Muscle-type Carnitine Palmitoyltransferase Research Articles

Fasting-induced changes in the expression of genes controlling substrate metabolism in the rat heart

During fasting, when overall metabolism changes, the contribution of glucose and fatty acids (FA) to cardiac energy production alters as well. Here, we examined if the heart is able to adapt to such fasting-induced changes by modulation of its gene expression. Rats were fed ad libitum or fasted for 46 h, resulting in reduced circulating glucose levels and a 3-fold rise in FA. Besides changes in the cardiac activity or content of proteins involved in glucose or FA metabolism, mRNA levels also altered. The cardiac expression of genes coding for glucose-handling proteins (glucose transporter GLUT4, hexokinase I and II) was up to 70% lower in fasted than in fed rats. In contrast, the mRNA levels of various genes involved in FA transport and metabolism (FA translocase/CD36, muscle-type carnitine palmitoyl transferase 1, long-chain acyl-CoA dehydrogenase) and of the uncoupling protein UCP-3 increased over 50% in hearts of fasted rats. Surprisingly, mRNA levels of the fatty acid-activated transcription factors PPARα and PPARβ/δ were reduced in hearts of fasted rats, whereas in livers, fasting led to a marked rise in PPARα mRNA. Reducing FA levels by nicotinic acid administration during the final 8 h of fasting did not affect the expression of the majority of metabolic genes, but totally abolished the induction of UCP-3. In conclusion, the adult rat heart responds to changes in nutritional status, as provoked by 46 h fasting, through adjustment of glucose as well as FA metabolism at the level of gene expression.—Van der Lee, K. A. J. M., P. H. M. Willemsen, S. Samec, J. Seydoux, A. G. Dulloo, M. M. A. L. Pelsers, J. F. C. Glatz, G. J. Van der Vusse, and M. Van Bilsen. Fasting-induced changes in the expression of genes controlling substrate metabolism in the rat heart.

Bezafibrate Reduces mRNA Levels of Adipocyte Markers and Increases Fatty Acid Oxidation in Primary Culture of Adipocytes

The molecular mechanisms by which peroxisome proliferator-activated receptor (PPAR) activation by fibrates reduces fat deposition and improves insulin sensitivity are not completely understood. We report that exposure of a rat primary culture of adipocytes for 24 h to the PPAR activator bezafibrate increased the mRNA levels of crucial genes involved in peroxisomal and mitochondrial beta-oxidation. The mRNA levels of the peroxisomal beta-oxidation rate-limiting enzyme acyl-CoA oxidase and of the muscle-type carnitine palmitoyl transferase I (M-CPT-I), which determines the flux of mitochondrial beta-oxidation, increased by 1.6-fold (P < 0.02) and 4.5-fold (P = 0.001), respectively. These changes were accompanied by an increase in the transcript levels of the uncoupling protein-2 (UCP-2; 1.5-fold induction; P < 0.05) and UCP-3 (3.7-fold induction; P < 0.001), mitochondrial proteins that reduce ATP yield and may facilitate the oxidation of fatty acids. Furthermore, bezafibrate increased the mRNA levels of the fatty acid translocase (2-fold induction; P < 0.01), suggesting a higher fatty acid uptake into adipocytes. In agreement with these changes, bezafibrate caused a 1.9-fold induction (P < 0.02) in 9,10-[(3)H]palmitate oxidation. Moreover, bezafibrate reduced the mRNA expression of several adipocyte markers, including PPARgamma (30% reduction; P = 0.05), tumor necrosis factor-alpha (33% reduction; P < 0.05), and the ob gene (26% reduction). In contrast, adipocyte fatty acid binding protein mRNA levels increased (1.5-fold induction; P < 0.01), pointing to a mobilization of fatty acids to mitochondria and peroxisomes. The reduction of the adipocyte markers caused by bezafibrate was accompanied by an increase in the mRNA levels of the preadipocyte marker Pref-1 (1.6-fold induction; P < 0.01). Some of the changes observed in the primary culture of rat adipocytes also were studied in the epididymal white adipose tissue of bezafibrate-treated rats for 7 days. In vivo, M-CPT-I mRNA levels increased (4.5-fold induction; P = 0.001) in epididymal white adipose tissue of bezafibrate-treated rats. Similarly, fatty acid translocase (2.6-fold induction; P = 0.002) and Pref-1 (5.6-fold induction) mRNA levels increased, although differences in the latter were not significant because of huge individual variations. These results indicate that exposure of adipocytes to bezafibrate, independent of its hepatic effects, increases the degradation of fatty acids, reducing their availability to synthesize triglycerides. As a result, some degree of dedifferentiation of adipocytes to preadipocyte-like cells is achieved. These changes may be involved in the reduction in fat depots and in the improvement of insulin sensitivity observed after bezafibrate treatment.

Identification of Positive and Negative Determinants of Malonyl-CoA Sensitivity and Carnitine Affinity within the Amino Termini of Rat Liver- and Muscle-type Carnitine Palmitoyltransferase I

The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonyl-CoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 --> Ala) or Delta(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant Delta(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19-82 of the enzyme. This was confirmed in the construct Delta(19-30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-Delta(19-30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-Delta(19-30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K(0.5) for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from Delta(3-18) to Delta(1-80). However, deletion of residues 3-18 from M-CPT I affected the K(m) for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme.

Novel Expression of Equivocal Messages Containing Both Regions of Choline/Ethanolamine Kinase and Muscle Type Carnitine Palmitoyltransferase I

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

Coordinated control of the human muscle-type carnitine palmitoyltransferase I (hM-CPT I) by myogenic factors and PPARs in skeletal muscle. Implications for the management of NIDDM

Coordinated control of the human muscle-type carnitine palmitoyltransferase I (hM-CPT I) by myogenic factors and PPARs in skeletal muscle. Implications for the management of NIDDM

Genomics of the Human Carnitine Acyltransferase Genes

Five genes in the human genome are known to encode different active forms of related carnitine acyltransferases: CPT1A for liver-type carnitine palmitoyltransferase I, CPT1B for muscle-type carnitine palmitoyltransferase I, CPT2 for carnitine palmitoyltransferase II, CROT for carnitine octanoyltransferase, and CRAT for carnitine acetyltransferase. Only from two of these genes (CPT1B and CPT2) have full genomic structures been described. Data from the human genome sequencing efforts now reveal drafts of the genomic structure of CPT1A and CRAT, the latter not being known from any other mammal. Furthermore, cDNA sequences of human CROT were obtained recently, and database analysis revealed a completed bacterial artificial chromosome sequence that contains the entire CROT gene and several exons of the flanking genes P53TG and PGY3. The genomic location of CROT is at chromosome 7q21.1. There is a putative CPT1-like pseudogene in the carnitine/choline acyltransferase family at chromosome 19. Here we give a brief overview of the functional relations between the different carnitine acyltransferases and some of the common features of their genes. We will highlight the phylogenetics of the human carnitine acyltransferase genes in relation to the fungal genes YAT1 and CAT2, which encode cytosolic and mitochondrial/peroxisomal carnitine acetyltransferases, respectively.

Characterization of a response element for peroxisomal proliferator activated receptor (PPRE) in human muscle-type carnitine palmitoyltransferase I.

Characterization of a response element for peroxisomal proliferator activated receptor (PPRE) in human muscle-type carnitine palmitoyltransferase I.

Expression and possible role of muscle-type carnitine palmitoyltransferase I during sperm development in the rat.

Because we had found whole testis from adult rats to be much richer in the messenger RNA for the muscle (M) than for the liver (L) form of mitochondrial carnitine palmitoyltransferase I (CPT I), we sought to determine which cell type(s) accounts for this expression pattern and how it might relate to reproductive function. Studies with immature (14-day-old) and adult animals included 1) Northern blot analysis of testis mRNA; 2) in situ hybridization with slices of testis; 3) enzyme assays for CPT I, CPT II, and carnitine acetyltransferase (CAT) in testicular germ cells and nongerm cells, together with measurement of the malonyl-coenzyme A (CoA) sensitivity and affinity for carnitine of CPT I; 4) labeling of testicular CPT I with [3H]etomoxir, a covalent inhibitor of the enzyme; and 5) the response of testicular and nontesticular CPT I to dietary etomoxir. The data established the following: 1) L-CPT I was the sole isoform detected in immature testis. 2) Expression of the M-CPT I gene was associated only with meiotic and postmeiotic germ cells. 3) Adult testis contains a mixture of the L- and M-CPT I enzymes, the L and M form dominating in extratubular cells and spermatids, respectively. Mature epididymal spermatozoa appear to be devoid of CPT I activity while possessing abundant levels of CPT II and CAT. 4) Five days of dietary etomoxir treatment at a dose that resulted in essentially complete inhibition of CPT I in liver, heart, skeletal muscle, and kidney was totally without effect on either the L- or M-type enzyme in the testis of mature rats. The data point to an important role for transient expression of M-CPT I, coupled with sustained activity of CAT, in the maturation and/or function of rat sperm. They also suggest that, at least in the case of one CPT I inhibitor (etomoxir), the testis is unusually resistant to the agent when given orally.

Fatty Acids Activate Transcription of the Muscle Carnitine Palmitoyltransferase I Gene in Cardiac Myocytes via the Peroxisome Proliferator-activated Receptor α

To explore the gene regulatory mechanisms involved in the metabolic control of cardiac fatty acid oxidative flux, the expression of muscle-type carnitine palmitoyltransferase I (M-CPT I) was characterized in primary cardiac myocytes in culture following exposure to the long-chain mono-unsaturated fatty acid, oleate. Oleate induced steady-state levels of M-CPT I mRNA 4.5-fold. The transcription of a plasmid construct containing the human M-CPT I gene promoter region fused to a luciferase gene reporter transfected into cardiac myocytes, was induced over 20-fold by long-chain fatty acid in a concentration-dependent and fatty acyl-chain length-specific manner. The M-CPT I gene promoter fatty acid response element (FARE-1) was localized to a hexameric repeat sequence located between 775 and 763 base pairs upstream of the initiator codon. Cotransfection experiments with expression vectors for the peroxisome proliferator-activated receptor alpha (PPARalpha) demonstrated that FARE-1 is a PPARalpha response element capable of conferring oleate-mediated transcriptional activation to homologous or heterologous promoters. Electrophoretic mobility shift assays demonstrated that PPARalpha bound FARE-1 with the retinoid X receptor alpha. The expression of M-CPT I in hearts of mice null for PPARalpha was approximately 50% lower than levels in wild-type controls. Moreover, a PPARalpha activator did not induce cardiac expression of the M-CPT I gene in the PPARalpha null mice. These results demonstrate that long-chain fatty acids regulate the transcription of a gene encoding a pivotal enzyme in the mitochondrial fatty acid uptake pathway in cardiac myocytes and define a role for PPARalpha in the control of myocardial lipid metabolism.

Control of Human Muscle-type Carnitine Palmitoyltransferase I Gene Transcription by Peroxisome Proliferator-activated Receptor

The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial beta-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5'-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy.

Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI-M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5′-region of the CPTI-M gene, we isolated cDNA clones corresponding to the 5′-region of its transcript by 5′-rapid amplification of cDNA ends (5′-RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5′-region of the human CPTI-M gene. The gene encoding human CPTI-M was found to consist of two 5′-non-coding exons, 18 coding exons and one 3′-non-coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5′-flanking region, a putative gene encoding a `choline kinase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. Comparison of the gene structure of human CPTI-M with the reported partial gene structure of human liver type CPTI (CPTI-L) showed that the intron insertion sites were completely conserved in these two genes.

Localization and intron usage analysis of the human CPT1B gene for muscle type carnitine palmitoyltransferase I

We isolated and sequenced cDNA and genomic DNA fragments of the human CPT1B gene, encoding muscle type carnitine palmitoyltransferase I. A recombinant P1 phage containing CPT1B was mapped to chromosome 22qter by fluorescent in situ hybridization. This finding supports the concept that `liver type' and `muscle type' isoforms of CPT I are encoded by different loci at separate chromosomal positions. Analysis of CPT1B cDNA sequences revealed the presence of an untranslated 5′ exon and differential processing of introns 13 and 19. The alternative splicing of intron 13 causes an in-frame deletion leading to a 10 amino acid residues smaller protein. Using different splice acceptor sites, intron 19 is spliced in the majority of cases, but 4 out of 14 sequenced CPT1B 3′ cDNA clones contain part of intron 19 in stead of exon 20. We found that differential polyadenylation is the mechanism behind the existence of these alternative 3′ CPT1B mRNA forms.

Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.