Results

4.1. Tissue Expression and Regulation of mRNA Levels

The tissue specific expression of CTE-I and MTE-I mRNA was carried out by Northern blot analysis. CTE-I was shown to be mainly expressed in kidney, brown adipose tissue and heart, but was also detectable in control liver, white adipose tissue, brain and testis. MTE-I was strongly expressed in heart and brown adipose tissue, with strong signals also detected in kidney and white adipose tissue. Weak expression of MTE-I was evident in liver and muscle but no detectable expression was seen in brain or testis.

4.2. Regulation of Acyl-CoA Thioesterases by Dietary Manipulation-

Previous studies by our group indicated a very strong induction of both CTE-I and MTE-I expression in mouse liver after fasting for 48 hours as compared to expression in untreated animals, where levels are barely detectable.7 We found that this induction was already maximal after 24 hours, and carried out a time course of fasting mice for 0, 6, 12, 18, 24, 36 and 48 hours to pinpoint more closely when induction took place. After 6 hours of fasting the expression of the cytosolic acyl-CoA thioesterase was already slightly increased, with maximum induction after only 12 hours fasting. mRNA levels of the MTE-I transcript were induced in line with its cytosolic counterpart. Due to the rapid induction of both CTE-I and MTE-I by fasting, it was of interest to examine how a refeeding effect would alter the induced expression of these enzymes. An experiment was carried out where mice were fasted for 24 hours and refed a normal chow diet for 0, 3, 6, 9, 12, 24 and 30 hours. The decrease in transcription of mCTE-I was again very rapid, being strongly reduced after only 6 hours of refeeding the normal chow diet with mRNA returning to control levels within 9 hours of this treatment. The apparent half-lives of the mRNAs were estimated to be about 3-4 hours.

In view of this different tissue specificity and the rapid regulation of expression of these enzymes in liver by fasting, we investigated the effects of fasting on CTE-I and MTE-I mRNA in tissues where the enzymes are constitutively expressed, such as heart, kidney and brown adipose tissue. Interestingly, fasting for 24 hours caused a very large upregulation of both CTE-I and MTE-I in mouse heart, which was not further increased at 48 hours. In kidney, mCTE-I transcription was much more strongly upregulated by fasting than the mitochondrial transcript. The mRNA levels of both these enzymes in brown adipose tissue were unaffected by fasting conditions.

Previous experiments at this laboratory had indicated that both mCTE-I and mMTE-I were downregulated at mRNA level by feeding a fat-free diet. We carried out a long-term time course of feeding mice a fat-free diet for 1,2,4 and 7 days. After two days of this treatment, both enzymes were suppressed at mRNA level.

4.3. Regulation of Acyl-CoA Thioesterases by the Peroxisome Proliferator Clofibrate

Previous results have indicated that both CTE-I and MTE-I are inducible at mRNA level in both rat and mouse liver by peroxisome proliferators such as clofibrate and di-(2-ethylhexyl)phthalate (DEHP). We examined the expression of CTE-I and MTE-I mRNA after various treatments with clofibrate-containing diets. In a time course carried out by feeding mice a 0.5% clofibrate-containing diet for 1, 2, 4 and 7 days, both CTE-I and MTE-I were already maximally induced after the first day of treatment and levels remained high for the duration of the experiment.

It was of interest to verify if the induction at the mRNA level was also reflected by an increase at the protein level. Western blot analysis of mouse total liver protein using an anti-MTE-I antibody, which labelled a strong band at the expected size of 45 kDa, indicated that there was a good correlation in the changes at the protein and mRNA levels. Fasting for 24 hours, treatment with a clofibrate-containing diet for 1 day or fasting and refeeding a clofibrate-containing diet all caused an increase of MTE-I and CTE-I proteins. Fasting and refeeding a chow diet for 24 hours normalised levels of the thioesterase protein. However, treatment with a fat-free diet for 4 days caused a suppression of both CTE-I and MTE-I at mRNA level, but this suppression was not evident at protein level.

4.4. Gene Structures of MTE-I and CTE-I

The screening of a mouse genomic library, using a cDNA probe corresponding to about 2/3 of the cDNA sequence (lacking the 5 -end) resulted in positive clones. After 3 more rounds of screening about 90% remained positive, suggesting that the probe recognized several genes of this apparent multi-gene family. Screening for MTE-I was carried out by generation of a specific cDNA probe containing the 5'-end of the sequence corresponding to the mitochondrial leader peptide, which was used to screen a "mini-library" consisting of a pool of the positive clones obtained in the first screening. This resulted in the isolation of two clones, one of which was completely sequenced. About 2.8kb of the 5-flanking region was also sequenced in both directions. This clone was found to contain the entire gene for MTE-I, and found to consist of 3 exons and 2 introns. The first exon contains 680 bp, the second exon contains 202 bp and the third exon about 695bp.

One of the clones obtained from the first screening, corresponding to CTE-I, was partially sequenced and the sequence information was used to design oligo oligonucleotides for Pl-clone screening. A P1 clone containing the entire CTE-I gene was obtained and completely sequenced and based on the sequence the gene structure was determined. Similarly to the MTE-I gene, it contains three exons and two introns, with the consensus sequences for the intron borders commencing with the sequence GTG and ending in the sequence CAG. The first exon, of 465 base pairs, is highly conserved between CTE-I and MTE-I (showing greater than 90% homology, differing mainly in that the mitochondrial variant contains a mitochondrial leader peptide of 42 amino acids coded for in the first exon). Similar to the MTE-I gene, the second exon contains 202 base pairs. The putative active site serine is encoded in the third exon in both genes.

4.5. Promoter Analysis

Approximately 2.4kb of the promoter region of CTE-I and 2.8kb of the promoter region of MTE-I were sequenced in the 5' direction from the ATG start site. The promoter sequences were analysed for the presence of various transcription factor sites, using TESS String Based Search, which identified putative peroxisome proliferator response elements (PPREs) in both promoters. These regions of the promoters have now been cloned into a luciferase expression vector, to be used in transfection experiments to examine for functional PPREs.

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