol/L cacodylate buffer) at room temperature and postfixed in an aqueous solution of 1% OsO4 and 1.5% K4(FeCN)6. The specimens then were embedded into Epon (Roth AG, Reinach, Switzerland) by routine procedures. Semithin sections (1 μm) were stained with toluidine blue and analyzed by light microscopy. Ultrathin sections (50 nm) were contrasted with lead citrate and uranyl acetate and studied with a CM100 Transmission Electron Microscope (Philips Electronics, Zurich, Switzerland).Analysis of Human SamplesHuman liver biopsy specimens were retrieved from the surgical pathology files of the Department of Pathology, University of Zurich, and were evaluated for steatosis, inflammation, and fibrosis according to the semiquantitative Kleiner et al27Kleiner 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 (6897) Google Scholar score, using H&E and Sirius red staining. Normal liver samples were obtained from patients without underlying liver disease undergoing liver surgery for metastases. Histologic analysis was performed by a single hepatopathologist (W.J.). The study of human liver tissue was approved by the ethical committee of the canton of Zurich. For each patient, RNA was extracted from ten 10-μm thick sections of paraffin-embedded liver biopsy specimens. Sections were placed in extraction buffer (20 mmol/L Tris, pH 7.5, 20 mmol/L EDTA, and 1% sodium dodecyl sulfate) and incubated for 10 minutes at 95°C. Tissue was digested with proteinase K (Roche Diagnostics, Rotkreuz, Switzerland). RNA subsequently was purified using the QIAshredder homogenizer (QIAGEN, Hilden, Germany), Trizol LS Reagent (Invitrogen), chloroform, and isopropanol according to the manufacturer’s instructions. Reverse transcription and real-time polymerase-chain-reaction (RT-PCR) analysis were performed as described earlier, using the TaqMan gene expression assays for human MAO-A (Hs00165140_m1) and SERT (Hs00169010_m1).Statistical AnalysisData are presented as means ± SD. Groups were compared with the Student t test for unpaired samples using Prism 4.0 (GraphPad Software, San Diego, CA). A 2-sided P value of less than .05 indicated statistical significance.ResultsDoes Serotonin Affect Hepatic Lipid Accumulation?Fat accumulation in hepatocytes represents the first hit in the pathogenesis of steatohepatitis. Therefore, we initially assessed the degree of lipid accumulation in wild-type (C57Bl/6) and Tph1−/− mice fed CMD or control diet for 6 weeks. In both wild-type and Tph1−/− mice, CMD diet led to hepatic fat accumulation, which remained negligible with control diet. More importantly, neither the area occupied by fat vacuoles on histologic liver sections, as assessed by image analysis (Figure 1A), nor biochemical quantification of total hepatic lipid content (Figure 1B) were significantly different in CMD-fed wild-type and serotonin-deficient animals. Thus, serotonin did not affect lipid accumulation in this model of steatohepatitis.Does Serotonin Increase Hepatocellular Injury in CMD-Fed Mice?NASH typically is accompanied by mild to moderate transaminase level increases,1Ioannou G.N. Boyko E.J. Lee S.P. The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999-2002.Am J Gastroenterol. 2006; 101: 76-82Crossref PubMed Scopus (252) Google Scholar reflecting hepatocellular injury. We therefore determined serum transaminase activity in wild-type and serotonin-deficient mice. Although CMD diet caused a 2- to 3-fold increase in AST (Figure 2A) and ALT (Figure 2B) levels in wild-type animals, this increase was mitigated significantly in Tph1−/− mice. This suggests that serotonin contributes to hepatocellular injury in nutritionally induced steatohepatitis.Figure 2Serum levels of transaminases. Wild-type mice (□) and Tph1−/− mice (■) were fed with control diet or CMD diet for 6 weeks. (A) AST, *P = .02. (B) ALT, *P < .001. There were 6 mice per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Does Serotonin Affect Liver Fibrogenesis in CMD-Fed Mice?Liver fibrosis is a fundamental component of NASH and serotonin recently was implicated as a possible inducer of liver fibrogenesis.28Ruddell R.G. Oakley F. Hussain Z. Yeung I. Bryan-Lluka L.J. Ramm G.A. Mann D.A. A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis.Am J Pathol. 2006; 169: 861-876Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Therefore, expression of the fibrogenic trigger transforming growth factor-β1 and the matrix protein collagen type I were analyzed by RT-PCR (Figures 3A and 3B). Both markers were up-regulated to a similar degree after 6 weeks of CMD diet in both wild-type and Tph1−/− mice. Accordingly, accumulation of collagen in liver tissue showed no significant difference between these 2 groups as assessed by Sirius red staining (Figure 3C).Figure 3Liver fibrosis. Wild-type and Tph1−/− mice were fed with control diet or CMD diet for 6 weeks. Expression levels of (A) transforming growth factor-β1 and (B) procollagen I α1 after control diet (□) or CMD diet (■). (C) Quantification of collagen deposition by morphometric analysis in wild-type (□) and Tph1−/− mice (■). There were 6 mice per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Does Serotonin Increase Hepatic Inflammation?Next, we wondered whether the inflammatory component of NASH, characterized by lobular infiltration with polymorphonuclear leukocytes, is affected by serotonin. Neutrophils infiltrating the hepatic parenchyma therefore were quantified using MPO immunostaining of liver sections (Figure 4A). The lack of serotonin in Tph1−/− mice significantly reduced the degree of neutrophil infiltration, although the numbers of B lymphocytes (Figure 4C), T lymphocytes (Figure 4D), and macrophages (Figure 4E) were not affected. This prompted us to analyze expression levels of the major proinflammatory cytokines (tumor necrosis factor-α, IL-6, and IL-1β) and chemokines (KC and CXCL2) in the liver using real-time PCR. In contrast to neutrophil infiltration, mRNA levels of tumor necrosis factor-α, IL-6, and IL-1β (data not shown), as well as mRNA levels of KC (Figure 4F) and CXCL2 (Figure 4G) were not altered significantly by the absence of peripheral serotonin. Thus, the reduction of hepatic inflammation in Tph1−/− mice cannot be attributed to a reduced expression of the major proinflammatory cytokines and chemokines.Figure 4Inflammation. Wild-type mice and Tph1−/− mice were fed with control diet or CMD diet for 6 weeks. (A) Neutrophils were immunostained for MPO and MPO-positive cell nests were counted on 2 entire liver sections in wild-type (□) and Tph1−/− mice (■). *P = .026. (B) Representative photomicrograph of a MPO-positive cell nest (400×). (C–E) Immunostainings and quantification of (C) B lymphocytes, (D) T lymphocytes, and (E) macrophages in wild-type (□) and Tph1−/− mice (■). (F and G) Expression levels of the chemokines (F) KC and (G) CXCL2 in mice fed with control diet (□) or CMD diet (■). There were 6 mice per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Does NASH Induce SERT and MAO-A Up-Regulation and Lead to Hepatic Lipid Peroxidation?CMD diet led to less hepatocyte injury and inflammation in mice lacking peripheral serotonin. To explore whether CMD diet–induced NASH is associated with an increased uptake of serotonin, we analyzed hepatic mRNA levels of the serotonin transporter SERT using real-time PCR (Figure 5A). Although CMD diet led to a significant up-regulation of SERT mRNA in both groups, this increase was dramatically more pronounced in mice lacking peripheral serotonin, which had a significantly lower expression at baseline (2-fold induction in wild-type and 13-fold induction in Tph1−/− animals). Collectively, these results point to an increased uptake of serotonin in CMD-induced steatohepatitis.Figure 5SERT and monoamine oxidase. Wild-type and Tph1−/− mice were fed with control diet (□) or CMD diet (■) for 6 weeks. Transcript levels of SERT and MAO-A were assessed by RT-PCR and normalized to wild-type control-fed animals. Protein levels of MAO-A were assessed in mitochondrial fractions by Western blotting, using placenta as positive control. (A) Expression of SERT, wt: *P = .039, Tph1−/−: **P = .001 compared with baseline, ***P = .003 Tph1−/− to wild-type baseline. (B) Expression of MAO-A, wt: *P = .025, Tph1−/−: *P = .047 compared with baseline. There were 6 mice per group. (C) Representative Western blot for MAO-A. Glyceraldehyde-3-phosphate dehydrogenase was used to ascertain equal loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The key enzyme for intracellular serotonin breakdown is mitochondrial MAO-A. Interestingly, MAO-A–mediated serotonin catabolism was implicated recently in cardiomyocyte death, through the production of ROS.21Bianchi P. Kunduzova O. Masini E. Cambon C. Bani D. Raimondi L. Seguelas M.H. Nistri S. Colucci W. Leducq N. Parini A. Oxidative stress by monoamine oxidase mediates receptor-independent cardiomyocyte apoptosis by serotonin and postischemic myocardial injury.Circulation. 2005; 112: 3297-3305Crossref PubMed Scopus (211) Google Scholar To explore whether the putative increase in hepatic serotonin uptake translates into increased ser
Referência(s)