studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 611A
localization of some of the identified P4-ATPase at the canalicular
membrane. Conclusions: Our data suggest that there
is not a large common proteome between the brush border
membrane of kidney proximal tubule cells and the canalicular
membrane. Furthermore, the identification of several P4-AT-
Pases in rat liver together with the previous findings on mice
with disrupted Atp11c displaying hyperbilirubinemia (PNAS
108:7890,2011) suggest that important proteins involved in
hepatocellular lipid homeostasis and canalicular lipid secretion
may await characterization. Acknowledgement: The expert
support by Dr. B Roschitzki from the Functional Genomics Center
in Zurich is greatly appreciated.
Disclosures:
The following authors have nothing to disclose: Pururawa M. Chaubey, Bruno
Stieger
807
TGR5 agonists improve pancreatic beta cell mass and
function by reprogramming alpha cells to produce GLP-
1: a novel mechanism of action of bile acids in diabetes
Divya P. Kumar 1 , Amon Asgharpour 2 , Faridoddin Mirshahi 2 , So
Hyun Park 3 , Sichen Liu 3 , Yumi Imai 3 , Jerry L. Nadler 3 , Karnam S.
Murthy 1 , Arun J. Sanyal 2 ; 1 Physiology and Biophysics, Virginia
Commonwealth University, Richmond, VA; 2 Internal Medicine, Virginia
Commonwealth University, Richmond, VA; 3 Internal Medicine,
Eastern Virginia Medical School, Norfolk, VA
BACKGROUND: Pancreatic α cells secrete glucagon (GLU)
under euglycemic conditions and GLP-1 under hyperglycemic
conditions. GLU and GLP-1 are derived from alternate splicing
of a common precursor by prohormone convertase 2 (PC2) and
PC1 respectively. Bile acids (BA) via their cognate receptors,
TGR5, stimulate GLP-1 release in intestinal L cells. Recently,
TGR5 have been identified on pancreatic α cells. The potential
effects of BA on pancreatic α cells and their physiological
relevance are unknown. HYPOTHESIS: BA, acting via TGR5
on pancreatic α cells, enhances hyperglycemia-induced PC1
expression thereby releasing GLP-1 which, in turn, acts as a
paracrine to increase pancreatic β cell mass and function.
AIMS: To examine (1) the in vivo effect (intraperitoneal) of
TGR5 specific agonist, INT-777 on glucose homeostasis, PC1
expression, GLP-1 release, β cell proliferation in control and
db/db mice (2) the effect of GLP-1 receptor antagonist on
TGR5-mediated insulin secretion in human islets, and (3) the in
vitro effect of INT-777 on β cell proliferation. METHODS: The
in vivo effect of INT-777 on body weight, glucose homeostasis
(glucose and insulin tolerance test), PC1 expression (immunofluorescence
and RT-PCR), GLP-1 release (ELISA) and β cell
mass and proliferation (morphometric analysis of pancreatic
sections) was examined in control and db/db mice. The in vitro
effect of INT-777 on β cell proliferation was measured by MTT
assay in MIN6 cells. Insulin secretion in response to INT-777
from human islets was measured in the presence or absence of
GLP-1 receptor antagonist, exendin (9-39) by ELISA. RESULTS:
Treatment with INT-777 for 7 weeks attenuated the increase
in body weight, and improved glucose tolerance and insulin
sensitivity in db/db mice compared to control. INT-777 augmented
PC1 expression in α cells and stimulated GLP-1 release
from islets of db/db mice compared to control. INT-777 also
increased pancreatic β cell proliferation and insulin synthesis
in vivo, but had no effect on β cell proliferation in vitro. Insulin
secretion by INT-777 in human islets cultured under high
(25mM) glucose was inhibited by exendin (9-39). These results
suggest that in diabetes, GLP-1 released from α cells augments
β cell proliferation and function. CONCLUSION: In diabetes,
BA reprogram α cells to switch from GLU synthesis to GLP-1
synthesis by increasing PC1 expression with associated trophic
effects on adjacent β cells and improvement in insulin sensitivity
and hyperglycemia. Thus, these results identify a novel mechanism
of action of TGR5 agonist INT-777 in glucose homeostasis
and its potential use in the treatment of insulin resistance and
type 2 diabetes.
Disclosures:
Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,
Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,
Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet
Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,
GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier
The following authors have nothing to disclose: Divya P. Kumar, Amon Asgharpour,
Faridoddin Mirshahi, So Hyun Park, Sichen Liu, Yumi Imai, Jerry L. Nadler,
Karnam S. Murthy
808
Higher order metabolites of heme increase under conditions
of hyperbilirubinemia, trigger cholestasis and
impair hepatocellular integrity
Raphael A. Seidel 1,5 , Franziska Schleser 1 , Christoph Sponholz 1 ,
Frans Cuperus 2,3 , Sandor Nietzsche 4 , Georg Pohnert 5 , Michael
Bauer 1 ; 1 Department of Anaesthesiology and Intensive Care
Medicine / Center for Sepsis Control and Care, Jena University
Hospital, Jena, Germany; 2 Hans Popper Laboratory of Molecular
Hepatology, Division of Gastroenterology and Hepatology,
Department of Internal Medicine III, Medical University of Vienna,
Vienna, Austria; 3 Pediatric Gastroenterology and Hepatology,
Department of Pediatrics, Center for Liver, Digestive, and Metabolic
Diseases, Beatrix Children’s Hospital - University Medical
Center Groningen, University of Groningen, Groningen, Netherlands;
4 Electron Microscopy Center, Jena University Hospital, Jena,
Germany; 5 Institute of Inorganic and Analytical Chemistry, Friedrich
Schiller University Jena, Jena, Germany
Background & Aim: Heme catabolism physiologically produces
large amounts of bilirubin that can function as potent and regenerative
scavenger of reactive oxygen species (ROS). Nonetheless,
ROS attacking heme, biliverdin or bilirubin can lead to the
formation of higher order metabolites of heme, among them the
regio-isomeric bilirubin oxidation products (BOXes) Z-BOX A
and Z-BOX B, with unknown implications for hepatic function.
The aim of this study was to clarify the appearance of Z-BOX
A and Z-BOX B under conditions of health and hyperbilirubinemia
as well as their fate and functional role in the liver as
a key-player in heme catabolism. Methods: Determination of
Z-BOX A and Z-BOX B was carried out via HPLC-MS/MS procedures
in patients suffering from liver failure, healthy human
controls, Gunn rats as animal model of hereditary bilirubin
conjugation failure, and Wistar rats. Pharmacokinetics, pharmacodynamics
and hepatic hemodynamics were studied in
Wistar rats. Cytoskeletal remodeling, cytotoxicity, and cellular
metabolism were investigated in cultured HepG2 and HepaRG
cells by means of fluorescence and scanning electron microscopy
in addition to biochemical assays. Results: The higher
order heme metabolites Z-BOX A and Z-BOX B were formed
under physiological conditions across species (combined levels
in blood: 25.4 ± 3.4 nmolL -1 , n = 10, healthy human;
19.1 ± 4.6 nmolL -1 , n = 12, Wistar rat) and were significantly
elevated under conditions of hyperbilirubinemia, i.e. human
liver failure (504.3 ± 30.3 nmolL -1 , n = 28; p < 0.0001) and
animal model of Crigler-Najjar syndrome type I (136.9 ± 15.1
nmolL -1 , Gunn rat, n = 14; p < 0.0001). Z-BOX A and Z-BOX
B appeared in an almost fixed ratio (~1.45:1) and highly correlated
with serum bilirubin (r > 0.86, p = 4.110 19 ). After
equimolar i.v. bolus application, the pharmacokinetic profiles
of these regio-isomers exhibited significantly shorter serum half-