Fatty acid synthase (FAS) is a key enzyme for denovo synthesis of long chain fatty acids in mammals. FAS activity is under transcriptional control by multiple hormones and nutrients, such as insulin, thyroid hormone, peroxisome proliferators, glucose, polyunsaturated fatty acids (PUFA) and cholesterol. Dietary PUFA suppresses FAS gene transcription in the liver of rats and mice. We tested this PUFA effect in the human hepatoma cell line HepG2 cells. Confluent HepG2 cells were serum starved for 48 h, and then treated with 100 μM of either oleic acid or arachidonic acid for another 48 h. Northern analysis of the extracted RNA showed that FAS mRNA in the arachidonic acid treated cells was reduced to about 40% of that in the oleic acid treated cells, which is comparable to the in vivo response to PUFA. Transfection analysis with FAS gene promoter from -2195 to +65 fused with chloramphenicol acetyl transferase (CAT) gene revealed that while CAT expression in the HepG2 cells responded to insulin in an in vivo fashion, it was not inhibited by arachidonic acid. Therefore, we extended the search for PUFA elements further upstream by screening a P-1 phage library of rat genome. A clone of about 90 kb was identified as containing the target region, and fragments covering up to -10 kb of the FAS gene have been subcloned into lambda phages and then into plasmids. Sequencing has been completed up to -6.5 kb, and functional analysis of the fragments is underway.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology