Acyl-CoA thioesterases belong to a novel gene family of peroxisome proliferator-regulated enzymes involved in lipid metabolism.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to the corresponding free fatty acid plus coenzyme A. The activity is strongly induced in rat and mouse liver after feeding the animals peroxisome proliferators (PPs). To elucidate the role of these enzymes in lipid metabolism, the authors have cloned the cDNAs corresponding to the inducible cytosolic and mitochondrial type I enzymes (CTE-I and MTE-I), and studied tissue expression and nutritional regulation of expression of the mRNAs in mice. The constitutive expression of both mRNAs was low in liver, with CTE-I expressed mainly in kidney and brown adipose tissue, and MTE-I expressed in brown adipose tissue and heart. As expected, the expression in liver of both the CTE-I and MTE-I mRNAs were strongly induced (> 50-fold) by treatment with clofibrate. A similar level of induction was observed by fasting and a time-course study showed that the CTE-I and MTE-I mRNAs were increased already at 6 h after removal of the diet. Refeeding normal chow diet to mice fasted for 24 h normalized the mRNA levels with a T1/2 of about 3-4 h. Feeding mice a fat-free diet further decreased the expression, possibly indicating repression of expression. The strong expression of MTE-I and CTE-I in the heart was increased about 10-fold by fasting. To further characterize these highly regulated enzymes, the authors have cloned the corresponding genes and promoter regions. The structures of the two genes were found to be very similar, consisting of three exons and two introns. Exon-intron borders conform to general consensus sequences, and, especially, the first exon appears to be highly conserved. The promoter regions of both the CTE-I and MTE-I genes contain putative PP response elements, suggesting an involvement of PP-activated receptors in the regulation of these genes.

Cloning and regulation of peroxisome proliferator-induced acyl-CoA thioesterases from mouse liver.

1.1. Acyl-CoA thioesterases hydrolyze acyl-CoAs to the corresponding free fatty acid plus CoASH. The activity is strongly induced in rat and mouse liver after feeding the animals peroxisome proliferators. To elucidate the role of these enzymes in lipid metabolism, we have cloned the cDNAs corresponding to the inducible cytosolic and mitochondrial type I enzymes (CTE-I and MTE-I) and studied tissue expression and nutritional regulation of expression of the mRNAs in mice. The constitutive expression of both mRNAs was low in liver, with CTE-I being expressed mainly in kidney and brown adipose tissue and MTE-I expressed in brown adipose tissue and heart. As expected, the expression in liver of both the CTE-I and MTE-I mRNAs was strongly induced (> 50-fold) by treatment with clofibrate. A similar level of induction was observed by fasting and a time-course study showed that both mRNAs were increased already at 6 hours after removal of the diet. Refeeding normal chow diet to mice fasted for 24 hours normalized the mRNA levels with a T1/2 of about 3-4 hours. Feeding mice a fat-free diet further decreased the expression, possibly indicating repression of expression. The strong expression of MTE-I and CTE-I in the heart was increased about 10-fold by fasting. To further characterize these highly regulated enzymes, we have cloned the corresponding genes and promoter regions. The structures of the two genes were found to be very similar, consisting of three exons and two introns. Exon-intron borders conform to general consensus sequences and especially the first exon appears to be highly conserved. The promoter regions of both the CTE-I and MTE-I genes contain putative peroxisome proliferator response elements (PPREs), suggesting an involvement of peroxisome proliferator-activated receptors in the regulation of these genes.

Molecular cloning of the peroxisome proliferator-induced 46-kDa cytosolic acyl-CoA thioesterase from mouse and rat liver--recombinant expression in Escherichia coli, tissue expression, and nutritional regulation.

Feeding clofibrate to rats and mice results in a strong induction of acyl-CoA thioesterase activity in the liver that is mainly due to increases in the enzyme activities in mitochondria and cytosol. The cytosolic acyl-CoA thioesterase protein of about 40 kDa, referred to as CTE-I, is strongly induced by the treatment. We report here the molecular cloning of the cDNA corresponding to the rat and mouse enzymes, and the further characterization of the mouse CTE-I by recombinant expression in bacteria and regulation of expression of the enzyme. The cDNAs corresponding to the rat and mouse enzymes contained open reading frames encoding proteins of 419 amino acids with calculated molecular masses of 45938 Da and 46135 Da, respectively. Sequence analysis revealed an active site serine consensus sequence commonly found in lipases and carboxylesterases. Recombinant expression of the mouse CTE-I cDNA in Escherichia coli resulted in production of immunoreactive protein that was mainly active with long-chain acyl-CoAs. Northern blot analysis showed that the full-length CTE-I cDNA probe hybridized to two major transcripts corresponding to CTE-I and MTE-I (mitochondrial acyl-CoA thioesterase I), respectively. The expression of both mRNA species was found to be highly regulated. As expected, both CTE-I and MTE-I were strongly upregulated (> 50-fold) by clofibrate treatment. Interestingly, fasting for 48 h resulted in a similar magnitude of induction as two days of clofibrate feeding. In addition, feeding a fat-free diet resulted in down-regulation of CTE-I mRNA. CTE-I mRNA was strongly expressed in kidney and brown adipose tissue and MTE-I mRNA was expressed mainly in brown adipose tissue and heart but was also expressed in kidney and white adipose tissue. Dietary regulation and tissue-specific expression suggest that CTE-I and MTE-I play important roles in lipid metabolism.

Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases.

Long-chain acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoAs to the corresponding free fatty acid and CoA. We recently cloned four members of a novel multi-gene family of peroxisome proliferator-induced genes encoding cytosolic (CTE-I), mitochondrial (MTE-I), and peroxisomal (PTE-Ia and PTE-Ib) acyl-CoA thioesterases (Hunt et al. 1999. J. Biol. Chem. 274: 34317-34326). As the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in regulating genes involved in lipid metabolism, we examined the involvement of this receptor in regulation of the thioesterases, particularly CTE-I and MTE-I. Northern blot analysis shows that the induction of these thioesterases by clofibrate is mediated through a strictly PPARalpha-dependent mechanism. All four acyl-CoA thioesterases are induced at mRNA level by fasting and using PPARalpha-null mice, it is evident that the increase in CTE-I due to fasting is mainly independent of the PPARalpha in liver and heart. The CTE-I gene responds rapidly to fasting, with induction of mRNA and protein evident after 6 h. This fasting effect is rapidly reversible, with CTE-I mRNA returning almost to control levels after 3 h refeeding, and being further repressed to 20% of control after 9 h refeeding. Although CTE-I mRNA shows a low basal expression in liver, it can be suppressed 90% by feeding a fat-free diet. These data demonstrate that the nutritional regulation of the thioesterases involves the PPARalpha and other signaling pathways responsible for activation and repression. Putative physiological functions for the acyl-CoA thioesterases are discussed.