GLUT4 expression at the plasma membrane is related to fibre volume in human skeletal muscle fibres

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.     

2型糖尿病小鼠骨骼肌细胞葡萄糖转运因子4转位变化的研究

目的：探讨2型糖尿病小鼠的骨骼肌细胞的葡萄糖转运因子4（GLUT4）转位变化。方法：6-8周龄的雌性C57BL/6J小鼠随机分成对照组和2型糖尿病组，每组各12只。对照组小鼠给予正常鼠膳食16周。2型糖尿病组给予高脂高糖膳食16周。用超速蔗糖梯度离心方法分离细胞内膜和外膜的匀浆，用Western blotting方法检测两组大鼠骨骼肌细胞内、外膜GLUT4的蛋白水平。结果：两组的内、外膜GLUT4蛋白总量相近，经胰岛素刺激后，2型糖尿病小鼠骨骼肌细胞GLUT4从内膜向外膜转位少于对照组。结论：在非肥胖2型糖尿病鼠，是胰岛素刺激的骨骼肌细胞GLUT4的转位下降，而不是GLUT4数量的下降，导致了GLUT4的下调,这种下调可能是导致2型糖尿病骨骼肌胰岛素抵抗的分子机制之一。     

Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.     

Characterization of GLUT4 and calpain expression in healthy human skeletal muscle during fasting and refeeding

Aims: Calpain‐10 and calpain‐3 and the diabetes ankyrin repeat protein (DARP) have all been linked to insulin resistance and type 2 diabetes. We set out to measure the expression of these genes in human skeletal muscle and relate them to functional measurements of insulin action during fasting (which induces insulin resistance) and refeeding (which reverses it). Methods: Ten healthy male volunteers underwent 48 h of starvation followed by 24 h of high carbohydrate refeeding. On three occasions, before and after starvation and after refeeding, subjects underwent a 16 min insulin tolerance test to quantify insulin sensitivity. Muscle biopsies were obtained before and after fasting and after refeeding for the analysis of calpain‐10 and calpain‐3, GLUT4 and DARP expression by Western blotting and real‐time PCR. Results: Fasting led to a marked reduction in whole body insulin sensitivity by approx. 45% (P < 0.01) and skeletal muscle GLUT4 gene expression by approx. 40% (P < 0.05). However, fasting had no effect on calpain‐10 and calpain‐3 mRNA or protein levels, or DARP mRNA expression. Refeeding only partly restored insulin sensitivity and GLUT4 gene expression to their pre‐fast values, but did not effect the expression of calpain‐10, calpain‐3 or DARP. Conclusions: These findings demonstrate that in healthy non‐diabetic humans induction of insulin resistance by fasting and its reversal by refeeding with a high CHO diet is mirrored by changes in skeletal muscle GLUT4 but not calpain‐10 and calpain‐3 expression.     

The expression of NFATc1 in adult rat skeletal muscle fibres

Although numerous studies have recently implicated the calcineurin-nuclear factor of activated T-cells (Cn-NFAT) signalling pathway in the regulation of activity-dependent fibre type switching in adult mammalian skeletal muscles, little is known about the endogenous expression of NFAT proteins in the various fibre types present in these muscles. In this study, the immunolocalization of NFATc1 (also known as NFATc or NFAT2) in the extensor digitorum longus (EDL; a mainly fast-twitch muscle) and the soleus (a predominantly slow-twitch muscle) muscles of adult ( approximately 90-day-old) Wistar rats was investigated. The results show that NFATc1 is expressed only in oxidative fibres (i.e. type I and type IIA fibres) that stain intensely for succinate dehydrogenase activity irrespective of whether they are from the fast- or slow-twitch muscle. Thus, 99 +/- 4% (n = 7 rats) of the muscle fibres in the soleus and 42 +/- 2% (n = 7 rats) of those in the EDL expressed NFATc1. In the soleus muscle fibres, NFATc1 was localized mainly in the fibre nuclei, whereas in the EDL fibres it was localized in both the cytoplasm and the nuclei. However, no difference in its localization was observed between type I and type IIA fibres in both muscles. Western blot experiments showed that the soleus expressed more NFATc1 proteins than the EDL. From these results, we suggest that NFATc1 controls the number and distribution of both type I and type IIA fibres, as well as the oxidative capacity of adult mammalian skeletal muscles.     

Gene expression of carnosine-related enzymes and transporters in skeletal muscle

Chronic oral beta-alanine supplementation can elevate muscle carnosine (beta-alanyl-l-histidine) content and improve high-intensity exercise performance. However, the regulation of muscle carnosine levels is poorly understood. The uptake of the rate-limiting precursor beta-alanine and the enzyme catalyzing the dipeptide synthesis are thought to be key steps. The aims of this study were to investigate the expression of possible carnosine-related enzymes and transporters in both human and mouse skeletal muscle in response to carnosine-altering stimuli. Human gastrocnemius lateralis and mouse tibialis anterior muscle samples were subjected to HPLC and qPCR analysis. Mice were subjected to chronic oral supplementation of beta-alanine and carnosine or to orchidectomy (7 and 30 days, with or without testosterone replacement), stimuli known to, respectively, increase and decrease muscle carnosine and anserine. The following carnosine-related enzymes and transporters were expressed in human and/or mouse muscles: carnosine synthase (CARNS), carnosinase-2 (CNDP2), the carnosine/histidine transporters PHT1 and PHT2, the beta-alanine transporters TauT and PAT1, beta-alanine transaminase (ABAT) and histidine decarboxylase (HDC). Six of these genes showed altered expression in the investigated interventions. Orchidectomy led to decreased muscle carnosine content, which was paralleled with decreased TauT expression, whereas CARNS expression was surprisingly increased. Beta-alanine supplementation increased both muscle carnosine content and TauT, CARNS and ABAT expression, suggesting that muscles increase beta-alanine utilization through both dipeptide synthesis (CARNS) and deamination (ABAT) and further oxidation, in conditions of excess availability. Collectively, these data show that muscle carnosine homeostasis is regulated by nutritional and hormonal stimuli in a complex interplay between related transporters and enzymes.     

Increased insulin-stimulated glucose uptake in athletes: the importance of GLUT4 mRNA,GLUT4 protein and fibre type composition of skeletal muscle

In the present study the expression of GLUT4 and fibre type composition were examined in biopsies from skeletal muscle in seven male athletes and eight male sedentary subjects. Estimated maximal oxygen uptake was increased in the trained group when compared with the sedentary group (74.0 ± 3.9 vs. 42.9±5.1 ml kg^-1 min^-1; P < 0.01). A biopsy of vastus lateralis muscle was taken in the fasting state, 36 h after the last bout of exercise. A second muscle biopsy was obtained following 4 h of a hyperinsulinaemic (2 mU kg^-1 min^-1), euglycaemic clamp. The rate of insulin-stimulated glucose uptake was increased in the trained subjects (17.34±0.53 vs. 13.53±0.79 mg kg^-1 min^-1, P < 0.01). In parallel, the steady state levels of GLUT4 protein and mRNA per DNA were higher in muscle biopsies obtained in the basal state from athletes than in sedentary controls, 21 and 71% respectively (P < 0.05). In the total group of participants, GLUT4 protein per DNA in the basal state and insulin-stimulated glucose uptake rate correlated positively, (r = 0.51, P = 0.05). In the insulin-stimulated state we did not find any significant correlation between GLUT4 protein per DNA and glucose uptake rate (r = 0.13, n.s.). No significant relationships between GLUT4 protein abundance per DNA and muscle fibre type distribution were observed. A significantly negative correladon was found between type 2B fibre area and insulin-stimulated glucose uptake (r =–0.63, P < 0.05). In conclusion, the abundance of GLUT4 protein and mRNA, respectively, is increased in skeletal muscle from endurance trained subjects compared to sedentary subjects. However, factors other than GLUT4 immunoreactive protein abundance seem to be determinant for the increased insulin-stimulated whole body glucose uptake in endurance trained subjects.     

高脂饮食喂养对大鼠骨骼肌细胞膜GLUT4含量的影响

目的:检测骨骼肌细胞膜GLUT4含量的改变,探讨高脂饮食喂养诱导胰岛素抵抗的受体后机制。方法:将动物分为3组:①正常对照组;②高脂饮食组;③高脂饮食+饮食控制组。通过8周高脂饮食喂养建立胰岛素抵抗大鼠模型,随后代以普通饮食继续喂养4周。用Westernblot方法检测骨骼肌细胞膜表面GLUT4蛋白表达。结果:在胰岛素刺激下,高脂饮食组大鼠骨骼肌细胞膜GLUT4蛋白表达显著少于正常对照组(减少约31%);饮食控制组骨骼肌细胞膜GLUT4蛋白表达明显高于高脂饮食组(约1.14倍)。结论:高脂喂养的方法可成功复制出胰岛素抵抗大鼠模型;高脂饮食可能通过影响胰岛素信号转导系统,使胰岛素刺激的GLUT4转位至细胞膜受阻,其在膜上的含量也降低,从而促进胰岛素抵抗的形成和发展。     

Passive stretching produces Akt- and MAPK-dependent augmentations of GLUT4 translocation and glucose uptake in skeletal muscles of mice

Muscle contraction is accompanied by passive stretching or deformation of cells and tissues. The present study aims to clarify whether or not acute passive stretching evokes glucose transporter 4 (GLUT4) translocation and glucose uptake in skeletal muscles of mice. Passive stretching mainly induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a plasma membrane-rich fraction (F2) and accelerated glucose uptake in hindlimb muscles; whereas electrical stimulation, which mimics physical exercise in vivo, and insulin, each induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a fraction rich in plasma membrane (F2), and to one rich in transverse tubules (PF3), along with subsequent glucose uptake. Mechanical stretching increased phosphorylation of Akt and p38 mitogen-activated protein kinase (p38 MAPK), but it had no apparent effect on the activity of AMP-activated protein kinase (AMPK). Electrical stimulation augmented the activity of not only AMPK but also phosphorylation of Akt and p38 MAPK. Our results suggest that passive stretching produces translocation of GLUT4 mainly from the fraction rich in intracellular membrane to that rich in plasma membrane, and that the glucose uptake could be Akt- and p38 MAPK-dependent, but AMPK-independent manners.     

Sarcolemmal ion channels in dystrophin-deficient skeletal muscle fibres

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and characterized by progressive skeletal muscle degeneration. A current hypothesis suggests that degeneration of dystrophin-deficient skeletal muscle results from a chronic intracellular Ca²⁺ overload. Ca²⁺ handling in skeletal muscle is tightly controlled by the membrane potential which is set by sarcolemmal ion channels activity. Also, with regard to the subsarcolemmal localization of dystrophin, it is reasonable to enquire if the distribution and function of ion channels might be affected by the absence of dystrophin. This paper briefly summarizes the current knowledge of the properties of sarcolemmal ion channels in fully differentiated dystrophin-deficient skeletal muscle fibres.     

"Giant" muscle fibres in skeletal muscle of normal pigs

Observations made during a growth and development study of the semitendinosus and trapezius muscles of 49 purebred Large White pigs between birth and 128 days of age revealed the presence of giant fibres. The occurrence, histochemical and ultrastructural properties of these giant fibres were investigated. A high proportion of the pigs (85 per cent) contained giant fibres in their muscles but these giant fibres usually represented less than 1 per cent of the total myofibre population. Giant fibres possessed enhanced adenosine triphosphatase activity and a high capacity for oxidative metabolism (indicated by succinate dehydrogenase activity) which was reflected ultrastructurally by the greatly heightened electron density of myofibrils and by an abnormally high percentage of mitochondria and lipid droplets. These deviations from normal muscle fibre composition, together with the reduced percentage volume of sarcoplasmic reticulum, were consistent with changes seen in functionally over-loaded muscle. It appears that giant fibre anomalies occur through increased activity stimulated in occasional muscle fibres, perhaps by a structural defect, such as an inadequate amount of sarcoplasmic reticulum, which causes hyper-contractile activity within the fibres and associated compensatory adaptations. Giant fibres did not appear to represent fibres undergoing degenerative changes.     

Gene expression in Huntington's disease skeletal muscle: a potential biomarker

Huntington's disease (HD) is an incurable and fatal neurodegenerative disorder. Improvements in the objective measurement of HD will lead to more efficient clinical trials and earlier therapeutic intervention. We hypothesized that abnormalities seen in the R6/2 mouse, a greatly accelerated HD model, might highlight subtle phenotypes in other mouse models and human HD. In this paper, we identify common gene expression changes in skeletal muscle from R6/2 mice, Hdh(CAG(150)) homozygous knock-in mice and HD patients. This HD-triggered gene expression phenotype is consistent with the beginnings of a transition from fast-twitch to slow-twitch muscle fiber types. Metabolic adaptations similar to those induced by diabetes or fasting are also present but neither metabolic disorder can explain the full phenotype of HD muscle. The HD-induced gene expression changes reflect disease progression. This raises the possibility that muscle gene expression may be used as an objective biomarker to complement clinical HD-rating systems. Furthermore, an understanding of the molecular basis of muscle dysfunction in HD should provide insight into mechanisms involved in neuronal abnormalities and neurodegeneration.