71 Hepatocyte-Specific Hypoxia-Inducible Factor 1α Mediates TNFα Secretion and Liver Injury in a...

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sgenotype, gender, age and ethnicity. To declare significance, we used a false discovery rate(FDR) threshold of 0.1. Results: Data from 61 patients were suitable for analysis: HCV-1 =90%; male = 67%; median age = 52 yrs; ethnicity = 62% Caucasian, 20% African Amer-ican,18% other; 70% were METAVIR F0-2, none cirrhotic. In the comparison of differentialintrahepatic mRNA expression, 164 genes were significant. ISGs were heavily over-repres-ented and showed lower expression levels in CC livers (OAS 1/2/3, MX1, IFIT 1/2/3 andISG15 all had >3 fold lower expression). IL28B and IL28A gene expression were detectableat low levels; there was no difference by IL28B-type. The IFN signaling pathway was themost enriched canonical pathway differentially expressed by IL28B-type (P-value <10-5).In 25 patients PEG/RBV response data was available (17 were NR prior to biopsy; 5/8 treatedpost-biopsy attained SVR). IL28B-type was strongly associated with SVR (P-value = 0.004).SVR was associated with lower intrahepatic ISG expression levels. Conclusions: The CCIL28B-type was strongly associated with reduced expression of intrahepatic ISGs. In a subset,SVR was associated with both CC IL28B-type and reduced ISG expression. This suggeststhat genetic variation in IL28B regulates the innate immune response to HCV in the liver,priming patients for a stronger ISG response to exogenous IFN-α therapy.

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Outcomes and Predictors of Simultaneous Liver-Kidney TransplantationAmong Patients With Kidney Dysfunction Who are Not on Dialysis on theLiver Transplant Waiting ListAyse L. Mindikoglu, Laurence S. Magder, Stephen L. Seliger, Jean-Pierre Raufman, CharlesD. Howell

Background: Glomerular filtration rate (GFR) <30 ml/min/1.73m2 is a consensus criterionfor simultaneous Liver-Kidney Transplantation (LKTx) in patients with End-Stage LiverDisease (ESLD) who are not on dialysis. We studied outcomes of subjects who met thiscriterion by GFR estimated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Methods: The analysis was based on Organ Procurement and TransplantationNetwork data as of August 25, 2009. Patients with ESLD who were not on dialysis throughoutthe period on the LT waiting list but with an estimated GFR <30 ml/min/1.73m2 wereanalyzed. Multiple logistic regression was used to identify variables associated with LKTxas opposed to Liver Transplantation alone (LTA). Results: From 2002-2009, 2436 patientswithGFR <30ml/min/1.73m2 underwent LT (86% LTA, 14% LKTx). Controlling for potentialconfounders, independent predictors for simultaneous LKTx were reduced GFR (loge GFR,OR:0.3, P<0.0001), diabetes (OR=1.6, P<0.0002), lower MELD score (OR=0.9, P<0.0001)and African-American (OR=2.4, P<0.0001). Compared to men, women were 47% less likelyto undergo LKTx rather than LTA (OR=0.5, P<0.0001). Patients with serum Na <135 mmol/L were 40% less likely to have LKTx compared to those with normal serum sodium (OR=0.6, p=0.0014). One-year estimated survival probabilities were 87% and 81% in patientswho underwent LKTx and LTA, respectively (Fig. 1). Conclusions: Our results suggest thatpatients with ESLD on the LT waiting list and a GFR estimated by CKD-EPI equation <30ml/min/1.73m2 might benefit from LKTx rather than undergoing LTA.

Figure 1. Among patients with an estimated GFR <30 ml/min/1.73m2 but not on dialysison the LT waiting list who underwent LT, those who had LKTx had significantly higherpost LT survival compared to LTA (P=0.027).

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Hepatocyte-Specific Hypoxia-Inducible Factor 1α Mediates TNFα Secretionand Liver Injury in a Mouse Model of Alcoholic SteatohepatitisBharath Nath, Ivan Levin, Gyongyi Szabo

Background: Alcoholic Steatohepatitis (ASH) is dependent on innate immune signaling,including the Toll-Like Receptor 4. Secretion of proinflammatory cytokines, including TumorNecrosis Factor-Alpha (TNFα) is greatly increased in chronic ethanol(CE)-fed mice afterlipopolysaccharide (LPS) challenge. TNFα secretion depend on Hypoxia Inducible Factor1-alpha (HIF1α), as HIF knockout in macrophages protected from LPS-induced sepsis. Wehypothesized that hepatocyte-specific or myeloid-specific HIF have different effects on ASH.Methods: A cre-lox system was used to engineer hepatocyte-specific (HepHIF(-)) or Myeloid-Specific(MyeHIF(-)) loss of HIF in C57Bl6 mice, HepHIF(-) mice, MyeHIF(-) and controlmice were fed with Lieber DeCarli ethanol (5.0% alcohol, v/v)or pair-fed (PF) diet for 5weeks. Some mice received LPS injections (50ug/kg) 18 hours prior to sacrifice and serumALT, HIF1 immunoblot, RT-PCR, and cytokines were (multiplex ) analyzed. For In Vitrostudies, Huh7 hepatoma cells were transfected with degradation-resistant HIF1α and chal-lenged with LPS. Results: HIF1α in liver nuclear extracts from WT mice was upregulatedwithin 4 hours of LPS challenge. CE and LPS injection upregulated serum ALT (p<0.05 vsEtOH-fed or PF/LPS injected groups). WT mice had increased HIF1α protein in liver nuclearextracts with CE (p<0.05) compared to PF or CE-fed HepHIF(-) mice. Interestingly, CE-fed HepHIF(-) mice had lower serum ALT than WT mice at 2 or 18 hours after LPS whereasMyeHIF(-) mice were less protected. WT, HepHIF(-) and MyeHIF(-) mice had comparable

S-774AASLD Abstracts

upregulation of RANTES after CE and LPS, but HepHIF(-) and MyeHIF(-) mice had lowerserum TNFα, IL-6, IL-10, and KC than WT mice. Moreover, mRNA levels of HIF1α targets,including PAI-1 and iNOS, were lower after LPS challenge in HepHIF(-) mice versus WT.Cytokine mRNA including KC and IL6, paralleled suppressed serum levels in HepHIF(-)mice versus WT after LPS injection. Based on these data, we turned to an In Vitro modeland determined that transfection of hepatoma cells with a degradation resistant HIF1αmutant increased TNFα mRNA levels (p<0.04), which was further upregulated with LPS.Conclusion: We conclude that the synergistic effect of LPS and chronic ethanol on pro-inflammatory cytokine secretion is prevented by suppression of hepatocyte HIF1α activation.The upregulation of TNFα mRNA with HIF expression in hepatocytes suggests a novel roleof the hepatocyte in the inflammatory response. Further work in this area may reveal anti-HIF1α therapies as modifying strategies for the treatment of alcoholic liver disease. Supportedby NIH R21 AA017544(GS) and F30 AA017030(BN).

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Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Attenuates HepatocyteLipoapoptosisSamar H. Ibrahim, Yuko Akazawa, Sophie Cazanave, Steven F. Bronk, Nafisa Elmi, DanielD. Billadeau, Gregory J. Gores

BACKGROUNDS AND AIMS: Saturated free fatty acids induce hepatocyte lipoapoptosis, apathologic feature of nonalcoholic steatohepatitis (NASH). The saturated free fatty acidpalmitate (PA) mediates hepatocyte lipoapoptosis via ER stress induced c-Jun-N-terminal(JNK) activation. GSK-3β is a pro-apoptotic kinase which can promote JNK activation. Thusour aim was to determine if GSK-3β inhibition suppresses PA induced JNK activation andlipoapoptosis. METHODS: We employed the human hepatocellular carcinoma cell line,Huh-7 cells for these studies. Cells were treated with 800 μM PA. Apoptosis was assessedby morphologically by assessing the nuclear changes of apoptosis by DAPI plus fluorescentmicroscopy and biochemically by caspase 3/7 activity. GSK-3β and JNK activation wereidentified by phospho-immunoblot analysis. The ER stress marker C/EBP homologous tran-scription factor (CHOP) was quantified by real time PCR. RESULTS: PA induced GSK-3βactivation as identified by dephosphorylation of serine 9 and 21 (dephosphorylation of theseresidues enhances enzymatic activity). The GSK-3β inhibitor, GSK-3 inhibitor IX 2 μM,significantly reduced JNK activation following treatment with PA as assessed by phospho-immunoblot analysis. Likewise, GSK-3β pharmacologic inhibition significantly reduced PA-mediated apoptosis (59% apoptosis in PA treated cells vs 12 % apoptosis in PA plus GSK-3 inhibitor treated cells, p<0.01). These findings were confirmed by the caspase 3/7 assay(26-fold increase in PA treated cells vs. 12-fold increase in cells treated with PA plus theGSK-3β inhibitor, p=0.02). More importantly, transfection with a GSK-3β targeted shRNAalso reduced PA-mediated apoptosis (57% apoptosis in PA treated wild cells vs 10% in PAtreated GSK-3β shRNA transfected cells p<0.01). We have recently reported that PA inducedapoptosis is associated with JNK-mediated induction of the pro-apoptotic Bcl-2 proteinPUMA. Consistent with GSK-3β mediated JNK activation, GSK-3β pharmacologic inhibitionalso reduced PUMA mRNA expression by PA (6-fold increase in PA treated cells vs. 2-foldincrease in cells treated with PA plus the GSK-3β inhibitor, p<0.01). On the other handthe GSK-3β inhibitor did not prevent PA induction of the ER stress marker CHOP (2.6-fold increase in PA treated cells vs 2.5-fold increase in PA plus GSK-3 inhibitor treated cells,p=NS). In CONCLUSION, our results suggest GSK-3β is activated downstream of free fattyacid mediated ER stress. Activation of this pro-apoptotic kinase initiates a JNK cytotoxicsignaling cascade culminating in lipoapoptosis. We speculate that GSK-3β is a potentialtherapeutic agent for the treatment of human NASH.

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Hepatic Fat Accumulation Promotes Postprandial Triglyceride-Enrichment ofHDL Particles in Association With Endothelial Dysfunction in Men With theMetabolic Syndrome and Men With Type 2 DiabetesMaarten E. Tushuizen, Peter G. Scheffer, Marcel H. Muskiet, Rick Vermue, CeesRustemeijer, Petra J. Pouwels, Michaela Diamant

Background and aims: Liver fat accumulation is common in type 2 diabetes (T2DM),and closely related with features of the metabolic syndrome (MetS) and increased risk ofcardiovascular disease (CVD). Liver fat associates with postprandial hypertriglyceridemia,thus potentially contributing to postprandial triglyceride-enrichment of HDL (HDL-TG),and subsequent enhanced HDL particle clearance and HDL dysfunction. LowHDL cholesterolis an independent CVD risk factor and also a feature of the MetS. Methods: We assessedpostprandial HDL composition changes and their association with liver fat and endothelialfunction, before and following 3 consecutive meals, given as breakfast (t=0h), lunch (t=4h)and diner (t=8h) during a 16h period in 12 men with uncomplicated T2DM (mean±SE: age55±1 yrs; BMI 32±1 kg/m2; HbA1c 7.2±0.3%), 12 normoglycemic men with the MetS, and12 age-matched controls. Blood sampling was performed before each meal, and 12h and16h after breakfast. Flow-mediated dilation (FMD) of the brachial artery was measured byultrasound before each blood collection. After ultracentrifugation, TG, cholesterol, andprotein content were determined in HDL. Liver fat content was measured by proton magneticresonance-spectroscopy. Results: Plasma TG increased significantly after the meals (P<0.001in all groups compared to baseline), and remained elevated after diner in T2DM and MetSmen only. Fasting HDL-TG was highest in T2DM, relative to MetS and controls (147.5±16,108.3±13, 78.3±8 nmol/g, respectively; P=0.002), and increased postprandially in all groups(P<0.001). FMD correlated inversely with HDL-TG, especially after diner (r=-0.57, P<0.001).Liver fat content was highest in T2DM compared to MetS and controls (24±6, 11±3, 7±2%,P<0.02, respectively) and associated with HDL-TG-content after 3 meals (r=0.64, P<0.001),after adjustment for age, metabolic state, TG and waist. Conclusion: We conclude that inmen with T2DM and the MetS, who had the highest liver fat content, exposure to 3consecutive meals produced exaggerated HDL-TG enrichment, which was closely associatedwith endothelial dysfunction.