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AIMS/HYPOTHESIS: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. METHODS: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. RESULTS: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). CONCLUSIONS/INTERPRETATION: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , RatonesRESUMEN
BACKGROUND AND PURPOSE: Activation of hepatic thyroid hormone receptor ß (THR-ß) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-ß agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-ß agonist, as a prototype to investigate the effects of THR-ß activation in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis. EXPERIMENTAL APPROACH: C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg-1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-ß activation. KEY RESULTS: Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis. CONCLUSION AND IMPLICATIONS: Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Hígado/patología , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/patología , Receptores beta de Hormona Tiroidea/genéticaRESUMEN
The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.
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Pre-clinical studies show that dietary protein restriction (DPR) improves healthspan and retards many age-related diseases such as type 2 diabetes. While mouse studies have shown that restriction of certain essential amino acids is required for this response, less is known about which amino acids are affected by DPR in humans. Here, using a within-subjects diet design, we examined the effects of dietary protein restriction in the fasted state, as well as acutely after meal feeding, on blood plasma amino acid levels. While very few amino acids were affected by DPR in the fasted state, several proteinogenic AAs such as isoleucine, leucine, lysine, phenylalanine, threonine, tyrosine, and valine were lower in the meal-fed state with DPR. In addition, the non-proteinogenic AAs such as 1- and 3-methyl-histidine were also lower with meal feeding during DPR. Lastly, using in silico predictions of the most limiting essential AAs compared with human exome AA usage, we demonstrate that leucine, methionine, and threonine are potentially the most limiting essential AAs with DPR. In summary, acute meal feeding allows more accurate determination of which AAs are affected by dietary interventions, with most essential AAs lowered by DPR.
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Aminoácidos/sangre , Dieta con Restricción de Proteínas , Proteínas en la Dieta , Adulto , Aminoácidos Esenciales/sangre , Diabetes Mellitus Tipo 2 , Ayuno , Humanos , Isoleucina , MasculinoRESUMEN
Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.
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Aminoácidos Esenciales/deficiencia , Alimentación Animal , Proteinuria/metabolismo , Animales , Proteínas en la Dieta/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Hormonas Gastrointestinales/metabolismo , Hepatocitos/metabolismo , Homeostasis , Hígado/metabolismo , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Fenotipo , Treonina/deficiencia , Triptófano/deficienciaRESUMEN
AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.
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Proteínas Quinasas Activadas por AMP/metabolismo , Diferenciación Celular , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Línea Celular , Linaje de la Célula , Factor de Transcripción GATA4/metabolismo , Proteína Homeótica Nkx-2.5/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , TranscriptomaRESUMEN
BACKGROUND: Epidemiological studies have shown that increased circulating branched-chain amino acids (BCAAs) are associated with insulin resistance and type 2 diabetes (T2D). This may result from altered energy metabolism or dietary habits. OBJECTIVE: We hypothesized that a lower intake of BCAAs improves tissue-specific insulin sensitivity. METHODS: This randomized, placebo-controlled, double-blinded, crossover trial examined well-controlled T2D patients receiving isocaloric diets (protein: 1 g/kg body weight) for 4 wk. Protein requirements were covered by commercially available food supplemented ≤60% by an AA mixture either containing all AAs or lacking BCAAs. The dietary intervention ensured sufficient BCAA supply above the recommended minimum daily intake. The patients underwent the mixed meal tolerance test (MMT), hyperinsulinemic-euglycemic clamps (HECs), and skeletal muscle and white adipose tissue biopsies to assess insulin signaling. RESULTS: After the BCAA- diet, BCAAs were reduced by 17% during fasting (P < 0.001), by 13% during HEC (P < 0.01), and by 62% during the MMT (P < 0.001). Under clamp conditions, whole-body and hepatic insulin sensitivity did not differ between diets. After the BCAA- diet, however, the oral glucose sensitivity index was 24% (P < 0.01) and circulating fibroblast-growth factor 21 was 21% higher (P < 0.05), whereas meal-derived insulin secretion was 28% lower (P < 0.05). Adipose tissue expression of the mechanistic target of rapamycin was 13% lower, whereas the mitochondrial respiratory control ratio was 1.7-fold higher (both P < 0.05). The fecal microbiome was enriched in Bacteroidetes but depleted of Firmicutes. CONCLUSIONS: Short-term dietary reduction of BCAAs decreases postprandial insulin secretion and improves white adipose tissue metabolism and gut microbiome composition. Longer-term studies will be needed to evaluate the safety and metabolic efficacy in diabetes patients.This trial was registered at clinicaltrials.gov as NCT03261362.
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Aminoácidos de Cadena Ramificada/administración & dosificación , Diabetes Mellitus Tipo 2/microbiología , Microbioma Gastrointestinal , Secreción de Insulina , Tejido Adiposo Blanco/metabolismo , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Masculino , Comidas , Persona de Mediana Edad , Mitocondrias/fisiología , Músculo Esquelético/metabolismoRESUMEN
The transcription factor Nrf2 controls pathways involved in oxidative-stress defense and is a potential pharmacological target for the treatment of chronic diseases. Activators of Nrf2 that have undergone clinical development are reactive molecules that are either associated with safety issues or for which it is unclear if their pharmacological efficacy depends on the activation of Nrf2. Therefore, the clinical validity of Nrf2 activation is not yet proven. The activity of Nrf2 is inhibited by Keap1 via a protein-protein interaction. Its structural characteristics allowed the identification of reversible small-molecule inhibitors of the Keap1-Nrf2 interaction that can hopefully elucidate the therapeutic potential of Nrf2 activation.
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Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Animales , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/química , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Dietary protein dilution (PD) has been associated with metabolic advantages such as improved glucose homeostasis and increased energy expenditure. This phenotype involves liver-induced release of FGF21 in response to amino acid insufficiency; however, it has remained unclear whether dietary dilution of specific amino acids (AAs) is also required. Circulating branched chain amino acids (BCAAs) are sensitive to protein intake, elevated in the serum of obese humans and mice and thought to promote insulin resistance. We tested whether replenishment of dietary BCAAs to an AA-diluted (AAD) diet is sufficient to reverse the glucoregulatory benefits of dietary PD. METHODS: We conducted AA profiling of serum from healthy humans and lean and high fat-fed or New Zealand obese (NZO) mice following dietary PD. We fed wildtype and NZO mice one of three amino acid defined diets: control, total AAD, or the same diet with complete levels of BCAAs (AAD + BCAA). We quantified serum AAs and characterized mice in terms of metabolic efficiency, body composition, glucose homeostasis, serum FGF21, and tissue markers of the integrated stress response (ISR) and mTORC1 signaling. RESULTS: Serum BCAAs, while elevated in serum from hyperphagic NZO, were consistently reduced by dietary PD in humans and murine models. Repletion of dietary BCAAs modestly attenuated insulin sensitivity and metabolic efficiency in wildtype mice but did not restore hyperglycemia in NZO mice. While hepatic markers of the ISR such as P-eIF2α and FGF21 were unabated by dietary BCAA repletion, hepatic and peripheral mTORC1 signaling were fully or partially restored, independent of changes in circulating glucose or insulin. CONCLUSIONS: Repletion of BCAAs in dietary PD is sufficient to oppose changes in somatic mTORC1 signaling but does not reverse the hepatic ISR nor induce insulin resistance in type 2 diabetes during dietary PD.
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Aminoácidos de Cadena Ramificada/metabolismo , Proteínas en la Dieta/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Adulto , Aminoácidos de Cadena Ramificada/sangre , Animales , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0166110.].
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BACKGROUND AND PURPOSE: The neutral amino acid transporter B0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B0 AT1 mediates the Na+ -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B0 AT1. EXPERIMENTAL APPROACH: A CHO-based cell line was generated, stably expressing collectrin and B0 AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. KEY RESULTS: We characterized a series of novel inhibitors of the B0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC50 of 44 ± 9 µM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine. CONCLUSION AND IMPLICATIONS: The tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry.
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Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Benzotropina/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animales , Benzotropina/química , Células CHO , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Drosophila , Femenino , Ensayos Analíticos de Alto Rendimiento , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Non-alcoholic fatty liver disease is a world-wide health concern and risk factor for cardio-metabolic diseases. Citrate uptake modifies intracellular hepatic energy metabolism and is controlled by the conserved sodium-dicarboxylate cotransporter solute carrier family 13 member 5 (SLC13A5, mammalian homolog of INDY: mINDY). In Drosophila melanogaster and Caenorhabditis elegans INDY reduction decreased whole-body lipid accumulation. Genetic deletion of Slc13a5 in mice protected from diet-induced adiposity and insulin resistance. We hypothesized that inducible hepatic mINDY inhibition should prevent the development of fatty liver and hepatic insulin resistance. METHODS: Adult C57BL/6J mice were fed a Western diet (60% kcal from fat, 21% kcal from carbohydrate) ad libitum. Knockdown of mINDY was induced by weekly injection of a chemically modified, liver-selective siRNA for 8 weeks. Mice were metabolically characterized and the effect of mINDY suppression on glucose tolerance as well as insulin sensitivity was assessed with an ipGTT and a hyperinsulinemic-euglycemic clamp. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry. RESULTS: Within the 8 week intervention, hepatic mINDY expression was suppressed by a liver-selective siRNA by over 60%. mINDY knockdown improved hepatic insulin sensitivity (i.e. insulin-induced suppression of endogenous glucose production) of C57BL/6J mice in the hyperinsulinemic-euglycemic clamp. Moreover, the siRNA-mediated mINDY inhibition prevented neutral lipid storage and triglyceride accumulation in the liver, while we found no effect on body weight. CONCLUSIONS: We show that inducible mINDY inhibition improved hepatic insulin sensitivity and prevented diet-induced non-alcoholic fatty liver disease in adult C57BL6/J mice. These effects did not depend on changes of body weight or body composition.
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Transportadores de Ácidos Dicarboxílicos/fisiología , Resistencia a la Insulina , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico , Interferencia de ARN , Simportadores/fisiología , Animales , Citratos , Ácido Cítrico , Dieta , Ratones , Ratones Endogámicos C57BLRESUMEN
The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti-oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice.
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Metabolismo de los Hidratos de Carbono/fisiología , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Transducción de Señal/fisiologíaRESUMEN
Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis.
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Diabetes Mellitus Tipo 2/metabolismo , Proteínas en la Dieta/administración & dosificación , Hígado/metabolismo , Tejido Adiposo/metabolismo , Adulto , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Metabolismo de los Hidratos de Carbono , Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucosa/metabolismo , Hepatocitos/metabolismo , Homeostasis , Humanos , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Neoplasias/metabolismo , Obesidad/metabolismo , Fenotipo , Proteína Desacopladora 1/metabolismoRESUMEN
The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of â¼6 µM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 µM RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Proteína 1 Asociada A ECH Tipo Kelch , Masculino , Ratones , Unión Proteica , Pirrolidinas/metabolismo , Sulfonamidas/metabolismoRESUMEN
Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B(0)AT1/3 fusion constructs, we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B(0)AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.
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Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animales , Sitios de Unión , Biotinilación , Células CHO , Catálisis , Cricetinae , Cricetulus , Drosophila melanogaster , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Mutagénesis , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Sintaxina 1/metabolismo , Xenopus laevisRESUMEN
OBJECTIVE: Type 2 diabetes arises from insulin resistance of peripheral tissues followed by dysfunction of ß-cells in the pancreas due to metabolic stress. Both depletion and supplementation of neutral amino acids have been discussed as strategies to improve insulin sensitivity. Here we characterise mice lacking the intestinal and renal neutral amino acid transporter B(0)AT1 (Slc6a19) as a model to study the consequences of selective depletion of neutral amino acids. METHODS: Metabolic tests, analysis of metabolite levels and signalling pathways were used to characterise mice lacking the intestinal and renal neutral amino acid transporter B(0)AT1 (Slc6a19). RESULTS: Reduced uptake of neutral amino acids in the intestine and loss of neutral amino acids in the urine causes an overload of amino acids in the lumen of the intestine and reduced systemic amino acid availability. As a result, higher levels of glucagon-like peptide 1 (GLP-1) are produced by the intestine after a meal, while the liver releases the starvation hormone fibroblast growth factor 21 (FGF21). The combination of these hormones generates a metabolic phenotype that is characterised by efficient removal of glucose, particularly by the heart, reduced adipose tissue mass, browning of subcutaneous white adipose tissue, enhanced production of ketone bodies and reduced hepatic glucose output. CONCLUSIONS: Reduced neutral amino acid availability improves glycaemic control. The epithelial neutral amino acid transporter B(0)AT1 could be a suitable target to treat type 2 diabetes.
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AIMS/HYPOTHESIS: Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage. METHODS: Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1ß, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice. RESULTS: Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines. CONCLUSIONS/INTERPRETATION: Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Inhibidores Enzimáticos/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pau d'arco is a plant-derived traditional medicine that acts by poorly understood molecular mechanisms. Here, we studied the effect of pau d'arco on the cytoprotective transcription factor Nrf2. An aqueous extract of pau d'arco stimulated Nrf2-dependent gene expression and led to nuclear localization of Nrf2 in vitro. Chromatographic separation and mass spectrometry of the extract identified benzene trioles or benzene tetraoles within the active fractions. The extract stimulated the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK1/2) pathway. The pharmacological inhibition of MEK, but not of p38 mitogen-activated protein kinase, glycogen synthase kinase-3 or phosphoinositide 3-kinase was required for the activation of Nrf2-dependent gene expression by pau d'arco, but not for the nuclear translocation of Nrf2. In vivo pau d'arco increased the expression of Nrf2-target genes in the intestine. The results suggest that the activation of Nrf2 could mediate beneficial effects of pau d'arco, in particular in the intestine.
Asunto(s)
Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Medicina Tradicional , Terapia Molecular Dirigida , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Fitoterapia , Extractos Vegetales/farmacología , Tabebuia , Transporte Activo de Núcleo Celular , Animales , Femenino , Hemo-Oxigenasa 1/metabolismo , Células Hep G2 , Humanos , Intestinos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína 1 Asociada A ECH Tipo Kelch , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estimulación Química , AguaRESUMEN
Acetyl CoA carboxylase isoforms 1 and 2 (ACC1/2) are key enzymes of fat utilization and their inhibition is considered to improve aspects of the metabolic syndrome. To identify pharmacological inhibitors of ACC1/2, a high throughput screen was performed which resulted in the identification of the lead compound 3 ( Gargazanli , G. ; Lardenois , P. ; Frost , J. ; George , P. Patent WO9855474 A1, 1998 ) as a moderate selective ACC2 inhibitor. Optimization of 3 led to 4m ( Zoller , G. ; Schmoll , D. ; Mueller , M. ; Haschke , G. ; Focken , I. Patent WO2010003624 A2, 2010 ) as a submicromolar dual ACC1/2 inhibitor of the rat and human isoforms. 4m possessed favorable pharmacokinetic parameters. This compound stimulated fat oxidation in vivo and reduced plasma triglyceride levels in a rodent model after subchronic administration. 4m is a suitable tool compound for the elucidation of the pharmacological potential of ACC1/2 inhibition.