Liver Carnitine Palmitoyltransferase Research Articles

Regulation of liver carnitine palmitoyltransferase I gene expression by hormones and fatty acids.

This brief review focuses on the transcriptional regulation of liver carnitine palmitoyltransferase I (L-CPT I) by pancreatic and thyroid hormones and by long-chain fatty acids (LCFA). Both glucagon and 3,3',5-tri-iodothyronine (T(3)) enhanced the transcription of the gene encoding L-CPT I, whereas insulin had the opposite effect. Interestingly, the transcriptional effect of T(3) required, in addition to the thyroid-responsive element, the co-operation of a sequence located in the first intron of L-CPT I gene. Non-esterified fatty acids rather than acyl-CoA ester or intra-mitochondrial metabolite were responsible for the transcriptional effect on the gene encoding L-CPT I. It was shown that LCFA and peroxisome proliferators stimulated L-CPT I gene transcription by distinct mechanisms. Peroxisome proliferator stimulated L-CPT I gene transcription through a peroxisome-proliferator-responsive element (PPRE) located at -2846 bp, whereas LCFA induced L-CPT I gene transcription through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent mechanism owing to a sequence located in the first intron of the gene.

Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent pathway.

Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor alpha (PPARalpha) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARalpha-null mice. These results suggest that the PPARalpha-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARalpha-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.

Long-chain fatty acids regulate liver carnitine palmitoyltransferase I gene (L-CPT I) expression through a peroxisome-proliferator-activated receptor α (PPARα)-independent pathway

Liver carnitine palmitoyltransferase I (L-CPT I) catalyses the transfer of long-chain fatty acid (LCFA) for translocation across the mitochondrial membrane. Expression of the L-CPT I gene is induced by LCFAs as well as by lipid-lowering compounds such as clofibrate. Previous studies have suggested that the peroxisome-proliferator-activated receptor α (PPARα) is a common mediator of the transcriptional effects of LCFA and clofibrate. We found that free LCFAs rather than acyl-CoA esters are the signal metabolites responsible for the stimulation of L-CPT I gene expression. Using primary culture of hepatocytes we found that LCFAs failed to stimulate L-CPT I gene expression both in wild-type and PPARα-null mice. These results suggest that the PPARα-knockout mouse does not represent a suitable model for the regulation of L-CPT I gene expression by LCFAs in the liver. Finally, we determined that clofibrate stimulates L-CPT I through a classical direct repeat 1 (DR1) motif in the promoter of the L-CPT I gene while LCFAs induce L-CPT I via elements in the first intron of the gene. Our results demonstrate that LCFAs can regulate gene expression through PPARα-independent pathways and suggest that the regulation of gene expression by dietary lipids is more complex than previously proposed.

Liporegulation in Diet-induced Obesity: THE ANTISTEATOTIC ROLE OF HYPERLEPTINEMIA

To test the hypothesis that the physiologic liporegulatory role of hyperleptinemia is to prevent steatosis during caloric excess, we induced obesity by feeding normal Harlan Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within 24 h and increased progressively to 26 ng/ml after 10 weeks, correlating with an approximately 150-fold increase in body fat (r = 0.91, p < 0.0001). During this time, the triacylglycerol (TG) content of nonadipose tissues rose only 1-2.7-fold implying antisteatotic activity. In rodents without leptin action (fa/fa rats and ob/ob and db/db mice) receiving a 6% fat diet, nonadipose tissue TG was 4-100 times normal. In normal rats on a 60% fat diet, peroxisome proliferator-activated receptor alpha protein and liver-carnitine palmitoyltransferase-1 (l-CPT-1) mRNA increased in liver. In their pancreatic islets, fatty-acid oxidation increased 30% without detectable increase in the expression of peroxisome proliferator-activated receptor-alpha or oxidative enzymes, whereas lipogenesis from [14C]glucose was slightly below that of the 4% fat-fed rats (p < 0.05). Tissue-specific overexpression of wild-type leptin receptors in the livers of fa/fa rats, in which marked steatosis is uniformly present, reduced TG accumulation in liver but nowhere else. We conclude that a physiologic role of the hyperleptinemia of caloric excess is to protect nonadipocytes from steatosis and lipotoxicity by preventing the up-regulation of lipogenesis and increasing fatty-acid oxidation.

The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Contains an Internal Mitochondrial Import Signal and Residues Essential for Folding of Its C-terminal Catalytic Domain

We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM). In the present study, we have identified the precise import sequence in this polytopic OMM protein. In vitro import studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence. Analysis of the regions flanking the TM segments revealed that residues 123-147, located immediately downstream of TM2, function as a noncleavable, matrix-targeting signal. They specify mitochondrial targeting, whereas the hydrophobic TM segment(s) acts as a stop-transfer sequence that stops and anchors the translocating CPT1 into the OMM. Heterologous expression in Saccharomyces cerevisiae of several deleted CPT1 proteins not only confirms the validity of the "stop-transfer" import model but also indicates that residues 1-82 of CPT1 contain a putative microsomal targeting signal whose cellular significance awaits further investigation. Finally, we identified a highly folded core within the C-terminal domain of CPT1 that is hidden in the entire protein by its cytosolic N-terminal residues. Functional analysis of the deleted CPT1 proteins indicates that this folded C-terminal core, which may belong to the catalytic domain of CPT1, requires TM2 for its correct folding achievement and is in close proximity to residues 1-47.

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.

Regulation of the Rat Liver Carnitine Palmitoyltransferase I Gene Transcription by Thyroid Hormone

L-CPT I isotype is the main locus of control for liver LCFA oxidation. T3 levels have been described as controlling L-CPT I gene expression, and in this paper we demonstrate that rat liver CPT I promoter responds to T3. Using deleted reporter constructs we located the thyroid hormone-responsive element between −2935 and −2918, consisting of a DR4. This response is mediated by the binding of the thyroid to this sequence as a monomer, homodimer, or heterodimer with RXR.

Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.

Identification by Mutagenesis of Conserved Arginine and Tryptophan Residues in Rat Liver Carnitine Palmitoyltransferase I Important for Catalytic Activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.

Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity

Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of β-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6×His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200-400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 °C compared with 4 °C (IC50 ≈ 11 μM versus 30 μM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver &amp;gt; fasted liver≈ liposomes prepared at 22 °C≈ P. pastoris mitochondria &amp;gt; liposomes prepared at 4 °C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6×His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.

Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity.

Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of beta-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process. The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions. A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes. The mechanism of this important regulatory effect is unknown and has aroused much interest. CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means. Here we describe the expression of a 6 x His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities. Reconstitution of the purified product into a liposomal environment yielded a 200--400-fold increase in enzymic activity and restored malonyl-CoA sensitivity. This is the first time that a CPT I protein has been available for study in a form that is both pure and active. Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour. The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 degrees C compared with 4 degrees C (IC(50) approximately 11 microM versus 30 microM, respectively). When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver>fasted liver approximately liposomes prepared at 22 degrees C approximately P. pastoris mitochondria>liposomes prepared at 4 degrees C. In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor. In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria. However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P. pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters. The kinetic data establish several novel points. First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form. Second, the kinetics of the reconstituted, 6 x His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P. pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme. Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins. In particular, the fluidity of the membrane had a direct influence on this parameter. These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme.

Expression of the rat liver carnitine palmitoyltransferase I (CPT-Iα) gene is regulated by Sp1 and nuclear factor Y: chromosomal localization and promoter characterization

Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the 'liver' isoform of the CPT-I gene (CPT-Ialpha) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Ialpha gene expression, we have characterized the proximal promoter of the CPT-Ialpha gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Ialpha gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Ialpha gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Ialpha promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Ialpha gene expression. Our studies demonstrate that CPT-Ialpha is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.

Expression of the rat liver carnitine palmitoyltransferase I (CPT-Iα) gene is regulated by Sp1 and nuclear factor Y: chromosomal localization and promoter characterization

Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the ‘liver’ isoform of the CPT-I gene (CPT-Iα) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Iα gene expression, we have characterized the proximal promoter of the CPT-Iα gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Iα gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Iα gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Iα promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Iα gene expression. Our studies demonstrate that CPT-Iα is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.

A Single Amino Acid Change (Substitution of Glutamate 3 with Alanine) in the N-terminal Region of Rat Liver Carnitine Palmitoyltransferase I Abolishes Malonyl-CoA Inhibition and High Affinity Binding

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

A sib-pair analysis study of 15 candidate genes in French families with morbid obesity: indication for linkage with islet 1 locus on chromosome 5q.

As part of an ongoing search for susceptibility genes in obese families, we performed linkage analyses in 101 French families between qualitative and quantitative traits related to morbid obesity and polymorphisms located in or near 15 candidate genes whose products are involved in body weight regulation. These included cholecystokinin A and B receptors (CCK-AR and CCK-BR), glucagon-like peptide 1 receptor (GLP-1R), the LIM/homeodomain islet-1 gene (Isl-1), the caudal-type homeodomain 3 (CDX-3), the uncoupling protein 1 (UCP-1), the beta3-adrenoceptor (beta3-AR), the fatty acid-binding protein 2 (FABP-2), the hormone-sensitive lipase (HSL), the lipoprotein lipase (LPL), the apoprotein-C2 (apo-C2), the insulin receptor substrate-1 (IRS-1), the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), tumor necrosis factor-alpha (TNF-alpha), and the liver carnitine palmitoyltransferase-1 (CPT-1). Phenotypes related to obesity such as BMI, adult life body weight gain, fasting leptin, insulin, fasting glycerol, and free fatty acids were used for nonparametric sib-pair analyses. A weak indication for linkage was obtained between the Isl-1 locus and obesity status defined by a z score over one SD of BMI (n = 226 sib pairs, pi = 0.54 +/- 0.02, P = 0.03). Moreover, a suggestive indication for linkage was found between the Isl-1 locus and BMI and leptin values (P = 0.001 and 0.0003, respectively) and leptin adjusted for BMI (P = 0.0001). Multipoint analyses for leptin trait with Isl-1 and two flanking markers (D5S418 and D5S407) showed that the logarithm of odds (LOD) score is 1.73, coinciding with the Isl-1 locus. Although marginally positive indications for linkage in subgroups of families were found with IRS-1, CPT-1, and HSL loci, our data suggested that these genes are not major contributors to obesity. Whether an obesity susceptibility gene (Isl-1 itself or another nearby gene) lies on chromosome 5q should be determined by further analyses.

Short-term regulation of carnitine palmitoyltransferase I in cultured rat hepatocytes: spontaneous inactivation and reactivation by fatty acids

Liver carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme of mitochondrial β-oxidation, rapidly loses its activity when hepatocytes are put in culture. 3-Thia fatty acids reactivate the enzyme and can increase its activity 3–4-fold in 5–10 min. Normal fatty acids are also able to stimulate CPT I, but to a limited extent, compared to 3-thia fatty acid. This activation does not affect malonyl-CoA sensitivity. CPT I in hepatocytes from both fasted and fasted-carbohydrate refed rats is inactivated and reactivated to a similar extent. Free dodecylthioacetic acid (DTA) is at least as efficient as DTA-CoA as activator. CPT I activity in isolated mitochondria is not influenced by incubation with DTA, suggesting that the regulation of CPT I depends on an extramitochondrial component(s) in the cell. It is concluded that fatty acids activate pre-existing, inactive CPT I without involvement of gene transcription and independently of malonyl-CoA.

Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components

The activity of hepatic carnitine palmitoyltransferase I (CPT-I) may be modulated by interactions with cytoskeletal components [Velasco et al. (1998) J. Biol. Chem. 273, 21497–21504]. We have studied whether the AMP-activated protein kinase (AMPK) is involved in this process. AMPK stimulated CPT-I in permeabilized hepatocytes but not in isolated liver mitochondria. In addition, AMPK abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytoskeletal fraction. These two effects of AMPK were not evident when the kinase was inactivated by pretreatment with protein phosphatase 2C. Cytokeratins 8 and 18 were phosphorylated by AMPK in vitro and by incubation of intact hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside, a cell-permeable activator of AMPK. These results provide the first evidence that AMPK stimulates CPT-I by direct phosphorylation of cytoskeletal components.

The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Mediates Import into the Outer Mitochondrial Membrane and Is Essential for Activity and Malonyl-CoA Sensitivity

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.

Deletion of the conserved first 18 N-terminal amino acid residues in rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA sensitivity and binding.

To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.

Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae

The rat liver carnitine palmitoyltransferase 1 (L-CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast-expressed L-CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl-CoA inhibition and affinity for carnitine. Normal Michaelis-Menten kinetics towards palmitoyl-CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure-function relationships of L-CPT 1.