Date on Master's Thesis/Doctoral Dissertation

12-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Barve, Shirish

Committee Co-Chair (if applicable)

McClain, Craig

Committee Member

McClain, Craig

Committee Member

Joshi-Barve, Swati

Committee Member

Cave, Matthew

Committee Member

Kidd, LaCreis

Abstract

Chronic alcohol consumption is a leading cause of liver disease and liver-related death worldwide. Alcoholic liver disease includes, hepatic steatosis, steatohepatitis and ultimately fibrosis and cirrhosis. Emerging evidence has established the important role of the “gut-liver” axis in the development of alcoholic liver disease (ALD). Our recent work indicated that chronic alcohol induced perturbations in the gut microbiome and consequent changes in fatty acids have a major impact on the development of intestinal barrier dysfunction and ALD. The aim of this study was to investigate whether treatment with tributyrin - a butyrate prodrug results in protection against ALD in terms of hepatic steatosis, inflammation and injury. Tributyrin is a triglyceride that is rapidly absorbed and metabolized to butyrate. Moreover, it has more favorable pharmacokinetics compared with butyrate with low toxicity. In the present study, we have used a mouse model of ALD to examine the effects of Tributyrin oral administration on ethanol-induced changes in intestinal permeability and hepatic steatosis, inflammation and injury. 8–10-week old C57BL/6 male mice were chronically pair-fed the Lieber-DeCarli liquid diet containing alcohol or isocaloric maltose dextrin. Tributyrin was administered to a sub-group of alcohol-fed animals by oral gavage (2g/kg) for 5 days/week to assess its effects. Tributyrin attenuated the ethanol-induced gut barrier dysfunction, as shown by the significant reduction in endotoxemia. Mechanisms of chronic alcohol-induced gut-barrier dysfunction were examined by immunohistochemical analyses of the ileum section of mice chronically fed alcohol. Specifically, alcohol induced a robust down-regulation of intestinal tight junction proteins ZO-1 and occludin which was markedly attenuated by tributyrin administration. Liver histological analysis showed a significant decrease in ethanol-induced hepatic steatosis. Tributyrin also prevented the ethanol-induced down-regulation of hepatic CPT-1, a key enzyme in free fatty acid b-oxidation, with a resultant decrease in hepatic triglycerides and free fatty acids. To further elucidate the mechanism by which tributyrin prevents hepatic steatosis, we performed experiments using primary hepatocytes. Hepatocytes were treated with alcohol and with/without pre-treatment with butyrate. Alcohol-induced downregulation of CPT-1 gene expression was significantly prevented by butyrate. Butyrate is a known HDAC inhibitor. Hence promoter histone acetylation state was investigated. In this regard, acetylation of histone 3 lysine 9 (H3K9), a critical feature of the active promoter state was significantly increased by butyrate and was essentially mediated by the p300-histone acetyltransferase. Increased acetylation leads to an open chromatin configuration causing increased recruitment of co-activators and TF such as PGC-1 and Sp-1 at the CPT-1 promoter. Additionally RNA-Pol II binding was also increased by butyrate pre-treatment in alcohol treated hepatocytes. These data suggest that butyrate, by acting as an HDAC inhibitor, induces promoter histone acetylation leading to the transcriptional activation of CPT-1 under pathologic conditions of alcohol exposure. Butyrate has been shown to have beneficial effects by decreasing obesity-associated inflammation, and insulin resistance. Hence, its effects on alcohol induced hepatic inflammation were also investigated. F4/80 staining of liver sections revealed increased macrophage infiltration in 2 weeks of alcohol feeding whereas tributryrin administration markedly blocked macrophage infiltration. Tributyrin administration attenuated the ethanol mediated pro-inflammatory cytokine TNF-a mRNA expression. Alcohol-induced neutrophil infiltration was also significantly suppressed by tributyrin administration. Neutrophil infiltration was further confirmed by MPO activity. Tributyrin administered mice had a significant drop in MPO activity as compared to alcohol-fed mice at 7 weeks but not in 2 weeks. Inflammatory chemokine and cytokine such as CXCL-2 (MCP-1) and CCL-2 (MIP-2) mRNA were significantly upregulated in alcohol-fed mice and tributryrin administration significantly attenuated alcohol-induced induction of these pro-inflammatory chemokines. Notably, tributryrin also significantly attenuated liver injury as seen by a decrease in serum AST & ALT levels. Hence, the present work demonstrates that tributyrin may be useful in preventing the ethanol-induced pathogenic changes in the “gut-liver” axis and may prove to be a useful therapy for the prevention/treatment of ALD. HIV protease inhibitors (HIV-PIs) are the major components of the highly active anti-retroviral therapy (HAART) and have been successfully used in the treatment of HIV-1 infection in the past two decades. However prolonged use of HAART is known to cause hepatotoxicity. Hence it is highly relevant to study the underlying mechanisms in HAART induced hepatotoxicity that can negatively impact treatment outcomes. One of the known mechanisms of hepatotoxicity caused due to HIV-PIs drugs is endoplasmic reticulum (ER) stress and subsequent activation of unfolded protein response (UPR) leading to dys-regulation of hepatic lipid metabolism. Our recent work shows that PDE4/cAMP metabolism plays a significant role in alcohol-induced hepatic steatosis and injury. Hence in the present study, we examined the potential mechanisms underlying HIV-PI induced hepatic ER stress and toxicity with a particular emphasis on the pathogenic role of PDE4 family of enzymes. The effects of clinically used HIV-PIs [Ritonavir and Lopinavir] were examined both in a rat hepatoma cell line (H4IIE). The data obtained from these studies demonstrated that Ritonavir and Lopinavir in combination led to a significant loss of hepatocyte survival. Notably, inhibition of PDE4 by a specific PDE4-inhibitor, rolipram, markedly attenuated hepatotoxicity induced by PI treatments. Examination of the mechanistic role of PDE4 showed that PDE4 inhibition significantly decreases the PIs-induced expression of the ER stress related proteins CHOP, ATF-4 and -3. Furthermore, examination of the PDE4/cAMP-regulated downstream signaling demonstrated the involvement of cAMP PKA (protein kinase A). Hence, effect of H-89 (PKA inhibitor) was examined in hepatocytes treated with HIV-PI and rolipram. PKA inhibition significantly reversed the rolipram protective effect leading to re-expression of ER-stress related genes. Fas/FasL signaling plays a critical role in drug-induced hepatotoxicity and cell death Hence activation of hepatotoxic Fas/FasL signaling in HIV-PI induced hepatotoxicity was investigated. Combination of Rit+Lop significantly up- regulated FasL and Fas expression in H4IIE rat hepatoma cell line. Commensurate with the increase in FasL and Fas, PDE4 expression was also increased with Rit+Lop treatment. Notably PDE4 inhibition down-regulated Fas and FasL mRNA and protein expression induced by HIV PIs whereas PKA inhibitor H-89 significantly reversed the rolipram effect on Fas and FasL expression. Taken together, these data strongly support and identify a pathogenic/mechanistic role for PDE4 regulated cAMP-PKA in the development of HIV-PI induced ER-stress and FasL mediated cell death leading to hepatic steatosis and injury.

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