RESUMEN
Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.
Asunto(s)
Alcaloides , Sarcopenia , Humanos , Masculino , Ratones , Animales , Sarcopenia/tratamiento farmacológico , Sarcopenia/prevención & control , Sarcopenia/metabolismo , NAD/metabolismo , Caenorhabditis elegans , Envejecimiento , Músculo Esquelético/metabolismo , Alcaloides/farmacología , Alcaloides/uso terapéutico , Alcaloides/metabolismoRESUMEN
Recent studies showed that germ-free (GF) mice are resistant to obesity when consuming a high-fat, high-carbohydrate Western diet. However, it remains unclear what mechanisms are involved in the antiobesity phenotype and whether GF mice develop insulin resistance and dyslipidemia with high-fat (HF) feeding. In the present study, we compared the metabolic consequences of HF feeding on GF and conventional (conv) C57BL/6J mice. GF mice consumed fewer calories, excreted more fecal lipids, and weighed significantly less than conv mice. GF/HF animals also showed enhanced insulin sensitivity with improved glucose tolerance, reduced fasting and nonfasting insulinemia, and increased phospho-Akt((Ser-473)) in adipose tissue. In association with enhanced insulin sensitivity, GF/HF mice had reduced plasma TNF-α and total serum amyloid A concentrations. Reduced hypercholesterolemia, a moderate accretion of hepatic cholesterol, and an increase in fecal cholesterol excretion suggest an altered cholesterol metabolism in GF/HF mice. Pronounced nucleus SREBP2 proteins and up-regulation of cholesterol biosynthesis genes indicate that enhanced cholesterol biosynthesis contributed to the cholesterol homeostasis in GF/HF mice. Our results demonstrate that fewer calorie consumption and increased lipid excretion contributed to the obesity-resistant phenotype of GF/HF mice and reveal that insulin sensitivity and cholesterol metabolism are metabolic targets influenced by the gut microbiota.
Asunto(s)
Colesterol/metabolismo , Grasas de la Dieta/efectos adversos , Resistencia a la Insulina/fisiología , Animales , Western Blotting , Peso Corporal/fisiología , Vida Libre de Gérmenes , Prueba de Tolerancia a la Glucosa , Glucógeno/metabolismo , Lípidos/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. METHODS: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and ß subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a ß3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. RESULTS: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and ß2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and ß2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. CONCLUSIONS: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for ß3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucemia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Tejido Adiposo Pardo/metabolismo , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/análogos & derivados , Animales , Bencimidazoles/administración & dosificación , Dieta Alta en Grasa , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Ratones , Ratones Noqueados , Modelos Animales , Piridinas/administración & dosificación , Ribonucleótidos/administración & dosificación , Termogénesis/efectos de los fármacosRESUMEN
The objective of this study was to investigate the effects of rosemary (Rosmarinus officinalis L.) leaf extract (RE) on the prevention of weight gain and associated metabolic disorders in mice fed a high-fat diet. For this purpose, RE was administered for 50 days at 20 or 200 mg/kg body weight (BW) to mice fed a high-fat diet. Body weight was monitored during the study and body composition was measured before and at the end of the intervention. Glucose tolerance, assessed by an intraperitoneal glucose tolerance test (IPGTT), and hepatic and faecal lipid contents were determined at the end of the study. Treatment with 200 mg/kg BW of RE induced a significant reduction of weight and fat mass gain (-64% and -57%, respectively) associated with an increase of faecal lipid excretion. This effect appears to be related to the inhibition of pancreatic lipase activity induced by RE, as demonstrated IN VITRO. While glucose tolerance and fasting glycaemia were not affected by RE treatment, hepatic triglyceride levels were decreased by 39% in RE-treated mice. Administration of the lower dose of RE (20 mg/kg BW) was ineffective on all the parameters measured. In conclusion, our results demonstrate that consumption of 200 mg/kg BW of RE can limit weight gain induced by a high-fat diet and protect against obesity-related liver steatosis.
Asunto(s)
Grasas de la Dieta/efectos adversos , Hígado Graso/prevención & control , Extractos Vegetales/farmacología , Hojas de la Planta/química , Rosmarinus/química , Aumento de Peso/efectos de los fármacos , Animales , Composición Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Heces/química , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/químicaRESUMEN
Recent data suggest that the gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type 2 diabetes. To assess this issue, we modulated gut microbiota via antibiotics administration in two different mouse models with insulin resistance. Results from dose-determination studies showed that a combination of norfloxacin and ampicillin, at a dose of 1 g/L, maximally suppressed the numbers of cecal aerobic and anaerobic bacteria in ob/ob mice. After a 2-wk intervention with the antibiotic combination, both ob/ob and diet-induced obese and insulin-resistant mice showed a significant improvement in fasting glycemia and oral glucose tolerance. The improved glycemic control was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the control ob/ob mice. Reduced liver triglycerides and increased liver glycogen correlated with improved glucose tolerance in the treated mice. Concomitant reduction of plasma lipopolysaccharides and increase of adiponectin further supported the antidiabetic effects of the antibiotic treatment in ob/ob mice. In summary, modulation of gut microbiota ameliorated glucose tolerance of mice by altering the expression of hepatic and intestinal genes involved in inflammation and metabolism, and by changing the hormonal, inflammatory, and metabolic status of the host.
Asunto(s)
Ampicilina/farmacología , Bacteroides/efectos de los fármacos , Bifidobacterium/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , Lactobacillus/efectos de los fármacos , Norfloxacino/farmacología , Animales , Antibacterianos/farmacología , Bacteroides/fisiología , Bifidobacterium/fisiología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/fisiopatología , Enterobacteriaceae/fisiología , Lactobacillus/fisiología , Ratones , Ratones Obesos , Pruebas de Sensibilidad Microbiana , Obesidad/microbiología , Obesidad/fisiopatologíaRESUMEN
GLUT8 is a glucose transporter isoform expressed at high levels in testis; at intermediate levels in the brain, including the hippocampus; and at lower levels in the heart and several other tissues. GLUT8 is located in an intracellular compartment and does not appear to translocate to the cell surface, except in blastocysts, where insulin has been reported to induce its surface expression. Here, we generated mice with inactivation of the glut8 gene. We showed that expression of GLUT8 was not required for normal embryonic development and that glut8-/- mice had normal postnatal development, glucose homeostasis, and response to mild stress. Adult glut8-/- mice showed increased proliferation of hippocampal cells but no defect in memory acquisition and retention. Absence of GLUT8 from the heart did not alter heart size and morphology but led to an increase in P-wave duration, which was not associated with abnormal Nav1.5 Na+ channel or connexin expression. Thus, absence of GLUT8 expression in the mouse caused complex but mild physiological alterations.
Asunto(s)
Desarrollo Embrionario , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Corazón/fisiología , Hipocampo/embriología , Organogénesis , Animales , Peso Corporal , Proliferación Celular , Conexinas/genética , Marcación de Gen , Genotipo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/deficiencia , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Corazón/embriología , Hipocampo/citología , Homeostasis/fisiología , Ratones , Ratones Noqueados , Miocardio/citología , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canales de Sodio/genética , XenopusRESUMEN
OBJECTIVE: The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 alpha-subunits assemble with KCNE1 beta-subunits forming a channel complex constituting the delayed rectifier current I(Ks). In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and beta-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention. METHODS AND RESULTS: Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of Nedd4-2, the isoform among the nine members of the Nedd4/Nedd4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of Nedd4-2, able to antagonize endogenous Nedd4-2 in guinea-pig cardiomyocytes, increased I(Ks) significantly, but did not modify I(K1). Concomitant with the reduction in current induced by Nedd4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that Nedd4-2 interacts with the C-terminal part of KCNQ1. The Nedd4/Nedd4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by Nedd4-2, and Nedd4-2 proteins were neither pulled-down nor co-immunoprecipitated. CONCLUSIONS: These results suggest that KCNQ1 internalization and stability is physiologically regulated by its Nedd4/Nedd4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell types.
Asunto(s)
Regulación hacia Abajo , Canal de Potasio KCNQ1/metabolismo , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Expresión Génica , Cobayas , Humanos , Ubiquitina-Proteína Ligasas Nedd4 , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb.
RESUMEN
The gut microbiome and lipid metabolism are both recognized as essential components in the maintenance of metabolic health. The mechanisms involved are multifactorial and (especially for microbiome) poorly defined. A strategic approach to investigate the complexity of the microbial influence on lipid metabolism would facilitate determination of relevant molecular mechanisms for microbiome-targeted therapeutics. E. coli is associated with obesity and metabolic syndrome and we used this association in conjunction with gnotobiotic models to investigate the impact of E. coli on lipid metabolism. To address the complexities of the integration of the microbiome and lipid metabolism, we developed transcriptomics-driven lipidomics (TDL) to predict the impact of E. coli colonization on lipid metabolism and established mediators of inflammation and insulin resistance including arachidonic acid metabolism, alterations in bile acids and dietary lipid absorption. A microbiome-related therapeutic approach targeting these mechanisms may therefore provide a therapeutic avenue supporting maintenance of metabolic health.
RESUMEN
The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1(st) week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice.
Asunto(s)
Dieta Alta en Grasa , Grasas de la Dieta/efectos adversos , Intolerancia a la Glucosa/metabolismo , Obesidad/metabolismo , Animales , Bacteroidetes/clasificación , Bacteroidetes/crecimiento & desarrollo , Trasplante de Microbiota Fecal , Firmicutes/clasificación , Firmicutes/crecimiento & desarrollo , Microbioma Gastrointestinal/fisiología , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/microbiología , Intolerancia a la Glucosa/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/microbiología , Obesidad/patología , Proteobacteria/clasificación , Proteobacteria/crecimiento & desarrolloRESUMEN
Development of NGS has revolutionized the analysis in microbial ecology contributing to our deeper understanding of microbiota in health and disease. However, the quality, quantity and confidence of summarized taxonomic abundances are in need of further scrutiny due to sample dependent and independent effects. In this article we introduce 'AVIT (Abundance and Variability In Taxonomy), an unbiased method to enrich for assigned members of microbial communities. As opposed to using a priori thresholds, 'AVIT uses inherent abundance and variability of taxa in a dataset to determine the inclusion or rejection of each taxa for further downstream analysis. Using in-vitro and in-vivo studies, we benchmarked performance and parameterized 'AVIT to establish a framework for investigating the dynamic range of microbial community membership in clinically relevant scenarios.
Asunto(s)
Microbiota , Algoritmos , Animales , Vida Libre de Gérmenes , Humanos , RatonesRESUMEN
Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3(PAR2/+)), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.
RESUMEN
BACKGROUND: Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. RESULTS: The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry. CONCLUSION: This work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages.
RESUMEN
In this study, we investigated the regulation of Interleukin-18 (IL-18) and caspase-1 mRNA and protein levels in adipose and liver tissue of obese (ob/ob) mice compared with ob/+ mice. In ob/ob mice, which have a twofold higher IL-18 plasma level as compared with lean mice, IL-18 mRNA expression was significantly reduced by 1.6-fold in adipose tissue, whereas protein level was enhanced fourfold as compared with ob/+ mice. However, caspase-1 mRNA expression and activity were significantly enhanced in adipose tissue of ob/ob mice. Conversely, both IL-18 mRNA and protein levels were slightly enhanced, but caspase-1 activity was reduced in liver of ob/ob mice as compared with lean mice. In conclusion, we show that adipose and hepatic IL-18 protein expressions are increased in obese mice. However, in contrast to liver, the adipose IL-18 protein level appears to be upregulated through a post-transcriptional mechanism probably involving caspase-1.
Asunto(s)
Tejido Adiposo/metabolismo , Interleucina-18/metabolismo , Obesidad/metabolismo , Regulación hacia Arriba , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Modelos Animales de Enfermedad , Interleucina-18/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Obesos , ARN Mensajero/metabolismoRESUMEN
Recent data suggest that gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type-2 diabetes. To address this issue, we modulated gut microbiota with two combinations of antibiotics in two different mouse models with insulin resistance. Treatment with norfloxacin and ampicillin for 2 weeks reduced the cecal bacterial DNA below the level of detection in ob/ob, diet-induced obese and insulin resistance (DIO) mice, and significantly improved fasting glycemia and oral glucose tolerance of the treated animals. The enhanced insulin sensitivity was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the untreated ob/ob mice. The reduced liver triglycerides, increased liver glycogen and improved glucose tolerance in the treated mice indicate broad impacts on metabolism by gut decontamination. The treatment with non-absorbable antibiotics polymyxin B and neomycin significantly modified cecal microbiota profile in the DIO mice, and the modified intestinal microbiota was associated with a gradual reduction in glycemia during a washout period. In summary, modulation of gut microbiota ameliorated glucose intolerance in mice and altered the hormonal, inflammatory and metabolic status of the host.