RESUMEN
Circadian clocks coordinate physiological, behavioral, and biochemical events with predictable daily environmental changes by a self-sustained transcriptional feedback loop. CLOCK and ARNTL are transcriptional activators that regulate Per and Cry gene expression. PER and CRY inhibit their own transcription, and their turnover allows this cycle to restart. The transcription factors BHLHB2 and BHLHB3 repress Per activation, whereas orphan nuclear receptors of the NR1D and ROR families control Arntl expression. Here we show the AMP-activated protein kinase (AMPK)gamma(3) subunit is involved in the regulation of peripheral circadian clock function. AMPKgamma3 knockout (Prkag3(-/-)) mice or wild-type littermates were injected with saline or an AMPK activator, 5-amino-4-imidazole-carboxamide riboside (AICAR), and white glycolytic gastrocnemius muscle was removed for gene expression analysis. Genes involved in the regulation of circadian rhythms (Cry2, Nr1d1, and Bhlhb2) were differentially regulated in response to AICAR in wild-type mice but remained unaltered in Prkag3(-/-) mice. Basal expression of Per1 was higher in Prkag3(-/-) mice compared with wild-type mice. Distinct diurnal changes in the respiratory exchange ratio (RER) between the light and dark phase of the day were observed in wild-type mice but not Prkag3(-/-) mice. In summary, the expression profile of clock-related genes in skeletal muscle in response to AICAR, as well as the diurnal shift in energy utilization, is impaired in AMPKgamma(3) subunit knockout mice. Our results indicate AMPK heterotrimeric complexes containing the AMPKgamma(3) subunit may play a specific role in linking circadian oscillators and energy metabolism in skeletal muscle.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Perfilación de la Expresión Génica , Músculo Esquelético/metabolismo , Transactivadores/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Glucemia/metabolismo , Proteínas CLOCK , Proteínas de Ciclo Celular/genética , Ritmo Circadiano/fisiología , Criptocromos , Proteínas de Unión al ADN/genética , Flavoproteínas/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 4/genética , Hexoquinasa/genética , Proteínas de Homeodominio/genética , Canales Iónicos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Proteínas Nucleares/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Circadianas Period , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Citoplasmáticos y Nucleares/genética , Ribonucleótidos/farmacología , Factores de Transcripción , Proteína Desacopladora 3RESUMEN
BACKGROUND: A polymorphism in the activating component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, neutrophil cytosolic factor 1 (NCF1), has previously been identified as a regulator of arthritis severity in mice and rats. This discovery resulted in a search for NADPH oxidase-activating substances as a potential new approach to treat autoimmune disorders such as rheumatoid arthritis (RA). We have recently shown that compounds inducing NCF1-dependent oxidative burst, e.g. phytol, have a strong ameliorating effect on arthritis in rats. However, the underlying molecular mechanism is still not clearly understood. The aim of this study was to use gene-expression profiling to understand the protective effect against arthritis of activation of NADPH oxidase in the immune system. RESULTS: Subcutaneous administration of phytol leads to an accumulation of the compound in the inguinal lymph nodes, with peak levels being reached approximately 10 days after administration. Hence, global gene-expression profiling on inguinal lymph nodes was performed 10 days after the induction of pristane-induced arthritis (PIA) and phytol administration. The differentially expressed genes could be divided into two pathways, consisting of genes regulated by different interferons. IFN-gamma regulated the pathway associated with arthritis development, whereas IFN-beta regulated the pathway associated with disease protection through phytol. Importantly, these two molecular pathways were also confirmed to differentiate between the arthritis-susceptible dark agouti (DA) rat, (with an Ncf-1DA allele that allows only low oxidative burst), and the arthritis-protected DA.Ncf-1E3 rat (with an Ncf1E3 allele that allows a stronger oxidative burst). CONCLUSION: Naturally occurring genetic polymorphisms in the Ncf-1 gene modulate the activity of the NADPH oxidase complex, which strongly regulates the severity of arthritis. We now show that the Ncf-1 allele that enhances oxidative burst and protects against arthritis is operating through an IFN-beta-associated pathway, whereas the arthritis-driving allele operates through an IFN-gamma-associated pathway. Treatment of arthritis-susceptible rats with an NADPH oxidase-activating substance, phytol, protects against arthritis. Interestingly, the treatment led to a restoration of the oxidative-burst effect and induction of a strikingly similar IFN-beta-dependent pathway, as seen with the disease-protective Ncf1 polymorphism.
Asunto(s)
Artritis Experimental/enzimología , Interferón beta/metabolismo , NADPH Oxidasas/genética , NADP/metabolismo , Animales , Artritis Experimental/genética , Artritis Experimental/prevención & control , Activación Enzimática , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , NADPH Oxidasas/metabolismo , Fitol/farmacocinética , Fitol/farmacología , ARN Mensajero/genética , Ratas , Estallido Respiratorio , Terpenos , Distribución TisularRESUMEN
Lactic acid bacteria are probiotics widely used in functional food products, with a variety of beneficial effects reported. Recently, intense research has been carried out to provide insight into the mechanism of the action of probiotic bacteria. We have used gene array technology to map the pattern of changes in the global gene expression profile of the host caused by Lactobacillus administration. Affymetrix microarrays were applied to comparatively characterize differences in gene transcription in the distal ileum of normal microflora (NMF) and germ-free (GF) mice evoked by oral administration of two Lactobacillus strains used in fermented dairy products today - Lactobacillus paracasei ssp. paracasei F19 (L. F19) or Lactobacillus acidophilus NCFB 1748. We show that feeding either of the two strains caused very similar effects on the transcriptional profile of the host. Both L. F19 and L. acidophilus NCFB 1748 evoked a complex response in the gut, reflected by differential regulation of a number of genes involved in essential physiological functions such as immune response, regulation of energy homeostasis and host defence. Notably, the changes in intestinal gene expression caused by Lactobacillus were different in the mice raised under GF v. NMF conditions, underlying the complex and dynamic nature of the host-commensal relationship. Differential expression of an array of genes described in this report evokes novel hypothesis of possible interactions between the probiotic bacteria and the host organism and warrants further studies to evaluate the functional significance of these transcriptional changes on the metabolic profile of the host.
Asunto(s)
Metabolismo Energético/genética , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Lactobacillus , Probióticos , Animales , Perfilación de la Expresión Génica/métodos , Homeostasis/genética , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimer important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMPK gamma3, has an important role in glucose uptake, glycogen synthesis, and fat oxidation in white skeletal muscle, as previously demonstrated by physiological characterization of AMPK gamma3 mutant (R225Q) transgenic (TgPrkag3(225Q)) and gamma3 knock-out (Prkag3(-/-)) mice. We determined AMPK gamma3-dependent regulation of gene expression by analyzing global transcription profiles in glycolytic skeletal muscle from gamma3 mutant transgenic and knock-out mice using oligonucleotide microarray technology. Evidence is provided for coordinated and reciprocal regulation of multiple key components in glucose and fat metabolism, as well as skeletal muscle ergogenics in TgPrkag3(225Q) and Prkag3(-/-) mice. The differential gene expression profile was consistent with the physiological differences between the models, providing a molecular mechanism for the observed phenotype. The striking pattern of opposing transcriptional changes between TgPrkag3(225Q) and Prkag3(-/-) mice identifies differentially expressed targets being truly regulated by AMPK and is consistent with the view that R225Q is an activating mutation, in terms of its downstream effects. Additionally, we identified a wide array of novel targets and regulatory pathways for AMPK in skeletal muscle.