RESUMEN
Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.
Asunto(s)
Resistencia a la Insulina , Fibras Musculares Esqueléticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Anciano , Animales , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Desarrollo de Músculos/genética , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética , Donantes de Tejidos , TransfecciónRESUMEN
A global health concern has emerged as a response to the recent SARS-CoV-2 pandemic. The identification and inhibition of drug targets of SARS-CoV-2 is a decisive obligation of scientists. In addition to the cell entry mechanism, SARS-CoV-2 expresses a complicated replication mechanism that provides excellent drug targets. Papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro) play a vital role in polyprotein processing, producing functional non-structural proteins essential for viral replication and survival in the host cell. Moreover, PLpro is employed by SARS-CoV-2 for reversing host immune responses. Therefore, if some particular compound has the potential to interfere with the proteolytic activities of 3CLpro and PLpro of SARS-CoV-2, it may be effective as a treatment or prophylaxis for COVID-19, reducing viral load, and reinstating innate immune responses. Thus, the present study aims to inhibit SARS-CoV-2 through 3CLpro and PLpro using marine natural products isolated from marine algae that contain numerous beneficial biological activities. Molecular docking analysis was utilized in the present study for the initial screening of selected natural products depending on their 3CLpro and PLpro structures. Based on this approach, Ishophloroglucin A (IPA), Dieckol, Eckmaxol, and Diphlorethohydroxycarmalol (DPHC) were isolated and used to perform in vitro evaluations. IPA presented remarkable inhibitory activity against interesting drug targets. Moreover, Dieckol, Eckmaxol, and DPHC also expressed significant potential as inhibitors. Finally, the results of the present study confirm the potential of IPA, Dieckol, Eckmaxol, and DPHC as inhibitors of SARS-CoV-2. To the best of our knowledge, this is the first study that assesses the use of marine natural products as a multifactorial approach against 3CLpro and PLpro of SARS-CoV-2.
Asunto(s)
Antivirales , COVID-19 , Polifenoles , SARS-CoV-2 , Replicación Viral , Humanos , Antivirales/química , Antivirales/aislamiento & purificación , Antivirales/farmacología , COVID-19/prevención & control , Simulación del Acoplamiento Molecular , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacos , Polifenoles/química , Polifenoles/aislamiento & purificación , Polifenoles/farmacologíaRESUMEN
Transcriptional coactivator with PDZ-binding motif (TAZ) plays crucial role in maintaining testicular structure and function via regulation of senescence of spermatogenic cells. However, it remains unclear whether TAZ is involved in testosterone biosynthesis in testicular Leydig cells. We found that TAZ deficiency caused aberrant Leydig cell expansion and increased lipid droplet formation, which was significantly associated with increased lipogenic enzyme expression. Additionally, the expression of key steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 (CYP) 11A1, CYP17A1, and 3ß-hydroxysteroid dehydrogenase, was greatly increased in TAZ-deficient testes and primary Leydig cells. Interestingly, the transcriptional activity of nuclear receptor 4 A1 (NR4A1) was dramatically suppressed by TAZ; however, the protein expression and the subcellular localization of NR4A1 were not affected by TAZ. TAZ directly associated with the N-terminal region of NR4A1 and substantially suppressed its DNA-binding and transcriptional activities. Stable expression of TAZ in the mouse Leydig TM3 cell line decreased the expression of key steroidogenic enzymes, whereas knockdown of endogenous TAZ in TM3 cells increased transcripts of steroidogenic genes induced by NR4A1. Consistently, testosterone production was enhanced within TAZ-deficient Leydig cells. However, TAZ deficiency resulted in decreased testosterone secretion caused by dysfunctional mitochondria and lysosomes. Therefore, TAZ plays essential role in NR4A1-induced steroidogenic enzyme expression and testosterone production in Leydig cells.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Células Intersticiales del Testículo/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Testosterona/metabolismo , Transactivadores/fisiología , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
Fucoidans are sulfated heteropolysaccharides found in the cell walls of brown seaweeds (Phaeophyceae) and in some marine invertebrates. Generally, fucoidans are composed of significant amounts of L-fucose and sulfate groups, and lesser amounts of arabinose, galactose, glucose, glucuronic acid, mannose, rhamnose, and xylose. In recent years, fucoidans isolated from brown seaweeds have gained considerable attention owing to their promising bioactive properties such as antioxidant, immunomodulatory, anti-inflammatory, antiobesity, antidiabetic, and anticancer properties. Inflammation is a complex immune response that protects the organs from infection and tissue injury. While controlled inflammatory responses are beneficial to the host, leading to the removal of immunostimulants from the host tissues and restoration of structural and physiological functions in the host tissues, chronic inflammatory responses are often associated with the pathogenesis of tumor development, arthritis, cardiovascular diseases, diabetes, obesity, and neurodegenerative diseases. In this review, the authors mainly discuss the studies since 2016 that have reported anti-inflammatory properties of fucoidans isolated from various brown seaweeds, and their potential as a novel functional material for the treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Polisacáridos/farmacología , Algas Marinas , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Organismos Acuáticos , Enfermedades Cardiovasculares/tratamiento farmacológico , Polisacáridos/química , Polisacáridos/uso terapéuticoRESUMEN
BACKGROUND AND AIM: Exogenous 8-hydroxydeoxyguanosine (8-OHdG) was suggested as an inhibitor of Rac1 and NADPH oxidase (NOX). The aim of this study was to evaluate the effects of the exogenous 8-OHdG on hepatic fibrogenesis in vitro and in vivo model of liver fibrosis. METHODS: Adult Sprague-Dawley rats were allocated to sham-operated rats (n = 7), rats that underwent bile duct ligation (BDL) (n = 6), and BDL rats treated with 8-OHdG (60 mg/kg/day by gavage, n = 6). All rats were sacrificed on day 21. Double immunofluorescence staining between either NOX1 or NOX2 and α-smooth muscle actin (SMA) in liver was performed. Hepatic fibrotic contents were assessed by hydroxyproline assay and quantified by Sirius red staining. In vitro, hepatic stellate cell (HSC) line LX-2 and HHSteC cells were stimulated by angiotensin II (10 µM). The reactive oxygen species (ROS) production was measured by confocal microscopy. The expressions of NOX1, NOX2, α-SMA, transforming growth factor (TGF)-ß1, and collagen Iα were analyzed by quantitative real-time polymerase chain reaction or immunoblotting. RESULTS: The 8-OHdG treatment in BDL rats reduced the NOX1 and NOX2 protein expression, which overlapped with α-SMA compared with BDL rats. The 8-OHdG treatment in BDL rats significantly decreased the mRNA expression of NOX1, NOX2, α-SMA, TGF-ß1, and collagen Iα, and fibrotic contents. Increases of ROS production, Rac1 activation, NOX1, NOX2, and fibronectin expression induced by angiotensin II in HSCs were attenuated by 8-OHdG. CONCLUSIONS: Rac1 activation and NOX-derived ROS are implicated to liver fibrosis. The 8-OHdG ameliorates liver fibrosis through the inhibition of Rac1 activation and NOX-derived ROS.
Asunto(s)
8-Hidroxi-2'-Desoxicoguanosina/farmacología , 8-Hidroxi-2'-Desoxicoguanosina/uso terapéutico , Actinas/genética , Actinas/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , NADPH Oxidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína de Unión al GTP rac1/metabolismo , Animales , Línea Celular , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Chronic alcohol feeding increases the levels of 2-arachidonoylglycerol (2-AG) in the liver, which activates hepatic cannabinoid receptor type 1 (CB1R), leading to oxidative liver injury. 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL). However, the mechanisms regulating hepatic DAGL gene expression and 2-AG production are largely unknown. In this study, we show that CB1R-induced estrogen-related receptor γ (ERRγ) controls hepatic DAGL gene expression and 2-AG levels. Arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1R agonist, significantly upregulated ERRγ, DAGLα, and DAGLß, and increased 2-AG levels in the liver (10 mg/kg) and hepatocytes (10 µM) of wild-type (WT) mice. ERRγ overexpression upregulated DAGLα and DAGLß expressions and increased 2-AG levels, whereas ERRγ knockdown abolished ACEA-induced DAGLα, DAGLß, and 2-AG in vitro and in vivo. Promoter assays showed that ERRγ positively regulated DAGLα and DAGLß transcription by binding to the ERR response element in the DAGLα and DAGLß promoters. Chronic alcohol feeding (27.5% of total calories) induced hepatic steatosis and upregulated ERRγ, leading to increased DAGLα, DAGLß, or 2-AG in WT mice, whereas these alcohol-induced effects did not occur in hepatocyte-specific CB1R knockout mice or in those treated with the ERRγ inverse agonist GSK5182 (40 mg/kg in mice and 10 µM in vitro). Taken together, these results indicate that suppression of alcohol-induced DAGLα and DAGLß gene expressions and 2-AG levels by an ERRγ-specific inverse agonist may be a novel and attractive therapeutic approach for the treatment of alcoholic liver disease.
Asunto(s)
Ácidos Araquidónicos/biosíntesis , Ácidos Araquidónicos/farmacología , Endocannabinoides/biosíntesis , Etanol/toxicidad , Glicéridos/biosíntesis , Lipoproteína Lipasa/genética , Receptores de Estrógenos/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Lipoproteína Lipasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptores de Estrógenos/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca2+ concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca2+ homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca2+ acts as a negative regulator of insulin signaling. Chronic intracellular Ca2+ overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca2+ inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca2+ abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 Finally, thermodynamic analysis of the binding interaction showed that Ca2+-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca2+ rather than PH domains to PIPs forming Ca2+-PIPs. Thus, Ca2+-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca2+-PIPs. Our findings provide a mechanistic link between intracellular Ca2+ dysregulation and Akt inactivation in insulin resistance.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Resistencia a la Insulina/fisiología , Fosfatidilinositoles/metabolismo , Dominios Homólogos a Pleckstrina/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Dieta Alta en Grasa , Intolerancia a la Glucosa/patología , Hiperinsulinismo/patología , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/patología , Fosfolipasa C delta/metabolismo , Fosforilación , Unión ProteicaRESUMEN
O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Esfuerzo Físico/fisiología , Acilación/fisiología , Animales , Glucemia/metabolismo , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Noqueados , Condicionamiento Físico Animal/métodosRESUMEN
Peripheral insulin resistance contributes to the development of type 2 diabetes. TCF7L2 has been tightly associated with this disease, although the exact mechanism was largely elusive. Here we propose a novel role of TCF7L2 in hepatic glucose metabolism in mammals. Expression of medium and short isoforms of TCF7L2 was greatly diminished in livers of diet-induced and genetic mouse models of insulin resistance, prompting us to delineate the functional role of these isoforms in hepatic glucose metabolism. Knockdown of hepatic TCF7L2 promoted increased blood glucose levels and glucose intolerance with increased gluconeogenic gene expression in wild-type mice, in accordance with the PCR array data showing that only the gluconeogenic pathway is specifically up-regulated upon depletion of hepatic TCF7L2. Conversely, overexpression of a nuclear isoform of TCF7L2 in high-fat diet-fed mice ameliorated hyperglycemia with improved glucose tolerance, suggesting a role of this factor in hepatic glucose metabolism. Indeed, we observed a binding of TCF7L2 to promoters of gluconeogenic genes; and expression of TCF7L2 inhibited adjacent promoter occupancies of CREB, CRTC2, and FoxO1, critical transcriptional modules in hepatic gluconeogenesis, to disrupt target gene transcription. Finally, haploinsufficiency of TCF7L2 in mice displayed higher glucose levels and impaired glucose tolerance, which were rescued by hepatic expression of a nuclear isoform of TCF7L2 at the physiological level. Collectively, these data suggest a crucial role of TCF7L2 in hepatic glucose metabolism; reduced hepatic expression of nuclear isoforms of this factor might be a critical instigator of hyperglycemia in type 2 diabetes.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Factores de Transcripción Forkhead , Resistencia a la Insulina , Hígado , Proteína 2 Similar al Factor de Transcripción 7 , Animales , Glucemia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Gluconeogénesis , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Humanos , Resistencia a la Insulina/genética , Hígado/metabolismo , Redes y Vías Metabólicas , Ratones , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
AIMS/HYPOTHESIS: Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca(2+)/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo. METHODS: The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4([Symbol: see text])) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic-euglycaemic clamp and isotopic measurements of glucose metabolism. RESULTS: The rate of insulin-stimulated whole-body glucose uptake was increased by â¼25% in CAMK4([Symbol: see text]) mice. This was largely attributed to an increase of â¼60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4([Symbol: see text]) mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4([Symbol: see text]) skeletal muscle. CONCLUSIONS/INTERPRETATION: Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.
Asunto(s)
Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Músculo Esquelético/enzimología , Animales , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Femenino , Masculino , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/genéticaRESUMEN
Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were increased by 12-13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.
Asunto(s)
Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Dióxido de Carbono/metabolismo , Proteínas Portadoras/genética , Desoxiglucosa/metabolismo , Ayuno/sangre , Femenino , Glucógeno/metabolismo , Proteínas de la Matriz de Golgi , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacología , Immunoblotting , Insulina/sangre , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacosRESUMEN
The world-wide prevalence of obesity and diabetes has increased sharply during the last two decades. Accordingly, the metabolic phenotyping of genetically engineered mouse models is critical for evaluating the functional roles of target genes in obesity and diabetes, and for developing new therapeutic targets. In this review, we discuss the practical meaning of metabolic phenotyping, the strategy of choosing appropriate tests, and considerations when designing and performing metabolic phenotyping in mice.
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Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Obesidad/metabolismo , Fenotipo , Animales , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Ratones , Ratones Transgénicos , Obesidad/diagnóstico , Obesidad/genética , Factores de RiesgoRESUMEN
Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.
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Estradiol/farmacología , Melanocortinas/metabolismo , Neuronas/efectos de los fármacos , Obesidad/fisiopatología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anorexia/inducido químicamente , Anorexia/fisiopatología , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/fisiología , Núcleo Arqueado del Hipotálamo/ultraestructura , Peso Corporal/efectos de los fármacos , Estradiol/administración & dosificación , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Inyecciones Intraventriculares , Leptina/genética , Leptina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Microscopía Electrónica , Neuronas/citología , Neuronas/metabolismo , Obesidad/genética , Ovariectomía , Proopiomelanocortina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET. METHODS: Male C57BL6J mice (9 weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA + RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using 2H2O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp. RESULTS: EAA + RET increased muscle mass (10%, P < 0.05) and strength (6%, P < 0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P < 0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA + RET) compared with sedentary groups (10%, P < 0.05), which was associated with increased AchR cluster size and MuSK activation (P < 0.05). EAA + RET also increased endurance capacity more than RET alone (26%, P < 0.05) by increasing both mitochondrial protein synthesis (53%, P < 0.05) and DRP1 activation (P < 0.05). Maximal respiratory capacity increased (P < 0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P < 0.05). CONCLUSIONS: Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.
RESUMEN
Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Metabolismo Energético/fisiología , Glucosa/metabolismo , Homeostasis/fisiología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Mitocondrias/metabolismo , Adiposidad/fisiología , Alelos , Animales , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Metabolismo Energético/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Gluconeogénesis/fisiología , Glucosa/genética , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Homeostasis/efectos de los fármacos , Resistencia a la Insulina/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Mitocondrias/genética , Complejos Multiproteicos , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Serina-Treonina Quinasas TOR , Vía de Señalización Wnt/fisiologíaRESUMEN
The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.
Asunto(s)
Ceramidas/metabolismo , Corazón/fisiopatología , Miocardio/enzimología , Serina C-Palmitoiltransferasa/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Células Cultivadas , Etiquetado Corte-Fin in Situ , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Serina C-Palmitoiltransferasa/genéticaRESUMEN
APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) has been established as an important mediator of insulin and adiponectin signaling. Here, we investigated the influence of transgenic (Tg) APPL1 overexpression in mice on high-fat diet (HFD)-induced cardiomyopathy in mice. Wild-type (WT) mice fed an HFD for 16 wk showed cardiac dysfunction, determined by echocardiography, with decreased ejection fraction, decreased fractional shortening, and increased end diastolic volume. HFD-fed APPL1 Tg mice were significantly protected from this dysfunction. Speckle tracking echocardiography to accurately assess cardiac tissue deformation strain and wall motion also indicated dysfunction in WT mice and a similar improvement in Tg vs. WT mice on HFD. APPL1 Tg mice had less HFD-induced increase in circulating nonesteridied fatty acid levels and myocardial lipid accumulation. Lipidomic analysis using LC-MS-MS showed HFD significantly increased myocardial contents of distinct ceramide, sphingomyelin, and diacylglycerol (DAG) species, of which increases in C16:0 and C18:0 ceramides plus C16:0 and C18:1 DAGs were attenuated in Tg mice. A glucose tolerance test indicated less peripheral insulin resistance in response to HFD in Tg mice, which was also apparent by measuring cardiac Akt phosphorylation and cardiomyocyte glucose uptake. In summary, APPL1 Tg mice exhibit improved peripheral metabolism, reduced cardiac lipotoxicity, and improved insulin sensitivity. These cellular effects contribute to protection from HFD-induced cardiomyopathy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cardiomiopatías/prevención & control , Dieta Alta en Grasa/efectos adversos , Resistencia a la Insulina/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Peso Corporal/fisiología , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Lípidos/sangre , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Complementary metal oxide semiconductor (CMOS) image sensors have been used previously in the analysis of biological samples. In the present study, a CMOS image sensor was used to monitor the concentration of oxidized mouse plasma glucose (86-322 mg dL(-1)) based on photon count variation. Measurement of the concentration of oxidized glucose was dependent on changes in color intensity; color intensity increased with increasing glucose concentration. The high color density of glucose highly prevented photons from passing through the polydimethylsiloxane (PDMS) chip, which suggests that the photon count was altered by color intensity. Photons were detected by a photodiode in the CMOS image sensor and converted to digital numbers by an analog to digital converter (ADC). Additionally, UV-spectral analysis and time-dependent photon analysis proved the efficiency of the detection system. This simple, effective, and consistent method for glucose measurement shows that CMOS image sensors are efficient devices for monitoring glucose in point-of-care applications.
Asunto(s)
Técnicas Biosensibles/métodos , Glucosa/análisis , Semiconductores , Animales , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Espectrofotometría Ultravioleta/métodosRESUMEN
Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved in the regulation of hepatic glucose metabolism in mice. Expression of hepatic SMEK1/2 is up-regulated during fasting or in mouse models of insulin-resistant conditions in a Peroxisome Proliferator-Activated Receptor-gamma Coactivator 1α (PGC-1α)-dependent manner. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas depletion of the SMEK proteins reduces hyperglycemia and enhances CRTC2 phosphorylation; the effect is blunted by S171A CRTC2, which is refractory to salt-inducible kinase (SIK)-dependent inhibition. Taken together, we would propose that mammalian SMEK/PP4C proteins are involved in the regulation of hepatic glucose metabolism through dephosphorylation of CRTC2.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Gluconeogénesis/fisiología , Hígado/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Transactivadores/metabolismo , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Inmunoprecipitación , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación , Subunidades de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TranscripciónRESUMEN
Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.