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308A AASLD ABSTRACTS HEPATOLOGY, October, 2015 196 A functional classification of ABCB4 missense variations identified in progressive familial intrahepatic cholestasis type 3 Jean-Louis Delaunay 1 , Anne-Marie Durand-Schneider 1 , Claire Dossier 1 , Thomas Falguières 1 , Julien Gautherot 1 , Tounsia Aït-Slimane 1 , Chantal Housset 1 , Emmanuel Jacquemin 2 , Michèle Maurice 1 ; 1 UMR_S 938, UPMC & Inserm, Paris Cedex 12, France; 2 Hépatologie Pédiatrique, APHP, Hôpital Bicêtre, Kremlin-Bicêtre, France Progressive familial intrahepatic cholestasis type 3 (PFIC3) is caused by bi-allelic ABCB4 variations. More than 70 % of disease-causing ABCB4 variations are missense variations. The aims of the study were i) to propose a functional classification of ABCB4 missense variations; ii) to test the rescue of trafficking-defective mutants by cyclosporins. Methods: Twelve single-nucleotide missense variations that were identified in 9 PFIC3 patients (6 homozygous, 3 composite heterozygous) and located in or close to transmembrane domains (F357L, S346I, P726L, T775M, Q855L, G954S), first intracellular loop (T175A), first nucleotide-binding domain (T424A, N510S, I541F, L556R), and linker region (R652G), were reproduced in the ABCB4 cDNA. The mutants thus obtained were expressed in HepG2 (transiently) and HEK 293 cells (stably), and compared with wild type ABCB4, to determine their impact on the ABCB4 expression or phosphatidylcholine transport activity, measured by a fluorimetric assay. Results: Four mutants were either fully (I541F and L556R) or partially (S346I and Q855L) retained in the endoplasmic reticulum, in an immature endoglycosidase H-sensitive form, of lower molecular weight than the mature N-glycosidase F-sensitive form. Phosphatidylcholine secretion by these mutants was less than 10 % that of wild type, except for Q855L (30 %). Rescue of these trafficking defects, i.e. exit from the endoplasmic reticulum and increase of the mature form at the bile canaliculi, was obtained by cell treatments with cyclosporins A or C, and to a lesser extent B, D or H. Four mutations with little or no effect on ABCB4 expression at the bile canaliculi, caused a significant reduction (F357L, T775M and G954S) or absence (P726L) of phosphatidylcholine secretion. Two mutants (T424A and N510S) were normally processed and expressed at the bile canaliculi but their stability, assessed by protein decay, was reduced. We found no defect of the T175A mutant, nor of R652G, previously described as a polymorphism, which herein co-existed with another variant (G954S) on the same allele, in two siblings. In patients, the most severe phenotypes appreciated by the duration of transplant-free survival, were caused by ABCB4 variants that were markedly retained in the endoplasmic reticulum, and expressed in a homozygous status. In conclusion, ABCB4 variations can be classified as follows: nonsense variations (I), and on the basis of current findings, missense variations that primarily affect the traffic (II), activity (III) or stability (IV) of the protein. Among missense variants, those in class II cause the most severe clinical phenotypes and can be rescued by pharmacological therapies. Disclosures: The following authors have nothing to disclose: Jean-Louis Delaunay, Anne-Marie Durand-Schneider, Claire Dossier, Thomas Falguières, Julien Gautherot, Tounsia Aït-Slimane, Chantal Housset, Emmanuel Jacquemin, Michèle Maurice 197 Liver-specific deletion of the insulin receptor profoundly reduces accumulation and proteotoxicity of misfolded α 1 -antitrypsin Z (ATZ) in a mouse model Andrew Chu 1 , Tunda Hidvegi 1 , Souvik Chakraborty 1 , Micheal Ewing 1 , Amitava Mukherjee 1 , Patrick Araya 2 , Pamela Hale 1 , Christine Dippold 1 , Yan Wang 1 , Jie LI 1 , Evelyn Akpadock 1 , Hou Ming Cai 1 , Stephen C. Pak 1 , Donna B. Stolz 2 , Gary A. Silverman 1 , David H. Perlmutter 1 ; 1 Pediatrics - Gastroenterology, Children’s Hospital of Pittsburgh of UPMC; University of Pittsburgh, Pittsburgh, PA; 2 University of Pittsburgh, Pittsburgh, PA Alpha-1-antitrypsin deficiency (ATD)-associated liver disease is an important cause of cirrhosis and hepatocellular carcinoma, with liver transplantation currently the only treatment for advanced disease. Insulin signaling is a key regulator of proteostasis mechanisms, with inhibition of insulin signaling linked to increased longevity in animals. The purpose of our study was to determine whether abrogation of insulin signaling reduces ATZ accumulation and proteotoxicity in a mouse model of ATD-associated liver disease. To investigate the effect of loss of insulin signaling, we generated double transgenic mice (IRZZ) by crossing liver-specific insulin receptor knockout mice (LIRKO) to the PiZ mouse model of ATD-associated liver disease. Histological analysis of livers from male mice at 6 months and 12-14 months demonstrated a 60 percent reduction of intracellular ATZ globules by PAS-D staining and a 50 percent reduction in hepatic fibrosis by Sirius Red staining in IRZZ mice compared with PiZ mice. There was also a complete elimination of nodular regeneration in the IRZZ liver. This is consistent with results from a C. elegans model of ATD in which RNAi corresponding to several intermediates in the insulin signaling pathway reduce intracellular ATZ accumulation. To determine the mechanism of reduced hepatic ATZ load in the IRZZ mice, we carried out pulse-chase radiolabelling on isolated hepatocytes and found a significant increase in intracellular degradation of ATZ in the IRZZ compared to the PiZ hepatocytes. To determine whether specific gene expression signatures or signaling pathways were involved in the effect of insulin receptor deletion, we used transcriptomic analysis to compare hepatic RNA levels in IRZZ to PiZ and control (LIRKO and wild-type) mice. Compared to PiZ, the gene expression pattern in IRZZ mice showed downregulation of collagen V consistent with the marked decrease in Sirius Red staining, upregulation of genes associated with hepatocellular proliferation/regeneration (prominin 1 and 2), and upregulation of genes involved in lysosomal function (lysosomal ATPase, H + transporting, V0 subunit D isoform, Atp6v0d2). Furthermore, using immunofluorescence for the lysosomal membrane protein LAMP2 and transmission electron microscopy, we found a marked increase in number of lysosomes in the IRZZ as compared to the PiZ liver, suggesting activation of the autophagolysosomal system in the IRZZ model. These results indicate that the insulin signaling pathway suppresses a key mechanism by which the liver attempts to protect itself from misfolded ATZ and suggest that activation of the autophagolysosomal system is the target of this mechanism. Disclosures: The following authors have nothing to disclose: Andrew Chu, Tunda Hidvegi, Souvik Chakraborty, Micheal Ewing, Amitava Mukherjee, Patrick Araya, Pamela Hale, Christine Dippold, Yan Wang, Jie LI, Evelyn Akpadock, Hou Ming Cai, Stephen C. Pak, Donna B. Stolz, Gary A. Silverman, David H. Perlmutter
HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 309A 198 Anti-inflammatory and Antifibrotic Activity of NGM282, A Novel Variant of FGF19, in an Mdr2-Deficient Mouse Model of Primary Sclerosing Cholangitis Lei Ling, Mei Zhou, Hong Yang, Marc Learned, Stephen Rossi, Alex M. DePaoli, Hui Tian; NGM Biopharmaceuticals, Inc., South San Francisco, CA Background and Aims: Primary sclerosing cholangitis (PSC) is a chronic inflammatory biliary disease characterized by periductal fibrosis and stricture formation. Recent data showed decreased expression and intraductular production of FGF19, which may worsen pre-existing inflammation and fibrosis. NGM282 is an engineered variant of FGF19 that inhibits Cyp7a1 but lacks the tumorigenic activity seen with FGF19. Mice deficient in phospholipid flippase multidrug resistant protein 2 (Mdr2-/-) are a preclinical model for PSC as they develop liver histopathology similar to human PSC. We studied FGF19 and NGM282 in Mdr2-/- mice to characterize the potential biologic activity in PSC. Methods: 12wk old Mdr2-/- mice with established hepatobiliary disease received 1 dose of adeno-associated virus carrying NGM282, FGF19 or control. Serum liver enzymes were analyzed at 4 and 24wks post-dose. Liver tissue was collected at 24wks and stained with Hematoxylin & Eosin and Sirius Red. Results: Significant decreases in alkaline phosphatase (ALP) were seen in male and female mice at 4wks with FGF19 (males=349+26 U/L to 107+16 U/L; females=565+49 U/L to 98+7 U/L; p
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1312A AUTHOR INDEX HEPATOLOGY, Octo
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1366A CATEGORY INDEX HEPATOLOGY, Oc
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