Adiponectin Regulation of Stellate Cell Activation via PPARγ-Dependent and -Independent Mechanisms
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.035
ISSN1525-2191
AutoresMahnoush S. Shafiei, Shoba Shetty, Philipp E. Scherer, Don C. Rockey,
Tópico(s)Liver physiology and pathology
ResumoIn this study, we elucidated the mechanism by which adiponectin modulates hepatic stellate cell activation and fibrogenesis. Adiponectin-overexpressing transgenic mice receiving thioacetamide were resistant to fibrosis, compared with controls. In contrast, adiponectin-null animals developed severe fibrosis. Expression of collagen α1(I) and α-smooth muscle actin (α-SMA) mRNAs were significantly lower in adiponectin-overexpressing mice, compared with controls. In wild-type stellate cells exposed to a lentivirus encoding adiponectin, expression of peroxisome proliferator-activated receptor-γ (PPARγ), SREBP1c, and CEBPα mRNAs was significantly increased (3.2-, 4.1-, and 2.2-fold, respectively; n = 3; P < 0.05, adiponectin virus versus control), consistent with possible activation of an adipogenic transcriptional program. Troglitazone, a PPARγ agonist, strongly suppressed up-regulation of collagen α1(I) and α-SMA mRNA in stellate cells isolated from wild-type mice; however, stellate cells from adiponectin-null animals failed to respond to troglitazone. Furthermore, in isolated stellate cells in which PPARγ was depleted using an adenovirus-Cre-recombinase system and in which adiponectin was also overexpressed, collagen α1(I) and α-SMA were significantly inhibited. We conclude that the PPARγ effect on stellate cell activation and the fibrogenic cascade appears to be adiponectin-dependent; however, the inhibitory effect of adiponectin on stellate cell activation was not dependent on PPARγ, suggesting the presence of PPARγ-dependent as well as independent pathways in stellate cells. In this study, we elucidated the mechanism by which adiponectin modulates hepatic stellate cell activation and fibrogenesis. Adiponectin-overexpressing transgenic mice receiving thioacetamide were resistant to fibrosis, compared with controls. In contrast, adiponectin-null animals developed severe fibrosis. Expression of collagen α1(I) and α-smooth muscle actin (α-SMA) mRNAs were significantly lower in adiponectin-overexpressing mice, compared with controls. In wild-type stellate cells exposed to a lentivirus encoding adiponectin, expression of peroxisome proliferator-activated receptor-γ (PPARγ), SREBP1c, and CEBPα mRNAs was significantly increased (3.2-, 4.1-, and 2.2-fold, respectively; n = 3; P < 0.05, adiponectin virus versus control), consistent with possible activation of an adipogenic transcriptional program. Troglitazone, a PPARγ agonist, strongly suppressed up-regulation of collagen α1(I) and α-SMA mRNA in stellate cells isolated from wild-type mice; however, stellate cells from adiponectin-null animals failed to respond to troglitazone. Furthermore, in isolated stellate cells in which PPARγ was depleted using an adenovirus-Cre-recombinase system and in which adiponectin was also overexpressed, collagen α1(I) and α-SMA were significantly inhibited. We conclude that the PPARγ effect on stellate cell activation and the fibrogenic cascade appears to be adiponectin-dependent; however, the inhibitory effect of adiponectin on stellate cell activation was not dependent on PPARγ, suggesting the presence of PPARγ-dependent as well as independent pathways in stellate cells. Altered hepatic pathology, function, or both are among the most common sequelae of the metabolic syndrome.1Kamada Y. Takehara T. Hayashi N. Adipocytokines and liver disease.J Gastroenterol. 2008; 43: 811-822Crossref PubMed Scopus (141) Google Scholar, 2Rombouts K. Marra F. Molecular mechanisms of hepatic fibrosis in non-alcoholic steatohepatitis.Dig Dis. 2010; 28: 229-235Crossref PubMed Scopus (48) Google Scholar Steatosis is generally the first step in this process, which may progress to development of inflammation, fibrosis, and even end-stage liver failure.3Farrell G.C. Larter C.Z. Nonalcoholic fatty liver disease: from steatosis to cirrhosis.Hepatology. 2006; 43: S99-S112Crossref PubMed Scopus (1991) Google Scholar The determinants of liver disease in metabolic syndrome are under intense investigation. A number of factors likely contribute significantly to the process, including dysregulated metabolic pathways, oxidative stress, and chronic inflammation.3Farrell G.C. Larter C.Z. Nonalcoholic fatty liver disease: from steatosis to cirrhosis.Hepatology. 2006; 43: S99-S112Crossref PubMed Scopus (1991) Google Scholar Activated stellate cells (myofibroblasts) are key effectors of the fibrogenic response in the liver.4Friedman S.L. Rockey D.C. Bissell D.M. Hepatic fibrosis 2006: report of the Third AASLD Single Topic Conference.Hepatology. 2007; 45: 242-249Crossref PubMed Scopus (98) Google Scholar Recent evidence has emphasized an adipogenic transcriptional program in stellate cells that is regulated by typical transcription factors in this pathway, including peroxisome proliferator-activated receptor-γ (PPARγ), SREBP1c, and CEBPα; this program appears to promote maintenance of stellate cells in a quiescent state.5Tsukamoto H. She H. Hazra S. Cheng J. Miyahara T. Anti-adipogenic regulation underlies hepatic stellate cell transdifferentiation.J Gastroenterol Hepatol. 2006; 21: S102-S105Crossref PubMed Scopus (85) Google Scholar, 6She H. Xiong S. Hazra S. Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells.J Biol Chem. 2005; 280: 4959-4967Crossref PubMed Scopus (262) Google Scholar During activation, stellate cells lose retinoids and transform into a myofibroblast-like appearance.5Tsukamoto H. She H. Hazra S. Cheng J. Miyahara T. Anti-adipogenic regulation underlies hepatic stellate cell transdifferentiation.J Gastroenterol Hepatol. 2006; 21: S102-S105Crossref PubMed Scopus (85) Google Scholar, 6She H. Xiong S. Hazra S. Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells.J Biol Chem. 2005; 280: 4959-4967Crossref PubMed Scopus (262) Google Scholar A critical corollary to the morphological transition is a set of remarkable functional changes that include production of extracellular matrix, as well as profibrotic mediators such as tissue inhibitors of matrix metalloproteinases (TIMPs).4Friedman S.L. Rockey D.C. Bissell D.M. Hepatic fibrosis 2006: report of the Third AASLD Single Topic Conference.Hepatology. 2007; 45: 242-249Crossref PubMed Scopus (98) Google Scholar Adiponectin has been suggested to play an important role in the pathogenesis of liver fibrosis.7Yoneda M. Iwasaki T. Fujita K. Kirikoshi H. Inamori M. Nozaki Y. Maeyama S. Wada K. Saito S. Terauchi Y. Nakajima A. Hypoadiponectinemia plays a crucial role in the development of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus independent of visceral adipose tissue.Alcohol Clin Exp Res. 2007; 31: S15-S21Crossref PubMed Scopus (38) Google Scholar, 8Wang J. Brymora J. George J. Roles of adipokines in liver injury and fibrosis.Expert Rev Gastroenterol Hepatol. 2008; 2: 47-57Crossref PubMed Scopus (16) Google Scholar, 9Shetty S. Kusminski C.M. Scherer P.E. Adiponectin in health and disease: evaluation of adiponectin-targeted drug development strategies.Trends Pharmacol Sci. 2009; 30: 234-239Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 10Asano T. Watanabe K. Kubota N. Gunji T. Omata M. Kadowaki T. Ohnishi S. Adiponectin knockout mice on high fat diet develop fibrosing steatohepatitis.J Gastroenterol Hepatol. 2009; 24: 1669-1676Crossref PubMed Scopus (86) Google Scholar, 11Fukushima J. Kamada Y. Matsumoto H. Yoshida Y. Ezaki H. Takemura T. Saji Y. Igura T. Tsutsui S. Kihara S. Funahashi T. Shimomura I. Tamura S. Kiso S. Hayashi N. Adiponectin prevents progression of steatohepatitis in mice by regulating oxidative stress and Kupffer cell phenotype polarization.Hepatol Res. 2009; 39: 724-738Crossref PubMed Scopus (72) Google Scholar, 12Arvaniti V.A. Thomopoulos K.C. Tsamandas A. Makri M. Psyrogiannis A. Vafiadis G. Assimakopoulos S.F. Labropoulou-Karatza C. Serum adiponectin levels in different types of non alcoholic liver disease Correlation with steatosis, necroinflammation and fibrosis.Acta Gastroenterol Belg. 2008; 71: 355-360PubMed Google Scholar, 13Nakayama H. Otabe S. Yuan X. Ueno T. Hirota N. Fukutani T. Wada N. Hashinaga T. Yamada K. Effects of adiponectin transgenic expression in liver of nonalcoholic steatohepatitis model mice.Metabolism. 2009; 58: 901-908Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 14Ma H. Gomez V. Lu L. Yang X. Wu X. Xiao S.Y. Expression of adiponectin and its receptors in livers of morbidly obese patients with non-alcoholic fatty liver disease.J Gastroenterol Hepatol. 2009; 24: 233-237Crossref PubMed Scopus (67) Google Scholar, 15Nannipieri M. Cecchetti F. Anselmino M. Mancini E. Marchetti G. Bonotti A. Baldi S. Solito B. Giannetti M. Pinchera A. Santini F. Ferrannini E. Pattern of expression of adiponectin receptors in human liver and its relation to nonalcoholic steatohepatitis.Obes Surg. 2009; 19: 467-474Crossref PubMed Scopus (40) Google Scholar Notably, in addition to adipose tissue, adiponectin is expressed in stellate cells.16Ding X. Saxena N.K. Lin S. Xu A. Srinivasan S. Anania F.A. 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 (223) Google Scholar Previous studies have shown that adiponectin has important effects specifically on stellate cells, and the mechanism of these effects remains an area of active investigation.6She H. Xiong S. Hazra S. Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells.J Biol Chem. 2005; 280: 4959-4967Crossref PubMed Scopus (262) Google Scholar, 17Hazra S. Xiong S. Wang J. Rippe R.A. Krishna V. Chatterjee K. Tsukamoto H. Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells.J Biol Chem. 2004; 279: 11392-11401Crossref PubMed Scopus (277) Google Scholar Here, we have hypothesized that the relationship between adiponectin and adipogenic signaling partners, and in particular the classic transcription factor PPARγ, is critical in modulating hepatic stellate cell activation and fibrogenesis. We examined the role of adiponectin in modulating hepatic stellate cell function in isolated primary cells and in genetic models that lack or overexpress adiponectin. Our findings highlight a complicated relationship between PPARγ and adiponectin in stellate cell activation. Homozygous adiponectin-deficient and heterozygous adiponectin-overexpressing mice (using aP2 as a promoter) and corresponding controls on FVB background were generated as described previously.18Nawrocki A.R. Rajala M.W. Tomas E. Pajvani U.B. Saha A.K. Trumbauer M.E. Pang Z. Chen A.S. Ruderman N.B. Chen H. Rossetti L. Scherer P.E. Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists.J Biol Chem. 2006; 281: 2654-2660Crossref PubMed Scopus (535) Google Scholar, 19Combs T.P. Pajvani U.B. Berg A.H. Lin Y. Jelicks L.A. Laplante M. Nawrocki A.R. Rajala M.W. Parlow A.F. Cheeseboro L. Ding Y.Y. Russell R.G. Lindemann D. Hartley A. Baker G.R. Obici S. Deshaies Y. Ludgate M. Rossetti L. Scherer P.E. A transgenic mouse with a deletion in the collagenous domain of adiponectin displays elevated circulating adiponectin and improved insulin sensitivity.Endocrinology. 2004; 145: 367-383Crossref PubMed Scopus (447) Google Scholar Male mice between the ages of 12 and 16 weeks were used for this study. All animals were maintained under temperature-controlled conditions (22 ± 2°C) in 12-hour light/dark cycles with unlimited access to food and water. All animals received humane care in compliance with University of Texas Southwestern Medical Center Institutional Animal Care and Use Guidelines. To induce liver fibrosis, the mice were given i.p. injections of 0.2 g/kg body weight of thioacetamide (TAA) dissolved in saline two times per week for 8 weeks as described previously.20Wu J.B. Chuang H.R. Yang L.C. Lin W.C. A standardized aqueous extract of Anoectochilus formosanus ameliorated thioacetamide-induced liver fibrosis in mice: the role of Kupffer cells.Biosci Biotechnol Biochem. 2010; 74: 781-787Crossref PubMed Scopus (24) Google Scholar At the end of the experimental period, liver samples were collected for histological and biochemical examinations. Animal protocols used to induce injury and fibrogenesis were approved by the University of Texas Southwestern Medical Center Animal Care and Use Committee. Mice containing PPARγ LoxP sites on a C57BL/6J mixed background (4 to 5 weeks of age) were kindly provided by Dr. Bruce Spiegelman (Dana Farber Cancer Institute, Boston, MA).21He W. Barak Y. Hevener A. Olson P. Liao D. Le J. Nelson M. Ong E. Olefsky J.M. Evans R.M. Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle.Proc Natl Acad Sci USA. 2003; 100: 15712-15717Crossref PubMed Scopus (798) Google Scholar Hepatic stellate cells were isolated by digestion of the liver with pronase (Roche Applied Science, Indianapolis, IN) followed by collagenase (Crescent Chemical, Hauppauge, NY). In brief, stellate cells were separated from other liver nonparenchymal cells by ultracentrifugation over gradients of 8.2% and 15.6% cell separation medium (Accident medium; Accurate Chemical & Scientific, Westbury, NY). The resulting upper layer consisted of >95% stellate cells. Cells were placed on uncoated plastic and were maintained in standard stellate cell growth medium (modified 199OR containing 10% fetal bovine serum and 10% calf serum) as described previously.22Rockey D.C. Chung J.J. Endothelin antagonism in experimental hepatic fibrosis Implications for endothelin in the pathogenesis of wound healing.J Clin Invest. 1996; 98: 1381-1388Crossref PubMed Scopus (185) Google Scholar Isolated stellate cells were seeded at a density of 3 × 102 cells/mm2. Cultures were incubated at 37°C in a humidified incubator (containing 95% O2 and 2.5% CO2), and the medium was changed every 24 hours. Cell viability was >90% in all cultures used. The cells were considered to be quiescent at 24 hours after plating (day 1). Cells at day 7 were considered activated, with >95% staining positive for α-SMA. Recombinant adenovirus expressing green fluorescent protein (Ad-GFP) was prepared as described previously.23Shafiei M.S. Rockey D.C. The role of integrin-linked kinase in liver wound healing.J Biol Chem. 2006; 281: 24863-24872Crossref PubMed Scopus (39) Google Scholar Adenovirus encoding Cre recombinase (Ad-Cre) was purchased from Vector Biolabs (Philadelphia, PA). Viruses were amplified and titered according to the manufacturer's instructions (BD Biosciences, San Jose, CA). Stellate cells were isolated as described above, plated on 35-mm dishes at approximately 85% confluency and at the specified stage of culture were routinely infected as described previously.23Shafiei M.S. Rockey D.C. The role of integrin-linked kinase in liver wound healing.J Biol Chem. 2006; 281: 24863-24872Crossref PubMed Scopus (39) Google Scholar Infection efficiency was monitored by the expression of GFP and typically reached 80% to 90% within 48 hours. Lentivirus vectors were kindly provided by Dr. Zhao Wang (UT Southwestern Medical Center, Dallas, TX). Stellate cells were isolated as above, plated on 35-mm dishes at approximately 85% confluency, and at the specified stage of culture, were routinely infected with lentivirus at a multiplicity of infection (MOI) of 100 or 350. Total RNA was extracted using TRIZOL reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The reverse-transcription reaction was performed by using 1 μg of RNA that was reverse transcribed using oligo(dt) primers and SuperScript (Invitrogen) reverse transcriptase. Amplification reactions were performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Five microliters of diluted cDNA samples (1:5 dilution) were used for quantitative two-step PCR (a 10-minute step at 95°C followed by 50 cycles of 15 seconds of 95°C and 1 second at 65°C) in the presence of 400 nmol/L specific forward and reverse primers and SYBR Green PCR master mix. Each sample was analyzed in triplicate. As negative controls, water was used as a template for each reaction. Primer sequences were as follows: type I collagen (COL1α1) forward, 5′-TTCCCTGGACCTAAGGGTACT-3′ and reverse, 5′-TTGAGCTCCAGCTTCGCC-3′; α-SMA forward, 5′-GTGGATCACCAAGCAGGAGT-3′ and reverse, 5′-CATAGCACGATGGTCGAT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse, 5′-CATCGAAGGTGGAAGAGTGG-3′; CEBPα forward, 5′-AAGAAGTCGGTGGATAAGAACAG-3′ and reverse, 5′-GTTGCGCTGTTTGGCTTTATCTC-3′; PPARγ forward, 5′-CCTGAAGCTCCAAGAATACCAAA-3′ and reverse, 5′-AGAGTTTTTCAGAATAATAAGG-3′; and SREBP1c forward, 5′-AGCTGTCGGGGTAGCGTCTG-3′ and reverse, 5′-GAGAGTTGGCACCTGGGCTG-3′. Cell lysates were prepared in buffer containing 1% Triton X-100, 150 mmol/L NaCl, 20 mmol/L Tris pH 7.5, 1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L sodium-2-glycerophosphate, 0.05 mmol/L Na3VO4, 10 μg leupeptin, 10% glycerol, and 100 mmol/L phenylmethylsulfonyl fluoride. Samples containing 50 g of total protein were subjected to SDS-PAGE, after which proteins were transferred to nitrocellulose membranes (Schleicher & Schuell Bioscience, Keene, NH). Membranes were incubated for 1 hour at room temperature in blocking buffer (10 mmol/L sodium phosphate, 0.5 mol/L NaCl, 0.05% Tween 20, and 2.5% dried milk) and then with primary antibody (1:1000) overnight at 4°C. Next, membranes were washed of excess primary antibody at room temperature in a phosphate-buffered saline Tween buffer (TBST: 10 mol/L 0.05% Tris pH 8, 0.9% sodium chloride, and Tween 20 0.05%) and then incubated for 1 hour at room temperature with secondary antibody. After the washing, specific signals were visualized using enhanced chemiluminescence detection according to the manufacturer's instructions (Thermo Fisher Scientific, Rockford. IL). Specific bands were scanned and data were collected over a narrow range of X-ray film (Eastman Kodak Co., Rochester, NY) linearity and then were quantitated by scanning densitometry. Livers were fixed in 10% phosphate-buffered formalin for 48 hours at 4°C, washed twice with water, stored in 70% ethanol at 4°C for 24 hours, and then embedded in paraffin. Sections 5-μm thick were then dehydrated and stained with 0.1% Sirius Red F3B in saturated picric acid and counterstained with Fast Green FCF (all from Sigma-Aldrich, St. Louis, MO). The proportion of tissue stained with picrosirus red was assessed by morphometric analysis using MetaView software (Universal Imaging, Downingtown, PA) as described previously.22Rockey D.C. Chung J.J. Endothelin antagonism in experimental hepatic fibrosis Implications for endothelin in the pathogenesis of wound healing.J Clin Invest. 1996; 98: 1381-1388Crossref PubMed Scopus (185) Google Scholar Data are reported as means ± SE. Significance was established using the Student's t-test and analysis of variance when appropriate. Differences were considered significant at P < 0.05. We initially characterized stellate cells from adiponectin-deficient and adiponectin-overexpressing mice. At early time points in culture, hepatic stellate cells from wild-type mice exhibited typical characteristics of quiescent cells, including abundant perinuclear retinoid, and a relatively rounded appearance (Figure 1A). Cells from adiponectin-deficient mice tended to spread more rapidly and had comparatively reduced amounts of retinoid droplets, consistent with the morphological appearance of activated stellate cells, whereas those from adiponectin-overexpressing mice retained features of quiescence. The differences were readily visible at 12, 24, and 48 hours (Figure 1A). At day 3 in culture, we quantified expression of activation and fibrosis markers in wild-type and adiponectin-null stellate cells (Figure 1, B–D) and found that both mRNA and protein levels of these indicators were significantly higher in stellate cells from adiponectin-null animals, compared with controls. These results suggest that lack of adiponectin accelerates the activation process and could explain the susceptibility of adiponectin-null mice to hepatic fibrosis. Next, we induced liver fibrosis in mice with TAA. Adiponectin-deficient animals weighed less than either wild-type or adiponectin-overexpressing mice, although the differences were not statistically significant (Table 1). After TAA administration, all mice lost weight, and the loss in weight was greatest in adiponectin-deficient mice. The exposure to TAA led to injury and inflammation, as expected. There were increases in alanine transaminase levels in each of the groups of mice; relative increases in alanine transaminase were similar among all groups (Table 1). After 8 weeks of exposure to TAA, liver sections stained with H&E revealed hepatocellular necrosis, which was focused predominantly in pericentral and intralobular areas and was associated with a mixed inflammatory infiltrate (Figure 2 and Table 2). When inflammatory changes were quantitated using the Knodell scoring system,24Knodell R.G. Ishak K.G. Black W.C. Chen T.S. Craig R. Kaplowitz N. Kiernan T.W. Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis.Hepatology. 1981; 1: 431-435Crossref PubMed Scopus (3459) Google Scholar there was slightly more necrosis in the knockout mice and slightly less necrosis in the transgenic mice, compared with wild-type mice. These data suggest that there was no biochemical evidence of differential injury to hepatocytes caused by TAA among the different groups, but that there were small differences in the degree of inflammation among the groups.Table 1Body Weight and Serum ALT and ALP Levels in Control and TAA-Injected MiceVariableWTKOTgWT+TAAKO+TAATg+TAABody weight (g)32.9 ± 2.930.4 ± 1.532.7 ± 3.232.7 ± 2.329.8 ± 1.732.5 ± 1.5Serum ALT (IU/L)45 ± 854 ± 8⁎P < 0.05 versus WT or Tg;43 ± 962 ± 12†P < 0.05 versus untreated animals;77 ± 14†P < 0.05 versus untreated animals;‡P < 0.001 versus WT+TAA or Tg+TAA mice.58 ± 9†P < 0.05 versus untreated animals;Serum ALP (IU/L)34 ± 737 ± 532 ± 737 ± 645 ± 736 ± 8Data are reported as means ± SE . n = 5 to 7 per group.ALT, alanine transaminase; ALP, alkaline phosphatase; KO, adiponectin knockout mice; TAA, thioacetamide; Tg, adiponectin transgenic mice; WT, wild type. P < 0.05 versus WT or Tg;† P < 0.05 versus untreated animals;‡ P < 0.001 versus WT+TAA or Tg+TAA mice. Open table in a new tab Table 2Liver Histological FindingsVariableWT+TAAKO+TAATg+TAAPeriportal–bridge necrosis0.3 ± 0.20.4 ± 0.20.2 ± 0.1Intralobular degeneration and focal necrosis2 ± 0.33 ± 0.51 ± 0.2Portal inflammation0.2 ± 0.10.3 ± 0.20.1 ± 0.1n = 5 to 7 per group.KO, adiponectin knockout mice; TAA, thioacetamide; Tg, adiponectin transgenic mice; WT, wild type. Open table in a new tab Data are reported as means ± SE . n = 5 to 7 per group. ALT, alanine transaminase; ALP, alkaline phosphatase; KO, adiponectin knockout mice; TAA, thioacetamide; Tg, adiponectin transgenic mice; WT, wild type. n = 5 to 7 per group. KO, adiponectin knockout mice; TAA, thioacetamide; Tg, adiponectin transgenic mice; WT, wild type. Notably, adiponectin-deficient mice exhibited greater fibrosis at baseline than did controls (Figure 3, A and B). Additionally, after exposure to TAA, adiponectin-deficient mice exhibited greater fibrosis than did matched controls (Figure 3, A and B). We further measured collagen α1(I) and α-SMA mRNA levels in these animals, and found that each was significantly up-regulated, compared with controls (Figure 3, C and D). We also explored a gain-of-function model in which adiponectin overexpression is driven by the aP2 promoter.19Combs T.P. Pajvani U.B. Berg A.H. Lin Y. Jelicks L.A. Laplante M. Nawrocki A.R. Rajala M.W. Parlow A.F. Cheeseboro L. Ding Y.Y. Russell R.G. Lindemann D. Hartley A. Baker G.R. Obici S. Deshaies Y. Ludgate M. Rossetti L. Scherer P.E. A transgenic mouse with a deletion in the collagenous domain of adiponectin displays elevated circulating adiponectin and improved insulin sensitivity.Endocrinology. 2004; 145: 367-383Crossref PubMed Scopus (447) Google Scholar After TAA-induced injury, collagen production in adiponectin-overexpressing mice was reduced, compared with controls (Figure 3, C and D). Additionally, expression of collagen α1(I) and α-SMA mRNA was significantly lower in adiponectin-overexpressing animals (Figure 3, C and D). Finally, we evaluated PPAR expression in the in vivo injury models (Figure 4). PPARγ was down-regulated in adiponectin-deficient and up-regulated in adiponectin-overexpressing mice. TAA appeared to blunt expression of PPARγ, which remained reduced in knockout mice and increased in overexpressing mice. The changes in PPARα mRNA expression were similar in their trends (Figure 4B).Figure 4PPAR expression in adiponectin-deficient and transgenic mice. Liver fibrosis was induced by repetitive intraperitoneal injection of 0.2 g/kg body weight of thioacetamide (TAA). Livers were harvested, total RNA was extracted, and real-time PCR was performed to detect mRNA expression of PPARγ (A) or PPARα (B) (means ± SE; n = 6). *P < 0.05 versus WT; **P < 0.05 versus WT and TAA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given that earlier studies showed that activation of stellate cells in culture is associated with a decline in expression of adiponectin,25Kamada Y. Tamura S. Kiso S. Matsumoto H. Saji Y. Yoshida Y. Fukui K. Maeda N. Nishizawa H. Nagaretani H. Okamoto Y. Kihara S. Miyagawa J. Shinomura Y. Funahashi T. Matsuzawa Y. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin.Gastroenterology. 2003; 125: 1796-1807Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar we asked whether activation could be reversed by re-expression of adiponectin in cultured cells. First, we demonstrated that infection of stellate cells with a lentivirus containing an adiponectin construct led to increased production of adiponectin (Figure 5A); additionally, a time-course experiment revealed that expression of adiponectin increased from 12 to 48 hours after infection (Figure 5B). Next, we found that exposure of stellate cells to this virus led to a reduction in α-SMA and collagen α1(I) mRNA, compared with control (Figure 6, A and B), consistent with retaining a more quiescent phenotype relative to controls.Figure 6Effects of adiponectin overexpression on stellate cell activation. Stellate cells from normal rats were isolated, plated at equivalent density, and allowed to undergo culture-induced activation for 3 days. Cells were transduced with a control lentivirus expressing GFP or lentivirus overexpressing adiponectin at 50 and 100 MOI (n = 3/group). Total RNA was harvested 72 hours later and was subjected to RT-PCR to detect mRNA expression of α-SMA (A), collagen α1(I) (B), PPARγ (C), SREBP1c (D), and CEBPα (E) (means ± SE; n = 3). White bars indicate control GFP; gray bars indicate adiponectin (50 MOI); black bars indicate adiponectin (100 MOI). *P < 0.05 versus control GFP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activation of hepatic stellate cells is associated with enhanced expression of the nuclear receptor PPARβ/δ, recognized for its role in energy homeostasis, particularly lipid oxidation,26Hellemans K. Michalik L. Dittie A. Knorr A. Rombouts K. De Jong J. Heirman C. Quartier E. Schuit F. Wahli W. Geerts A. Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.Gastroenterology. 2003; 124: 184-201Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar and with reduced expression of PPARγ, known for its role in regulation of adipocyte differentiation.6She H. Xiong S. Hazra S. Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells.J Biol Chem. 2005; 280: 4959-4967Crossref PubMed Scopus (262) Google Scholar, 27Yang L. Chan C.C. Kwon O.S. Liu S. McGhee J. Stimpson S.A. Chen L.Z. Harrington W.W. Symonds W.T. Rockey D.C. Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G902-G911Crossref PubMed Scopus (108) Google Scholar We next examined whether adiponectin overexpression modulated the expression of specific members of the adipogenic program in stellate cells. PPARγ, SREBP1c, and CEBPα were all up-regulated after overexpression of adiponectin in stellate cells (Figure 6, C–E). Given that stellate cell activation is associated with significant decline in PPARγ and that forced expression of PPARγ leads to reversal of stellate cell activation,27Yang L. Chan C.C. Kwon O.S. Liu S. McGhee J. Stimpson S.A. Chen L.Z. Harrington W.W. Symonds W.T. Rockey D.C. Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G902-G911Crossref PubMed Scopus (108) Google Scholar, 28Da Silva Morais A. Abarca-Quinones J. Horsmans Y. Stärkel P. Leclercq I.A. Peroxisome proliferated-activated receptor gamma ligand, pioglitazone, does not prevent hepatic fibrosis in mice.Int J Mo
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