Hepatic inflammation caused by dysregulated bile acid synthesis is reversible by butyrate supplementation.

Dysregulated bile acid (BA) synthesis or reduced farnesoid X receptor (FXR) levels are found in patients having metabolic diseases, autoimmune hepatitis, and liver cirrhosis or cancer. The objective of this study was to establish the relationship between butyrate and dysregulated BA synthesis-induced hepatitis as well as the effect of butyrate in reversing the liver pathology. Wild-type (WT) and FXR knockout (KO) male mice were placed on a control (CD) or western diet (WD) for 15 months. In the presence or absence of butyrate supplementation, feces obtained from 15-month-old WD-fed FXR KO mice, which had severe hepatitis and liver tumors, were transplanted to 7-month-old WD-fed FXR KO for 3 months. Hepatic phenotypes, microbiota profile, and BA composition were analyzed. Butyrate-generating bacteria and colonic butyrate concentration were reduced due to FXR inactivation and further reduced by WD intake. In addition, WD-fed FXR KO male mice had the highest concentration of hepatic ß-muricholic acid (ß-MCA) and bacteria-generated deoxycholic acid (DCA) accompanied by serious hepatitis. Moreover, dysregulated BA and reduced SCFA signaling co-existed in both human liver cancers and WD-fed FXR KO mice. Microbiota transplantation using butyrate-deficient feces derived from 15-month-old WD-fed FXR KO mice increased hepatic lymphocyte numbers as well as hepatic ß-MCA and DCA concentrations. Furthermore, butyrate supplementation reduced hepatic ß-MCA as well as DCA and eliminated hepatic lymphocyte infiltration. In conclusion, reduced butyrate contributes to the development of hepatitis in the FXR KO mouse model. In addition, butyrate reverses dysregulated BA synthesis and its associated hepatitis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

S-adenosylmethionine prevents Mallory Denk body formation in drug-primed mice by inhibiting the epigenetic memory.

UNLABELLED: In previous studies, microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridine decarboxylate (DDC) for 10 weeks followed by 1 month of drug withdrawal (drug-primed mice) and then 7 days of drug refeeding showed an increase in the expression of numerous genes referred to here as the molecular cellular memory. This memory predisposes the liver to Mallory Denk body formation in response to drug refeeding. In the current study, drug-primed mice were refed DDC with or without a daily dose of S-adenosylmethionine (SAMe; 4 g/kg of body weight). The livers were studied for evidence of oxidative stress and changes in gene expression with microarray analysis. SAMe prevented Mallory Denk body formation in vivo. The molecular cellular memory induced by DDC refeeding lasted for 4 months after drug withdrawal and was not manifest when SAMe was added to the diet in the in vivo experiment. Liver cells from drug-primed mice spontaneously formed Mallory Denk bodies in primary tissue cultures. SAMe prevented Mallory Denk bodies when it was added to the culture medium. CONCLUSION: SAMe treatment prevented Mallory Denk body formation in vivo and in vitro by preventing the expression of a molecular cellular memory induced by prior DDC feeding. No evidence for the involvement of oxidative stress in induction of the memory was found. The molecular memory included the up-regulation of the expression of genes associated with the development of liver cell preneoplasia.

Hepatocyte retinoid X receptor alpha-dependent regulation of lipid homeostasis and inflammatory cytokine expression contributes to alcohol-induced liver injury.

Hepatocyte retinoid X receptor alpha (RXRalpha)-deficient mice are more sensitive to ethanol toxicity than wild-type mice. Because RXRalpha-mediated pathways are implicated in lipid homeostasis and the inflammatory response, we hypothesized that a compromise in lipid metabolism and associated production of proinflammatory mediators are responsible for the hepatotoxicity observed in ethanol-treated hepatocyte RXRalpha-deficient mice. Wild-type and hepatocyte RXRalpha-deficient mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. After ethanol treatment, serum ALT levels increased significantly (4-fold) in hepatocyte RXRalpha-deficient mice, but not in the wild-type mice. Hepatic liver fatty acid binding protein (L-FABP) mRNA and protein levels were reduced due to RXRalpha deficiency. Ethanol induced L-FABP mRNA and protein in wild-type mice and provided protection against nonesterified fatty acid toxicity; however, this effect was absent in the mutant mice. Accordingly, hepatic nonesterified fatty acid level was increased in ethanol-fed mutant mice. Ethanol increased nuclear factor (NF)-kappaB binding activity in hepatocyte RXRalpha-deficient mice, but not in wild-type mice. In agreement, hepatic mRNA levels of proinflammatory cytokines and chemokines were increased to a greater extent in the mutant than in wild-type mice. Furthermore, signal transducer and activator of transcription factor (STAT) 3 and associated Bcl-xL induction was observed in ethanol-fed wild-type mice but not in ethanol-fed hepatocyte RXRalpha-deficient mice. Taken together, after ethanol treatment, hepatocyte RXRalpha deficiency results in lack of L-FABP induction, increased hepatic free fatty acids, NF-kappaB activation, and proinflammatory cytokines production and a lack of STAT3 activation, which in part may contribute to alcohol-induced liver damage.

Ethanol withdrawal induced CYP2E1 degradation in vivo, blocked by proteasomal inhibitor PS-341.

The aim of this study was to characterize CYP2E1 degradation in vivo using PS-341, a potent proteasome inhibitor. Previously, only in vitro evidence showed that CYP2E1 induced by ethanol is degraded by the proteasome. Male Wistar rats were given ethanol intragastrically for 30 d. Ethanol was withdrawn at the same time that PS-341 was injected, 24 h before the rats were sacrificed. The liver proteasomal chymotrypsin-like activity (ChT-L) in rats fed ethanol was inhibited. After ethanol withdrawal, the proteasomal ChT-L activity returned to control levels. In the ethanol-withdrawn rats injected with PS-341, the ChT-L activity was significantly inhibited before withdrawal (p <.001). Ethanol treatment induced a 3-fold increase in CYP2E1 levels determined by Western blot. When ethanol was withdrawn, CYP2E1 decreased to control levels. In ethanol-withdrawn rats injected with PS-341, CYP2E1 remained at the induced level. These results show, for the first time, that the proteasome is responsible for ethanol-induced CYP2E1 degradation in vivo.

Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared.

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.

The role of the ubiquitin-proteasome pathway in the formation of mallory bodies.

The dynamics of Mallory body (MB) formation are difficult to follow in vivo. Because of the lack of an in vitro mouse hepatocyte culture model, a cellular extract approach was developed. In this model an immunoprecipitate was obtained using an antibody to cytokeratin-8 (CK-8). The isolate contained a large number of compounds: CK-8, ubiquitin, a frameshift mutation of ubiquitin (UBB(+1)), proteasomal subunits beta5 (a catalytic subunit of the 20S proteasome) and Tbp7 (an ATPase subunit of the 26S proteasome), transglutaminase, tubulin, heat shock proteins 90 and 70, and MBs. In Western blots, CK-8 immunoprecipitates showed colocalization of these components in a complex of proteins colocalized in a high-molecular-weight smear. When the CK-8 immunoprecipitate was incubated with the isolate of proteasomes and an energy generating source (ATP), the components of the ubiquitinated protein smear increased. These observations taken together with the in vivo observation that these proteins colocalized at the edge of the MB shown in the present study suggest that these proteins form aggregates through covalent binding of CK-8, ubiquitin, and the proteasomes. Covalent aggregation is suggested by the fact that the protein complex found in the high-molecular-weight smear that forms in vitro fails to dissociate in SDS. This protein complex is present in the CK-8 immunoprecipitates of livers forming MBs but not in control livers. In conclusion, the results support the concept that Mallory bodies are aggresomes which form as the result of the failure of the ubiquitin-proteasome complex to adequately eliminate cytokeratins destined for proteolysis.