RESUMEN
γ-Butyrobetaine hydroxylase (γ-BBH) is the last limiting enzyme of the l-carnitine biosynthesis pathway and plays an important role in catalyzing the hydroxylation of γ-butyrobetaine (γ-BB) to l-carnitine. To study the developmental effect of substrate concentration on the enzyme's specific activity, kinetics of γ-BBH were measured in liver and kidney from newborn and 1-, 7-, 21-, 35-, 56-, and 210-day-old domestic pigs. Fresh tissue homogenates were assayed under nine concentrations of γ-BB from 0 to 1.5 mM. Substrate inhibition associated with age was observed at ≥0.6 mM of γ-BB. Hepatic activity was low at birth but increased after 1 day. By 21 days, the activity rose by 6.6-fold (P < 0.05) and remained constant after 56 days. Renal activity was higher than in liver at birth but remained constant through 35 days. By 56 days, the velocity increased by 44% over the activity at birth (P < 0.05). The apparent Km for γ-BB at birth on average was 2.8-fold higher than at 1 day. The Km value was 60% higher in kidney than liver during development but showed no difference in adult pigs. The total organ enzyme activity increased by 130-fold for liver and 18-fold for kidney as organ weight increased from birth to 56 days. In conclusion, age and substrate affect γ-BBH specific activity and Km for γ-BB in liver and kidney. Whereas the predominant organ for carnitine synthesis is likely the kidney at birth, the liver appears to predominate after the pig exceeds 7 days of age.
Asunto(s)
Carnitina/biosíntesis , gamma-Butirobetaína Dioxigenasa/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Peso Corporal , Inhibidores Enzimáticos/farmacología , Riñón/enzimología , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Cinética , Hígado/enzimología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Tamaño de los Órganos , Sus scrofa , Porcinos , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidoresRESUMEN
BACKGROUND & AIMS: Nonalcoholic fatty liver disease is characterized by excessive hepatic accumulation of triglycerides. We aimed to identify metabolites that differ in plasma of patients with liver steatosis vs healthy individuals (controls) and investigate the mechanisms by which these might contribute to fatty liver in mice. METHODS: We obtained blood samples from 15 patients with liver steatosis and 15 controls from a single center in China (discovery cohort). We performed untargeted liquid chromatography with mass spectrometry analysis of plasma to identify analytes associated with liver steatosis. We then performed targeted metabolomic analysis of blood samples from 2 independent cohorts of individuals who underwent annual health examinations in China (1157 subjects with or without diabetes and 767 subjects with or without liver steatosis; replication cohorts). We performed mass spectrometry analysis of plasma from C57BL/6J mice, germ-free, and mice given antibiotics. C57BL/6J mice were given 0.325% (m/v) N,N,N-trimethyl-5-aminovaleric acid (TMAVA) in their drinking water and placed on a 45% high-fat diet (HFD) for 2 months. Plasma, liver tissues, and fecal samples were collected; fecal samples were analyzed by 16S ribosomal RNA gene sequencing. C57BL/6J mice with CRISPR-mediated disruption of the gene encoding γ-butyrobetaine hydroxylase (BBOX-knockout mice) were also placed on a 45% HFD for 2 months. Hepatic fatty acid oxidation (FAO) in liver tissues was determined by measuring liberation of 3H2O from [3H] palmitic acid. Liver tissues were analyzed by electron microscopy, to view mitochondria, and proteomic analyses. We used surface plasmon resonance analysis to quantify the affinity of TMAVA for BBOX. RESULTS: Levels of TMAVA, believed to be a metabolite of intestinal microbes, were increased in plasma from subjects with liver steatosis compared with controls, in the discovery and replication cohorts. In 1 replication cohort, the odds ratio for fatty liver in subjects with increased liver plasma levels of TMAVA was 1.82 (95% confidence interval [CI], 1.14-2.90; P = .012). Plasma from mice given antibiotics or germ-free mice had significant reductions in TMAVA compared with control mice. We found the intestinal bacteria Enterococcus faecalis and Pseudomonas aeruginosa to metabolize trimethyllysine to TMAVA; levels of trimethyllysine were significantly higher in plasma from patients with steatosis than controls. We found TMAVA to bind and inhibit BBOX, reducing synthesis of carnitine. Mice given TMAVA had alterations in their fecal microbiomes and reduced cold tolerance; their plasma and liver tissue had significant reductions in levels of carnitine and acyl-carnitine and their hepatocytes had reduced mitochondrial FAO compared with mice given only an HFD. Mice given TMAVA on an HFD developed liver steatosis, which was reduced by carnitine supplementation. BBOX-knockout mice had carnitine deficiency and decreased FAO, increasing uptake and liver accumulation of free fatty acids and exacerbating HFD-induced fatty liver. CONCLUSIONS: Levels of TMAVA are increased in plasma from subjects with liver steatosis. In mice, intestinal microbes metabolize trimethyllysine to TMAVA, which reduces carnitine synthesis and FAO to promote steatosis.
Asunto(s)
Bacterias/metabolismo , Microbioma Gastrointestinal , Intestinos/microbiología , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Valeratos/metabolismo , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Dieta Alta en Grasa , Disbiosis , Ácidos Grasos no Esterificados/metabolismo , Heces/microbiología , Femenino , Humanos , Lipólisis/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/microbiología , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Regulación hacia Arriba , Valeratos/sangre , Valeratos/toxicidad , Adulto Joven , gamma-Butirobetaína Dioxigenasa/genética , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
The final step in the biosynthesis of l-carnitine in humans is catalysed by the 2-oxoglutarate and ferrous iron dependent oxygenase, γ-butyrobetaine hydroxylase (BBOX). 1H and 19F NMR studies inform on the BBOX mechanism including by providing evidence for cooperativity between monomers in substrate/some inhibitor binding. The value of the 19F NMR methods is demonstrated by their use in the design of new BBOX inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Espectroscopía de Resonancia Magnética , gamma-Butirobetaína Dioxigenasa/metabolismo , Betaína/análogos & derivados , Betaína/síntesis química , Betaína/química , Betaína/metabolismo , Carnitina/biosíntesis , Carnitina/síntesis química , Carnitina/química , Carnitina/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Flúor/química , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidoresRESUMEN
BACKGROUND AND PURPOSE: The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury. KEY RESULTS: In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC50 3 µM) and organic cation transporter 2 (OCTN2, IC50 3 µM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20 mg·kg(-1)) attenuated the infarct size by 45% and improved 24 h survival of rats by 20-30%. CONCLUSIONS AND IMPLICATIONS: Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.
Asunto(s)
Carnitina/biosíntesis , Ácidos Grasos/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Ácido gamma-Aminobutírico/análogos & derivados , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Estructura Molecular , Infarto del Miocardio/prevención & control , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico , Oxidación-Reducción , Compuestos de Amonio Cuaternario/síntesis química , Compuestos de Amonio Cuaternario/química , Ratas , Ratas Wistar , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/síntesis química , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/farmacología , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II) dependent oxygenase that catalyses an essential step during carnitine biosynthesis in animals. BBOX is inhibited by ejection of structural zinc by a set of selenium containing analogues. Previous structural analyses indicated that an undisrupted N-terminal zinc binding domain of BBOX is required for catalysis. Ebselen is a relatively potent BBOX inhibitor, an observation which may in part reflect its cardioprotective properties.
Asunto(s)
Azoles/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos de Organoselenio/farmacología , Zinc/metabolismo , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Catálisis , Humanos , Isoindoles , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
l-Carnitine is a cofactor in the energy metabolism pathways where it drives the uptake and oxidation of long chain fatty acids (LCFA) by mitochondria. LCFA lipotoxicity causes mitochondrial damage and results in an insufficient energy supply and a decrease in l-carnitine content limits LCFA flux and protects mitochondria. Here, we tested whether the inhibition of GBB dioxygenase (BBOX) or organic cation transporter 2 (OCTN2) is the most effective strategy to decrease l-carnitine content. The activity of 51 compounds was tested and we identified selective inhibitors of OCTN2. In contrast to selective inhibitors of BBOX, OCTN2 inhibitors induced a 10-fold decrease in l-carnitine content in the heart tissues and a significant 35% reduction of myocardial infarct size. In addition, OCTN2 inhibition correlated with the inhibitor content in the heart tissues, and OCTN2 could potentially be an efficient target to increase drug transport into tissues and to reduce drug elimination by urine. In conclusion, the results of this study confirm that selective inhibition of OCTN2, compared to selective inhibition of BBOX, is a far more effective approach to decrease l-carnitine content and to induce cardioprotective effects. OCTN2 could potentially be an efficient tool to increase drug transport in tissues and to reduce drug elimination via urine.
Asunto(s)
Cardiotónicos/uso terapéutico , Carnitina/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Animales , Cardiotónicos/farmacología , Carnitina/sangre , Carnitina/orina , Masculino , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Ratas Wistar , Miembro 5 de la Familia 22 de Transportadores de SolutosRESUMEN
γ-Butyrobetaine hydroxylase (BBOX) catalyzes the conversion of gamma butyrobetaine (GBB) to l-carnitine, which is involved in the generation of metabolic energy from long-chain fatty acids. BBOX inhibitor 3-(1,1,1-trimethylhydrazin-1-ium-2-yl)propanoate (mildronate), which is an approved, clinically used cardioprotective drug, is a relatively poor BBOX inhibitor and requires high daily doses. In this paper we describe the design, synthesis, and properties of 51 compounds, which include both GBB and mildronate analogues. We have discovered novel BBOX inhibitors with improved IC50 values; the best examples are in the nanomolar range and about 2 orders of magnitude better when compared to mildronate. For six inhibitors, crystal structures in complex with BBOX have been solved to explain their activities and pave the way for further inhibitor design.
Asunto(s)
Carnitina/antagonistas & inhibidores , Carnitina/biosíntesis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Calorimetría , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Indicadores y Reactivos , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Metilhidrazinas/química , Metilhidrazinas/farmacología , Modelos Moleculares , Conformación Molecular , Unión Proteica , Proteínas Recombinantes/química , Relación Estructura-Actividad , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
Mildronate has been used as antianginal drug in parts of Europe for many years, but its pharmacokinetic (PK) properties in humans remain unclear. This study was designed to assess and compare the PK properties of mildronate capsules after single escalating oral dose and multiple doses in healthy Chinese volunteers. Volunteers were randomly assigned to receive a single dose of 250, 500, 1000, 1250 or 1500 mg of mildronate capsules. Those who received the 500-mg dose continued on the multiple-dose phase and received 500 mg three times a day for 13 days. Plasma drug concentrations were analysed by ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Tolerability was assessed throughout the study. A total of 40 Chinese volunteers were enrolled in the study. No period or sequence effect was observed. Area under the concentration and C(max) were increased proportionally with the dose levels, whereas t(1/2) and V(d)/f were dependent on the dose. Nonlinear PK properties were found at doses of 250-1500 mg. There was an accumulation after multiple-dose administration. No serious adverse events (AEs) were reported in the PK study. The formulation was well tolerated.
Asunto(s)
Cardiotónicos/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Metilhidrazinas/farmacocinética , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Adulto , Calibración , Cápsulas , Cardiotónicos/administración & dosificación , Cardiotónicos/efectos adversos , Cardiotónicos/sangre , China , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/sangre , Femenino , Semivida , Humanos , Límite de Detección , Masculino , Tasa de Depuración Metabólica , Metilhidrazinas/administración & dosificación , Metilhidrazinas/efectos adversos , Metilhidrazinas/sangre , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II)-dependent oxygenase that catalyses the final step of L-carnitine biosynthesis in animals. BBOX catalyses the oxidation of 3-(2,2,2-trimethylhydrazinium)propionate (THP), a clinically used BBOX inhibitor, to form multiple products including 3-amino-4-(methyamino)butanoic acid (AMBA), which is proposed to be formed via a Stevens type rearrangement mechanism. We report the synthesis of AMBA and confirm that it is a product of the BBOX catalysed oxidation of THP. AMBA reacts with formaldehyde, which is produced enzymatically by BBOX, to give a cyclic adduct.
Asunto(s)
Butiratos/química , Inhibidores Enzimáticos/química , Metilaminas/química , Metilhidrazinas/química , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Butiratos/síntesis química , Catálisis , Inhibidores Enzimáticos/síntesis química , Metilaminas/síntesis química , Metilhidrazinas/síntesis química , Oxidación-Reducción , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
2-Oxoglutarate (2OG) dependent oxygenases are ubiquitous iron enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. In humans their roles include collagen biosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, and hypoxic sensing. Commercial applications of 2OG oxygenase inhibitors began with plant growth retardants, and now extend to a clinically used pharmaceutical compound for cardioprotection. Several 2OG oxygenases are now being targeted for therapeutic intervention for diseases including anaemia, inflammation and cancer. In this critical review, we describe studies on the inhibition of 2OG oxygenases, focusing on small molecules, and discuss the potential of 2OG oxygenases as therapeutic targets (295 references).
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/metabolismo , Colágeno/biosíntesis , Etilenos/biosíntesis , Giberelinas/metabolismo , Humanos , Metales/farmacología , Oxigenasas de Función Mixta/química , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Ácido Succínico/metabolismo , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidoresRESUMEN
Analysis of medical treatment was conducted with justified using of the metabolic component in a complex therapy of ischemic heart disease with chronic heart failure in hemodynamically formed insignificant aortic stenoses. The effect of metabolic correction is shown for pharmaceutical compounds Meldoniya in the form of Vasonat manufactured by "OlainFarm" (Latvia). Positive results of maintenance of systolic activity and prevention of diastolic dysfunction of myocardium were presented. The application of Vasonat in appropriate for the stabilization of adaptive properties of the myocardium and prophylaxis of the development of critical indicators of heart failure in this combined.
Asunto(s)
Estenosis de la Válvula Aórtica/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Isquemia Miocárdica/tratamiento farmacológico , Vasodilatadores/uso terapéutico , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , Aorta/patología , Estenosis de la Válvula Aórtica/complicaciones , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/fisiopatología , Enfermedad Crónica , Diástole/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Hemodinámica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Miocardio/patología , Sístole/efectos de los fármacos , Vasodilatadores/administración & dosificación , gamma-Butirobetaína Dioxigenasa/metabolismoRESUMEN
Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0A resolution. The GBBH monomer consists of a catalytic double-stranded beta-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes. The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered. The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His-X-Asp-X(n)-His triad, which is found in all related enzymes.
Asunto(s)
gamma-Butirobetaína Dioxigenasa/química , Dominio Catalítico , Cristalografía , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Metilhidrazinas/farmacología , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Zinc/química , gamma-Butirobetaína Dioxigenasa/antagonistas & inhibidores , gamma-Butirobetaína Dioxigenasa/genéticaRESUMEN
The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.