studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 645A
881
Regulation of Adaptive Thermogenesis by the Gut
Microbiome: Implications for Non-Alcoholic Fatty Liver
Disease (NAFLD) Pathogenesis
Tibor I. Krisko 1,2 , Hayley T. Nicholls 1 , Katherine B. Leclair 1 , Alexander
S. Banks 1 , David E. Cohen 1 ; 1 Medicine, Brigham and Women’s
Hospital, Boston, MA; 2 Medicine, VA Medical Center, Boston,
MA
Background: The gut microbiome plays a critical role in the
pathophysiology of both obesity and non-alcoholic fatty liver
disease (NAFLD). While an altered energy balance is known
to be associated with liver injury, the mechanisms by which the
gut microbiome controls energy homeostasis and thus may contribute
to NAFLD are incompletely understood. Recent studies
have shown that increasing thermogenesis ameliorates hepatic
steatosis via both direct and indirect mechanisms (Wang, Nat
Med 2014;20:1436). Aim: This study was designed to elucidate
the contribution of the gut microbiome to adaptive thermogenesis,
as reflected by alterations in energy expenditure
under conditions of varied thermal stress. Methods: We utilized
a temperature-controlled comprehensive lab animal monitoring
system that was custom-designed to maintain sterile conditions.
Rates of O 2
consumption (VO 2
) and CO 2
production (VCO 2
)
were quantified by indirect calorimetry in control and germ-free
C57BL/6J mice as functions of variations in ambient temperatures
that ranged from thermoneutrality (30 °C) to severe cold
stress (4 °C). Energy expenditure (kcal/72 h) was estimated
according to the formula VO 2
(3.82 + 1.22RER) and respiratory
exchange ratio (RER) was calculated as (VCO 2
/VO 2
).
Physical activity was recorded as cumulative beam breaks per
hour over 72 h. Body composition was determined by NMR
spectroscopy and blood glucose concentrations using a glucometer.
Germ-free status was confirmed at the beginning and
end of experiments by quantitative culture and gram staining of
stool. Results: Compared to controls, germ-free mice exhibited
lower energy expenditure, both at thermoneutrality (18.9 ± 0.2
vs 21.1 ± 0.3, p < 0.01) and in the cold (54.7 ± 0.5 vs 58.2
± 0.2, p < 0.05). This occurred despite increased physical
activity in germ-free mice that was most pronounced at 4 °C
(1023 ± 54 vs 1542 ±127, p < 0.05). Germ-free mice exhibited
a greater percentage of adipose tissue mass compared
to controls. Plasma glucose concentrations were reduced and
values of RER were lower in germ-free mice at 4 °C, indicating
increased lipid utilization. Conclusions: Reductions in energy
expenditure and increased adiposity despite increased physical
activity support a key role for the gut microbiome in promoting
adaptive thermogenesis across a broad range of ambient
temperatures. In addition, reduced RER values and plasma
glucose concentrations in germ-free mice indicate that the gut
microbiome promotes the utilization of dietary carbohydrate as
an energy substrate. We speculate that impaired thermogenesis
in the setting of dysbiosis contributes to the pathogenesis
of NAFLD.
Disclosures:
David E. Cohen - Advisory Committees or Review Panels: Merck, Aegerion,
Genzyme; Consulting: Intercept
The following authors have nothing to disclose: Tibor I. Krisko, Hayley T. Nicholls,
Katherine B. Leclair, Alexander S. Banks
882
The dual FXR/TGR5 agonist INT-767 counteracts nonalcoholic
steatohepatitis in a rabbit model of high fat
diet-induced metabolic syndrome
Linda Vignozzi 1 , Ilaria Cellai 1 , Sandra Filippi 4 , Paolo Comeglio 1 ,
Erica Sarchielli 2 , Annamaria Morelli 2 , Elena Maneschi 1 , Gabriella
Barbara Vannelli 2 , Luciano Adorini 3 , Mario Maggi 1 ; 1 Dep.
of Experimental and Clinical Biomedical Sciences, University of
Florence, Florence, Italy; 2 Dep. of Experimental and Clinical Medicine,
University of Florence, Florence, Italy; 3 Intercept Pharmaceuticals,
New York, NY 10011, NY; 4 Interdepartmental Laboratory
of Functional and Cellular Pharmacology of Reproduction, Dep. of
NEUROFARBA and Dep. of Experimental and Clinical Biomedical
Sciences, University of Florence, Florence, Italy
Farnesoid X receptor (FXR) and Takeda G protein-coupled
receptor 5 (TGR5) are interesting pharmacological targets for
the treatment of liver and metabolic diseases. FXR-deficient
mice on a high-fat diet (HFD) exhibit massive hepatic steatosis,
necro-inflammation and fibrogenesis. Moreover, pharmacological
activation of TGR5 in mice promotes protective mechanisms
in biliary epithelial cells, inhibits hepatic and systemic
inflammation. Thus, we hypothesized that a FXR/TGR5 dual
agonist would ameliorate features of nonalcoholic steatohepatitis
(NASH) in a rabbit model of metabolic syndrome (MetS).
Treatment with increasing doses of the dual FXR/TGR5 agonist
INT-767 (3,10,30mg/Kg/day, 5 days a week for 12 weeks)
in a rabbit model of HFD-induced MetS, characterized also
by NASH, dose-dependently reduced several MetS-associated
alterations, including hepatomegaly, insulin resistance,
increase of ALT, glucose and cholesterol levels, while significantly
increasing HDL levels. ALT was positively associated
with all MetS parameters; however introducing all MetS factors
in a multivariate analysis, only total cholesterol levels resulted
positively associated with ALT level (Adj.r: 0.493, p=0.014).
High macrophage M1pro-inflammatory/M2 anti-inflammatory
ratio was observed in MetS-induced NASH, which was
independently associated with serum ALT levels (Adj.r: 0.322,
p=0.032). HFD-induced increase in M1/M2 ratio was reduced
by INT-767 treatment and M2 macrophage markers (IL-10,T-
GFβ) were increased. Genes related to neutrophil apoptosis/
apoptotic-neutrophil clearance (lactoferrin, eNOS, RAGE)
and to extracellular matrix degradation (MMP2, TIMP2) were
also increased by INT-767 treatment. INT-767 also reduced
liver expression of IL-6, which preferentially skews the Th cell
response towards a Th17-phenotype, while increasing Foxp3
expression, a Treg cell marker. Thus these data indicate that
INT-767 can promote the neutrophil and macrophage-driven
resolution phase of inflammation and fibrosis regression. In
addition, INT-767 increased genes related to hepatic fatty acid
metabolism (PPARa, AR, CD36) and lipid droplet formation
(SNAP23, VAMP4,syntaxin5, perilipin) therefore suggesting
that INT-767 counteracts excess fatty acid mediated lipotoxicity
in the liver. Genes related to insulin signaling (IRS1, SREBP1,
G6Pase, and PEPCK) were also increased by INT-767. Finally,
immunohistochemical studies demonstrated that INT-767 treatment
significantly reduced both HFD-induced liver inflammation
and fibrosis. In conclusion, INT-767 treatment counteracts
NASH in a rabbit model of HFD-induced MetS by promoting
insulin sensitivity, resolution of inflammation and fibrosis regression.
Disclosures:
Luciano Adorini - Employment: Intercept Pharmaceuticals
Mario Maggi - Consulting: BAYER, ELI LILLY, MENARINI, PROSTRAKAN, INTER-
CEPT
The following authors have nothing to disclose: Linda Vignozzi, Ilaria Cellai,
Sandra Filippi, Paolo Comeglio, Erica Sarchielli, Annamaria Morelli, Elena Maneschi,
Gabriella Barbara Vannelli