RESUMO
Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression.
Assuntos
Trifosfato de Adenosina/metabolismo , Biotina/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Glucoquinase/metabolismo , Guanilato Ciclase/fisiologia , Ilhotas Pancreáticas/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais , Animais , Comunicação Autócrina , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucoquinase/genética , Guanilato Ciclase/antagonistas & inibidores , Insulina/fisiologia , Antagonistas da Insulina/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Masculino , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase SolúvelRESUMO
During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.
En décadas recientes, diversas investigaciones han demostrado que las vitaminas afectan la expresión genética. Los casos mejor estudiados son los de las vitaminas A y D. Existe menos información para las vitaminas hidrosolubles sobre su efecto en la expresión de los genes, sin embargo, se sabe que éstas también los modifican. La biotina es una vitamina hidrosoluble que actúa como grupo prostético de las carboxilasas. Además de su función como cofactor de enzimas, participa en el desarrollo embrionario, en la proliferación celular, en funciones inmunológicas y en el metabolismo. Ha habido un notable avance en la identificación de genes cuya expresión está regulada por la biotina. Asimismo, se han investigado los mecanismos moleculares a través de los cuales la biotina efectúa estas acciones. Estos estudios brindan nuevas claves para entender el papel de la biotina en la expresión genética, en el metabolismo, y en otras funciones biológicas de esta vitamina.
Assuntos
Animais , Humanos , Biotina/fisiologia , Regulação da Expressão Gênica , MetabolismoRESUMO
During the last few decades, an increasing number of vitamin-mediated effects has been discovered at the level of gene expression in addition to their well-known roles as substrates and cofactors; the best recognized examples are the lipophilic vitamins A and D. Although little is known about water-soluble vitamins as genetic modulators, there are increasing examples of their effect on gene expression. Biotin is a hydro soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase cofactor, biotin affects several systemic functions such as development, immunity and metabolism. In recent years, significant progress has been made in the identification of genes that are affected by biotin at the transcriptional and post-transcriptional levels as well as in the elucidation of mechanisms that mediate the effects of biotin on the gene expression. These studies bring new insights into biotin mediated gene expression and will lead to a better under-standing of biotin roles in the metabolism and in systemic functions.
Assuntos
Biotina/fisiologia , Regulação da Expressão Gênica , Metabolismo , Animais , HumanosRESUMO
Biotin, a water-soluble vitamin, is used as cofactor of enzymes involved in carboxylation reactions. In humans, there are five biotin-dependent carboxylases: propionyl-CoA carboxylase; methylcrotonyl-CoA carboxylase; pyruvate carboxylase, and two forms of acetyl-CoA carboxylase. These enzymes catalyze key reactions in gluconeogenesis, fatty acid metabolism, and amino acid catabolism; thus, biotin plays an essential role in maintaining metabolic homeostasis. In recent years, biotin has been associated with several diseases in humans. Some are related to enzyme deficiencies involved in biotin metabolism. However, not all biotin-responsive disorders can be explained based on the classical role of the vitamin in cell metabolism. Several groups have suggested that biotin may be involved in regulating transcription or protein expression of different proteins. Biotinylation of histones and triggering of transduction signaling cascades have been suggested as underlying mechanisms behind these non-classical biotin-deficiency manifestation in humans.
Assuntos
Biotina/metabolismo , Biotina/fisiologia , Sequência de Aminoácidos , Carbono-Nitrogênio Ligases/metabolismo , Catálise , Feminino , Regulação Enzimológica da Expressão Gênica , Histonas/metabolismo , Humanos , Masculino , Modelos Biológicos , Modelos Químicos , Dados de Sequência Molecular , Deficiência Múltipla de Carboxilase/genética , Gravidez , Transdução de Sinais , Transcrição GênicaRESUMO
Se realizaron dos experimentos para evaluar los cambios en la digestibilidad de paja de cártamo (PAC) usando un suplemento melaza-urea (MU) y un probiótico (PRO; Saccharomyces cerevisiae). En el experimento 1, se determinó la degradabilidad in situ de fibra (FDN) con cánula ruminal en 12 ovinos (45 ñ 5.8 kg). Se incubaron in situ 4 g de muestra en base seca por periodos de 0, 6, 24, 48, 72 y 96 h, y se muestreó líquido ruminal a 0, 2, 4 y 6 h pospandrial para determinar pH y concentración de N amoniacal. Los resultados se analizaron usando un diseño completamente al azar con arreglo factorial de tratamiento (2 x 2 x 3), donde los factores fueron dos niveles de PRO (o vs .5 g/d intrarruminal), MU (0 vs 10 por ciento) y tres niveles de PAC (50, 60 y 70). La digestibilidad de FDN se incrementó (P< .05) por PRO (3.2 por ciento promedio) y MU (3 por ciento promedio) en todos los tiempos de incubación; también hubo diferencias (P< .05) en la digestión de FDN para 50 por ciento de PAC (35.9 por ciento) con respeco a 60 y 70 por ciento (31.82 y 30.75 por ciento, respectivamente). En el experimento 2 se midió la digestibilidad in vivo (DIV) usando 70 por ciento de PAC, dos niveles de pro (0 vs .05 g/animal/d) y MU (0 vs .10 por ciento) y una ración testigo (T5:40 por ciento PAC, 30 por ciento heno de alfalfa y 30 por ciento concentrado) en un diseño completamente al azar con arreglo factorial (2 x 2 + 1). Los tratamientos fueron: T1, MU 0 por ciento + PRO 0 por ciento; T2, MU 0 por ciento + PRO .5 por ciento; T3, MU 10 por ciento + PRO 0 por ciento y T4, MU 10 por ciento + PRO .5 por ciento. La DIVFDN (por ciento) fue diferente (P< .05) entre tratamientos: 66.8a (T4), 61.9ab (T5),52.2bc (T3),52.1bc (T2) y 41.5c (T1). No hubo cambios en comsumo y pH ruminal (P>.05). El número de protozoarios/ml x 10 3 fue diferente (P< .05) entre tratamientos: 1128a (T4), 950ab (T5), 900ab (T3), 764b (T2) y 771b (T1). Los resultados indican que el probiótico y el suplemento melaza-urea incrementaron la digestibilidad de la fibra y la población de protozoarios, los cuales pudieran haber influido en la digestibilidad de la fibra