Fausto G. Hegardt

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 β-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.

Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control.

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine dimunition was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete β-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.

CPT1c Is Localized in Endoplasmic Reticulum of Neurons and Has Carnitine Palmitoyltransferase Activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol·mg-1·min-1, respectively, which is ∼50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20–300 times lower catalytic efficiency.

Isolation and structural characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) catalyses the synthesis of mevalonate, the specific precursor of all isoprenoid compounds present in plants. We have characterized two overlapping cDNA clones that encompass the entire transcription unit of an HMG-CoA reductase gene from Arabidopsis thaliana. The transcription product has an upstream non-coding sequence of 70 nucleotides preceding an open reading frame of 1776 bases and a 3′ untranslated region in which two alternative polyadenylation sites have been found. The analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 592 residues with a molecular mass of 63 605 Da. The hydropathy profile of the protein indicates the presence of two highly hydrophobic domains near the N-terminus. A sequence of 407 amino acids corresponding to the C-terminal part of the protein (residues 172–579), which presumably contains the catalytic site, shows a high level of similarity to the region containing the catalytic site of the hamster, human, yeast and Drosophila enzymes. The N-terminal domain contains two putative membrane-spanning regions, in contrast to the enzyme from other organisms which has seven trans-membrane regions. A. thaliana contains two different HMG-CoA reductase genes (HMG1 and HMG2), as estimated by gene cloning and Southern blot analysis. Northern blot analysis reveals a single transcript of 2.4 kb in leaves and seedlings, which presumably corresponds to the expression of the HMG1 gene.

Molecular therapy for obesity and diabetes based on a long-term increase in hepatic fatty-acid oxidation †‡

Obesity‐induced insulin resistance is associated with both ectopic lipid deposition and chronic, low‐grade adipose tissue inflammation. Despite their excess fat, obese individuals show lower fatty‐acid oxidation (FAO) rates. This has raised the question of whether burning off the excess fat could improve the obese metabolic phenotype. Here we used human‐safe nonimmunoreactive adeno‐associated viruses (AAV) to mediate long‐term hepatic gene transfer of carnitine palmitoyltransferase 1A (CPT1A), the key enzyme in fatty‐acid β‐oxidation, or its permanently active mutant form CPT1AM, to high‐fat diet‐treated and genetically obese mice. High‐fat diet CPT1A‐ and, to a greater extent, CPT1AM‐expressing mice showed an enhanced hepatic FAO which resulted in increased production of CO2, adenosine triphosphate, and ketone bodies. Notably, the increase in hepatic FAO not only reduced liver triacylglyceride content, inflammation, and reactive oxygen species levels but also systemically affected a decrease in epididymal adipose tissue weight and inflammation and improved insulin signaling in liver, adipose tissue, and muscle. Obesity‐induced weight gain, increase in fasting blood glucose and insulin levels, and augmented expression of gluconeogenic genes were restored to normal only 3 months after AAV treatment. Thus, CPT1A‐ and, to a greater extent, CPT1AM‐expressing mice were protected against obesity‐induced weight gain, hepatic steatosis, diabetes, and obesity‐induced insulin resistance. In addition, genetically obese db/db mice that expressed CPT1AM showed reduced glucose and insulin levels and liver steatosis. Conclusion: A chronic increase in liver FAO improves the obese metabolic phenotype, which indicates that AAV‐mediated CPT1A expression could be a potential molecular therapy for obesity and diabetes. (HEPATOLOGY 2011)

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for &bgr;-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

Mutations and Variants in the Cohesion Factor Genes NIPBL, SMC1A, and SMC3 in a Cohort of 30 Unrelated Patients With Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) manifests facial dysmorphic features, growth and cognitive impairment, and limb malformations. Mutations in three genes (NIPBL, SMC1A, and SMC3) of the cohesin complex and its regulators have been found in affected patients. Here, we present clinical and molecular characterization of 30 unrelated patients with CdLS. Eleven patients had mutations in NIPBL (37%) and three patients had mutations in SMC1A (10%), giving an overall rate of mutations of 47%. Several patients shared the same mutation in NIPBL (p.R827GfsX2) but had variable phenotypes, indicating the influence of modifiers in CdLS. Patients with NIPBL mutations had a more severe phenotype than those with mutations in SMC1A or those without identified mutations. However, a high incidence of palate defects was noted in patients with SMC1A mutations. In addition, we observed a similar phenotype in both male and female patients with SMC1A mutations. Finally, we report the first patient with an SMC1A mutation and the Sandifer complex.

Important roles of brain-specific carnitine palmitoyltransferase and ceramide metabolism in leptin hypothalamic control of feeding

Brain-specific carnitine palmitoyltransferase-1 (CPT-1c) is implicated in CNS control of food intake. In this article, we explore the role of hypothalamic CPT-1c in leptins anorexigenic actions. We first show that adenoviral overexpression of CPT-1c in hypothalamic arcuate nucleus of rats increases food intake and concomitantly up-regulates orexigenic neuropeptide Y (NPY) and Bsx (a transcription factor of NPY). Then, we demonstrate that this overexpression antagonizes the anorectic actions induced by central leptin or compound cerulenin (an inhibitor of fatty acid synthase). The overexpression of CPT-1c also blocks leptin-induced down-regulations of NPY and Bsx. Furthermore, the anorectic actions of central leptin or cerulenin are impaired in mice with brain CPT-1c deleted. Both anorectic effects require elevated levels of hypothalamic arcuate nucleus (Arc) malonyl-CoA, a fatty acid-metabolism intermediate that has emerged as a mediator in hypothalamic control of food intake. Thus, these data suggest that CPT-1c is implicated in malonyl-CoA action in leptins hypothalamic anorectic signaling pathways. Moreover, ceramide metabolism appears to play a role in leptins central control of feeding. Leptin treatment decreases Arc ceramide levels, with the decrease being important in leptin-induced anorectic actions and down-regulations of NPY and Bsx. Of interest, our data indicate that leptin impacts ceramide metabolism through malonyl-CoA and CPT-1c, and ceramide de novo biosynthesis acts downstream of both malonyl-CoA and CPT-1c in mediating their effects on feeding and expressions of NPY and Bsx. In summary, we provide insights into the important roles of malonyl-CoA, CPT-1c, and ceramide metabolism in leptins hypothalamic signaling pathways.

Hypothalamic Ceramide Levels Regulated by CPT1C Mediate the Orexigenic Effect of Ghrelin

Recent data suggest that ghrelin exerts its orexigenic action through regulation of hypothalamic AMP-activated protein kinase pathway, leading to a decline in malonyl-CoA levels and desinhibition of carnitine palmitoyltransferase 1A (CPT1A), which increases mitochondrial fatty acid oxidation and ultimately enhances the expression of the orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY). However, it is unclear whether the brain-specific isoform CPT1C, which is located in the endoplasmic reticulum of neurons, may play a role in this action. Here, we demonstrate that the orexigenic action of ghrelin is totally blunted in CPT1C knockout (KO) mice, despite having the canonical ghrelin signaling pathway activated. We also demonstrate that ghrelin elicits a marked upregulation of hypothalamic C18:0 ceramide levels mediated by CPT1C. Notably, central inhibition of ceramide synthesis with myriocin negated the orexigenic action of ghrelin and normalized the levels of AgRP and NPY, as well as their key transcription factors phosphorylated cAMP-response element–binding protein and forkhead box O1. Finally, central treatment with ceramide induced food intake and orexigenic neuropeptides expression in CPT1C KO mice. Overall, these data indicate that, in addition to formerly reported mechanisms, ghrelin also induces food intake through regulation of hypothalamic CPT1C and ceramide metabolism, a finding of potential importance for the understanding and treatment of obesity.

Structural model of a malonyl-CoA-binding site of carnitine octanoyltransferase and carnitine palmitoyltransferase I: Mutational analysis of a malonyl-CoA affinity domain

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an “in silico” macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001–45008). The putative malonyl-CoA domain contained His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubı́, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.