Signal transduction and nuclear receptors


Retinoid acid related orphan receptor α (RORα ) regulates circadian rhythm and fasting induction of sterol 12α -hydroxylase (CYP8B1) in bile acid synthesis

Preeti Pathak1, Tiangang Li1,2, John Chiang1

1Department of Integrative Medical Sciences, Northeast Ohio Medical University, Roofstown, OH; 2Pharmacology, Toxicology and Therpeutics' University of Kansas Medical Center, Kansas City, KS

Sterol 12α-hydroxylase (CYP8B1) is involved in cholic acid synthesis and plays a key role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone disease, dyslipidemia, fatty liver disease and atherosclerosis. In this study, we investigated the underlying mechanism of a fasting-induced and cholesterol activated nuclear receptor and core clock gene RORa in regulation of circadian rhythm and fasting induction of CYP8B1 expression. In free fed mice, CYP8B1 mRNA expression was reduced to the lowest level at the onset of the dark cycle when RORα mRNA expression was the lowest. Fasting stimulated, while re-feeding reduced expression of CYP8B1 mRNA and protein. Interestingly, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting and cAMP increased RORα protein stability by protein kinase A-mediated phosphorylation. Adenovirus-mediated transduction of RORα to mice strongly induced CYP8B1 gene expression, increased 12α-hydroxylated bile acids, and serum and liver cholesterol. Reporter assay, mutagenesis and EMSA identified a functional RORα response element in CYP8B1 promoter. Mammalian two-hybrid assay shows RORα interacts with cAMP response element binding protein-binding protein (CBP). The interaction was stimulated by cAMP but inhibited by 7ahydroxycholesterol, an antagonist of RORα. ChlP assay showed that RORα recruited CBP to the CYP8B1 promoter to stimulate histone acetylation and induce CYP8B1. In conclusion, RORα is a key regulator of circadian expression and fasting induction of CYP8B1 to increase 12a-hydroxylated-bile acids and regulate bile acid composition, which plays a role in intestinal lipid absorption and maintaining lipid homeostasis. Glucagon/cAMP signaling phosphorylates and stabilizes RORα protein to induce CYP8B1 in response to fasting and circadian rhythm to regulate cholic acid synthesis and bile acid composition, which regulates hepatic metabolic homeostasis, dyslipidemia, diabetes and obesity. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.


The following people have nothing to disclose: Preeti Pathak, Tiangang Li, John Chiang


The novel function of small heterodimer partner (SHP) in integrating circadian rhythm and metabolism networks in the liver

Songmin Lee1, Yuxio Zhang1, Hiroyuki Tsuchiya1, Rana Smalling1, Anton M. Jeffen2, Li Wang1

1Gl-lnternal medicine, University of Utah, Salt Lake City, UT; 2Division of Intamuro Research, National Institutes of Health, Research Triangle Park, NC

Purpose/Background: In mammals, circadian rhythms play an essential role in coordinating the timing of various physiological processes including glucose, lipid and mitochondrial oxidative metabolism. Small heterodimer partner (Shp, Nrob2) is an atypical orphan nuclear receptor and exhibits unique features of interaction with many nuclear receptors to regulate the expression of multiple genes. Given that SHP is the master regulator of metabolism in the liver, it is unknown whether SHP is necessary to coordinate metabolism and circadian rhythms. Methods: Shp+/+ and Shp-/- mice on a C57BL/6 background (n=35/group) were fed a standard chow diet and water ad libitum. Serum and livers were collected at zeitgeber time (ZT) 2, 6, 10, 14, 18 and 22. RNA-sequencing (RNA-seq) and qPCR were used to examine the rhythmic expression of liver clock, glucose and lipid metabolic genes. GC/MS and LC/MS were used to identify the oscillation of metabolites in serum and liver, respectively. In vitro assays include: transient transfection, luciferase reporter assay, ChIP assay, gel-shift assay, Co-IP, Western blots, adenovirus overexpression and siRNA knockdown. Results: RNA-seq and qPCR revealed a drastic impact of Shp-deletion on the circadian rhythmic expression of glucose, bile acid and lipid metabolic genes. A significant global alteration in the oscillation of intermediate metabolites in pathways of glucose catabolism, fatty acid, triglyceride, cholesterol, and bile acid synthesis and metabolism, amino acid metabolism, as well as TCA cycle, was observed in the Shp-/- mice. Such alteration was accompanied by marked changes in the generation of NAD, NADP, NADH and NADPH. The major components of the molecular clock in the liver, including Npas2, Rora/y, and Pgc1 a, exhibited sharp increases in their peak expression in Shp-/- liver. We showed that Shp inhibited Npas2 transcription by two mechanisms: through a direct interaction with Rory to repress Rory transactivation of the Npas2 promoter and through a direct interaction with Rev-erba to enhance Rev-erba inhibition on the Npas2 promoter activation by Rora. 〇n the other hand, the rhythmic expression of Shp was tightly controlled by Npas2 through its rhythmic binding to the Shp promoter and two intergenic enhancers in the Shp gene via cross-talk with Clock and Bmal1, as demonstrated by ChlP-seq and ChlP-assays. Conclusion: These novel findings identified Shp as a central player in integrating circadian rhythm and metabolism networks in the liver.


The following people have nothing to disclose: Sangmin Lee, Yuxia Zhang, Hiroyuki Tsuchiya, Rana Smalling, Anton M. Jetten, Li Wang


Cardiomyocyte specific deletion of bile acid membrane receptor-TGR5 induces contractile failure, exercise intolerance, inotropic insensitivity and alteration in cell signaling in mice

Moreshwar S. Desai1, Zeena Eblimit1, Corey Reynolds2, Saul J. Karpen3, David D. Moore4, Daniel J. Fenny5

1 Pediatric Critical Care Medicine, Baylor College of Medicine, Houston, TX; 2Mouse Phenotype Core, Baylor College of Medicine, Houston, TX; 3Pediatric Gastroenterology, Emory School of Medicine, Atlanta, GA; 4Cell and Molecular Biology, Baylor College of Medicine, Houston, TX; 5Pediatric Cardiology, Baylor College of Medicine, Houston, TX

Background: Dysregulation in cardiac metabolism precedes overt contractile failure and provides an early manipulatable target for intervention, prevention and cure. Bile acid membrane receptor-TGR5 regulates metabolism, function and cell signaling pathways in brown adipose tissue and skeletal muscles in mice. TGR5 is expressed in the hearts of mice, rodents and humans, but its role in cardiac function and cell biology is unknown. Objective: Using mice with cardiomyocyte specific deletion of TGR5, we tested the hypothesis that TGR5 deletion impairs myocardial contractility, exercise tolerance, catecholamine response and alters cell signaling pathways. Methods: Cardiomyocyte specific TGR5 deleted mice (TGR5flox/flox/cre+ [CKO])were generated from TGR5 floxed and transgenic αMHCcre mice. After confirmation of successful deletion by PCR, 12 week old male CK〇 mice and their age matched littermates (TGR5flox/flox/cre-[WT]) underwent echocardiography (ECHO), treadmill stress tests and acute catecholamine (isoprenaline [20mcg/kg i. p]) challenge with stress ECHO. Hearts were analyzed for gene expression of stress signaling pathways. Results: Mean±SD; Stats: Mann-Whitney or AN〇VA; p<0.05 is significant; n=5/group unless specified. Results: Compared to WT mice, CKO mice demonstrated a modest but significant decrease in cardiac output (18. 5±1. 5 vs. 15±3ml/kg/min; n=10-15/grp), ejection fraction (65±12 vs. 43±17%; n = 10-15/grp) and shortening fraction (36±8 vs. 22±9%; n=10-15/grp). ECHO showed an increase in LV End diastolic diameter (3. 6±0.3 vs. 4. 2±0.5mm; n = 10-15/grp) and a decrease in diastolic LV wall thickness (0.8±0.1 vs. 0.7±0.05mm; n=10-15/grp). 〇n challenge with isoprenaline, peak shortening (70±9 vs. 47±15%) and peak ejection fractions (95±5 vs. 77±14%) were significantly attenuated in the CK〇 mice compared to controls. 〇n the treadmill, CK〇 mice showed exercise intolerance compared to WT, as evidenced by 50% lesser distance covered (400±100 vs. 200±65 m) and earlier fatigue (30±10 vs. 15±5 mins). At the RNA level, there was robust (10X) induction of MYH7 and more modest upregulation of BNP (2x), with a 50% downregulation of UCP3, suggesting activation of “fetal” stress signaling pathways. Moreover, cardiac output negatively correlated with the degree of TGR5 deletion in the heart. Conclusions: Cardiomyocyte specific deletion of TGR5 impairs myocardial function, stress response and cell signaling. This study emphasizes a novel role for TGR5 in myocardial cell biology. Further studies into the role of cardiac TGR5 are warranted as TGR5 receptor modulation may provide an attractive therapy for heart failure. [P30 DK056338(MD)].


The following people have nothing to disclose: Moreshwar S. Desai, Zeena Eblimit, Corey Reynolds, Saul J. Karpen, David D. Moore, Daniel J. Penny


The Hippo Tumor Suppressor Links Autophagy to Hepatic Growth Regulation

Youngmin A. Lee1, Tingfang Lee1, Luke A. Noon1, Elisabeth G. Kramer2, Gareth John2, Cathie Pfleger3, M. Isabel Fiel4, Scott L. Friedman1

1 Liver Diseases, Icahn School of Medicine of Mount Sinai, New York, NY; 2lnstitute of Neurosciences' Icahn School of Medicine of Mount Sinai, New York, NY; 3Department for Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY; 4Department for Pathology, Icahn School of Medicine of Mount Sinai, New York, NY

Background: The Hippo Tumor Suppressor Pathway plays a highly conserved role from flies to mammals in establishing organ size. In particular, the Hippo Pathway is a critical determinant of liver size, since hepatocyte-specific deletion of Hippo signaling components (e. g. Mst1/2, Sav1, Mer) leads to significant hepatomegaly. Similarly, hepatomegaly is a prominent feature of models where autophagy is genetically deleted, yet the underlying mechanisms are obscure. Methods: 〇lig1Cre: Atg7f/f mice were generated and the specificity of the 〇lig1 promoter determined by crossing 〇lig1Cre mice with a reporter mouse line. Livers of Atg7-deficient mice were characterized for weight and proliferative activity by Ki67 staining. Hippo Pathway activity was analyzed by western blotting for core pathway components and qPCR for expression of Yap downstream targets, as well as by immunostaining for nuclear Yap. Results: We exploited a new promoter, 〇lig1, to broadly drive Cre recombinase expression in livers of mice with floxed alleles of Atgz (a required autophagy regulator). 〇lig1 and Cre staining was evident in hepatocytes, biliary epithelial cells and hepatic stellate cells. 〇lig1Cre: Atg7f/f mice displayed dramatic, symmetric hepatomegaly with a 3-fold increase in liver-to-body weight ratio. Serum AST and ALT were markedly elevated (2800 U/ml and 2200 U/ml respectively). H&E staining documented hypertrophy as well as hyperplasia of hepatocytes, with increased pancellular proliferative activity as determined by Ki67 staining (12. 8 pos. nuclei/field vs. x 76. 5 pos. nuclei per field, n=3) at 3 months of age. There was significantly increased nuclear localization of Yap in hepatocytes and biliary epithelium, consistent with Yap activation and decreased Yap targeting by the core components of the Hippo Pathway. This finding was complemented by: (1)reduced levels of both p-Yap, the inhibited form and of p-Mst1, the activated form of Mst1, and (2) decreased expression of the upstream Hippo Pathway component, Merlin (NF2). There was also significantly increased mRNA expression of downstream Yap targets in whole liver lysates (Ctgf, Birc5, Itgb2, Afp and Areg), further linking autophagy deficiency to Yap activation. Conclusion: We have uncovered a new link between autophagy and the Hippo Pathway, mediated by its downstream transcriptional co-activator Yap. Hepatic autophagy deficiency leads to significant hyperplasia and pancellular proliferation via suppression of Hippo Pathway mediated Yap inhibition, linking two important homeostatic pathways for the first time. These findings have implications for unraveling the basis for hepatic growth regulation and regeneration.


Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical, Sanoti-Aventis; Consulting: Abbott Laboratories, Conatus Pharm, Exalenz, Genenetch, Glaxo Smith Kline, Hoffman-La Roche, Intercept Pharma, Isis Pharmaceuticals, Melior Discovery, Nitto Denko Corp., Debio Pharm, Synageva, Gilead Pharm., Ironwood Pharma, Alnylam Pharm, Tokai Pharmaceuticals, Bristol Myers Squibb, Takeda Pharmaceuticals, Nimbus Discovery, Isis Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira Pharm, Vaccinex Therapeutics; Stock Shareholder: Angion Biomedica

The following people have nothing to disclose: Youngmin A. Lee, Tingfang Lee, Luke A. Noon, Elisabeth G. Kramer, Gareth John, Cathie Pfleger, M. Isabel Fiel


Hypoxia-inducible factor 2alpha activation disrupts cholesterol metabolism homeostasis of liver and accelerates atherosclerosis in apoE-null mice

Aijuan Qu1, Changtao Jiang1, Fei Li1, Naoki Tanaka1, Bin Gao2, Yatrik M. Shah3, Frank J. Gonzolez1

1 Laboratory of Metabolism, NCI/NIH, Bethesda, MD; 2Laboratory of Liver Diseases, NIAAA/NIH/ Bethesda, MD; 3Department of Molecular and Infegrative Physiology and Internal Medicine, Division of Gastroenterology, University of Michigan School of Medicine, Ann Arbor, MI

An increased incidence of hypercholesterolemia and atherosclerosis has been observed in patients with obstructive sleep apnea (OSA), which has been attributed to liver dysfunction induced by chronic intermittent hypoxia (CIH). However, the underlying mechanism remains elusive. Cellular adaptive response to hypoxia is mainly mediated by hypoxia-inducible factor (HIF), which consists of a ubiquitously expressed beta subunit (HIF1beta) and two related oxygen sensitive alpha subunits, HIF1alpha and HIF2alpha. Under normoxia, HIFalpha subunits are rapidly degraded via the von Hippel-Lindau tumor suppressor protein (VHL) E3 ubiquitin ligase complex. In the present study, CIH resulted in increased hepatic HIF2alpha expression and elevated cholesterol accumulation in liver and serum. Hepatocyte-specific disruption of VHL resulted in constitutive activation of HIF signaling, and the resultant increased HIF expression augmented liver cholesterol accumulation, disturbed serum lipoprotein profiles and hypercholesterolemia. These effects were completely abolished in mice with compound knockout of VHL and HIF2alpha, thus demonstrating that HIF2alpha plays an essential role in control of cholesterol metabolism in liver and serum hypercholesterolemia. Mechanistica丨丨y, HIF2alpha activation repressed the expression of Cyp7a1 and Gyp2/a1 to block the conversion of free cholesterol to bile acids, downregulated acyl-GoA: cholesterolacyltransferase 2 (Acat2) to suppress the synthesis of cholesterol ester from free cholesterol, and inhibited ATP-binding cassette g5 (Abcg5) and Abcg8 to decrease the transport of cholesterol from liver to small intestine, thus leading to the accumulation of cholesterol in liver. In vivo luciferase assays via the Xenogen imaging system revealed that HIF2alpha activation could inhibit the transcriptional activity of the farnesoid X receptor (FXR), which is a critical transcription factor controlling liver cholesterol homeostasis. Moreover, disruption of HlF2alpha in apoEnull mice could decrease serum cholesterol levels and alleviate the development of atherosclerosis as revealed by reduced oil red O staining in aortic sinus sections and entire aortas. Together, these findings demonstrate that HIF2alpha activation in liver is critical for liver cholesterol dysregulation, hypercholesterolemia and atherosclerosis, thus providing a potential target for the therapeutics for hypercholesterolemia and atherosclerosis.

Disclosures:The following people have nothing to disclose: Aijuan Qu, Changtao Jiang, Fei Li, Naoki Tanaka, Bin Gao, Yatrik M. Shah, Frank J. Gonzalez


FGF21 promotes cirrhosis associated angiogenesis through endocytosis dependent activation of FGFR1 in endothelial cells

Usman Yaqoob, Sheng Cao, Thiago de Assuncoo, Vijay Shah

Gastroenterology Research Unit, Mayo Clinic, Rochester, MN

Background/Aims: Endothelial cell (EG) angiogenesis is associated with liver fibrosis. Fibroblast growth factor (FGF) 21 is a nonclassical, endocrine FGF family member that requires the βKlotho co-receptor to activate FGF receptor-1 (FGFR1) and its downstream signaling molecule ERK. Prior studies indicate that FGF21 production from hepatocytes correlates with liver injury. We hypothesized that FGF21 might link liver injury with angiogenesis and fibrosis. Method/Results: FGF21, βKlotho and FGFR1 levels were increased in liver tissues from patients with NASH cirrhosis compared to controls by Western blot and immunostaining. FGFR1 expression was increased in liver sinusoids in bile duct ligated mice as compared to sham by immunostaining. In vivo, FGF21 promoted angiogenesis in a murine Matrigel assay. In vitro, FGF21 promoted ERK activation and tubulogenesis in EC, which was enhanced by 2-fold with adenoviral overexpression of βKlotho (p<0.05). Biotinylation and immunostaining studies for receptor endocytosis revealed endocytosis of βKlotho with FGFR1 upon FGF21 stimulation. Overexpression of an adenoviral construct encoding a dominant negative form of the endocytosis regulatory protein dynamin-2 (dynK44A) or siRNA targeting the Rab5 GTPase that regulates early endosome formation, attenuated FGF21 mediated activation of ERK and tubulogenesis in liver EC by 2fold indicating that FGF21/FGFR1 activation and angiogenesis requires endocytosis and endosomal targeting (p<0.05). Genetically modified mice harboring endothelial selective overexpression of dynK44A, revealed impaired angiogenesis in a modified Matrigel assay. Conclusion: FGF21 is a novel angiogenic ligand for FGFR1 and acts through dynamin dependent endocytosis and Rab dependent endosomal targeting of FGFR1. These studies extend our understanding of endosomal signaling through receptor tyrosine kinase activation in liver EC and angiogenesis.


The following people have nothing to disclose: Usman Yaqoob, Sheng Cao, īhiago de Assuncao, Vijay Shah