RESUMO
The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP-stimulated state 3 respiration and increased the apparent Km for ADP twofold. The half maximal inhibitory concentration (IC50 ) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose-dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31 P-magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Isquemia , Mitocôndrias Cardíacas/metabolismo , Miocárdio , Palmitoil Coenzima A , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Metabolismo Energético , Isquemia/metabolismo , Isquemia/patologia , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Consumo de Oxigênio , Palmitoil Coenzima A/farmacologia , Palmitoil Coenzima A/fisiologia , Ratos , Ratos WistarRESUMO
Cytochrome c (cyt-c) release from the mitochondria to the cytosol is a key process in the initiation of hepatocyte apoptosis involved in the progression of non-alcoholic fatty liver disease (NAFLD) to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatocyte apoptosis may be related to lipotoxicity due to the accumulation of palmitic acid and palmitoyl-CoA (Pal-CoA). Therefore, the aim of this study is to examine whether Pal-CoA induces cyt-c release from liver mitochondria of sucrose-fed rat (SF). Pal-CoA-induced cyt-c release was sensitive to cyclosporine A indicating the involvement of the mitochondrial membrane permeability transition (mMPT). In addition, cyt-c release from SF mitochondria remains significantly lower than C mitochondria despite the increased rate of H2O2 generation in SF mitochondria. The decreased cyt-c release from SF may be also related to the increased proportion of the palmitic acid-enriched cardiolipin, due to the high availibilty of palmitic acid in SF liver. The enrichment of cardiolipin molecular species with palmitic acid makes cardiolipin more resistant to peroxidation, a mechanism involved in the dissociation of cyt-c from mitochondrial inner membrane. These results suggest that Pal-CoA may participate in the progression of NAFLD to more severe disease through mechanisms involving cyt-c release and mMPT, a key process of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Citocromos c/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Obesidade/metabolismo , Palmitoil Coenzima A/farmacologia , Animais , Sacarose Alimentar , Peróxido de Hidrogênio/metabolismo , Fígado/efeitos dos fármacos , Masculino , Membranas Mitocondriais/efeitos dos fármacos , Obesidade/induzido quimicamente , Permeabilidade/efeitos dos fármacos , Ratos WistarRESUMO
Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60â min post I.P.) increased submaximal (100-1000â µM ADP) mitochondrial respiration â¼2-fold without altering maximal (>1000â µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration â¼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.
Assuntos
Difosfato de Adenosina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Insulina/farmacologia , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Injeções Intraperitoneais , Insulina/administração & dosagem , Insulina/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacologiaRESUMO
Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.
Assuntos
Carnitina/metabolismo , Carnitina/farmacologia , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Animais , Citocromos c/metabolismo , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Palmitoil Coenzima A/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Overnutrition can lead to oxidative stress, but its underlying mechanism remains unclear. In this study, we report that human liver-derived HepG2 cells utilize cytosolic thioredoxin reductase (TrxR1) and thioredoxin (hTrx1) to defend against the high glucose/palmitate-mediated increase in reactive oxygen species. However, enhanced TrxR1/hTrx1 palmitoylation occurs in parallel with a decrease in their activities under the conditions studied here. An autoacylation process appears to be the major mechanism for generating palmitoylated TrxR1/Trx1 in HepG2 cells. A novel feature of this post-translational modification is the covalent inhibition of TrxR1/hTrx1 by palmitoyl-CoA, an activated form of palmitate. The palmitoyl-CoA/TrxR1 reaction is NADPH-dependent and produces palmitoylated TrxR1 at an active site selenocysteine residue. Conversely, S-palmitoylation occurs at the structural Cys62/Cys69/Cys72 residues but not the active site Cys32/Cys35 residues of hTrx1. Palmitoyl-CoA concentration and the period of incubation with TrxR1/hTrx1 are important factors that influence the inhibitory efficacy of palmitoyl-CoA on TrxR1/hTrx1. Thus, an increase in TrxR1/hTrx1 palmitoylation could be a potential consequence of high glucose/palmitate. The time-dependent inactivation of the NADPH-TrxR1-Trx1 system by palmitoyl-CoA occurs in a biphasic manner - a fast phase followed by a slow phase. Kinetic analysis suggests that the fast phase is consistent with a fast and reversible association between TrxR1/hTrx1 and palmitoyl-CoA. The slow phase is correlated with a slow and irreversible inactivation, in which selenolate/thiolate groups nucleophilically attack the α-carbon of bound palmitoyl-CoA, leading to the formation of thioester/selenoester bonds. hTrx1 can enhance rate of fast phase but limits the rate of slow phase when it is present in a preincubation mixture containing NADPH, TrxR1 and palmitoyl-CoA. Therefore, hTrx1 may provide palmitoylation sites or partially protect the TrxR1 active site selenol/thiol group(s) from palmitoylation. Our data suggest that Se/S-palmitoylation acts as an important modulator of TrxR1/hTrx1 activities, representing a novel potential mechanism that underlies overnutrition-induced events.
Assuntos
Citosol/enzimologia , Lipoilação , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Bovinos , Glucose/farmacologia , Células Hep G2 , Humanos , Lipoilação/efeitos dos fármacos , Dados de Sequência Molecular , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Palmitoil Coenzima A/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/química , Fatores de TempoRESUMO
Nicotinamide adenine dinucleotide (NAD) belongs to the family of naturally occurring adenine dinucleotides, best known for their various intracellular roles. However, there is evidence that they can also be released from cells to act as novel extracellular signalling molecules. Relatively little is known about the extracellular actions of NAD, especially in the cardiovascular system. The present study investigated the actions of NAD in the rat thoracic aorta, porcine coronary artery and porcine mesenteric arteries, mounted in organ baths for isometric tension recording. In the rat thoracic aorta and porcine coronary artery, NAD caused endothelium-independent concentration-dependent vasorelaxations which were unaffected by palmitoylCoA, a P2Y1 receptor antagonist, but which were blocked by CGS15943, a non-selective adenosine receptor antagonist. In the porcine coronary artery, NAD-evoked relaxations were abolished by SCH58261, a selective A2A receptor antagonist. In the rat thoracic aorta, NAD-evoked relaxations were attenuated by A2A receptor antagonism with SCH58261 but were unaffected by an A2B receptor antagonist, MRS1754. In contrast, in the porcine mesenteric artery, NAD-evoked endothelium-independent contractions, which were unaffected by a P2 receptor antagonist, suramin, or by NF449, a P2X1 receptor antagonist, but were attenuated following P2X receptor desensitisation with αß-meATP. In conclusion, the present results show that NAD can alter vascular tone through actions at purine receptors in three different arteries from two species; its molecular targets differ according to the type of blood vessel.
Assuntos
Aorta Torácica/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , NAD/farmacologia , Receptores Purinérgicos/metabolismo , Acetamidas/farmacologia , Animais , Aorta Torácica/metabolismo , Vasos Coronários/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Palmitoil Coenzima A/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Purinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Suínos , Triazóis/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
BACKGROUND AND PURPOSE: Acyl derivatives of CoA have been shown to act as antagonists at human platelet and recombinant P2Y1 receptors, but little is known about their effects in the cardiovascular system. This study evaluated the effect of these endogenous nucleotide derivatives at P2Y1 receptors natively expressed in rat and porcine blood vessels. EXPERIMENTAL APPROACH: Isometric tension recordings were used to evaluate the effects of CoA, acetyl CoA, palmitoyl CoA (PaCoA) and 3'-dephospho-palmitoyl-CoA on concentration relaxation-response curves to ADP and uridine triphosphate (UTP). A FlexStation monitored ADP- and UTP-evoked calcium responses in HEK293 cells. KEY RESULTS: Acetyl CoA and PaCoA, but not CoA, inhibited endothelium-dependent relaxations to ADP with apparent selectivity for P2Y1 receptors (over P2Y(2/4) receptors) in rat thoracic aorta; PaCoA was more potent than acetyl CoA (331-fold vs. fivefold shift of ADP response curve evoked by 10 µM PaCoA and acetyl CoA, respectively); the apparent pA2 value for PaCoA was 6.44. 3'-dephospho-palmitoyl-CoA (10 µM) was significantly less potent than PaCoA (20-fold shift). In porcine mesenteric arteries, PaCoA and the P2Y1 receptor antagonist MRS2500 blocked ADP-mediated endothelium-dependent relaxations; in contrast, they were ineffective against ADP-mediated endothelium-independent relaxation in porcine coronary arteries (which does not involve P2Y1 receptors). Calcium responses evoked by ADP activation of endogenous P2Y1 receptors in HEK293 cells were inhibited in the presence of PaCoA, which failed to alter responses to UTP (acting at endogenous P2Y(2/4) receptors). CONCLUSIONS AND IMPLICATIONS: Acyl derivatives of CoA can act as endogenous selective antagonists of P2Y1 receptors in blood vessels, and this inhibitory effect critically depends on the palmitate and 3'-ribose phosphate substituents on CoA.
Assuntos
Acil Coenzima A/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Aorta Torácica/fisiologia , Relaxamento Muscular , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Difosfato de Uridina/metabolismo , Acetilcoenzima A/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Vasos Coronários/fisiologia , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Artérias Mesentéricas/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Palmitoil Coenzima A/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y1/metabolismo , SuínosRESUMO
Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the cassette mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl- CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA.
Assuntos
Cisteína/química , Glutamato Desidrogenase/efeitos dos fármacos , Glutamato Desidrogenase/metabolismo , Palmitoil Coenzima A/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Sítios de Ligação , Glutamato Desidrogenase/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismoRESUMO
Cardiovascular risks are frequently accompanied by high serum fatty acid levels. Although recent studies have shown that fatty acids affect mitochondrial function and induce cell apoptosis, L-carnitine is essential for the uptake of fatty acids by mitochondria, and may attenuate the mitochondrial dysfunction and apoptosis of cardiocytes. This study aimed to elucidate the activity of L-carnitine in the prevention on fatty acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoyl-CoA-induced mitochondrial respiration that was observed with L-carnitine was inhibited with oligomycin. The palmitoyl-CoA-induced mitochondrial membrane depolarization and swelling were greatly inhibited by the presence of L-carnitine. In ultrastructural observations, terminally swollen and ruptured mitochondria with little or no distinguishable cristae structures were induced by treatment with palmitoyl-CoA. However, the severe morphological damage in cardiac mitochondria was dramatically inhibited by pretreatment with L-carnitine. Treatment with L-carnitine also attenuated 4-hydroxy-L-phenylglycine- and rotenone-induced mitochondrial swelling even when the L-carnitine could not protect against the decrease in oxygen consumption associated with these inhibitors. Furthermore, L-carnitine completely inhibited palmitoyl-CoA-induced cytochrome c release. We concluded that L-carnitine is essential for cardiac mitochondria to attenuate the membrane permeability transition, and to maintain the ultrastructure and membrane stabilization, in the presence of high fatty acid ß-oxidation. Consequently, the cells may be protected against apoptosis by L-carnitine through inhibition of the fatty acid-induced cytochrome c release.
Assuntos
Carnitina/farmacologia , Ácidos Graxos/farmacologia , Dilatação Mitocondrial/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Complexo Vitamínico B/farmacologia , Animais , Mitocôndrias Cardíacas/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Palmitoil Coenzima A/farmacologia , Permeabilidade/efeitos dos fármacos , RatosRESUMO
Preparations of rat liver mitochondria, but not of brain and heart mitochondria, produce large quantities of reactive oxygen species (ROS) in the presence of palmitoyl-CoA and other long-chain acyl-CoAs. Palmitoyl-CoA inhibited respiration of rat liver mitochondria with glutamate plus malate or with succinate as substrate. However, ROS production induced by acyl-CoA was independent of respiration inhibition, as it was also observed in antimycin A- and rotenone-inhibited mitochondria and in submitochondrial particles in the absence of respiratory substrates (other than acyl-CoA). Increased ROS production by acyl-CoA in rat liver mitochondrial preparations was observed when measured in the external medium using Amplex red as a probe, but not inside mitochondria using the internal fluorescent probe MitoSOX or aconitase activity as the "intrinsic" indicator of ROS generation in the matrix compartment. Stimulation by acyl-CoA of ROS generation was higher in "light" mitochondrial preparations that were enriched in peroxisomes, as assayed by urate oxidase. It is concluded that stimulation of ROS production in preparations of rat liver mitochondria could be ascribed to contaminating peroxisomes. Preparations of rat brain and heart mitochondria were not or were much less contaminated with peroxisomes, as indicated by low urate oxidase activity.
Assuntos
Acil Coenzima A/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Peroxissomos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Encéfalo/metabolismo , Fracionamento Celular , Respiração Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/química , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Palmitoil Coenzima A/farmacologia , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Ratos , Ratos Wistar , Extratos de Tecidos/metabolismo , Urato Oxidase/metabolismoRESUMO
We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.
Assuntos
Plaquetas/efeitos dos fármacos , Coenzima A/farmacologia , Fibrinolíticos/farmacologia , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Plaquetas/citologia , Plaquetas/fisiologia , Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Forma Celular/efeitos dos fármacos , Coenzima A/química , Humanos , Proteínas dos Microfilamentos/metabolismo , Palmitoil Coenzima A/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12RESUMO
The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L(-1) for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity.
Assuntos
Carnitina/análogos & derivados , Acetonitrilas/química , Animais , Carnitina/análise , Carnitina/farmacologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Cromatografia Líquida de Alta Pressão , Formiatos/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Células PC12 , Palmitoil Coenzima A/farmacologia , Ratos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.
Assuntos
Proteínas de Bactérias/genética , Bdellovibrio/enzimologia , Membrana Celular/química , Proteínas de Membrana/genética , Serina C-Palmitoiltransferase/genética , Sphingobacterium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico/genética , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/química , DNA Bacteriano/genética , Dimerização , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/enzimologia , Expressão Gênica , Cinética , Espectrometria de Massas , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Palmitoil Coenzima A/farmacologia , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/isolamento & purificação , Espectrofotometria , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.
Assuntos
Acil Coenzima A/farmacologia , Substituição de Aminoácidos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Diazóxido/farmacologia , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Obesos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Deltapsi) and cytosolic ATP concentrations. We here show that 5 microm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Deltapsi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, palmitoyl-CoA induced concentration-dependent H(2)O(2) formation, which can only partly be explained by its effect on Deltapsi. Although all measured fluxes and intermediate concentrations were affected by palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Deltapsi (C = -0.01) was small. Quantitative assessment of the effects of palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Obesidade/metabolismo , Palmitoil Coenzima A/farmacologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Masculino , Mitocôndrias Hepáticas/enzimologia , Modelos Biológicos , Oxirredução , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The acyl-CoA-dependent modulation of hepatic microsomal UDP-glucuronosyltransferase (UGT) function in rats was studied. Oleoyl- and palmitoyl-CoAs inhibited UGT activity toward 4-methylumbelliferone in the presence of Brij 58. However, acyl-CoAs enhanced UGT activity in untreated microsomes. A maximum activation of about 8-fold over the control was observed at 15 microM oleoyl-CoA, whereas 50 microM or more oleoyl-CoA had an inhibitory effect on UGT function. Medium- and long-chain acyl-CoAs also exhibited similar effects. On the basis of resistance to tryptic digestion of UGTs, oleoyl-CoA at 15 microM has no ability to change the permeability of the endoplasmic reticulum (ER) membrane, although perturbation of the membrane occurred with 50 microM oleoyl-CoA. N-Ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) abolished the oleoyl-CoA (15 microM)-dependent activation of microsomal UGT. These results suggest that: (1) acyl-CoAs play a role as an endogenous activator of UGTs, and (2) a sulfhydryl group is required for the activation of UGT by physiological concentrations of acyl-CoAs.
Assuntos
Acil Coenzima A/metabolismo , Glucuronosiltransferase/metabolismo , Acil Coenzima A/farmacologia , Animais , Cetomacrogol/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Etilmaleimida/farmacologia , Ácidos Graxos/farmacologia , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacologia , Ratos , Ratos Sprague-Dawley , Reagentes de Sulfidrila/farmacologiaRESUMO
Visual perception is mediated by a family of G protein-coupled receptors called the opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde upon absorption of a photon. Restoration of light sensitivity to the photobleached opsin requires chemical re-isomerization of the chromophore. This is carried out by an enzymatic pathway called the visual cycle in retinal pigment epithelial cells. The isomerase in this pathway uses fatty-acyl esters of all-trans-retinol as substrate. A retinyl-ester synthase that produces these esters, called lecithin:retinol acyltransferase (LRAT), has been extensively characterized. Based on prior biochemical studies and the phenotype in lrat(-/-) knockout mice, it has been assumed that LRAT is the sole or dominant retinyl-ester synthase in the retinal pigment epithelium. Here we demonstrate the presence of a second ester synthase activity in these cells called acyl CoA:retinol acyltransferase (ARAT). We show that this activity uses palmitoyl coenzyme A as an acyl donor, unlike LRAT which uses phosphatidylcholine. Similar to LRAT, ARAT esterifies both all-trans-retinol and 11-cis-retinol. LRAT and ARAT are both potently inhibited by the retinyl-ester analog, all-trans-retinylbromoacetate, but only ARAT is inhibited by progesterone. Unexpectedly, the maximum turnover rate (V(max)) of ARAT was similar to that of LRAT. However, the Michaelis constant (K(M)) of ARAT was 10-fold higher than the K(M) of LRAT for all-trans-retinol. These observations suggest that ARAT may complement LRAT to provide additional retinyl-ester synthase activity under conditions of high all-trans-retinol. These conditions occur in the retina following exposure to bright light.
Assuntos
Epitélio Pigmentado Ocular/enzimologia , Retinol O-Graxo-Aciltransferase/análise , Aciltransferases/metabolismo , Animais , Bovinos , Linhagem Celular , Membrana Celular/química , Clonagem Molecular , Humanos , Immunoblotting , Camundongos , Microssomos/química , Palmitoil Coenzima A/farmacologia , Retinol O-Graxo-Aciltransferase/genética , Estimulação Química , Transfecção/métodosRESUMO
The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 microM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [(14)C]palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at approximately 35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide (232)VPTPNVSVVDLTRC*R(245) was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.
Assuntos
Cisteína/metabolismo , Ácidos Graxos/efeitos adversos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/antagonistas & inibidores , Resistência à Insulina/fisiologia , Palmitoil Coenzima A/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Esterificação , Membranas Intracelulares/metabolismo , Lipossomos/metabolismo , Microssomos/ultraestrutura , Palmitatos/metabolismo , Palmitoil Coenzima A/farmacologia , Coelhos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.
Assuntos
Acil Coenzima A/farmacologia , Adipócitos/enzimologia , Ilhotas Pancreáticas/enzimologia , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Animais , Citosol/enzimologia , Inibidor da Ligação a Diazepam/farmacologia , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/metabolismo , Insulina/metabolismo , Secreção de Insulina , Lipase/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Palmitoil Coenzima A/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/deficiência , Esterol Esterase/metabolismo , Triglicerídeos/metabolismoRESUMO
AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and inactivate skeletal muscle acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 and fatty acid oxidation. Contraction-induced activation of AMPK with subsequent phosphorylation/inactivation of ACC has been postulated to be responsible in part for the increase in fatty acid oxidation that occurs in muscle during exercise. These studies were designed to answer the question: Does phosphorylation of ACC by AMPK make palmitoyl-CoA a more effective inhibitor of ACC? Purified rat muscle ACC was subjected to phosphorylation by AMPK. Activity was determined on nonphosphorylated and phosphorylated ACC preparations at acetyl-CoA concentrations ranging from 2 to 500 microM and at palmitoyl-CoA concentrations ranging from 0 to 100 microM. Phosphorylation resulted in a significant decline in the substrate saturation curve at all palmitoyl-CoA concentrations. The inhibitor constant for palmitoyl-CoA inhibition of ACC was reduced from 1.7 +/- 0.25 to 0.85 +/- 0.13 microM as a consequence of phosphorylation. At 0.5 mM citrate, ACC activity was reduced to 13% of control values in response to the combination of phosphorylation and 10 muM palmitoyl-CoA. Skeletal muscle ACC is more potently inhibited by palmitoyl-CoA after having been phosphorylated by AMPK. This may contribute to low-muscle malonyl-CoA values and increasing fatty acid oxidation rates during long-term exercise when plasma fatty acid concentrations are elevated.