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Excerpt
We have previously reported that hepatic fatty acid synthase (FAS) activity and mRNA abundance are significantly down-regulated by an acute bout of prolonged exercise in rats fasted and refed a high-carbohydrate (CHO) diet. To elucidate the molecular mechanisms involved, we investigated nuclear protein binding to end-labeled oligonucleotide probes corresponding to the insulin response element (IRE) and CHO response element (ChoRE) of the FAS gene, using the gel mobility shift assay. Male Sprague-Dawley rats (N=18) were randomly divided into 3 groups: fasted for 48 h (F); fasted for 48 h and refed a high-fructose diet for 8 h and killed at rest (R); or fast-refed the same diet and run to exhausion (E). Liver nuclear extracts from F rats showed minimal DNA binding. The slow mobility protein-IRE/ChoRE complexes were intensified in R rats, whereas E severely attenuated the bindings by 32 and 42%, respectively. Competition gel experiments using unlabeled probes with increasing concentrations (5-100 fold) verified that nuclear protein bindings to IRE and ChoRE were specific. Using antibodies to the upstream stimulatory factors (USF1+USF2), we showed in gel supershift experiments that these complexes contain both USF1 and USF2. UV-cross linking indicated that these complexes contain several proteins of variable sizes, including USF1 and USF2. It is concluded that dietary regulation of the FAS gene is at the level of transcription and that prolonged exercise down-regulates this enzyme by decreasing transcription factor binding to IRE and ChoRE.
