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• W2802528664 abstract "Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, SHP) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide in vitro, we demonstrated that the suppression of Shp expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun–mediated transcriptional repression of Shp. Interestingly, in vivo induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C–C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH. Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide, ranging from nonalcoholic fatty liver (NAFL, steatosis without hepatocellular injury) to the more aggressive nonalcoholic steatohepatitis (NASH, steatosis with ballooning, inflammation, or fibrosis). Although many studies have greatly contributed to the elucidation of NAFLD pathogenesis, the disease progression from NAFL to NASH remains incompletely understood. Nuclear receptor small heterodimer partner (Nr0b2, SHP) is a transcriptional regulator critical for the regulation of bile acid, glucose, and lipid metabolism. Here, we show that SHP levels are decreased in the livers of patients with NASH and in diet-induced mouse NASH. Exposing primary mouse hepatocytes to palmitic acid and lipopolysaccharide in vitro, we demonstrated that the suppression of Shp expression in hepatocytes is due to c-Jun N-terminal kinase (JNK) activation, which stimulates c-Jun–mediated transcriptional repression of Shp. Interestingly, in vivo induction of hepatocyte-specific SHP in steatotic mouse liver ameliorated NASH progression by attenuating liver inflammation and fibrosis, but not steatosis. Moreover, a key mechanism linking the anti-inflammatory role of hepatocyte-specific SHP expression to inflammation involved SHP-induced suppression of NF-κB p65-mediated induction of chemokine (C–C motif) ligand 2 (CCL2), which activates macrophage proinflammatory polarization and migration. In summary, our results indicate that a JNK/SHP/NF-κB/CCL2 regulatory network controls communications between hepatocytes and macrophages and contributes to the disease progression from NAFL to NASH. Our findings may benefit the development of new management or prevention strategies for NASH. Nonalcoholic fatty liver disease (NAFLD) 3The abbreviations used are: NAFLDnonalcoholic fatty liver diseaseNAFLnonalcoholic fatty liverNASHnonalcoholic steatohepatitisSHPsmall heterodimer partnerHFCFhigh fat, cholesterol, and fructoseALTalanine aminotransferaseASTaspartate aminotransferaseTGtriglycerideH&Ehematoxylin and eosinTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingVLDLvery-low-density lipoproteinMCDmethionine/choline–deficientJNKc-Jun N-terminal kinaseHSChepatic stellate cellKCKupffer cellqPCRquantitative PCRPApalmitic acidLPSlipopolysaccharidePI3Kphosphatidylinositol 3-kinaseTRE12-O-tetradecanoylphorbol-13-acetate response elementCMconditioned mediumFXRfarnesoid X–activated receptorFBSfetal bovine serumNASNAFLD activity scoreGTTglucose tolerance test. affects 25.24% (95% confidence interval: 22.10–28.65) of the general population (1Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease: Meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64 (26707365): 73-8410.1002/hep.28431Crossref PubMed Scopus (5294) Google Scholar) and is rapidly becoming a major health concern because of significant increases in the prevalence of obesity, insulin resistance diabetes, and hyperlipidemia (2Bettermann K. Hohensee T. Haybaeck J. Steatosis and steatohepatitis: Complex disorders.Int. J. Mol. Sci. 2014; 15 (24897026): 9924-994410.3390/ijms15069924Crossref PubMed Scopus (30) Google Scholar, 3Temple J.L. Cordero P. Li J. Nguyen V. Oben J.A. A guide to non-alcoholic fatty liver disease in childhood and adolescence.Int. J. Mol. Sci. 2016; 17 (27314342): E974Crossref PubMed Scopus (107) Google Scholar). Encompassing the entire spectrum of fatty liver disease in individuals without significant alcohol consumption, NAFLD is histologically categorized into nonalcoholic fatty liver (NAFL; steatosis without hepatocellular injury) and nonalcoholic steatohepatitis (NASH; steatosis with ballooning, inflammation, with or without fibrosis) (4Chalasani N. Younossi Z. Lavine J.E. Diehl A.M. Brunt E.M. Cusi K. Charlton M. Sanyal A.J. The diagnosis and management of non-alcoholic fatty liver disease: Practice Guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association.Hepatology. 2012; 55 (22488764): 2005-202310.1002/hep.25762Crossref PubMed Scopus (2530) Google Scholar). The chances of developing more serious diseases such as cirrhosis, hepatocellular carcinoma, and cardiovascular diseases are increased in patients with NASH (5Farrell G.C. van Rooyen D. Gan L. Chitturi S. NASH is an inflammatory disorder: Pathogenic, prognostic and therapeutic implications.Gut Liver. 2012; 6 (22570745): 149-17110.5009/gnl.2012.6.2.149Crossref PubMed Scopus (299) Google Scholar). NASH is characterized by hepatocyte damage due to lipotoxicity as well as macrophage-associated liver inflammation, a process in which the cross-talk between hepatocytes and macrophages is crucial. Emerging evidence highlights that lipid accumulation in hepatocytes stimulates the production of proinflammatory cytokines and chemokines, thereby potentially contributing to the initiation of hepatic inflammation and subsequent liver injury (6Ibrahim S.H. Hirsova P. Tomita K. Bronk S.F. Werneburg N.W. Harrison S.A. Goodfellow V.S. Malhi H. Gores G.J. Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.Hepatology. 2016; 63 (26406121): 731-74410.1002/hep.28252Crossref PubMed Scopus (146) Google Scholar, 7Joshi-Barve S. Barve S.S. Amancherla K. Gobejishvili L. Hill D. Cave M. Hote P. McClain C.J. Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes.Hepatology. 2007; 46 (17680645): 823-83010.1002/hep.21752Crossref PubMed Scopus (277) Google Scholar). Despite this knowledge, what controls the release of proinflammatory cytokines and chemokines leading to the NASH transition is still obscure and requires elucidation. nonalcoholic fatty liver disease nonalcoholic fatty liver nonalcoholic steatohepatitis small heterodimer partner high fat, cholesterol, and fructose alanine aminotransferase aspartate aminotransferase triglyceride hematoxylin and eosin terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling very-low-density lipoprotein methionine/choline–deficient c-Jun N-terminal kinase hepatic stellate cell Kupffer cell quantitative PCR palmitic acid lipopolysaccharide phosphatidylinositol 3-kinase 12-O-tetradecanoylphorbol-13-acetate response element conditioned medium farnesoid X–activated receptor fetal bovine serum NAFLD activity score glucose tolerance test. Nuclear receptor small heterodimer partner (Nr0b2, Homo sapiens SHP; Mus musculus Shp) is highly expressed in normal hepatocytes and acts as an important transcriptional regulator for bile acid, glucose, and lipid metabolism (8Zhang Y. Hagedorn C.H. Wang L. Role of nuclear receptor SHP in metabolism and cancer.Biochim. Biophys. Acta. 2011; 1812 (20970497): 893-90810.1016/j.bbadis.2010.10.006Crossref PubMed Scopus (169) Google Scholar). In support of a critical role for SHP in metabolic diseases, SHP mutation is associated with an increase in body weight and morbidity risk of type 2 diabetes in Japanese populations (9Nishigori H. Tomura H. Tonooka N. Kanamori M. Yamada S. Sho K. Inoue I. Kikuchi N. Onigata K. Kojima I. Kohama T. Yamagata K. Yang Q. Matsuzawa Y. Miki T. et al.Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects.Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11136233): 575-58010.1073/pnas.98.2.575Crossref PubMed Scopus (137) Google Scholar). More recently, SHP has been shown to suppress toll-like receptor 4 (TLR4)-induced (10Yuk J.M. Shin D.M. Lee H.M. Kim J.J. Kim S.W. Jin H.S. Yang C.S. Park K.A. Chanda D. Kim D.K. Huang S.M. Lee S.K. Lee C.H. Kim J.M. Song C.H. et al.The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors.Nat. Immunol. 2011; 12 (21725320): 742-75110.1038/ni.2064Crossref PubMed Scopus (148) Google Scholar) and NLRP3 inflammasome-mediated (11Yang C.S. Kim J.J. Kim T.S. Lee P.Y. Kim S.Y. Lee H.M. Shin D.M. Nguyen L.T. Lee M.S. Jin H.S. Kim K.K. Lee C.H. Kim M.H. Park S.G. Kim J.M. et al.Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome.Nat. Commun. 2015; 6 (25655831): 611510.1038/ncomms7115Crossref PubMed Scopus (88) Google Scholar) inflammatory responses in monocytes, which suggests a connection between SHP and inflammation. Now, hepatocytes are gradually being recognized as important players involved in the initiation of inflammation in NASH (6Ibrahim S.H. Hirsova P. Tomita K. Bronk S.F. Werneburg N.W. Harrison S.A. Goodfellow V.S. Malhi H. Gores G.J. Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes.Hepatology. 2016; 63 (26406121): 731-74410.1002/hep.28252Crossref PubMed Scopus (146) Google Scholar, 7Joshi-Barve S. Barve S.S. Amancherla K. Gobejishvili L. Hill D. Cave M. Hote P. McClain C.J. Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes.Hepatology. 2007; 46 (17680645): 823-83010.1002/hep.21752Crossref PubMed Scopus (277) Google Scholar). However, whether hepatocyte SHP plays a role in this process remains unexplored. Inflammatory chemokine (C–C motif) ligand 2 (CCL2; or monocyte chemoattractant protein 1 (MCP1)) is responsible for attracting monocytes and T cells during liver injury (12Ansari A.W. Heiken H. Meyer-Olson D. Schmidt R.E. CCL2: A potential prognostic marker and target of anti-inflammatory strategy in HIV/AIDS pathogenesis.Eur. J. Immunol. 2011; 41 (22076814): 3412-341810.1002/eji.201141676Crossref PubMed Scopus (41) Google Scholar). There are many types of liver cells that produce CCL2, including hepatocytes, stellate cells, and Kupffer cells (13Saiman Y. Friedman S.L. The role of chemokines in acute liver injury.Front. Physiol. 2012; 3 (22723782): 213Crossref PubMed Scopus (118) Google Scholar, 14Weiskirchen R. Tacke F. Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology.Hepatobiliary Surg. Nutr. 2014; 3 (25568859): 344-363PubMed Google Scholar). Studies have shown that high levels of CCL2 in NAFLD contribute to the conversion of NAFL to NASH (15Haukeland J.W. Damås J.K. Konopski Z. Løberg E.M. Haaland T. Goverud I. Torjesen P.A. Birkeland K. Bjøro K. Aukrust P. Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2.J. Hepatol. 2006; 44 (16618517): 1167-117410.1016/j.jhep.2006.02.011Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), and pharmacological inhibition of CCL2 reduces liver macrophage infiltration in NASH (16Baeck C. Wehr A. Karlmark K.R. Heymann F. Vucur M. Gassler N. Huss S. Klussmann S. Eulberg D. Luedde T. Trautwein C. Tacke F. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.Gut. 2012; 61 (21813474): 416-42610.1136/gutjnl-2011-300304Crossref PubMed Scopus (386) Google Scholar), which makes CCL2 a good therapeutic target for NASH prevention and treatment. However, how CCL2 is produced during the disease progression from NAFL to NASH is incompletely understood. A recent study has demonstrated that the activation of SHP by a small-molecule activator inhibits liver cancer cell migration by blocking CCL2 signaling (17Yang Z. Koehler A.N. Wang L. A novel small molecule activator of nuclear receptor SHP inhibits HCC cell migration via suppressing Ccl2.Mol. Cancer Ther. 2016; 15 (27486225): 2294-230110.1158/1535-7163.MCT-16-0153Crossref PubMed Scopus (31) Google Scholar), which potentially links SHP to CCL2 production. Here, we show that SHP was markedly decreased in the livers of patients with NASH and in diet-induced mouse NASH. The loss of SHP in hepatocytes resulted in NF-κB p65-mediated induction of CCL2, leading to macrophage activation. Meanwhile, overexpressing SHP in hepatocytes prevented NAFL progression to NASH by attenuating liver Inflammation and fibrosis. Taken together, our study has uncovered a novel regulatory network in hepatocytes consisting of JNK/SHP/NF-κB/CCL2, which controls macrophage recruitment during the disease progression from NAFL to NASH. The findings from our study may benefit the development of new management or prevention strategies for NASH. To determine whether the expression of SHP is associated with NAFLD pathogenesis, we examined SHP mRNA levels in two sets of human liver specimens. The first set was obtained through the University of Kansas Liver Center. The liver histology of human NAFL and NASH is shown in Fig. 1A. NAFL is characterized by the deposition of triglycerides as lipid droplets in hepatocytes. NASH is distinguished from NAFL by the presence of hepatocyte injury (hepatocyte ballooning and cell death), inflammation, and/or collagen deposition (fibrosis). Perisinusoidal/pericellular (chicken wire) fibrosis is the characteristic pattern of liver fibrosis in NASH, which typically begins in zone 3 due to the deposition of collagen along the sinusoids and around the hepatocytes. As shown in Fig. 1A, picrosirius red staining showed the chicken-wire pattern of perisinusoidal/pericellular fibrosis and periportal fibrosis in human NASH. Although there were similar SHP mRNA levels in the liver of normal and NAFL samples, a significant decrease in SHP mRNA was observed in NASH samples compared with NAFL samples (Fig. 1A). Consistently, the analysis of microarray data set GSE48452 also revealed a significant decrease in SHP mRNA levels in patients with NASH compared with NAFL and normal controls (Fig. 1B). Western blot analysis confirmed the decrease in SHP protein in NASH samples compared with NAFL and normal livers (Fig. 1C). Collectively, our results strongly suggest the biological relevance of SHP down-regulation during the disease progression from NAFL to NASH in humans. To more precisely examine SHP expression during the development of NAFLD, we developed a mouse model that carries the disease progression from NAFL to NASH with obesity and insulin resistance, the two common features of NAFLD in humans. Diet enriched in high fat and fructose has been implicated in the development of obesity and NASH in humans (18Ouyang X. Cirillo P. Sautin Y. McCall S. Bruchette J.L. Diehl A.M. Johnson R.J. Abdelmalek M.F. Fructose consumption as a risk factor for non-alcoholic fatty liver disease.J. Hepatol. 2008; 48 (18395287): 993-99910.1016/j.jhep.2008.02.011Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 19Abdelmalek M.F. Suzuki A. Guy C. Unalp-Arida A. Colvin R. Johnson R.J. Diehl A.M. Nonalcoholic Steatohepatitis Clinical Research Network Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease.Hepatology. 2010; 51 (20301112): 1961-197110.1002/hep.23535Crossref PubMed Scopus (528) Google Scholar). Recently, a diet enriched in high fat, cholesterol, and fructose (research diet D09100301: 40 kcal% fat, 2% cholesterol, 20 kcal% fructose; hereafter referred to as HFCF diet) was utilized to induce mouse NASH (20Trevaskis J.L. Griffin P.S. Wittmer C. Neuschwander-Tetri B.A. Brunt E.M. Dolman C.S. Erickson M.R. Napora J. Parkes D.G. Roth J.D. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 302 (22268099): G762-G77210.1152/ajpgi.00476.2011Crossref PubMed Scopus (182) Google Scholar, 21Clapper J.R. Hendricks M.D. Gu G. Wittmer C. Dolman C.S. Herich J. Athanacio J. Villescaz C. Ghosh S.S. Heilig J.S. Lowe C. Roth J.D. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 305 (23886860): G483-G49510.1152/ajpgi.00079.2013Crossref PubMed Scopus (176) Google Scholar). In this diet, excess fat alone contributes to the development of mild steatosis, whereas the addition of elevated fructose and cholesterol levels increases hepatic oxidative stress; combined, these dietary components predispose animals to necroinflammation and fibrogenesis (22Kohli R. Kirby M. Xanthakos S.A. Softic S. Feldstein A.E. Saxena V. Tang P.H. Miles L. Miles M.V. Balistreri W.F. Woods S.C. Seeley R.J. High-fructose, medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis.Hepatology. 2010; 52 (20607689): 934-94410.1002/hep.23797Crossref PubMed Scopus (268) Google Scholar). We fed 2-month-old C57Bl/6J male mice with either a chow or HFCF diet for 1 and 5 months. We chose to study male mice based on our previous observation that male but not female mice developed NASH after 5 months of HFCF diet, 4A. Zou, N. Magee, F. Deng, S. Lehn, C. Zhong, and Y. Zhang, unpublished data. which also has been reported by another group (23Ganz M. Bukong T.N. Csak T. Saha B. Park J.K. Ambade A. Kodys K. Szabo G. Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice.J. Transl. Med. 2015; 13 (26077675): 19310.1186/s12967-015-0552-7Crossref PubMed Scopus (81) Google Scholar). The observation that males are more susceptible to NASH is supported by human epidemiology studies showing that NAFLD cases more commonly arise and frequently progress in males, as females possess a resistance to NAFLD attributed to higher levels of estrogen (24Suzuki A. Abdelmalek M.F. Nonalcoholic fatty liver disease in women.Women's Health (Lond.). 2009; 5 (19245356): 191-203Crossref PubMed Scopus (109) Google Scholar). Mice on the HFCF diet developed rapid weight gain and obesity compared with chow-fed controls (Fig. 2A). Although 1 month of HFCF feeding did not significantly change the liver weight compared with controls, the liver weight was significantly increased in mice on the HFCF diet for 5 months (Fig. 2B). This was accompanied by an increase in the liver to body weight ratio (Fig. 2B). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), two markers of liver injury, both increased after mice were fed the HFCF diet for 5 months (Fig. 2C). Additionally, 5 months of HFCF feeding led to a significant increase in fasting glucose levels and total cholesterol levels (Fig. 2D). Meanwhile, mice fed for 1 month on the HFCF diet developed hypertriglyceridemia, but the levels of serum triglycerides (TG) at the 5-month time point declined to levels that were similar to those noted in chow-fed controls (Fig. 2D). Moreover, the mice displayed glucose intolerance after 5 months on the HFCF diet (Fig. 2E). Collectively, these results indicated that mice fed a HFCF diet developed liver injury with obesity, dyslipidemia, and hyperglycemia with impaired glucose tolerance. We next examined the extent of steatosis, cell death, inflammation, and fibrosis in the livers of mice fed chow or HFCF diets. Liver sections stained with hematoxylin and eosin (H&E) and oil red O revealed liver steatosis in mice fed a HFCF diet for 1 and 5 months, which was not observed in chow-fed controls (Fig. 2F). However, the cell death detected by TUNEL staining was only observed in the livers of mice fed a HFCF diet for 5 months (Fig. 2F). Consistently, immunohistochemistry staining with the macrophage-specific antibody F4/80 showed a dramatic increase in macrophage infiltration in the livers of mice on the HFCF diet for 5 months (Fig. 2F). Moreover, liver sections stained with Picosirius red detected an apparent collagen deposition in 5-month HFCF diet-fed mice, indicative of fibrosis development (Fig. 2F). The summary in Fig. 2G shows that the grades of steatosis, inflammation, necrosis, and fibrosis became apparent in the livers of mice fed the HFCF diet for 5 months. At the mRNA levels, peroxisome proliferator-activated receptor γ (Pparγ), a master control of lipid synthesis, sustained an increase in the livers of mice fed a HFCF diet (Fig. 3A). However, the expression of both apolipoprotein B (ApoB) and microsomal triglyceride transfer protein (Mttp), two genes involved in the very-low-density lipoprotein (VLDL) synthesis that carries TG in the plasma, was decreased in mice fed the HFCF diet for 5 months (Fig. 3A), consistent with the changes in serum TG levels shown in Fig. 2D. Tumor necrosis factor α (Tnfα) and chemokine Ccl2, two important inflammatory mediators contributing to the inflammatory cell infiltration in the liver, were both significantly increased over time in mice on the HFCF diet (Fig. 3A). In addition, a progressive increase in the expression of macrophage M1 marker nitric-oxide synthase 2 (Nos2) and a decrease in the expression of M2 markers arginase-1 (Agr1) and CD163 were observed in the livers of mice on the HFCF diet for 5 months (Fig. 3A). Similarly, the liver expression of collagen 1α1 (Col1A1) increased in 5-month HFCF diet-fed animals (Fig. 3A), supporting the increase in collagen deposition shown in Fig. 2F. Additionally, the serum levels of CCL2 were markedly elevated in the 5-month HFCF diet-fed mice (Fig. 3B). Overall, these results indicate the progression of NAFL to NASH in mice on the HFCF diet for 1 and 5 months as evidenced by increases in hepatic cell death, macrophage infiltration, and liver fibrosis. We next examined SHP expression in the liver of the HFCF-dietary mouse model. As shown in Fig. 3C, 1 month of HFCF feeding did not change liver Shp mRNA levels compared with those of the chow-fed controls. However, a significant decrease in Shp mRNA was observed after mice were on the HFCF diet for 5 months. SHP protein is a rapidly degraded protein with a very short half-life (25Miao J. Xiao Z. Kanamaluru D. Min G. Yau P.M. Veenstra T.D. Ellis E. Strom S. Suino-Powell K. Xu H.E. Kemper J.K. Bile acid signaling pathways increase stability of small heterodimer partner (SHP) by inhibiting ubiquitin-proteasomal degradation.Genes Dev. 2009; 23 (19390091): 986-99610.1101/gad.1773909Crossref PubMed Scopus (109) Google Scholar). We employed two anti-SHP antibodies in Western blotting to determine SHP protein levels in the liver. SHP (H-160) is a rabbit polyclonal antibody, whereas SHP (H-5) is a mouse mAb. Both antibodies recognized the epitope corresponding to amino acids 1–160 mapping at the N terminus of SHP protein. As shown in Fig. 3D, both antibodies detected a significant decrease in SHP protein expression in the livers of mice fed a HFCF diet for 5 months. A methionine/choline–deficient (MCD) diet induces NASH-like liver pathology including liver steatosis, inflammation, and fibrosis, despite weight loss and insulin sensitivity (26Hebbard L. George J. Animal models of nonalcoholic fatty liver disease.Nat. Rev. Gastroenterol. Hepatol. 2011; 8 (21119613): 35-4410.1038/nrgastro.2010.191Crossref PubMed Scopus (346) Google Scholar). Next, we explored SHP expression in the livers of mice fed an MCD diet. As shown in Fig. 3E, the liver morphology indicated the development of liver steatosis, inflammation, and fibrosis in mice fed an MCD diet for 1 month. Consistently, the expression of genes involved in liver inflammation and fibrosis such as F4/80, Tnfα, Ccl2, IL-1β, and Col1A1 was increased in mice fed an MCD diet (Fig. 3F). Importantly, we observed a significant decrease in Shp mRNA level in the livers of mice fed an MCD diet compared with chow-fed controls (Fig. 3F). Collectively, our results indicate that the expression of Shp is decreased dramatically in the liver of mouse NASH. Moreover, our HFCF dietary mouse studies provide convincing evidence that SHP is suppressed during NAFL progression to NASH. Because SHP is highly expressed in hepatocytes (27Suh J.H. Huang J. Park Y.Y. Seong H.A. Kim D. Shong M. Ha H. Lee I.K. Lee K. Wang L. Choi H.S. Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-β signaling by repressing SMAD3 transactivation.J. Biol. Chem. 2006; 281 (17074765): 39169-3917810.1074/jbc.M605947200Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), we speculated that SHP suppression during NAFL transition to NASH results mainly from the decrease of SHP in hepatocytes. To begin to test our hypothesis, we isolated hepatocytes, hepatic stellate cells (HSC), and resident macrophage cells (KC) from mouse liver and compared Shp expression among these cells. Cell purification was confirmed by the detection of various cell-specific markers by quantitative PCR (qPCR), including hepatocyte marker albumin (Alb), quiescent HSC marker Hh-interacting protein (Hhip), and KC marker F4/80. As expected, Shp mRNA is abundantly expressed in hepatocytes and low is in KCs and HSCs (Fig. 4A). We next sought to investigate the potential mechanisms of SHP suppression in NASH. Lipotoxicity, the major mechanism underlying hepatocyte dysfunction in NAFLD, occurs in the setting of excessive free fatty acid traffic in hepatocytes, especially saturated fatty acids (28Leamy A.K. Egnatchik R.A. Young J.D. Molecular mechanisms and the role of saturated fatty acids in the progression of non-alcoholic fatty liver disease.Prog. Lipid Res. 2013; 52 (23178552): 165-17410.1016/j.plipres.2012.10.004Crossref PubMed Scopus (288) Google Scholar). Palmitic acid (PA) is one of the most abundant of the saturated fatty acids presented in diets and in serum (29Baylin A. Kabagambe E.K. Siles X. Campos H. Adipose tissue biomarkers of fatty acid intake.Am. J. Clin. Nutr. 2002; 76 (12324287): 750-75710.1093/ajcn/76.4.750Crossref PubMed Scopus (264) Google Scholar). PA binds to TLR4 leading to JNK activation (30Huang S. Rutkowsky J.M. Snodgrass R.G. Ono-Moore K.D. Schneider D.A. Newman J.W. Adams S.H. Hwang D.H. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways.J Lipid Res. 2012; 53 (22766885): 2002-201310.1194/jlr.D029546Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 31Win S. Than T.A. Le B.H. García-Ruiz C. Fernandez-Checa J.C. Kaplowitz N. Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity.J. Hepatol. 2015; 62 (25666017): 1367-137410.1016/j.jhep.2015.01.032Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We next employed a primary mouse hepatocyte culture as an in vitro model and explored whether PA or TLR4 ligand lipopolysaccharide (LPS) could alter SHP expression. Treatment with PA (0.5 mm) or LPS (100 ng/ml) for 6 h significantly decreased Shp mRNA expression (Fig. 4B). To further investigate the potential mechanisms of SHP suppression by PA and LPS, we superimposed various signaling pathway inhibitors, including JNK inhibitor SP600125 (50 μm), NF-κB inhibitor BAY 11-7082 (5 μm), and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μm) on a PA or LPS treatment regimen. Interestingly, co-treatment with the JNK inhibitor completely obviated the decrease of Shp mRNA by PA or LPS (Fig. 4B). Thus, our results indicate that JNK activation mediates the suppression of SHP by PA and LPS in hepatocytes. Moreover, JNK activation was observed in the livers of mice fed a HFCF diet for 5 months as evidenced by the induction of phosphorylated JNK (Fig. 4C, p-JNK, activated form of JNK). Importantly, the activation of JNK correlated positively with SHP suppression in the livers of 5-month HFCF diet-fed mice (Fig. 3D). Collectively, our in vitro and in vivo results indicate, for the first time, that JNK activation suppresses Shp expression in NASH. JNK was originally identified because of its capability of specifically phosphorylating c-Jun on its N-terminal transactivation domain at two serine residues, Ser-63 and Ser-73 (32Hibi M. Lin A. Smeal T. Minden A. Karin M. Identification of an oncoprotein- and UV-responsiv" @default.
• W2802528664 created "2018-05-17" @default.
• W2802528664 creator A5004376157 @default.
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• W2802528664 date "2018-06-01" @default.
• W2802528664 modified "2023-10-17" @default.
• W2802528664 title "Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis" @default.
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