ROLE OF CYP2E1 IN THE PATHOGENESIS OF ALCOHOLIC LIVER DISEASE:MODIFICATIONS BY cAMP AND UBIQUITIN-PROTEASOME PATHWAY

Gouillon Z-Q¹, Miyamoto K², Donohue TM³, Wan Y-J Y ¹ , French BA ¹, Nagao Y ¹, Fu P ¹, Reitz RC ⁴ , Hagbjork A ⁵, Yap C ¹, Yuan QX ¹, Ingelman-Sundberg M ⁵, French SW ¹

1 Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509, ² Miyamoto Medical Clinic, Kanagawa, Japan ³ University of Nebraska and VA Medical Center, Omaha, NE, ⁴Department of Biochemistry, University of Nevada, Reno, NE,^{, 5} Institute of Environmental Medicine, Division of Molecular Toxicology, Karolinska Institute 17177, Stockholm, Sweden

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Methods

3.1. Animals
3.2. Biochemical and liver morphology analysis
3.3. Hepatic lipid analysis
3.4. Western blot analysis
3.5. Northern blot analysis
3.6. Electrophoretic mobility shift analysis (EMSA) of NFkappaB
3.7. Assay of proteasomal proteolytic enzymes
3.8. Statistical analysis

4. Results

4.1. Animals and liver pathology
4.2. Fatty acid composition
4.3. Liver regeneration
4.4. Cyclic AMP levels
4.5. CYP2E1 levels
4.6. CYP2E1 expression
4.7. Ubiquitin levels
4.8. Proteasome peptidase activty
4.9. NFkappaB activation

5. Discussion

5.1. Proteolytic and synthetic pathways of CYP2E1 regulation
5.2. Influence of cAMP on ethanol-induced liver injury

6. Acknowledgements

7. References

1. ABSTRACT

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.