RIP140-targeted repression of gene expression in adipocytes.

Ligand-dependent repression of nuclear receptor activity forms a novel mechanism for regulating gene expression. To investigate the intrinsic role of the corepressor RIP140, we have monitored gene expression profiles in cells that express or lack the RIP140 gene and that can be induced to undergo adipogenesis in vitro. In contrast to normal white adipose tissue and in vitro-differentiated wild-type adipocytes, RIP140-null cells show elevated energy expenditure and express high levels of the uncoupling protein 1 gene (Ucp1), carnitine palmitoyltransferase 1b, and the cell-death-inducing DFF45-like effector A. Conversely, all these changes are abrogated by the reexpression of RIP140. Analysis of the Ucp1 promoter showed RIP140 recruitment to a key enhancer element, demonstrating a direct role in repressing gene expression. Therefore, reduction in the levels of RIP140 or prevention of its recruitment to nuclear receptors may provide novel mechanisms for the control of energy expenditure in adipose cells.

Post-transcriptional regulation of rat carnitine octanoyltransferase.

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.

Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter.

The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70,301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5' untranslated region (UTR), 791 bp of 3' UTR, two putative polyadenylation sites, and a poly(A19-23) tail. Screening of a rat genomic DNA library in the lambda phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position -1140 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing -1140 to +84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at -284 and -93), two GC boxes for Sp1 binding (at -160 and -68), two AP2 sites (at -359 and -25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at -737 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified.

Regulation of carnitine acyltransferase synthesis in lean and obese Zucker rats by dehydroepiandrosterone and clofibrate.

The effects of dehydroepiandrosterone (DHEA) and clofibrate on mitochondrial and peroxisomal proliferation and carnitine acyltransferases [mitochondrial carnitine palmitoyltransferase (CPT) and peroxisomal carnitine octanoyltransferase (COT)] were measured in lean and obese female Zucker rats. DHEA increased total hepatic mitochondrial protein twofold; clofibrate increased total hepatic peroxisomal protein more than fivefold. Both DHEA and clofibrate administration increased enzyme activities, immunoreactive protein, messenger RNA levels and transcription rates for the carnitine acyltransferases. Transcription rates and messenger RNA concentration for both carnitine acyltransferases correlated with the increases in activity. These data suggest that the hepatic CPT and COT in female Zucker rats are regulated primarily at the transcriptional level by DHEA and clofibrate.

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis.

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The present studies examined the effect of agents that induce peroxisomal and mitochondrial beta-oxidation on hepatic mitochondrial carnitine palmitoyltransferase (CPT) and peroxisomal carnitine acyltransferase [CPTs of Ramsay (1988) Biochem. J. 249, 239-245; COT of Farrell & Bieber (1983) Arch. Biochem. Biophys. 222, 123-132 and Miyazawa, Ozasa, Osumi & Hashimoto (1983) J. Biochem. 94, 529-542]. In the first studies, high fat diets containing corn oil or fish oil were used to induce peroxisomal and mitochondrial enzymes. Rats were fed one of three diets for 4 weeks: (1) low fat, with corn oil as 11% of energy (kJ); (2) high fat, with corn oil as 45% of kJ; (3) high fat, with fish oil as 45% of kJ. At the end of 4 weeks, both mitochondrial CPT and peroxisomal CPTs exhibited increases in activity, immunoreactive protein, mRNA levels and transcription rates in livers of rats fed either high-fat diet compared to the low fat diet. Riboflavin deficiency or starvation for 48 h also increased the peroxisomal CPTs mRNA. A second set of studies used the plasticizer 2-(diethylhexyl)phthalate (DEHP), 0.5% clofibrate or 1% acetylsalicylic acid (fed for 3 weeks) to alter peroxisomal and mitochondrial fatty acid oxidation. With DEHP, the mitochondrial CPT and peroxisomal CPTs activity, immunoreactive protein, mRNA levels and and transcription rate were all increased by 3-5-fold. The peroxisomal CPTs activity, immunoreactive protein, mRNA levels and transcription rate were increased 2-3-fold by clofibrate and acetylsalicylic acid, again similar to mitochondrial CPT. The results of the combined studies using both diet and drugs to cause enzyme induction suggest that the synthesis of the carnitine acyltransferases (mitochondrial CPT and peroxisomal CPTs) may be co-ordinated with each other; however, the co-ordinate regulatory factors have not yet been identified.

Cloning, sequencing, and regulation of rat liver carnitine octanoyltransferase: transcriptional stimulation of the enzyme during peroxisome proliferation.

Several complementary DNAs for the peroxisomal enzyme carnitine octanoyltransferase (COT), cloned in the expression vector lambda gt11, have been isolated. Together, these clones cover 2143 bp of the COT cDNA sequence with an open reading frame for 523 amino acids. Northern analysis showed the mRNA size for this enzyme to be 3.5 kilobases. The 523 residue long amino acid sequence amounts to a molecular mass of 60,269 daltons, indicating that the cloned cDNAs contain most or all of the coding sequence for COT (Mr approximately 62,000). Hybridization studies showed that the increased COT activity in the liver of rats, fed the potent peroxisome-proliferating drug Wy-14,643, is associated with a more than 40-fold rise in the steady-state level of the COT mRNA.

Biosynthesis of carnitine octanoyltransferase and carnitine palmitoyltransferase.

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine octanoyltransferase (COT) in the liver was increased 23.5-fold in rats given DEHP. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rate of synthesis of COT was 14.1-fold higher and that of its degradation was 1.5-fold lower in the DEHP group. COT was translated much more effectively in free polysomes than in membrane-bound polysomes. The molecular size of the in vitro product was the same as that of the mature enzyme. The translation activity of mRNA coding for COT measured with total hepatic RNA was 16.6-fold higher in the DEHP group. Carnitine palmitoyltransferase (CPT) was increased 5.9-fold after administration of DEHP. The rate of synthesis of CPT measured in the in vivo experiment was 5.0-fold higher in the DEHP group. The rate of its degradation was the same in the two groups. CPT was also translated much more effectively in free polysomes. The size of the preenzyme was larger than that of the subunit of the mature enzyme by about 2,400 daltons. In contrast to COT, the increase in the translation activity of mRNA for CPT by administration of DEHP was markedly higher than the increase in the rate of its synthesis measured in the in vivo experiment.