The major hypotheses of this competing continuation application are: Ethanol exposure down-regulates alpha-2,6-sialyltransferase (2,6-ST) at the molecular level leading to defective glycosylation of apolipoprotein E (ApoE) and its association with HDL. Ethanol also affects sphingomyelin (SPM) and other phospholipids (PL) of HDL. These defects lead to impaired reverse cholesterol transport (RCT) that are reversed by low dietary omega 3-faty acids (omega 3FA). This laboratory has the following published (10 publication & 1 review) and preliminary data in support of the above hypotheses: 1. Ethanol impaired sialylation of both transferring (Tf) and ApoE by down-regulating 2,6-ST and its mRNA. 2. Both 2,6-ST-mRNA and GAPDH mRNA levels seemed to be intact in two autopsy liver samples from non-alcoholic controls. 3. Ethanol at 50 and 100mM decreased ST mRNA in human HepG2 cells stably expressing Cyp2E (Cyp2E cells) and high alcohol dehydrogenase (ADH cells), but not in Wild type. 4. However, acetaldehyde (Ach) at 50muM concentration decreased 2,6-ST mRNA even in Wilde type. 5. Ethanol destabilized liver 2,6-STmRNA, presumably via cis and trans- acting factors. 6. Desialylated ApoE had low affinity for HDL resulting in its impaired RCT function in both human and rat. 7. HDLs from both chronic ethanol-fed rats and human alcoholics showed defective RCT. 8. Very low dietary omega 3FA restored ethanol-mediated inhibition of RCT capacity of HDL. 9. Both 20:5 and 22:6 omega 3FA contents of HDL increased 4-4.7-fold in omega 3FA-fed group compared to the control group. 10. Sphingomyelin (SPM) content of HDL was decreased in ethanol group with concomitant inhibition of RCT. 11. HDLs from chronic human alcoholics also had lower HDL SPM compared to non-drinkers. 12. HDL devoid of SPM showed impaired RCT function. Therefore, the following specific aims are proposed to test hypotheses to understand the mechanisms of actions of alcohol and omega 3FA: Glycosylation:
Specific Aim 1 : Are protein factors involved in the ethanol-mediated destabilization of liver 2,6-ST mRNA? How does ethanol affect the distribution of 2,6-ST protein & its mRNA? Do livers from human alcoholics also exhibit decreased 2,6-ST protein & its mRNA? Specific Aim 2: Can the effects of ethanol in vivo be mimicked in human liver cell systems? Is active metabolism of ethanol a prerequisite for its effects in these cell systems? What are the cis and trans factors responsible for destabilizing 2,6-ST mRNA? Is Ach responsible for these effects at clinically relevant levels? Functional Consequences:
Specific Aim 3 : How do omega 3 FA alter ethanol-mediated defects in the RCT function of HDL? Specific Aim 4: Does ethanol affect the HDL SPM and other PL? Do human alcoholics have altered SPM and other PL levels in their HDL? Does ethanol-induced loss of SPM and other PL? Do human alcoholics have altered SPM and other PL levels in their HDL? Does ethanol-induced loss of SPM and other PL from HDL affect its RCT function? The laboratory will accomplish these in human and animal systems using molecular biology, immunochemical and biochemical approaches.
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