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Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/radioterapia , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/radioterapia , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/cirugía , Terapia Combinada , Femenino , Neoplasias de Cabeza y Cuello/cirugía , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Neoplasias Cutáneas/cirugía , Resultado del TratamientoRESUMEN
BACKGROUND AND PURPOSE: The sarcoplasmic reticulum Ca²âº-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²âº transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents. EXPERIMENTAL APPROACH: Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. KEY RESULTS: Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E1-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²âº at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS: NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²âº-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.
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Adenosina Trifosfato/metabolismo , Ácidos Araquidónicos/farmacología , Dopamina/análogos & derivados , Fibras Musculares Esqueléticas/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Adulto , Animales , Calcio/metabolismo , Células Cultivadas , Dopamina/farmacología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
The perilipin proteins enclose intracellular lipid droplets. We describe the mRNA expression of the five perilipins in human skeletal muscle in relation to fatty acid supply, exercise and energy balance. We observed that all perilipins were expressed in skeletal muscle biopsies with the highest mRNA levels of perilipin 2, 4 and 5. Cultured myotubes predominantly expressed perilipin 2 and 3. In vitro, incubation of myotubes with fatty acids enhanced mRNA expression of perilipin 1, 2 and 4. In vivo, low fat diet increased mRNA levels of perilipin 3 and 4. Endurance training, but not strength training, enhanced the expression of perilipin 2 and 3. Perilipin 1 mRNA correlated positively with body fat mass, whereas none of the perilipins were associated with insulin sensitivity. In conclusion, all perilipins mRNAs were expressed in human skeletal muscle. Diet as well as endurance exercise modulated the expression of perilipins.
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Proteínas Portadoras/metabolismo , Ácidos Grasos/farmacología , Expresión Génica/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fosfoproteínas/metabolismo , ARN Mensajero/biosíntesis , Tejido Adiposo , Anciano , Proteínas Portadoras/genética , Técnicas de Cultivo de Célula , Dieta , Grasas de la Dieta/metabolismo , Metabolismo Energético/fisiología , Ejercicio Físico/fisiología , Femenino , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Especificidad de Órganos , Perilipina-1 , Fosfoproteínas/genética , Resistencia Física/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
CONTEXT: Increased oxidative stress and mitochondrial dysfunction have been implicated in the development of insulin resistance in type 2 diabetes. To date, it is unknown whether increased mitochondrial reactive oxygen species (ROS) production in skeletal muscle from patients with type 2 diabetes is primarily increased or a secondary adaptation to environmental, lifestyle, and hormonal factors. OBJECTIVE: This study investigates whether ROS production is primarily increased in isolated diabetic myotubes. SETTING: Mitochondrial membrane potential, hydrogen peroxide (H(2)O(2)), superoxide, and mitochondrial mass were determined in human myotubes precultured under normophysiological conditions. Furthermore, the corresponding ATP synthesis was measured in isolated mitochondria. PATIENTS: Muscle biopsies were taken from 10 lean subjects, 10 obese subjects, and 10 subjects with type 2 diabetes; satellite cells were isolated, cultured, and differentiated to myotubes. RESULTS: Mitochondrial mass, membrane potential/mitochondrial mass, and superoxide-production/mitochondrial mass were not different between groups. In contrast, H(2)O(2) production/mitochondrial mass and ATP production were significantly reduced in diabetic myotubes compared to lean controls (P < 0.05). The ATP/H(2)O(2) ratios were not significantly different between groups. CONCLUSIONS: Our result indicates that the ROS production is not primarily increased in diabetic myotubes but rather is reduced. Moreover, the comparable ATP/H(2)O(2) ratios indicate that the reduced ROS production in diabetic myotubes parallels the reduced ATP production because ROS production in diabetic myotubes must be considered to be in a proportion comparable to lean. Thus, the increased ROS production seen in skeletal muscle of type 2 diabetic patients is an adaptation to the in vivo conditions.
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Diabetes Mellitus Tipo 2/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Diferenciación Celular , Células Cultivadas , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Persona de Mediana Edad , Obesidad/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.
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Factor de Transcripción Activador 6/metabolismo , Retículo Endoplásmico/metabolismo , Glucosamina/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Factor de Transcripción Activador 6/genética , Análisis de Varianza , Animales , Western Blotting , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Glucosamina/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Transportador de Glucosa de Tipo 4/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Resistencia a la Insulina/fisiología , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Persona de Mediana Edad , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Fibras Musculares Esqueléticas/fisiología , Factor Nuclear 1 de Respiración/genética , Fosforilación Oxidativa , Factores de Transcripción/genética , Transcripción Genética , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/complicaciones , Humanos , Persona de Mediana Edad , Músculo Esquelético/fisiología , Obesidad/complicaciones , Obesidad/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
AIMS/HYPOTHESIS: Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). METHODS: Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). CONCLUSIONS/INTERPRETATION: T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased.
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Proteínas de Unión al ADN/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/fisiopatología , Glucosa/metabolismo , Lípidos/fisiología , Obesidad/fisiopatología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Glucemia/metabolismo , Índice de Masa Corporal , Células Cultivadas , Diabetes Mellitus Tipo 2/complicaciones , Hemoglobina Glucada/metabolismo , Humanos , Insulina/sangre , Receptores X del Hígado , Persona de Mediana Edad , Obesidad/complicaciones , Receptores Nucleares Huérfanos , Delgadez/sangreRESUMEN
BACKGROUND: Alterations in glucocorticoid hormone metabolism in skeletal muscle have been suggested to contribute to the pathogenesis of the metabolic syndrome. Circulating glucocorticoids consist of inactive cortisone and active cortisol interconverted in various tissues by the enzyme 11beta hydroxysteroid dehydrogenase (HSD). This study aims to investigate whether human myotubes established from healthy obese and matched obese type 2 diabetic (T2D) subjects reveal differences in the expression level of glucocorticoid receptor (GR) and 11beta hydroxysteroid dehydrogenase (HSD1 and HSD2), and to investigate whether chronic exposure to cortisone affects glucose transport. METHODS: In myotubes established from T2D and healthy control subjects we determined the mRNA expression of HSD1, HSD2, GR and determined basal and insulin-stimulated glucose uptake in myotubes precultured with cortisone, cortisol and the HSD1 inhibitor, carbenoxolone for four days. RESULTS: Myotubes established from T2D subjects showed an increased expression of HSD1 mRNA, but with no differences in mRNA of GRalpha, LXRalpha and LXRbeta, whereas HSD2 mRNA was not expressed. Cortisone reduced glucose uptake in diabetic myotubes and the cortisone effect could be abolished by the HSD1 inhibitor carbenoxolone. CONCLUSIONS: Our study shows that cortisone reduces glucose uptake in diabetic myotubes and that this effect seems mediated by an increased mRNA HSD1 expression emphasizing that the local conversion of inactive to active glucocorticoids may be important in the pathogenesis of insulin resistance.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Fibras Musculares Esqueléticas/enzimología , Obesidad/enzimología , Receptores de Glucocorticoides/metabolismo , Carbenoxolona/farmacología , Cortisona/farmacología , Proteínas de Unión al ADN/metabolismo , Humanos , Hidrocortisona/farmacología , Resistencia a la Insulina/fisiología , Receptores X del Hígado , Persona de Mediana Edad , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.
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Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/metabolismo , Adulto , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Western Blotting , Diabetes Mellitus Tipo 2/metabolismo , Feto/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Obesidad/metabolismo , Polimiositis/metabolismoRESUMEN
We have recently reported a progressive decline in the expression of glucose transporter isoform 4 (GLUT4) from control subjects through obese non-diabetics to obese type 2 diabetic subjects, indicating that the reduced GLUT4 in slow twitch fibres could be secondary to obesity. In this study we investigate the association of GLUT4 expression with the intracellular triglyceride (TG) content in the same muscle fibres and with plasma lipid parameters. We used histochemistry and stereology to study the relationship between TG content and GLUT4 expression in muscle fibres from obese, obese type 2 diabetic subjects, and young lean controls. TG density was significantly higher in slow compared to fast fibres in all studied subjects (p<0.05). We found an increased TG density in slow twitch fibres of obese diabetic subjects compared to obese (p<0.05) and lean controls (p<0.008). Intracellular TG densities in slow and fast fibres did not correlate with the corresponding GLUT4 density in the same fibres in our study groups (p>0.05). Plasma TG and FFA did not correlate with GLUT4 expression in slow or fast fibres (p>0.05). In conclusion, TG content was increased in diabetic slow fibres with a reduced GLUT4 expression. The GLUT4 expression was not associated with an increased intracellular triglyceride content or with increased plasma FFA levels. Thus, intracellular TG content and circulating FFA may not influence glucose transport directly through GLUT4 expression.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus/metabolismo , Resistencia a la Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Triglicéridos/metabolismo , Adulto , Biomarcadores/análisis , Glucemia/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus Tipo 2/sangre , Transportador de Glucosa de Tipo 4 , Humanos , Insulina/sangre , Lípidos/sangre , Persona de Mediana Edad , Fibras Musculares de Contracción Lenta/citología , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/patología , Músculo Esquelético/citología , Músculo Esquelético/patología , Miosinas/metabolismo , Obesidad/sangre , Valores de ReferenciaRESUMEN
The presence of the GLUT3 glucose transporter protein in human muscle cells is a matter of debate. The present study was designed to establish whether GLUT3 is expressed in mature human skeletal muscle fibres and, if so, whether its expression changes under different conditions, such as metabolic stress (obesity, obese non-insulin-dependent diabetes mellitus), hypertrophy (training), de- and reinnervation (amyotrophic lateral sclerosis) or regeneration (polymyositis). We used an immunohistochemical approach to detect and localise GLUT3. GLUT3 immunoreactivity was not detectable in adult skeletal muscle fibres, nor did metabolic stress, training or de- and re-innervation induce GLUT3 expression, while a few GLUT3 expressing fibres were seen in some cases of polymyositis. In contrast, GLUT4 was expressed in all investigated muscle fibres. GLUT3 immunoreactivity was found in perineural and endoneural cells, indicating that GLUT3 is important for glucose transport into nerves through the perineurium. Taken together, these data suggest that GLUT3 expression is restricted to regenerating muscle fibres and nerves in adult human muscle. Although the significance of GLUT3 in adult human muscle fibres appears limited, GLUT3 may be of importance for the glucose supply in fetal muscle fibres and regenerating adult muscle fibres.
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Esclerosis Amiotrófica Lateral/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/embriología , Músculo Esquelético/fisiopatología , Proteínas del Tejido Nervioso , Polimiositis/fisiopatología , Regeneración/fisiología , Adulto , Preescolar , Feto/metabolismo , Transportador de Glucosa de Tipo 3 , Humanos , Inmunohistoquímica , Lactante , Persona de Mediana Edad , Músculo Esquelético/metabolismoRESUMEN
In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane, GLUT4 was more abundantly expressed in slow as compared to fast fibres at the same fibre diameter (p < 0.01) and the GLUT4 expression increased with increasing fibre radius independently of fibre type (p < 0.01). The GLUT4 density at the surface of slow fibres of both diabetic and obese was reduced compared to control subjects at the same diameter (p < 0.001). Fast fibres in obese and type 2 diabetic subjects expressed a fibre-volume-dependent GLUT4 expression (p < 0.001), while this did not reach significance in slow fibres (obese p = 0.18 and diabetic p = 0.06). Our results show that increasing fibre volume is associated with increasing GLUT4 expression in both slow and fast fibres. Based on the possible dependency of GLUT4 expression on volume, we hypothesize that the reduced GLUT4 expression in obesity and type 2 diabetes may partly be compensated for by physical activity.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Adulto , Transporte Biológico , Tamaño de la Célula , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Inmunohistoquímica , Masculino , Matemática , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/análisis , Proteínas de Transporte de Monosacáridos/biosíntesis , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/ultraestructura , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/ultraestructura , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Lenta/ultraestructura , Músculo Esquelético/citología , Músculo Esquelético/ultraestructura , Miosinas/análisis , Obesidad/metabolismoRESUMEN
There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased the sensitivity of GS to the allosteric activator glucose 6-phosphate (A(0.5)) and increased the sensitivity of GS to its substrate UDPG (K(m(0.1))) when myotubes were precultured at low insulin with/without high glucose conditions. However, this effect of acute insulin stimulation was abolished in myotubes precultured at high insulin with or without high glucose. Furthermore, we found significant correlations between the fractional velocities FV(0.1) of GS and K(m(0.1)) (rho=-0.72, P<0.0001), between FV(0.1) and A(0.5) (rho=-0.82, P<0.0001) and between K(m(0.1)) and A(0.5) values (rho=0.71, P<0.0001). Our results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin stimulation to decrease A(0.5) and K(m(0.1)) in human skeletal muscle.
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Glucógeno Sintasa/metabolismo , Insulina/farmacología , Fibras Musculares Esqueléticas/metabolismo , Biopsia , Células Cultivadas , Glucosa/farmacología , Glucógeno/metabolismo , Humanos , Inmunohistoquímica , Resistencia a la Insulina , Cinética , Fibras Musculares Esqueléticas/efectos de los fármacos , Miosinas/metabolismoRESUMEN
The influence of training on GLUT4 expression in slow- and fast-twitch skeletal muscle fibres was studied in male endurance-trained athletes and control subjects. The trained state was ensured by elevated maximal oxygen uptake (29%), as well as citrate synthase (60%) and 3-hydroxy-acyl-CoA dehydrogenase (38%) activities in muscle biopsy samples of the vastus lateralis. GLUT4 densities in slow- and fast-twitch fibres were measured by the use of a newly developed, sensitive method combining immunohistochemistry with morphometry, and no effect of training was found. GLUT4 density was higher in slow-twitch fibres compared to fast-twitch fibres (P<0.05) when biopsy samples from untrained subjects were examined. In athletes GLUT4 density was identical in slow- and fast-twitch fibres. Slow-twitch fibre diameters were 10% larger in the athletes (P<0.01), and slow-twitch fibre fractions were 140% of the fraction in the control group. Thus, GLUT4 originating from slow-twitch fibres was increased by 30% (P<0.02) in athletes. We conclude that long-lasting endurance training increases slow-twitch fibre GLUT4 expression by means of an elevated slow-twitch fibre mass in human skeletal muscle.
Asunto(s)
Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Musculares , Educación y Entrenamiento Físico , Deportes , Adulto , Transportador de Glucosa de Tipo 4 , Humanos , Inmunohistoquímica , Masculino , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/ultraestructura , Fibras Musculares de Contracción Lenta/ultraestructuraRESUMEN
To gain further insight into the mechanisms underlying muscle insulin resistance, the influence of obesity and type 2 diabetes on GLUT4 immunoreactivity in slow and fast skeletal muscle fibers was studied. Through a newly developed, very sensitive method using immunohistochemistry combined with morphometry, GLUT4 density was found to be significantly higher in slow compared with fast fibers in biopsy specimens from lean and obese subjects. In contrast, in type 2 diabetic subjects, GLUT4 density was significantly lower in slow compared with fast fibers. GLUT4 density in slow fibers from diabetic patients was reduced by 9% compared with the weight-matched obese subjects and by 18% compared with the lean control group. The slow-fiber fraction was reduced to 86% in the obese subjects and to 75% in the diabetic subjects compared with the control group. Estimated GLUT4 contribution from slow fibers was reduced to 77% in the obese subjects and to 61% in type 2 diabetic patients compared with the control subjects. We propose that a reduction in the fraction of slow-twitch fibers, combined with a reduction in GLUT4 expression in slow fibers, may reduce the insulin-sensitive GLUT4 pool in type 2 diabetes and thus contribute to skeletal muscle insulin resistance.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Musculares , Adulto , Western Blotting , Diabetes Mellitus Tipo 2/fisiopatología , Transportador de Glucosa de Tipo 4 , Humanos , Resistencia a la Insulina , Masculino , Persona de Mediana Edad , Fibras Musculares de Contracción Rápida/metabolismo , Obesidad/metabolismo , Valores de ReferenciaRESUMEN
Primary satellite cell cultures have become an important tool as a model system for skeletal muscles. A common problem in human satellite cell culturing is fibroblast overgrowth. We combined N-CAM (Leu19) immunocytochemical staining of satellite cells (Sc) with stereological methods to estimate the fraction of Sc in culture. Evaluation of different culture conditions allowed us to find proliferation conditions preferentially for Sc: a) Sc should be cultured on surfaces coated with ECM-gel. b) Primary cell culture should be inoculated in DMEM supplemented with 10% fetal calf serum to increase cell adherence. c) Change of media to DMEM supplemented with 2% Ultroser-G and 2% FCS after 24 h.d) Before subcultivation, cells should be preplated for 30 min. The fractional content of Sc in passage four when applying this method of cultivation was 0.82 +/- 0.07 (mean +/- SE, N = 10). Our method enabled us to establish culture conditions which resulted in high Sc content despite several subcultivations. Estimation of the fractional cell content could be a useful tool for optimizing not only Sc-culturing but all cultures initially containing more cell types.
Asunto(s)
Músculo Esquelético/citología , Células Madre/citología , Técnicas de Cultivo de Célula/métodos , División Celular , Separación Celular/métodos , Células Cultivadas , Medios de Cultivo , Humanos , Inmunohistoquímica , Músculo Esquelético/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células Madre/metabolismoRESUMEN
The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc-cultures. In order to identify the differentiation conditions which give a good survival of myotubes and a high grade of differentiation, Sc-cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK-activities in cultures differentiating in FCS-supplemented media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between days 4-10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8-myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with clear cross striation. We recommend the use of differentiated Sc-cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation-related changes will take place in the cells during this period of time.
Asunto(s)
Modelos Biológicos , Músculo Esquelético/citología , Células Madre/citología , Adenosina Trifosfato/metabolismo , Diferenciación Celular , Células Cultivadas , Creatina Quinasa/metabolismo , Forma MM de la Creatina-Quinasa , ADN/metabolismo , Humanos , Inmunohistoquímica , Isoenzimas/metabolismo , Microscopía Electrónica , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Células Madre/metabolismoRESUMEN
Prompted by our recent observations that GLUT-1 is expressed in fetal muscles, but not in adult muscle fibers, we decided to investigate whether GLUT-1 expression could be reactivated. We studied different stimuli concerning their ability to induce GLUT-1 expression in mature human skeletal muscle fibers. Metabolic stress (obesity, non-insulin-dependent diabetes mellitus), contractile activity (training), and conditions of de- and reinnervation (amyotrophic lateral sclerosis) could not induce GLUT-1 expression in human muscle fibers. However, regenerating muscle fibers in polymyositis expressed GLUT-1. In contrast to GLUT-1, GLUT-4 was expressed in all investigated muscle fibers. Although the significance of GLUT-1 in adult human muscle fibers appears limited, GLUT-1 may be of importance for the glucose supplies in immature and regenerating muscle.
Asunto(s)
Proteínas de Transporte de Monosacáridos/biosíntesis , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Adulto , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ejercicio Físico/fisiología , Femenino , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/análisis , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/química , Músculo Esquelético/fisiología , Obesidad/metabolismo , Resistencia Física , RegeneraciónRESUMEN
The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels.
Asunto(s)
Proteínas de Transporte de Monosacáridos/biosíntesis , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Adulto , Western Blotting , Preescolar , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Lactante , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Polivinilos , EmbarazoRESUMEN
GLUT-4 expression in individual fibers of human skeletal muscles in younger and older adults was studied. Furthermore, the dependency of insulin-stimulated glucose uptake on fiber type distribution was investigated. Fiber type distribution was determined in cryosections of muscle biopsies from 8 younger (29 yr) and 8 older (64 yr) healthy subjects, and estimates of GLUT-4 expression in individual fibers were obtained by combining immunohistochemistry and stereology. GLUT-4 was more abundantly expressed in slow compared with fast muscle fibers in both younger (P < 0.007) and older (P < 0. 001) subjects. A 25% reduction of GLUT-4 density in fast fibers (P < 0.001) and an unchanged GLUT-4 density in slow fibers were demonstrated in older compared with younger subjects. Insulin-stimulated glucose uptake rates measured by hyperinsulinemic, euglycemic clamp were not correlated with the fraction of slow fibers in the young (r = -0.45, P > 0.25) or in the elderly (r = 0. 11, P > 0.75) subjects. In conclusion, in human skeletal muscle, GLUT-4 expression is fiber type dependent and decreases with age, particularly in fast muscle fibers.