Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction

Recent studies have shown that hepatic mRNAS14 responds rapidly to thyroid hormone administration. Moreover, this mRNA is known to increase in mass with the administration of a high carbohydrate fat-free diet. Therefore, it appears to share many of the same properties of the known hepatic lipogenic enzymes. Because the lipogenic enzymes display a synergistic interaction between thyroid hormones and carbohydrates, we investigated the kinetics of response of mRNAS14 to carbohydrate feeding, as well as its interaction with triiodothyronine (T3). We found that mRNAS14 responds rapidly to the dietary administration of sucrose in euthyroid rats, with a 2-fold increase within 30 min, and a 25-fold increase by 4 h. On the other hand, when given to hypothyroid rats, sucrose ultimately lead to only a 2-3-fold increase in the level of mRNAS14, attaining a level less than that found in starved euthyroid rats. The diminished response of mRNAS14 to sucrose in hypothyroidism could not be enhanced by insulin administration. However, administration of replacement doses of T3 (400 ng/100 g of body weight) immediately restored the rapid response to sucrose feeding. The response of sucrose and T3 was synergistic. Dose-response studies with T3 indicated that the rapid interaction between T3 and sucrose was limited by the occupancy of the T3 nuclear receptor. A similar synergistic response to T3 and glucose was noted in primary hepatocyte cultures, thus indicating that the synergism between these two stimuli is not due to changes in extrahepatic hormones or metabolites. Our data are most consistent with the hypothesis that the T3-nuclear receptor complex multiplies a signal generated by carbohydrate metabolism to induce hepatic mRNAS14. The interaction does not appear to require the preliminary induction of carbohydrate-metabolizing enzymes and their mRNAs.     

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.     

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.