Date on Master's Thesis/Doctoral Dissertation

5-2016

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)

Gobejishvili, Leila

Committee Member

Gobejishvili, Leila

Committee Member

McCain, Craig

Committee Member

Arteel, Gavin

Committee Member

Cole, Marsha

Committee Member

Clark, Geoffrey

Author's Keywords

Alcoholic liver diseases; Neuro-inflammation, Alcohol--Physiological effect; Phosphodiesterases

Abstract

Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the U.S., and there is still no FDA-approved therapy. Alcohol metabolism leads to generation of free radicals and oxidative stress with a resultant formation of lipid peroxidation products, which, in turn, contribute to the development of ALD. Alcohol induced hepatic steatosis is the earliest and most frequent manifestation of ALD and a significant risk factor for progressive liver disease. Cyclic adenosine monophosphate (cAMP) signaling has been shown to significantly regulate lipid metabolism. Moreover, agents that increase cAMP have been shown to effectively mitigate oxidative stress both in vivo and in vitro. Hence, the role of hepatic PDE4 and a resultant dysregulation of cAMP signaling in alcohol induced hepatic steatosis and lipid peroxidation was examined. C57BL/6 wild type (WT) and Pde4b knockout (Pde4b-/-) mice were pair-fed control and ethanol liquid diets. One group of wild type mice received Rolipram, a PDE4 specific inhibitor, during alcohol feeding. Alcohol feeding resulted in a significant fat accumulation and oxidative stress in WT mice as demonstrated by increased hepatic free fatty acid levels and lipid peroxidation. This alcohol effect was associated with a significant decrease in hepatic carnitine palmitoyltransferase 1A (CPT1A) expression, a rate limiting enzyme in fatty acid β-oxidation. Additionally, hepatic F4/80 staining was markedly increased in alcohol fed WT mice, indicating Kupffer cell activation. Importantly, alcohol feeding significantly increased hepatic PDE4 enzyme expression as early as in one week with the concomitant decrease in cAMP/pCREB levels. PDE4 inhibition in alcohol fed mice prevented the decrease in hepatic CPT1A expression and lipid accumulation. This effect on CPT1A expression was mediated by preventing the decrease in a critical transcription factor for CPT1A expression, peroxisome proliferator-activated receptor (PPARα) and increase in PPARα co-activators, peroxisome proliferator-activated receptor gamma coactivator 1α and sirtuin 1(PGC-1α and SIRT1). Moreover, compared to wild type mice, Pde4b knockout and Rolipram treated alcohol fed mice had higher levels of antioxidant enzymes SOD1/2, and GPx1/2 and decreased 4HNE and F4/80 staining. In summary, these results demonstrate that the alcohol- induced increase in hepatic PDE4, specifically PDE4B expression, and compromised cAMP signaling predisposes the liver to impaired fatty acid oxidation and increased oxidative stress. These data also suggest that hepatic PDE4 is a clinically relevant therapeutic target for the treatment of alcoholic fatty liver disease. Chronic ethanol consumption significantly increases brain TLR4 expression and downstream inflammatory gene expression, contributing to microglial activation and neuro-inflammation. Our group has previously shown that TLR4 inducible PDE4 expression plays a major role in regulating inflammatory cytokine production in alcohol exposed monocytes/macrophages. We have also shown that inhibition of PDE4 markedly down-regulates endotoxin inducible TNF expression and alcohol mediated priming of monocytes/macrophages. In the present study we examined the potential role of PDE4 in alcohol induced activation of glial cells and neuro-inflammation using both in vitro and in vivo models. Primary mouse microglial cells were treated in vitro with ethanol followed by endotoxin stimulation. Protein and gene expression analysis showed that alcohol treatment increased TNF and PDE4B expression and primed microglial cells to increase production of TNF in response to endotoxin. The PDE4 inhibitor, Rolipram, significantly attenuated TNF expression indicating the role of PDE4B in alcohol mediated effect on microglial TNF expression. To examine the role of PDE4B in alcohol induced neuro-inflammation in vivo, C57Bl/6 and pde4b knockout (Pde4b-/-) mice were pair-fed control and ethanol liquid diets for 4 weeks. Additionally, one group of mice received Rolipram to pharmacologically inhibit PDE4 activity. Examination of brain tissues from alcohol fed mice showed increased PDE4B protein expression compared to pair-fed mice. Along with PDE4B, alcohol was also observed to: (i) increase COX-2 expression; and (ii) induce activation of glial cells, as indicated by an increase in GFAP (glial fibrillary acidic protein) positive astrocytes, and IBA-1 (ionized calcium-binding adapter molecule 1) positive microglial cells. Importantly, both alcohol-induced activation of glial cells and inflammation were markedly attenuated in Pde4b-/- mice and by pharmacologic inhibition of PDE4. Overall, these data identify the critical role of PDE4B in regulating alcohol-induced neuro-inflammation that can be targeted for effective therapeutic intervention.

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