The Lecom Developmental Gene Expression Laboratory investigates the control of gene expression in mammalian cells and the consequences of altered gene expression. The effects of ethanol on gene expression in liver cells are of primary interest at the moment. When exposed to ethanol, the liver increases expression of mitochondrial aspartate aminotransferase, and the increase in the amount of this enzymatic activity in serum is a clinical marker for alcoholic liver disease. Interestingly, this protein also serves a second function in the body, facilitating fatty acid uptake into cells. Another hallmark of alcoholic liver disease is that fat accumulates in liver cells, causing hepatic steatosis. As one gene may be involved in both functions, we are concentrating our efforts on this particular gene. The questions of how ethanol increases expression of the gene, and how it functions in fatty acid uptake, are being investigated.

As there are many other genes which may alter fat metabolism in the liver, we are also investigating changes in expression of a number of candidate genes. The transcription factor SREBP1 is significantly increased when hepatoma cells are exposed to ethanol, so the genes it regulates are also targets of interest. We will be extending the studies to other genes and using real-time PCR to assess expression levels in various conditions, with short-term and long-term exposure to ethanol.

We are also investigating the role of ethanol in increasing fatty acid uptake. The hepatoma cells, when adapted to cultures in 40 mM ethanol, show a significant increase in the rate of fatty acid uptake. This increase is maintained during exposure to ethanol, and for some time after ethanol is withdrawn. We have recently demonstrated that the uptake rate can decline to the level shown by untreated cells in time.

Further studies are being pursued to examine the role of aspartate aminotransferase in fatty acid uptake. The putative fatty acid binding site has been identified. Mutagenesis of key residues has suggested that fatty acid binding does occur in this region. Transfection of expression constructs into fibroblasts show that wild-type protein expression increases fatty acid uptake, while mutated constructs do not. The contribution of various amino acids to fatty acid binding and uptake will be further assessed. New methods of measuring fatty acid uptake will be employed, such as uptake of fluorescent analogs using a fluorescent-luminescent plate reader. Further mutants will be prepared and studied, to further refine the parameters of fatty acid binding.

Other areas that will be investigated involve the early development of mammals and development of the germ cells. Observations made in previous studies may allow for new and simple methods of tracing early germ cell development and delineation of specific structures during spermatogenesis. Advanced embryo culture techniques are also under investigation.