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• W1988293732 abstract "Adiponectin receptors play a key role in steatosis and inflammation; however, very little is known about regulation of adiponectin receptors in liver. Here, we examined the effects of palmitate loading, endoplasmic reticulum (ER) stress, and the hypolipidemic agent fenofibrate on adiponectin receptor R2 (AdipoR2) levels and AMP-activated protein kinase (AMPK) in human hepatoma Huh7 cells and in Huh.8 cells, a model of hepatitis C-induced steatosis. Palmitate treatment reduced AdipoR2 protein and basal AMPK phosphorylation in Huh7 cells. Fenofibrate treatment preserved AdipoR2 and phosphorylated AMPK (pAMPK) levels in palmitate-treated cells accompanied by reduced triglyceride (TG) accumulation and less activation of ER stress markers CCAAT/enhancer binding (C/EBPβ) and eukaryotic translation initiation factor 2 α. ER stress agents thapsigargin and tunicamycin suppressed AdipoR2 and pAMPK levels in Huh7 cells, while fenofibrate and the chemical chaperone 4-phenylbutyrate (PBA) prevented these changes. AdipoR2 levels were lower in Huh.8 cells and fenofibrate treatment increased AdipoR2 while reducing activation of c-Jun N-terminal kinase and C/EBPβ expression without changing TG levels. Taken together, these results suggest that fatty acids and ER stress reduce AdipoR2 protein and pAMPK levels, while fenofibrate and PBA might be important therapeutic agents to correct lipid- and ER stress-mediated loss of AdipoR2 and pAMPK associated with nonalcoholic steatohepatitis. Adiponectin receptors play a key role in steatosis and inflammation; however, very little is known about regulation of adiponectin receptors in liver. Here, we examined the effects of palmitate loading, endoplasmic reticulum (ER) stress, and the hypolipidemic agent fenofibrate on adiponectin receptor R2 (AdipoR2) levels and AMP-activated protein kinase (AMPK) in human hepatoma Huh7 cells and in Huh.8 cells, a model of hepatitis C-induced steatosis. Palmitate treatment reduced AdipoR2 protein and basal AMPK phosphorylation in Huh7 cells. Fenofibrate treatment preserved AdipoR2 and phosphorylated AMPK (pAMPK) levels in palmitate-treated cells accompanied by reduced triglyceride (TG) accumulation and less activation of ER stress markers CCAAT/enhancer binding (C/EBPβ) and eukaryotic translation initiation factor 2 α. ER stress agents thapsigargin and tunicamycin suppressed AdipoR2 and pAMPK levels in Huh7 cells, while fenofibrate and the chemical chaperone 4-phenylbutyrate (PBA) prevented these changes. AdipoR2 levels were lower in Huh.8 cells and fenofibrate treatment increased AdipoR2 while reducing activation of c-Jun N-terminal kinase and C/EBPβ expression without changing TG levels. Taken together, these results suggest that fatty acids and ER stress reduce AdipoR2 protein and pAMPK levels, while fenofibrate and PBA might be important therapeutic agents to correct lipid- and ER stress-mediated loss of AdipoR2 and pAMPK associated with nonalcoholic steatohepatitis. Hepatic steatosis is a hallmark of nonalcoholic fatty liver disease; however, it is a frequent histological finding in other metabolic liver diseases, including chronic hepatitis C (1Powell E.E. Cooksley W.G. Hanson R. Searle J. Halliday J.W. Powell L.W. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years.Hepatology. 1990; 11: 74-80Crossref PubMed Scopus (1326) Google Scholar, 2Hourigan L.F. Macdonald G.A. Purdie D. Whitehall V.H. Shorthouse C. Clouston A. Powell E.E. Fibrosis in chronic hepatitis C correlates significantly with body mass index and steatosis.Hepatology. 1999; 29: 1215-1219Crossref PubMed Scopus (607) Google Scholar). Nonalcoholic fatty liver disease encompasses a wide range of liver injury, from simple steatosis to nonalcoholic steatohepatitis (NASH) or even cirrhosis or fibrosis (3Farrell G.C. Larter C.Z. Nonalcoholic fatty liver disease: from steatosis to cirrhosis.Hepatology. 2006; 43: S99-112Crossref PubMed Scopus (1961) Google Scholar, 4Marion A.W. Baker A.J. Dhawan A. Fatty liver disease in children.Arch. Dis. Child. 2004; 89: 648-652Crossref PubMed Scopus (87) Google Scholar). The pathogenesis of NASH begins with increased synthesis and/or decreased oxidation of triglycerides (TG) in the hepatocyte, thereby leading to increased fat accumulation. Because steatosis may render the liver more vulnerable to inflammation and fibrosis (5Adinolfi L.E. Gambardella M. Andreana A. Tripodi M.F. Utili R. Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity.Hepatology. 2001; 33: 1358-1364Crossref PubMed Scopus (957) Google Scholar), there is increasing interest in the role of steatosis-induced liver injury. Excess TG accumulation activates a number of cellular stress signaling and inflammatory pathways, such as c-Jun N-terminal kinase (JNK) and nuclear factor κB, that may eventually lead to apoptosis, giving rise to the phenomenon termed lipotoxicity (6Unger R.H. Orci L. Lipoapoptosis: its mechanism and its diseases.Biochim. Biophys. Acta. 2002; 1585: 202-212Crossref PubMed Scopus (299) Google Scholar, 7Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J. Clin. Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (3182) Google Scholar). NASH is associated with a significant decrease in plasma adiponectin levels, which correlates with the severity of liver histology (8Tsochatzis E. Papatheodoridis G.V. Archimandritis A.J. The evolving role of leptin and adiponectin in chronic liver diseases.Am. J. Gastroenterol. 2006; 101: 2629-2640Crossref PubMed Scopus (163) Google Scholar). The metabolic effects of adiponectin are mediated by two putative receptors: adiponectin receptor R1 (AdipoR1) and adiponectin receptor R2 (AdipoR2). The former is primarily expressed in muscle, while the latter is expressed in liver (9Lin H.V. Kim J.Y. Pocai A. Rossetti L. Shapiro L. Scherer P.E. Accili D. Adiponectin resistance exacerbates insulin resistance in insulin receptor transgenic/knockout mice.Diabetes. 2007; 56: 1969-1976Crossref PubMed Scopus (76) Google Scholar). At the cellular/molecular level, AdipoR2 mRNA expression is decreased in most (10Kaser S. Moschen A. Cayon A. Kaser A. Crespo J. Pons-Romero F. Ebenbichler C.F. Patsch J.R. Tilg H. Adiponectin and its receptors in non-alcoholic steatohepatitis.Gut. 2005; 54: 117-121Crossref PubMed Scopus (367) Google Scholar, 11Jonsson J.R. Moschen A.R. Hickman I.J. Richardson M.M. Kaser S. Clouston A.D. Powell E.E. Tilg H. Adiponectin and its receptors in patients with chronic hepatitis C.J. Hepatol. 2005; 43: 929-936Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 12Liu C.J. Chen P.J. Jeng Y.M. Huang W.L. Yang W.S. Lai M.Y. Kao J.H. Chen D.S. Serum adiponectin correlates with viral characteristics but not histologic features in patients with chronic hepatitis C.J. Hepatol. 2005; 43: 235-242Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 13Ma 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) but not all (14Vuppalanchi R. Marri S. Kolwankar D. Considine R.V. Chalasani N. Is adiponectin involved in the pathogenesis of nonalcoholic steatohepatitis? A preliminary human study.J. Clin. Gastroenterol. 2005; 39: 237-242Crossref PubMed Scopus (85) Google Scholar, 15Nannipieri M. Cecchetti F. Anselmino M. Mancini E. Marchetti G. Bonotti A. Baldi S. Solito B. Giannetti M. Pinchera A. et al.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) liver samples from NASH patients as compared with control patients, with no difference in AdipoR1 mRNA expression (10Kaser S. Moschen A. Cayon A. Kaser A. Crespo J. Pons-Romero F. Ebenbichler C.F. Patsch J.R. Tilg H. Adiponectin and its receptors in non-alcoholic steatohepatitis.Gut. 2005; 54: 117-121Crossref PubMed Scopus (367) Google Scholar). Yamauchi et al. (16Yamauchi T. Nio Y. Maki T. Kobayashi M. Takazawa T. Iwabu M. Okada-Iwabu M. Kawamoto S. Kubota N. Kubota T. et al.Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.Nat. Med. 2007; 13: 332-339Crossref PubMed Scopus (1050) Google Scholar) have shown that adenovirus-mediated overexpression of either AdipoR1 or AdipoR2 in liver reversed steatosis-linked insulin resistance in diabetes. This was recently confirmed by Tomita et al. (17Tomita K. Oike Y. Teratani T. Taguchi T. Noguchi M. Suzuki T. Mizutani A. Yokoyama H. Irie R. Sumimoto H. et al.Hepatic AdipoR2 signaling plays a protective role against progression of nonalcoholic steatohepatitis in mice.Hepatology. 2008; 48: 458-473Crossref PubMed Scopus (108) Google Scholar) in a methionine-deficient and choline-deficient mouse model of NASH. Conversely, simultaneous disruption of these two receptors resulted in glucose intolerance and insulin resistance. AdipoRl is reportedly linked with AMP-activated protein kinase (AMPK) activation and AdipoR2 with peroxisome proliferator-activated receptor α (PPARα) (16Yamauchi T. Nio Y. Maki T. Kobayashi M. Takazawa T. Iwabu M. Okada-Iwabu M. Kawamoto S. Kubota N. Kubota T. et al.Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.Nat. Med. 2007; 13: 332-339Crossref PubMed Scopus (1050) Google Scholar). In addition, a hepatoprotective role of adiponectin is now emerging in which adiponectin acts as an antiinflammatory hormone participating in the repair process of liver injury (18Aygun C. Kocaman O. Senturk O. Adiponectin: Is it a novel treatment modality to alleviate liver injury in non-alcoholic fatty liver disease (NAFLD)?.Med. Hypotheses. 2008; 70: 457-458Crossref PubMed Scopus (4) Google Scholar, 19Sheikh M.Y. Choi J. Qadri I. Friedman J.E. Sanyal A.J. Hepatitis C virus infection: molecular pathways to metabolic syndrome.Hepatology. 2008; 47: 2127-2133Crossref PubMed Scopus (212) Google Scholar). Thus, this raises the possibility that the loss of hepatic AdipoR2 receptor in NASH patients may play an important role in worsening many of the metabolic, enzymatic, and gene regulatory changes that characterize human NASH patients (8Tsochatzis E. Papatheodoridis G.V. Archimandritis A.J. The evolving role of leptin and adiponectin in chronic liver diseases.Am. J. Gastroenterol. 2006; 101: 2629-2640Crossref PubMed Scopus (163) Google Scholar, 20Capeau J. The story of adiponectin and its receptors AdipoR1 and R2: to follow.J. Hepatol. 2007; 47: 736-738Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). However, very little information is currently available about the interaction between steatosis and loss of adiponectin receptor expression in hepatic cells. Emerging evidence suggests that the ability to counter-regulate inflammatory responses plays an important role in metabolic control through regulation of endoplasmic reticulum (ER) stress (21Wu J. Kaufman R.J. From acute ER stress to physiological roles of the unfolded protein response.Cell Death Differ. 2006; 13: 374-384Crossref PubMed Scopus (722) Google Scholar, 22Ozcan U. Yilmaz E. Ozcan L. Furuhashi M. Vaillancourt E. Smith R.O. Gorgun C.Z. Hotamisligil G.S. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes.Science. 2006; 313: 1137-1140Crossref PubMed Scopus (1985) Google Scholar). Metabolic disorders including obesity and alcohol ingestion can cause protein misfolding or overloading in the ER, triggering a stress cascade with pathological consequences including inflammation and cell death (23Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science. 2004; 306: 457-461Crossref PubMed Scopus (2973) Google Scholar, 24Ji C. Mehrian-Shai R. Chan C. Hsu Y.H. Kaplowitz N. Role of CHOP in hepatic apoptosis in the murine model of intragastric ethanol feeding.Alcohol. Clin. Exp. Res. 2005; 29: 1496-1503Crossref PubMed Scopus (146) Google Scholar). When mammalian cells are subjected to protein overload in the ER, an immediate response is activation of the pancreatic eukaryotic translation initiation factor α kinase (eIF2α) to inhibit protein biosynthesis through phosphorylation of eIF2α (21Wu J. Kaufman R.J. From acute ER stress to physiological roles of the unfolded protein response.Cell Death Differ. 2006; 13: 374-384Crossref PubMed Scopus (722) Google Scholar). If the overload of unfolded or misfolded proteins in the ER is not resolved, prolonged activation leads to programmed cell death requiring increased CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP/GADD153), a transcription factor that potentiates apoptosis (25Day C.P. James O.F. Steatohepatitis: a tale of two “hits”?.Gastroenterology. 1998; 114: 842-845Abstract Full Text Full Text PDF PubMed Scopus (3462) Google Scholar). The transcription factor C/EBPβ is also activated in the liver through the actions of cytokines, hormones, nutrients, and ER stress (26Poli V. The role of C/EBP isoforms in the control of inflammatory and native immunity functions.J. Biol. Chem. 1998; 273: 29279-29282Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 27Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem. J. 2002; 365: 561-575Crossref PubMed Google Scholar) and has been suggested to play an important role in proinflammatory and apoptotic pathways (28Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. Jiang H. Qadri I. Maclean K.N. Friedman J.E. CCAAT/enhancing binding protein beta deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (126) Google Scholar). The main aim of the present study was to investigate the effects of lipid loading and the therapeutic effects of the PPARα agonist fenofibrate on expression of AdipoR2 and levels of phosphorylated AMPK (pAMPK) in Huh7 human hepatoma cells and in the Huh.8 cell line, which stably expresses a hepatitis C virus (HCV) subgenome replicon (29Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1276) Google Scholar). These cells display many features of HCV-induced fatty liver disease including increased intracellular lipid accumulation and activation of JNK by a reactive oxygen species-ER stress pathway (30Qadri I. Iwahashi M. Kullak-Ublick G.A. Simon F.R. Hepatocyte nuclear factor (HNF) 1 and HNF4 mediate hepatic multidrug resistance protein 2 up-regulation during hepatitis C virus gene expression.Mol. Pharmacol. 2006; 70: 627-636Crossref PubMed Scopus (44) Google Scholar, 31Qadri I. Iwahashi M. Capasso J.M. Hopken M.W. Flores S. Schaack J. Simon F.R. Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1.Biochem. J. 2004; 378: 919-928Crossref PubMed Scopus (132) Google Scholar). Fenofibrate is widely used as a hypolipidemic drug that activates PPARα and thereby regulates the expression of a number of genes critical for lipid and lipoprotein metabolism (32Schoonjans K. Staels B. Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression.J. Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar, 33Staels B. Dallongeville J. Auwerx J. Schoonjans K. Leitersdorf E. Fruchart J.C. Mechanism of action of fibrates on lipid and lipoprotein metabolism.Circulation. 1998; 98: 2088-2093Crossref PubMed Scopus (1409) Google Scholar, 34Kliewer S.A. Lehmann J.M. Willson T.M. Orphan nuclear receptors: shifting endocrinology into reverse.Science. 1999; 284: 757-760Crossref PubMed Scopus (426) Google Scholar), leading to lipid catabolism in the liver and reduction in total body fat as well as circulating plasma lipids (35Mancini F.P. Lanni A. Sabatino L. Moreno M. Giannino A. Contaldo F. Colantuoni V. Goglia F. Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats.FEBS Lett. 2001; 491: 154-158Crossref PubMed Scopus (118) Google Scholar). We also investigated whether ER stress played a causal role in the loss of AdipoR2 and pAMPK in response to lipid overload. We found that ER stress inducers thapsigargin (TPG) and tunicamycin (TUN) caused a loss of AdipoR2 and pAMPK in Huh7 cells, while treatment with the chemical chaperone 4-phenylbutyrate (PBA), an agent used to reduce ER stress, restored palmitate- and TPG-mediated reduction of AdipoR2 expression and AMPK phosphorylation in both the parental Huh7 cell line and in Huh.8 derived cells. Our results demonstrate that ER stress is associated with decreased AdipoR2 and pAMPK levels and suggest that fenofibrate and PBA might be important therapeutic agents to prevent metabolic abnormalities by reducing ER stress associated with fatty liver disease. The cell lines used in this study, Huh7 and Huh.8, were provided by Dr. Charles Rice (Rockefeller University, New York, NY). The Huh.8 cell line was derived from the parental Huh7 cells and contains an HCV-derived expression vector stably integrated into the Huh7 background. The expression vector includes the HCV proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B linked to the antibiotic selection marker G418 (29Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1276) Google Scholar). The Huh7 parental cell lines were cultured in DMEM containing high glucose (25 mM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (all from Sigma-Aldrich, St. Louis, MO). Huh.8 cells were maintained in complete DMEM (25 mM glucose) supplemented with 10% heat-inactivated fetal bovine serum, nonessential amino acids, and 1 mg/ml G418 (Gemini Bio-Products, West Sacramento, CA). Cells were maintained at 37°C for two to three passages in a humidified environment containing 5% CO2. For experimental treatments, Huh7 and Huh.8 cells were serum-starved overnight and treated with different concentrations of palmitate, fenofibrate (Sigma), TUN (Sigma), or PBA (Calbiochem) as indicated in the appropriate figure legends. The conjugation of palmitate and albumin was prepared to deliver a final concentration of 200 μM palmitate in the tissue culture medium. One mol of palmitic acid was conjugated to 3 mol of albumin (both from Sigma). A 200 mM palmitic acid solution was prepared by dissolving 0.256 g palmitic acid in 5 ml of 100% ethanol and separately, a 27% (wt/v) albumin solution was prepared in PBS. From there, the conjugate (palmitate:albumin) was prepared by adding 4.5 ml of 27% albumin to 250 μl of 200 mM palmitate solution. The final volume was brought to 5 ml with PBS after adjusting to pH 7.4. The 10 mM palmitate stock was added directly to the culture without further dilution. The appropriate controls for experiments using palmitate contained 0.1% ethanol and 0.5% albumin. Cytosolic and nuclear extracts were prepared as previously reported (28Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. Jiang H. Qadri I. Maclean K.N. Friedman J.E. CCAAT/enhancing binding protein beta deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (126) Google Scholar). For analysis of AdipoR2 protein levels, membrane fractions were prepared by centrifuging the cytosolic extract in a Beckman ultracentrifuge at 60,000 rpm for 2 h. The pellets were resuspended in 50 μl of hypotonic buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, 2 μg/ml each of aprotinin and leupeptin, and 0.5 mg/ml benzamidine). For phospho-protein assays, cells were prepared and quantitated as described previously (28Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. Jiang H. Qadri I. Maclean K.N. Friedman J.E. CCAAT/enhancing binding protein beta deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (126) Google Scholar). Lysates were subjected to SDS-PAGE and Western blot analysis as previously described (28Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. Jiang H. Qadri I. Maclean K.N. Friedman J.E. CCAAT/enhancing binding protein beta deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (126) Google Scholar). Primary antibodies used in this study were C/EBPβ, CHOP, PPARα, actin, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated JNK (pJNK), pAMPK (Thr172), AMPK, phospho-eIF2α, and eIF2α (Cell Signaling Technology, Danvers, MA), JNK (R and D Systems, Minneapolis, MN), and AdipoR2 (Phoenix Pharmaceuticals, Burlingame, CA). Lipids were extracted from cells using the procedure of Bligh and Dyer (36Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). Quantitative enzymatic measurements of TG content were performed in triplicate using TG determination reagents (Sigma). We checked the effects of palmitate and palmitate in combination with fenofibrate on pAMPK and AdipoR2 levels in Huh7 human hepatoma cells. Huh7 cells were grown in complete DMEM (25 mM glucose) treated with either 200 μM palmitate alone or in combination with two concentrations of fenofibrate (50 and 100 μM), a known hypolipidemic agent. Palmitate significantly suppressed both pAMPK and AdipoR2 levels and coincubation with fenofibrate prevented palmitate-induced suppression of pAMPK and AdipoR2 expression in Huh7 cells (Fig. 1, A and B). In addition, we found that fenofibrate treatment alone (100 μM) for 24 h increased pAMPK levels and AdipoR2 protein expression in Huh7 cells (Fig. 1, C and D). The ER stress response has recently been recognized in the pathogenesis of liver disease, including apoptosis, fat accumulation, and inflammation (37Ji C. Dissection of endoplasmic reticulum stress signaling in alcoholic and non-alcoholic liver injury.J. Gastroenterol. Hepatol. 2008; 23: S16-S24Crossref PubMed Scopus (117) Google Scholar). To determine whether the positive effects of fenofibrate had any effect on reducing ER stress in palmitate-treated Huh7 cells, we monitored the expression of C/EBPβ and phospho-eIF2α, two key proteins induced by two different arms of the ER stress pathway (38Harding H.P. Zhang Y. Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.Nature. 1999; 397: 271-274Crossref PubMed Scopus (2519) Google Scholar, 39Chen C. Dudenhausen E.E. Pan Y.X. Zhong C. Kilberg M.S. Human CCAAT/enhancer-binding protein beta gene expression is activated by endoplasmic reticulum stress through an unfolded protein response element downstream of the protein coding sequence.J. Biol. Chem. 2004; 279: 27948-27956Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Palmitate treatment (200 μM) significantly increased phospho-eIF2α levels while coincubation with fenofibrate (100 μM) markedly reduced phospho-eIF2α expression (Fig. 2A), consistent with a reduction in ER stress. Palmitate alone induced a marked 100% increase in nuclear C/EBPβ, an inducer of fatty liver and ER stress in hepatic cells (28Rahman S.M. Schroeder-Gloeckler J.M. Janssen R.C. Jiang H. Qadri I. Maclean K.N. Friedman J.E. CCAAT/enhancing binding protein beta deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis.Hepatology. 2007; 45: 1108-1117Crossref PubMed Scopus (126) Google Scholar, 40Schroeder-Gloeckler J.M. Rahman S.M. Janssen R.C. Qiao L. Shao J. Roper M. Fischer S.J. Lowe E. Orlicky D.J. McManaman J.L. et al.CCAAT/enhancer-binding protein beta deletion reduces adiposity, hepatic steatosis, and diabetes in Lepr(db/db) mice.J. Biol. Chem. 2007; 282: 15717-15729Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), while addition of fenofibrate significantly decreased C/EBPβ protein expression and increased PPARα levels in palmitate-treated Huh7 cells (Fig. 2B). Palmitate treatment has been shown to induce sustained JNK activation and insulin resistance in mouse hepatocytes (41Solinas G. Naugler W. Galimi F. Lee M.S. Karin M. Saturated fatty acids inhibit induction of insulin gene transcription by JNK-mediated phosphorylation of insulin-receptor substrates.Proc. Natl. Acad. Sci. USA. 2006; 103: 16454-16459Crossref PubMed Scopus (227) Google Scholar). To determine whether fenofibrate treatment could prevent JNK activation in Huh7 cells, cells were treated with 200 μM palmitate or 200 μM palmitate plus 100 μM fenofibrate for 24 h. pJNK was significantly induced in palmitate-treated Huh7 cells, while fenofibrate reduced palmitate-induced pJNK by >50% (Fig. 2C). Huh7 cells incubated with palmitate showed a 6-fold increase in TG concentration, while fenofibrate alone or coincubation with palmitate significantly lowered TG levels compared with levels in control or palmitate-treated Huh7 cells, respectively (Fig. 2D). These results demonstrate that fenofibrate treatment prevented TG accumulation, reduced pJNK activation, and reduced phospho-eIF2α and C/EBPβ protein levels, suggesting the ability to lower ER stress levels. Having established an effect of fenofibrate on lowering markers of ER stress and restoration of AdipoR2, we then went on to ask whether treatment with two ER stress-inducing agents, TPG and TUN, might be causative for reduced AdipoR2 and pAMPK levels in Huh7 cells and to test the efficacy of fenofibrate. Huh7 cells treated with 1 μM TPG increased C/EBPβ and CHOP protein levels (Fig. 3, A and B), indicative of ER stress. Huh7 cells were then pretreated with 1 μM TPG and incubated with or without 100 μM fenofibrate for 24 h. Both AMPK phosphorylation and AdipoR2 expression were significantly lower in Huh7 cells pretreated with TPG; however, fenofibrate treatment significantly increased AMPK phosphorylation and AdipoR2 expression (Fig. 3, C and D). Similar results were found when Huh7 cells were pretreated with 10 μg/ml TUN for 6 h and then incubated with or without 100 μM fenofibrate for 24 h (Fig. 3, E and F). Treatment of obese and diabetic ob/ob mice with PBA or tauroursodeoxycholate, both chemical chaperones, has been shown to restore glucose tolerance and insulin sensitivity and resolve fatty liver in parallel with marked inhibition of ER stress markers pancreatic eIF2α kinase, inositol-requiring enzyme-1 alpha, and JNK activation (22Ozcan U. Yilmaz E. Ozcan L. Furuhashi M. Vaillancourt E. Smith R.O. Gorgun C.Z. Hotamisligil G.S. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes.Science. 2006; 313: 1137-1140Crossref PubMed Scopus (1985) Google Scholar, 42Vilatoba M. Eckstein C. Bilbao G. Smyth C.A. Jenkins S. Thompson J.A. Eckhoff D.E. Contreras J.L. Sodium 4-phenylbutyrate protects against liver ischemia reperfusion injury by inhibition of endoplasmic reticulum-stress mediated apoptosis.Surgery. 2005; 138: 342-351Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To determine whether PBA can restore AMPK phosphorylation and AdipoR2 expression in TPG- and palmitate-loaded Huh7 cells, we coincubated Huh7 cells with the chemical chaperone PBA. PBA prevented TPG- and palmitate-mediated reduction of AMPK phosphorylation and AdipoR2 expression (Fig. 4, A–D). PBA also reduced palmitate-mediated induction of phospho-eIF2α expression (Fig. 4E), suggesting a reduction in ER stress. However, PBA did not reduce palmitate-mediated induction of TG levels in Huh7 cells (Fig. 4F). This result provides evidence that ER stress, rather than lowering TG, might contribute to reduced AMPK phosphorylation and AdipoR2 expression and that suppression of ER stress can restore AMPK phosphorylation and AdipoR2 expression. Hepatitis C is well known to induce insulin resistance and steatosis (43Tellinghuisen T.L. Evans M.J. von Hahn T. You S. Rice C.M. Studying hepatitis C virus: making the best of a bad virus.J. Virol. 2007; 81: 8853-8867Crossref PubMed Scopus (116) Google Scholar, 44Bjornsson E. Angulo P. Hepatitis C and steatosis.Arch. Med. Res. 2007; 38: 621-627Crossref PubMed Scopus (44) Google Scholar) and is associated with loss of AdipoR2 mRNA expression in human liver in vivo (11Jonsson J.R. Moschen A.R. Hickman I.J. Richardson M.M. Kaser S. Clouston A.D. Powell E.E. Tilg H. Adiponectin and its receptors in patients with chronic hepatitis C.J. Hepatol. 2005; 43: 929-936Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Furthermore, it is well recognized that HCV invades the ER and induces ER stress with profound effects on normal ER functions (45Tardif K.D. Waris G. Siddiqui A. Hepatitis C virus, ER stress, and oxidative stress.Trends Microbiol. 2005; 13: 159-163Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In the Huh7-derived cell line Huh.8 engineered with HCV, AdipoR2 expression was significantly lower compared with Huh7 cells (Fig. 5A). Interestingl" @default.
• W1988293732 created "2016-06-24" @default.
• W1988293732 creator A5011610840 @default.
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• W1988293732 title "Fenofibrate and PBA prevent fatty acid-induced loss of adiponectin receptor and pAMPK in human hepatoma cells and in hepatitis C virus-induced steatosis" @default.
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