studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 861A
1323
Ethanol-Induced Alterations In The Intestinal Methionine
Metabolic Pathway Promote Tight Junction Disruption
Dan Feng 1,2 , Paul G. Thomes 3 , Natalia A. Osna 1,2 , Dean J.
Tuma 1,2 , Kusum K. Kharbanda 1,2 ; 1 Research Service, Veterans
Affairs Nebraska-Western Iowa Health Care System, Omaha, NE;
2 Department of Internal Medicine, University of Nebraska Medical
Center, Omaha, NE; 3 Liver Pathobiology Laboratory, Cannon
Research Center, Carolinas Healthcare System, Charlotte, NC
The gut-liver interaction has emerged as a critical component
in the pathogenesis of alcoholic liver disease (ALD). The central
mediator of ALD progression is the gut luminal antigens, especially
endotoxins that translocate to the liver via compromised
gut epithelial tight junctions (TJ). We have previously shown
that ethanol consumption can alter the liver methionine metabolic
pathway to impair several methylation reactions leading
to the generation of many hallmark features of alcoholic liver
injury. This study was undertaken to examine whether alcohol
exposure alters the intestinal methionine metabolic pathway
and to explore whether these alterations play a causal role in
disrupting the gut barrier integrity. Adult male Wistar rats were
pair-fed the Lieber DeCarli control or ethanol diet for 8 weeks.
At the end of the feeding regimen, intestines were removed and
analyzed. We observed the level of methionine synthase (MS,
an enzyme of the methionine metabolic pathway that prevents
buildup of a harmful metabolite, S-adenosylhomocysteine,
SAH) was decreased by 50% in the ileum of ethanol-fed rats
compared to controls. This decrease was accompanied by a
compensatory increase in betaine homocysteine methyltransferase
(BHMT), another enzyme that maintains the level of a vital
methylating agent, S-adenosylmethionine (SAM), and prevents
SAH buildup. However, while the adaptive increase in BHMT
maintained SAM levels in the ileum of the ethanol-fed rat, there
was a significant elevation in SAH levels ultimately lowering
the SAM:SAH ratio compared to controls. All these changes
were similar to those previously reported in the livers of ethanol-fed
rats. Concomitant with the changes in the crucial components
of the methionine metabolic pathway that controls the
cellular methylation potential, we saw the disassembly of TJs
in the ileum of the ethanol-fed rats. There was a disorganized
localization of the TJ proteins, occludin and claudin -1, which
normally seal the intercellular spaces to prevent the paracellular
translocation of luminal antigens into portal circulation. Further
studies using an inhibitor of lysine methyltransferase identified
that the methylation reactions catalyzed by this enzyme
were important for maintaining the barrier integrity. We further
observed that occludin is methylated and this post-translational
modification occurred on the lysine and not arginine residue(s).
Taken together, our results indicate that alcohol-induced alterations
in the intestinal methionine metabolic pathway and the
resulting impairments in methylation reactions contribute to the
disruption of the TJs, thereby promoting gut leakiness and progressive
liver injury.
Disclosures:
The following authors have nothing to disclose: Dan Feng, Paul G. Thomes,
Natalia A. Osna, Dean J. Tuma, Kusum K. Kharbanda
1324
Role of Pre-mRNA Splicing Regulator SLU7 in Mediating
Detrimental Action of Ethanol in the Liver
Jiayou Wang 1,2 , Xudong Hu 1,3 , Alvin Jogasuria 1 , Kwangwon Lee 1 ,
Jiashin Wu 1 , Min You 1 ; 1 Pharmaceutical Sciences, Northeast Ohio
Medical University (NEOMED), Rootstown, OH; 2 Department of
Anatomy, Guangzhou University of Chinese Medicine, Guangzhou,
China; 3 Department of Biology, Shanghai University of Traditional
Chinese Medicine, Shanghai, China
Precursor mRNA (pre-mRNA) splicing is a critical step in gene
expression that removes intronic sequences from immature
mRNA, producing mature mRNA that can be translated into
protein. Errors in splicing contribute to at least 15% of human
genetic disorders. Aberrant pre-mRNA splicing plays a significant
role in liver diseases, but very little is known about its
role in alcoholic steatosis/steatohepatits. Here, we identified
a new target of ethanol action in the liver, namely, pre-mRNA
splicing regulator SLU7. The effects of ethanol on splicing factor
SLU7 were assessed in cultured hepatocytes and in animal
liver. Feeding mice a modified Lieber-DeCarli ethanol-containing
liquid diet for 4-wks resulted in significant decreases in the
mRNA and protein expression levels of hepatic SLU7. More
importantly, ethanol exposure robustly reduced the amount of
splicing factor SLU7 in the nucleus in cultured hepatocytes and
in mouse liver. Slu7 governs the splicing and/or expression of
multiple genes essential for hepatic lipid metabolism, including
serine/arginine-rich splicing factor 3 (SRSF3) and hepatocyte
nuclear factor 4α (HNF4α). SRSF3 pre-mRNA alternative splicing
generates the full-length isoform lacking exon 4 (Iso1) and
an alternative isoform including exon 4 (Iso2). Ethanol-mediated
disruption of splicing factor SLU7 triggered altered SRSF3
pre-mRNA splicing by inhibiting the mRNAs of total SRSF3,
SRSF3_Iso 1, or SRSF3_ Iso2 and HNF-4α in the livers of ethanol-fed
mice. Remarkably, the alteration of hepatic SLU7-SRSF3
axis by ethanol exposure was closely associated with the development
of excess accumulation of hepatic triglycerides in mice.
Taken together, our findings suggest that splicing regulator
SLU7 may play an essential role in regulating the effects of ethanol
on hepatic lipid metabolism and development of alcoholic
fatty liver.
Disclosures:
The following authors have nothing to disclose: Jiayou Wang, Xudong Hu, Alvin
Jogasuria, Kwangwon Lee, Jiashin Wu, Min You
1325
Impaired Proteasome Function is Associated with
Alcohol-Induced Hepatic Receptor Interacting Protein
3-Mediated Necroptosis, Steatosis and Liver Injury
Wen-Xing Ding, Shaogui Wang, Hong-Min Ni; Pharmacology,
Toxicology and Therapeutics, The University of Kansas Medical
Center, Kansas City, KS
Both hepatocyte apoptosis and necrosis are hallmarks of alcohol-induced
liver injury. Recent evidence suggests that receptor-interacting
protein kinase (RIP) 3-mediated necroptosis is
important in chronic alcohol feeding-induced liver injury in
mice. However, the mechanisms by which alcohol activates
RIP3-mediated necroptosis are unclear. Using the recently established
chronic alcohol feeding plus binge (Gao-binge) model,
we demonstrated that Gao-binge alcohol treatment increased
RIP3 but not RIP1 protein levels in mouse livers based on both
western blot analysis and immunohistochemical staining for
RIP1 and RIP3. 7-OCl-Nec 1, a recently developed specific
RIP1 inhibitor, failed to protect against Gao-binge alcohol-induced
steatosis and liver injury, suggesting alcohol-induced