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A novel phosphospecific RXRα Ser260 antibody uncovers new inflammation-associated cytoplasmic and submembrane localizations for the essential nuclear receptor RXRα

Hong Tang1, Zhining Den2, Astrid Kosters1, Daniel A. Moore1, Saul J. Karpen1
1Pediatrics, Emory University School of Medicine, Atlanta, GA; 2The University of Texas MD Anderson Cancer Center, Houston, TX

The retinoid X receptor a (RXRα; NR2B1), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Studies indicated that post-translational modification of RXRα, in particular, inflammation-induced phosphorylation of several sites, can lead to further post-translational modification (e. g., ubiquitination and SUMOylation), as well as altered RXRa stability and subce l l ular localization. We have found that inflammation-induced reduction in RXRα nuclear quantities involves JNK-dependent phosphorylation at Ser260.Details regarding the fate, induction, localization and function of RXRα where Ser260 is phosphorylated are poorly understood, due to the lack of specific detecting reagents, cell lines and mouse models. To begin to address these important issues, we developed and characterized an anti-pRXRα Ser260 antibody (p260 Ab) and verified its specificity and sensitivity with shRNA knockdown, immunoblotting and confocal immunofluorescence assays in both human and mouse models. The phosphorylation of RXRα Ser260 is significantly increased in both nuclear and cytoplasmic compartments of IL-1 β-treated Huh-7 cells and LPS-treated mouse liver, with a novel finding of a submembrane localization at baseline which is increased in response to inflammation. Moreover, the JNK inhibitor, SP600125, inhibits IL-1 –β-induced upregulation of pRXRα Ser260 in Huh-7 cells, suggesting that JNK is a necessary upstream kinase involved in RXRα Ser260 phosphorylation. Initial explorations with confocal immunofluorescence microscopy and co-immunoprecipitation (Co-IP) assays identified submembrane phospho-RXRα interactions with the submembrane protein β-catenin. Moreover, Co-IP and immunofluorescence assays revealed that inflammation increases the interaction between phospho-RXRα and β-catenin in IL-1 β treated Huh-7 cells and LPS treated mouse livers in both cytoplasmic and subplasma membrane locales extend our knowledge of the potential biological roles played by RXRα species. We conclude that inflammatory stimuli induce JNK-dependent RXRα Serine260 phosphorylation, the interaction between p-catenin and RXRα, and the subcellular redistribution of RXRα, including heretofore novel cytoplasmic and submembrane locales.

Disclosures:

The following people have nothing to disclose: Hong Tang, Zhining Den, Astrid Kosters, Daniel A. Moore, Saul J. Karpen

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Uncovering a novel regulation of Bcl2 in bile acid homeostasis and cholestatic liver fibrosis

Yuxia Zhang1, Hiroyuki Tsuchiya1, Rana Smalling1, James Cox2, Don Delker1, Curt H. Hagedorn1, Li Wang1
1Internal Medicine, University of Utah, Salt Lake City, UT; 2Metabolomics Core Facility, University of Utah, Salt Lake City, UT

[Purpose] Bcl2 is a well established anti-apoptotic protein but its involvement in bile acid metabolism has never been studied. Nuclear receptor SHP (Nr0b2) is critical in feedback regulation of bile acid (BA) synthesis. This study investigated the role of Bcl2 in BA homeostasis and cholestatic liver fibrosis. [Methods] Experimental groups: GFP, Bcl2, GFP+CA, Bcl2+CA. GFP control and Bcl2 adenoviruses were subjected to 8 weeks old, male C57BL6 mice via tail vein injection for two weeks. For the GFP+CA and Bcl2+CA groups, mice received adenoviruses for one week then were fed 1% cholic acid (CA)-containing diet for seven days. Serum, liver and ileum were collected by the end of two weeks. Serum BA, BA pool size, fecal BA excretion, serum AST and ALT levels, and serum FGF15 were measured. Liver morphology, fibrosis, and inflammation were analyzed using H&E, picro sirius red and F4/80 staining, respectively. RNA sequencing (RNA-seq) was employed to identify transcriptome alterations. Metabolomics by gas chromatography/mass spectrometry (GC/MS) was used to identify changes in small metabolites in serum and liver extracts. Q-PCR, Western blots, and Co-IP assays were used to determine the molecular mechanisms. [Results] Hepatic overexpression of Bcl2 in mice caused yellowish appearance of liver and serum, and led to a significant increase in serum BA and FGF15 levels and a decrease in total BA pool size and fecal BA output. CA feeding further enhanced the effect of Bcl2 on the increase of serum BA, as well as ALT and AST levels. Severe hepatocyte necrosis, liver fibrosis, and Kupffer cell activation were observed in mice overexpressing Bcl2, which was accompanied by the increased PCNA protein and TGR5 expression. RNA-seq identified 1091 upregulated and 1073 downregulated genes in Bcl2 overexpressed mice. In particular, genes involved in bile acid synthesis and transport were decreased, and genes in collagen formation and inflammatory responses were significantly increased, as validated by qPCR analysis. The most drastic changes in metabolites, as determined by GC/MS, were the increases of intermediate metabolites in TCA cycle. Using a series of cell based biochemistry and molecular biology approaches, we found that the interaction of Bcl2 with SHP induced a fast SHP protein degradation via activation of the caspase 8-caspase 3 pathway. Downregulation of SHP by Bcl2 resulted in a diminished feedback inhibition of BA synthesis. The disturbances in bile formation by Bcl2 contributed to the development of cholestatic liver fibrosis. [Conclusions] Our results uncovered a unique metabolic regulatory axis that couples Bcl2 with SHP to control BA homeostasis.

Disclosures:

The following people have nothing to disclose: Yuxia Zhang, Hiroyuki Tsuchiya, Rana Smalling, James Cox, Don Delker, Curt H. Hagedorn, Li Wang

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Fibroblast Growth Factor 15 is Critical for Liver Regeneration after Partial Hepatectomy in Mice

Bo Kong1, Jiansheng Huang6, Yan Zhu3, Guodong Li4, Jessica A. Williams5, Steven H. Shen5, Lauren M. Aleksunes1, Jason R. Richardson2, Udayan Apte5, David A. Rudnick6, Grace L. Guo1;
1Department of Pharmacology and Toxicology, Rutgers University,Piscataway, NJ; 2Department of Environmental & Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ; 3Department of General Sugery, Xuonwu Hospital, Capital Medical University, Beijing, China; 4Department of Sugical Oncology, Cancer Treatment Center, Fourth Affiliated Hospital of Harbin Medical University, Harbin, China; 5Department of Pharmacology/ Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 6Department of Pediatrics and Developmental Biology and Genetics Unit, Washington University School of Medicine, St. Louis, MO

Fibroblast growth factor 15 (FGF15) is highly expressed in the small intestine of mice and is one of the strongest target genes of farnesoid X receptor, the master regulator of bile acid homeostasis. FGF15 and its human orthologue, FGF19, are endocrine FGFs and are critical in regulating liver functions, including suppressing bile acid synthesis, promoting protein synthesis, regulating glucose homeostasis, and promoting hepatocyte proliferation. To determine the roles of FGF15 in liver regeneration, liver regrowth in FGF15 knockout (KO)mice after 2/3 partial hepatectomy (PHx) was studied. Data demonstrated that mouse genetic background greatly impacted the outcome of liver regeneration in FGF15 KO mice after PHx. FGF15 KOmice on a pure C57BL/6J genetic background were embryonic lethal. Under 75% C57BL/6J and 25% 129SvJ genetic background, KO mice exhibited higher rates of liver necrosis and death within 48 hrs following PHx compared to WT or KO mice with 25% C57BL/6J and 75% 129SvJ genetic background. Furthermore, FGF15 KO mice with 75% C57BL/6J and 25% 129SvJ genetic background showed increased bile acid and bilirubin levels and impaired expression of markers of DNA synthesis and cell cycle progression. Specifically, the cell signaling pathways critical for liver regeneration priming andmouse survival, including signal transducer and activator of transcription 3 (STAT3), nuclear factor kB (NFkB), mitogen-activated protein kinase (MAPK), protein kinase B (PKB/AKT) and mammalian target of rapamycin (mTOR), were either interrupted or deactivated at 30 mins and/or 3 hrs following PHx. Additionally, 10 and 30 mins after PHx, there was a delayed and reduced induction of immediate-early genes, including growth-control transcription factors that are critical for regenerative response in the post-hepatectomized liver. In summary, the results suggest that FGF15 is critical for liver regeneration after PHx, likely by maintaining bile acid homeostasis and/or being involved in the initiation of liver regeneration, and its effects are modified by genetic background.

Disclosures:

The following people have nothing to disclose: Bo Kong, Jiansheng Huang, Yan Zhu, Guodong Li, Jessica A. Williams, Steven H. Shen, Lauren M. Aleksunes, Jason R. Richardson, Udayan Apte, David A. Rudnick, Grace L. Guo

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Effect of small bowel bacterial overgrowth on hepatobiliary transporter expression and bile composition in a jejunal self-filling blind loop model in mouse

Qingqing Wang1, Vijay Saxena1, Bin Wang1, Lili Miles2, Jaimie D. Nathan1
1Division of Pediatric General & Thoracic Surgery, Cincinnati Children΄s Hospital Medical Center, Cincinnati, OH; 2Division of Pathology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH

Purpose: The pathogenic link between gut microbiota and chronic liver disease requires elucidation. In the present study, we hypothesized that small bowel bacterial overgrowth and translocation induces changes in bile composition through reduction in hepatobiliary transporter expression, which eventually lead to liver injury. Methods: In order to study this hypothesis, a 3 cm jejunal self-filling blind loop (SFBL) was surgically created in C57BL/6 mice, 5 cm distal to the ligament of Treitz. Control mice underwent laparotomy (sham). Mice were sacrificed three weeks after surgery. Aerobic and anaerobic bacterial cultures of SFBL luminal content, peritoneal cavity, mesenteric lymph node, liver and blood were performed to evaluate bacterial overgrowth and translocation. Histology scoring was performed on H&E-stained slides by a pathologist blinded to experimental design to evaluate intestinal and liver inflammation and injury. Bile and portal vein blood was collected and total bile acid, phospholipid, and cholesterol concentrations were measured by commercial assays. Portal vein serum anti-flagellin antibody was assessed by ELISA. Hepatobiliary transporter mRNA expression in the liver was measured by RT-PCR. Results: Creation of a SFBL induced a dramatic increase in intraluminal bacterial counts compared to sham mice. 100% of SFBL mice had mesenteric lymph node translocation, compared to 9% of sham mice. SFBL mice had significantly higher histological scores for intrahepatic cholangitis and hepatocellular injury, as well as for jejunal barrier disruption parameters, consistent with ongoing injury. Creation of SFBL resulted in decrease in bile flow rate, but increase in total biliary bile acid concentration. Significant reductions in bile phospholipid and cholesterol output, but not bile acid output were observed in the SFBL group, which resulted in a significant elevation in bile acid/phospholipid ratio, suggestive of the formation of toxic bile. Portal vein serum bile composition exhibited no difference between SFBL and sham mice. A significant reduction in hepatic expression of hepatobiliary transporters involved in biliary canalicular export (Abcg8, Bsep, Mrp2 and Mdr2), as well as basolateral uptake (Ntcp, Oatp1, Oatp2 and Oatp4), was observed in SFBL mice. Conclusion: Taken together, the above data suggest that small bowel bacterial overgrowth alters bile composition with formation of toxic bile via changes in the expression of hepatobiliary transporters, which may play a potential pathogenic role in liver inflammation and cholestatic injury.

Disclosures:

The following people have nothing to disclose: Qingqing Wang, Vijay Saxena, Bin Wang, Lili Miles, Jaimie D. Nathan

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A genetic variant mimicking the effect of ezetimibe associates with increased risk of symptomatic gallstone disease

Bo K. Lauridsen1, 3, Stefan Stender1, 3, Ruth Frikke-Schmidt1, 3, Børge G. Nordesfgaard2, 3, Anne Tybjærg-Hansen1, 3
1Dept. of Clinical Biochemisty, Rigshospitalet, Copenhagen Ø, Denmark; 2Dept. of Clinical Biochemistry, Herlev Hospital, Herlev, Denmark; 3Faculty of Health and Medical Sciences, Copenhagen University, Copenhagen N, Denmark

Objective: We tested the hypothesis that a common genetic variant in Niemann-Pick C1-Like protein 1(NPC1L1) is associated with decreased risk of ischemic vascular disease and with increased risk of symptomatic gallstone disease. Background: NPC1L1 mediates cholesterol uptake from the intestine and bile into enterocytes and hepatocytes, respectively. An NPCIL1 genetic variant mimicking the effect of ezetimibe, an inhibitor of N PC 1L1, s associated with reduced low-density l ipoprotein(LDL) cholesterol and possibly with increased biliary cholesterol, a risk factor for gallstone disease. Methods: We genotyped 73, 457 individuals from the Danish general population, including 10, 481 with ischemic vascular disease and 3, 874 with symptomatic gallstone disease, for NPC1L1 rs2072183.Results: NPC1L1 genotype was associated with stepwise reductions in plasma levels of LDL cholesterol of up to 1.6%(0.05 mmol/L) for CC versus GG-homozygotes(Ptrend<0.001). Multifactorially adjusted hazard ratios(HRs) for ischemic vascular disease were 0.95(95% confidence interval, 0.87-1.03) for CG-heterozygotes and 0.93(0.86-1.01) for CChomozygotes versus GG-homozygotes(P-trend=0.07). Corresponding HRs for symptomatic gallstone disease were 1. 14(0.98-1.33) and 1.19(1.03-1.38)(P-trend=0.02). Conclusion: A common genetic variant in NPC1L1, mimicking the effects of ezetimibe on LDL cholesterol, was associated with increased risk of symptomatic gallstone disease and with a trend towards reduced risk of ischemic vascular disease. These results raise the question whether long-term treatment with ezetimibe might increase the risk of symptomatic gallstone disease.

Disclosures:

The following people have nothing to disclose: Bo K. Lauridsen, Stefan Stender, Ruth Frikke-Schmidt, Bøfrge G. Nordestgaard, Anne Tybjærg-Hansen

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Ileocecal resection (ICR) in patients with Crohn΄s disease is associated with lower FGF19 and higher 7α-OH-cholesterol levels in comparison to ulcerative colitis

Dana Friedrich1, Dieter Luetjohann3, Frank Lammert1, Christoph Reichel2
1Departmentof Medicine II, Saarland Medical Center, Homburg, Germany; 2Rehabilitation Center Bad Brückenau, Clinic Hartwald, Bad Brückenau, Germany; 3lnstitute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany

Background / Aim: The secretion of the intestinal hormone FGF19 is induced by binding of bile acid (BA) to the bile acidnuclear farnesoid X receptor (FXR) in the terminal ileum. On the other side FGF19 suppresses hepatic bile acid biosynthesis. We hypothesized that patients with Crohn΄s disease (CD) show lower FGF19 levels as compared to patients with ulcerative colitis (UC). Patients and Methods: In total, we recruited 12 CD patients after ileocecal resection (ICR), 12 nonoperated CD patients and 12 UC patients as controls in remission. Serum FGF19 levels were determined by ELISA after 10 hrs of overnight fasting. All individuals received orally 1g fat (Calogen®) per kg body weight, and FGF19 levels were measured after 2, 4 and 6 hrs. Serum concentrations of BA and 7α-OHcholesterol levels, which is a valid marker of BA biosynthesis, were determined by GC-MS after 2, 4 and 6 hrs. Results: Basal FGF19 levels are significantly lower in CD patients (± ICR) as compared to UC patients. The increase of FGF19 levels 2, 4 und 6 hrs after the oral fat load differs between UC and CD (ICR+) patients, with highest levels after 4 hrs in UC patients (p<0.05). CD (ICR+) patients display the lowest FGF19 levels at all time points. Fasting and postprandial levels of BA are not significantly different between CD and UC patients. However at all time points, serum 7α-OH-cholesterol levels are significantly higher in CD (ICR+) in comparison to UCpatients. In the whole study cohort basal FGF19 and basal 7 α- O H-cholesterol levels are inversely correlated (r=0.397, p=0.017). Conclusions: Low FGF19 levels in CD (± ICR) patients could be the consequence of persistent inflammation and impaired bile acid signaling in the ileum. CD (ICR+) patients display lowest FGF19 levels, consistent with highest 7α-O H-cholesterol levels and lack of repression of BA synthesis. We speculate that this observation results from insufficient intestinal FXR activity in CD.

Disclosures:

The following people have nothing to disclose: Dana Friedrich, Dieter Luetjohann, Frank Lammert, Christoph Reichel