1. ETHANOL RESPONSIVE ELEMENTS IN THE MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE PROMOTER
When exposed to ethanol, hepatocytes increase their expression of mitochondrial aspartate aminotransferase. This leads to an increase in the activity of this enzyme in serum and is the biological basis for the characteristic rise in AST seen in alcoholic liver disease. However, this enzyme has been found to be identical to plasma membrane fatty acid binding protein, the first protein identified as promoting facilitated uptake of long-chain free fatty acids into cells. The increase in expression may also lead to hepatic steatosis seen in alcoholic liver disease. This dual role makes this gene of extreme interest in the study of alcoholic liver disease. Analysis of the promoter is underway, to locate and define transcription factor binding sites that may be involved in the response to ethanol. Reporter constructs with various portions of the promoter regulating expression of secreted alkaline phosphatase (SEAP) are employed, and transfected into human hepatoma cells. After selection, cells are exposed to medium with 0 or 40 mM ethanol for 24 hours and medium is assayed for SEAP activity using a fluorescent assay. Regions responsive to ethanol will be analyzed for transcription factor binding sites using bioinformatics, DNA Footprinting, EMSA, and various newer assay formats becoming available to determine what factors alter promoter function. Comparisons may be made with promoters of other genes known to be responsive to ethanol.

2. DELINEATION OF THE FATTY ACID BINDING SITE OF MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE
Molecular modeling of mitochondrial aspartate aminotransferase has identified a specific region in which a large number of hydrophobic residues face a cleft in the surface. The volume of the cleft is suitable for binding a long-chain fatty acid. Also, an arginine residue (R201) is present at one end, similar to the placement of arginine or lysine residues in the fatty acid binding sites of albumin. A rat cDNA clone has been mutated to alter specific residues to their cognate forms found in the cytoplasmic isozyme, which has no significant fatty acid binding capacity. Although the cytoplasmic and mitochondrial forms catalyze the same reaction, they are only ~50% identical at the amino acid sequence level. There are numerous residues that are conserved in either form, where all mammals have a specific amino acid at one position in the mitochondrial form, while all cytoplasmic forms share a different residue. In the cleft region defined by just over 100 residues, 23 of these conserved substitutions are present. Preliminary findings indicate that the substitutions R201T and A219P decrease fatty acid binding and/or uptake substantially. A construct with a complete replacement of the binding site region with cytoplasmic sequence is also under investigation, as well as double mutants and a mutation, R201K, which retains a basic residue at the key site. These structure-function studies will help us determine how this single protein participates in two disparate cellular processes.

3. EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON HEPATOCYTE FATTY ACID UPTAKE
This project is based upon previous observations that hepatocytes (liver cells) and hepatoma cells (liver tumor cells) in culture increase their rate of fatty acid uptake in the presence of ethanol. This phenomenon may be part of the mechanism of hepatic steatosis, or fat accumulation in the liver, seen in alcoholic liver disease. Using the HuH7 human hepatoma line, we find that fatty acid uptake increases significantly after adaptation to growth in the presence of 40 mM ethanol. This change is maintained during culture in ethanol, and we have investigated whether the change is reversible. When ethanol is removed from the culture medium, the rate of uptake is maintained for a time, but does decline to a rate similar to untreated cells with time. The rate of change in both the increase and decrease in uptake kinetics is now being defined.

4. GENE EXPRESSION CHANGES IN HEPATOCYTES DUE TO ETHANOL

One of the most common consequences of alcoholic liver disease is steatosis, or an increase in the amount of fat in liver cells. This may be due to changes in fatty acid uptake or other aspects of fat metabolism, whether synthesis of fat from excess carbohydrates, decreased metabolism of fat for energy, or changes in the export of fat as VLDL for use in other tissues. In order to examine the role of these various pathways that influence the flux of fat through the liver, we are examining the expression of genes involved in each of these pathways. After either short or long-term exposure to ethanol, the amount of messenger RNA (mRNA) that codes for various proteins is being measured by reverse transcriptase PCR, to identify those genes where there is a significant change in level of expression. This analysis will be followed by real-time PCR of selected genes that appear to be altered by ethanol. As the transcription factor SREBP1 has shown a significant change, gene which it regulates are likely targets of future investigation.