studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 237A
58
Hepatocytes Release Ceramide-rich Proinflammatory
Extracellular Vesicles in an IRE1alpha-dependent manner
Eiji Kakazu 1,2 , Amy S. Mauer 1 , Harmeet Malhi 1 ; 1 Gastroenterology
and Hepatology, Mayo Clinic, Rochester, MN; 2 Department of
Community Medical Supports, Tohoku University, Aobaku, Japan
Palmitate (PA), a lipotoxic free fatty acid, is implicated in
hepatocyte apoptosis, macrophage-mediated liver inflammation,
and activation of the IRE1alpha branch of the endoplasmic
reticulum (ER) stress response - all key features of
progressive nonalcoholic steatohepatitis (NASH). Recently,
extracellular vesicles (EVs) have been implicated in NASH.
However, the exact pathways of hepatocyte EV generation
under lipotoxic conditions and their downstream effects on
macrophages are undefined. Therefore, we hypothesized that
hepatocytes release proinflammatory PA-induced EVs via an
ER stress response. To test this hypothesis we employed immortalized
mouse hepatocytes (IMH) from wild-type (WT) or IRE1a
knockout (KO) mice, Huh 7 and primary mouse hepatocytes
(PMH). Cells were treated with 400μM PA or thapsigargin
(Tg) to induce ER stress. EVs were isolated from cell culture
supernatants by ultracentrifugation and characterized by immunofluorescence
and western blotting for EV markers, electron
microscopy (EM) and by nanoparticle tracking analysis (NTA)
for morphology and size. Ceramides were measured by mass
spectrometry. CRISPR/Cas9 technology was used to delete
XBP1. Results: PA and Tg significantly increased EV release
in all three hepatocyte cells tested (IMH, Huh7 and PMH). The
released EVs were 100nm in size, decorated with EV markers
CD63, TSG101 and LAMP1, and demonstrated characteristic
cup-shaped morphology on EM. As PA and Tg activate an ER
stress response, we next tested cells lacking in each of the three
canonical ER stress sensors, IRE1alpha, ATF6alpha and PERK.
Both PA and Tg induced a significant EV release in cells lacking
ATF6 alpha or PERK; however, PA and Tg-induced EV release
was significantly suppressed in IRE1alpha KO cells. We tested
the involvement of XBP1 signaling in this phenomenon and
found a reduction in EV response in cells lacking XBP1. Next
we measured ceramides in PA-induced EVs as they are implicated
in EV biogenesis. PA-induced EVs were enriched in C16
ceramide; this enrichment occurred in an IRE1alpha-dependent
manner. Inhibition of de novo ceramide synthesis by myriocin
ameliorated PA-induced EV release, and exogenous C16 ceramide
resulted in EV release in IRE1alpha KO and WT cells.
Lastly, PA-stimulated EVs were chemotactic to macrophages.
Conclusions: PA induces C16 ceramide-enriched EV release
in an IRE1alpha-dependent manner. PA-induced EVs stimulate
macrophage chemotaxis and this may be a mechanism for the
recruitment of macrophages to the liver under lipotoxic conditions.
We hypothesize that interference with this macrophage
recruitment response may be a therapeutic avenue in NASH.
Disclosures:
The following authors have nothing to disclose: Eiji Kakazu, Amy S. Mauer,
Harmeet Malhi
59
Deficiency of intestinal mucin-2 protects mice from
diet-induced fatty liver disease and obesity
Phillipp Hartmann, Caroline T. Seebauer, Magdalena Mazagova,
Angela Horvath, Lirui Wang, Cristina Llorente-Izquierdo, Katharina
Brandl, Samuel B. Ho, David A. Brenner, Bernd Schnabl; University
of California San Diego, La Jolla, CA
Background: Non-alcoholic fatty liver disease (NAFLD) and
obesity are characterized by altered gut microbiota, inflammation,
and gut barrier dysfunction. The aim of this study was to
investigate the role of mucin-2 (Muc2) as the major component
of the intestinal mucus layer in the development of fatty liver
disease and obesity. Methods and Results: We studied experimental
fatty liver disease and obesity induced by high-fat diet
(HFD) feeding for 16 weeks in wild-type (WT) littermate and
mucin-2 knockout mice. Muc2 deficiency protected mice from
HFD-induced obesity and fatty liver disease as evidenced by a
significantly reduced body weight and lower hepatic triglyceride
concentrations, respectively. Furthermore, Muc2 -/- mice displayed
significantly decreased weights of subcutaneous and
epididymal white adipose tissue and brown adipose tissue
relative to WT mice on HFD. There was no difference in food
intake and fecal triglyceride content between the HFD groups
that would explain differences in phenotype. HFD-fed Muc2 -/-
mice exhibited improved glucose tolerance and responsiveness
to insulin, as evidenced by intraperitoneal glucose tolerance
and insulin tolerance tests, as well as reduced inflammation
in white adipose tissue than WT littermates. Furthermore, they
showed an upregulated expression of key genes involved in
lipolysis, such as adipose triglyceride lipase (ATGL) and hormone-sensitive
lipase (HSL), and in fatty acid β-oxidation, e.g.
palmitoyl acyl-CoA oxidase 1 (ACOX1), in white adipose tissue
compared with WT mice after 16 weeks of HFD feeding.
Interleukin-22 (IL-22) is not only crucial for maintaining intestinal
homeostasis, but administration of IL-22 to obese mice has
been shown to improve insulin sensitivity, decrease chronic
inflammation and reduce body weight. Most interestingly,
Muc2 -/- mice showed increased intestinal gene expression
of IL-22 and IL-22 target genes Reg3b and Reg3g relative to
WT mice fed HFD. Plasma levels of IL-22 were significantly
higher in HFD-fed Muc2 -/- mice as compared with WT mice.
After bypassing the mucus layer by intraperitoneal injections
of bacteria-derived flagellin, differences in IL-22 plasma levels
between HFD-fed WT and Muc2 -/- mice vanished, suggesting
that the mucosal immune system produces increased amounts
of IL-22 in the absence of a protective mucus barrier. Conclusion:
An impaired gut barrier elicits a strong intestinal immune
response in Muc2 deficient mice, which is characterized by the
induction of IL-22. Increased systemic IL-22 mediates beneficial
metabolic and anti-inflammatory effects, and protects against
obesity and fatty liver disease.
Disclosures:
Angela Horvath - Grant/Research Support: Instutut Allergosan
Samuel B. Ho - Grant/Research Support: Gilead, Genentech; Speaking and
Teaching: Prime Education, Inc
The following authors have nothing to disclose: Phillipp Hartmann, Caroline T.
Seebauer, Magdalena Mazagova, Lirui Wang, Cristina Llorente-Izquierdo, Katharina
Brandl, David A. Brenner, Bernd Schnabl