RESUMEN
Administration of the fatty acid analogue tetradecylthioacetic acid (TTA) to rodents up-regulates peroxisomal and mitochondrial lipid-metabolizing enzymes and induces a proliferation of these organelles in hepatocytes. We show here that male NMRI mice fed a diet containing 0.3% (w/w) TTA revealed a transient two-fold increase in the incorporation of [3H]thymidine into the liver mtDNA followed by a 1.6-fold increase in the content of mtDNA. In addition, a transient three-fold increase in the mitochondrial thymidine kinase (TK2) activity and a slight increase in the DNA polymerase gamma activity was observed, indicating that the TTA induced mitochondrial proliferation is linked to an up-regulation of the mitochondrial thymidine kinase activity.
Asunto(s)
Mitocondrias Hepáticas/efectos de los fármacos , Timidina Quinasa/metabolismo , Regulación hacia Arriba , Animales , Secuencia de Bases , Peso Corporal/efectos de los fármacos , ADN Polimerasa gamma , Cartilla de ADN , Replicación del ADN , ADN Mitocondrial/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Masculino , Ratones , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/fisiología , Tamaño de los Órganos/efectos de los fármacos , Sulfuros/farmacologíaRESUMEN
The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.
Asunto(s)
Acilcoenzima A/farmacología , Coenzima A/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Acil-CoA Oxidasa , Animales , Línea Celular , Cromatografía de Afinidad , Proteínas de Unión al ADN/efectos de los fármacos , Dimerización , Genes Reporteros , Glutatión Transferasa/genética , Histona Acetiltransferasas , Ligandos , Ratones , Modelos Moleculares , Coactivador 1 de Receptor Nuclear , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Ratas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Receptores X Retinoide , Spodoptera , Transactivadores/metabolismo , Factores de Transcripción/efectos de los fármacos , Transcripción Genética , TransfecciónRESUMEN
A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.
Asunto(s)
Acilcoenzima A/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Núcleo Celular/química , Hígado/química , Animales , Especificidad de Anticuerpos , Células COS , Proteínas Portadoras/inmunología , Compartimento Celular , Fraccionamiento Celular , Núcleo Celular/ultraestructura , Cromatografía Líquida de Alta Presión , Citoplasma/química , Inhibidor de la Unión a Diazepam , Ácidos Grasos/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Hígado/ultraestructura , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sulfuros/metabolismo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
We introduced methyl or ethyl groups to the 2- or 3-position of the eicosapentaenoic acid (EPA) molecule to investigate whether the branching of EPA could influence its hypolipidemic effect in rats. The most effective branching involved two methyl groups in the 2-position and one methyl group in the 3-position. These EPA derivatives increased hepatic mitochondrial and peroxisomal beta-oxidation and decreased plasma lipids concomitant with suppressed acetyl-coenzyme A (CoA) carboxylase (EC 6.4.1.2) and fatty acid synthase (EC 2.3.1.85) activities. This was followed by elevated activities of camitine O-palmitoyltransferase (EC 2.3.1.21) and possibly 2,4-dienoyl-CoA reductase (EC 1.3.1.34), as well as induced mRNA levels of these enzymes and fatty acyl-CoA oxidase. The fatty acid composition in liver changed, with an increased 18:1 n-9 content, whereas the expression of delta9-desaturase remained unchanged. We investigated the flux of fatty acids in cultured hepatocytes, and found that oxidation of [1-14C]-labeled palmitic acid increased but the secretion of palmitic acid-labeled triglycerides decreased after addition of 2-methyl-EPA. The fatty acyl-CoA oxidase (EC 1.3.3.6) activity in these cells remained unchanged. A significant negative correlation was obtained between palmitic acid oxidation and palmitic acid-labeled synthesized triglycerides. To investigate whether the hypolipidemic effect occurred independently of induced peroxisomal beta-oxidation, we fed rats 2-methyl-tetradecylthioacetic acid. This compound increased the peroxisomal but not the mitochondrial beta-oxidation, and the plasma lipid levels were unchanged. In conclusion, EPA methylated in the 2- or 3-position renders it more potent as a hypolipidemic agent. Furthermore, this study supports the hypothesis that the mitochondrion is the primary site for the hypolipidemic effect.
Asunto(s)
Antioxidantes/farmacología , Ácido Eicosapentaenoico/farmacología , Ácidos Grasos/metabolismo , Microcuerpos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Sulfuros/farmacología , Animales , Células Cultivadas , Ácido Eicosapentaenoico/metabolismo , Lípidos/sangre , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Metilación , Microcuerpos/enzimología , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/genética , Oxidación-Reducción , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Sulfuros/metabolismoRESUMEN
1. We investigated the nature and roles of various xenobiotic acyl-CoA hydrolases in liver subcellular fractions from rat treated with sulphur-substituted (thia) fatty acids. To contribute to our understanding of factors influencing enzymes involved in the degradation of activated fatty acids, the effects on these activities of the oppositely acting thia fatty acid analogues, the peroxisome proliferating 3-thia fatty acids (tetradecylthioacetic acid and 3-dithiacarboxylic acid), which are blocked for beta-oxidation, and a non-peroxisome-proliferating 4-thia fatty acid (tetradecylthiopropionic acid), which undergoes one cycle of beta-oxidation, were studied. 2. The hepatic subcellular distributions of palmitoyl-CoA, tetradecylthioacetyl-CoA and tetradecylthiopropionyl-CoA hydrolase activities were similar to each other in the control and 3-thia fatty acid-treated rat. In control animals, most of these hydrolases were located in the microsomal fraction, but after treatment with the 3-thia fatty acids, the specific activities of the mitochondrial, peroxisomal, and cytosolic palmitoyl-CoA, tetradecylthioacetyl-CoA, and tetradecylthiopropionyl-CoA hydrolase activities were significantly increased. This increase in activity was seen mostly for the enzymes using tetradecylthiopropionyl-CoA and tetradecylthioacetyl-CoA as substrates. The increased mitochondrial activities for these two substrates were seen already after 1 day of treatment, whereas the peroxisomal activities increased after 3 days. No stimulation was seen after treatment with the 4-thia fatty acid analogue, tetradecylthiopropionic acid, but a decrease in peroxisomal hydrolase activities for all three substrates was observed. 3. The cellular distributions of clofibroyl-CoA, POCA-CoA, and sebacoyl-CoA hydrolase activities were different from those of the 'long-chain acyl-CoA' hydrolases mentioned above both in the normal and 3-thia fatty acid treated rat. This group of hydrolases was found in the mitochondrial, peroxisomal, and cytosolic fractions. 3-Thia fatty acid treatment increased the activities of clofibroyl-CoA and sebacoyl-CoA hydrolases in all three fractions. Clofibroyl-CoA and sebacoyl-CoA hydrolase activities were increased after 1 day of treatment. Only the cytosolic POCA-CoA hydrolase was stimulated after 3-thia fatty acid treatment after only 1 day of treatment, whereas treatment with the 4-thia fatty acid led to an increase of enzyme activity in the mitochondrial and peroxisomal fractions. 4. Based on the subcellular distributions and specific activities, we suggest that several enzymes exist which may act as regulators of intracellular acyl-CoA levels.
Asunto(s)
Acilcoenzima A/metabolismo , Ésteres/metabolismo , Ácidos Grasos/farmacología , Hígado/enzimología , Palmitoil-CoA Hidrolasa/metabolismo , Sulfuros/farmacología , Animales , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/metabolismo , Citosol/enzimología , Hígado/ultraestructura , Masculino , Microcuerpos/enzimología , Microsomas Hepáticos/enzimología , Mitocondrias Hepáticas/enzimología , Palmitoil Coenzima A/metabolismo , Propionatos/metabolismo , Ratas , Ratas Wistar , Especificidad por Sustrato , Sulfuros/metabolismoRESUMEN
Acyl-CoA-binding protein (ACBP) is an ubiquitously expressed 10-kDa protein which is present in high amounts in cells involved in solute transport or secretion. Rat ACBP is encoded by a gene containing the typical hallmarks of a housekeeping gene. Analysis of the promoter region of the rat ACBP gene by electrophoretic mobility shift assay (EMSA) revealed specific binding of proteins from rat liver nuclear extracts to potential recognition sequences of NF-1/CTF, Sp1, AP-1, C/EBP and HNF-3. In addition, specific binding to a DR-1 type element was observed. By using in vitro translated peroxisome proliferator activated receptors (PPAR) and a retinoid X receptor alpha (RXRalpha), we demonstrated that this DR-1 element was capable of binding PPARalpha/RXRalpha, PPARdelta/RXRalpha and PPARgamma2/RXRalpha heterodimers. The PPARgamma2/RXRalpha heterodimer appeared to have the highest affinity for the ACBP DR-1 element. Addition of peroxisome proliferators (PP) to H4IIEC3 rat hepatoma cells led to an increase in the ACBP mRNA level, indicating that the DR-1 element could be a functional peroxisome proliferator responsive element (PPRE). Analysis of the ACBP promoter by transient transfection showed that deletion of the region containing the DR-1 element reduced transcriptional activity, and further indicated that three AP-2 sites and one NF-1/CTF site in the proximal promoter are of importance for basal promoter activity.