Regenerating human muscle fibres express GLUT3 protein

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.     

The GLUT4 density in slow fibres is not increased in athletes. How does training increase the GLUT4 pool originating from slow fibres?

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.     

Relationship between muscle fibre composition,glucose transporter protein 4 and exercise training: possible consequences in non‐insulin‐dependent diabetes mellitus

Skeletal muscle is composed of different fibre types, which differ in contractile as well as in metabolic properties. The myosin molecule, which exists in several different isoforms, is of major importance in determining the contractile properties of the muscle cell. The plasticity of skeletal muscle is reflected in this tissue’s adaptability to changes in the functional demand. In both rats and humans, a decrease in activity level will in most cases change the muscle fibre composition towards faster myosin isoforms and an increase in activity level (such as seen with exercise training) will induce an increase in slower myosin isoforms. The glucose transporter protein 4 (GLUT4), which is the major insulin regulatable glucose transporter in mammalian skeletal muscle, is found in larger amounts in slow muscle fibres compared with fast muscle fibres. An increase in activity level will increase the GLUT4 protein expression and a decrease in activity level will in most cases decrease GLUT4. Thus, there seems to be some kind of relationship between the muscle fibre type and GLUT4. However, the main factor regulating both the GLUT4 protein expression and the muscle fibre composition seems to be the activity level of the muscle fibre. Patients suffering from non‐insulin‐dependent diabetes mellitus (NIDDM) are insulin resistant in their skeletal muscles but are generally normal when it comes to skeletal muscle fibre composition and the GLUT4 protein expression. There is good evidence that exercise training beneficially impacts on insulin sensitivity in healthy individuals and in patients with type II diabetes. An increase in the GLUT4 protein expression in skeletal muscle may at least partly explain this effect of training.     

Dissociation of the effects of training on oxidative metabolism,glucose utilisation and GLUT4 levels in skeletal muscle of streptozotocin-diabetic rats

The effects of long-term, moderate physical exercise on in vivo glucose uptake, levels of two glucose transporter proteins (GLUT1 and GLUT4) and activities of various key enzymes of energy metabolism were measured in skeletal muscle from streptozotocin-diabetic rats. Diabetes (12–16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI) in muscle containing mainly type I fibres by 55% but had no effect in muscles containing mainly type IIa and IIb fibres. GMI was increased in the diabetic white skeletal muscle (mainly type IIb fibres) by more than 120%. In contrast to the complex changes in GMI, GLUT4 levels were reduced in all types of skeletal muscle from diabetic rats with no change in GLUT1 levels. Exercise training had no effects on GMI or the glucose transporter levels. Streptozotocin induced diabetes significantly reduced the oxidative capacity of skeletal muscle assayed as the activities of citrate synthase, succinate dehydrogenase and cytochrome c oxidase. Training increased the activities of oxidative enzymes, with this increase being more prominent in the diabetic animals. The present data indicate that long-term streptozotocin-induced diabetes decreases oxidative metabolic capacity and GLUT4 protein levels in skeletal muscle, but that the changes of glucose transport largely depend on the fibre type composition. Moderate training fully reverses the effect of insulinopenia and hyperglycaemia on muscle oxidative metabolism. In contrast to the previous suggestions, the expression of GLUT4 is not correlated with the capacity of oxidative metabolism in skeletal muscle of streptozotocin-diabetic rats.     

GLUT1 and p63 expression in endometrial intraepithelial and uterine serous papillary carcinoma

AIMS: To analyse the expression patterns of GLUT1, p63 and p53 and the possible correlation between these markers in uterine serous papillary carcinoma (USPC) and endometrial intraepithelial carcinoma (EIC). METHODS AND RESULTS: Fourteen cases of USPC, 12 of which also showed EIC, were examined for GLUT1, p63 and p53 immunoreactivity. Four-micrometre sections from formalin-fixed paraffin-embedded tissue were immunostained using commercially available primary antibodies and Dako Envision Plus reagents for visualization. Membranous GLUT1 immunoreactivity was observed in all cases, including all 14 invasive tumours (100%) and 11 of 12 associated EICs (92%), and was confined to neoplastic cells. In USPC, staining tended to be strongest in superficial antistromal regions. p63 positivity was found in 8/14 (57%) USPCs and 9/12 (75%) associated EICs. In 11 cases p53 was overexpressed in both invasive USPC (11/14; 79%) and EIC (11/12; 92%). p53+ USPCs tended to be positive for p63, whereas p53- USPCs were also negative for p63. CONCLUSIONS: GLUT1 is expressed in the vast majority of USPC and EIC, suggesting a biological role during the early steps of carcinogenesis in endometrial serous neoplasms. GLUT1 expression may be induced by hypoxia-related as well as other mechanisms.     

Glucose transporters GLUT4 and GLUT8 are upregulated after facial nerve axotomy in adult mice

Peripheral nerve axotomy in adult mice elicits a complex response that includes increased glucose uptake in regenerating nerve cells. This work analyses the expression of the neuronal glucose transporters GLUT3, GLUT4 and GLUT8 in the facial nucleus of adult mice during the first days after facial nerve axotomy. Our results show that whereas GLUT3 levels do not vary, GLUT4 and GLUT8 immunoreactivity increases in the cell body of the injured motoneurons after the lesion. A sharp increase in GLUT4 immunoreactivity was detected 3 days after the nerve injury and levels remained high on Day 8, but to a lesser extent. GLUT8 also increased the levels but later than GLUT4, as they only rose on Day 8 post‐lesion. These results indicate that glucose transport is activated in regenerating motoneurons and that GLUT4 plays a main role in this function. These results also suggest that metabolic defects involving impairment of glucose transporters may be principal components of the neurotoxic mechanisms leading to motoneuron death.     

Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.     

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.     

Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach

We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.     

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.     

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.     

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.     

Expression during rat fetal development of GLUT12--a member of the class III hexose transporter family

Glucose is an essential molecule for most mammalian cells, and is particularly important during fetal development, when cells are rapidly dividing and differentiating. In rats, GLUT1 is present at high levels in most fetal tissues, with levels decreasing after birth. We used immunohistochemistry to localise GLUT12 protein, a recently identified member of the sugar transporter family, and GLUT1 during rat fetal development. GLUT12 staining was observed in heart muscle from gestational days 15 to 21. GLUT12 staining in skeletal muscle increased from gestational days 17 to 21, and GLUT12 was also detected in brown adipose tissue. The expression of GLUT12 in insulin-responsive tissues supports a potential role for GLUT12 in the provision of glucose to these tissues before the appearance of GLUT4. GLUT12 protein was also expressed in fetal chondrocytes from gestational day 15 onward, in kidney distal tubules and collecting ducts from day 19, and in lung bronchioles from day 19. The specific pattern of expression observed in the rat fetus suggests that GLUT12 may be important in hexose delivery to developing tissues.     

Muscle energy metabolism: structural and functional features in different types of porcine striated muscles

Striated muscles exhibit a wide range of metabolic activity levels. Heart and diaphragm are muscles with continuous contractile performance, which requires life-long function. In contrast, skeletal muscles like longissimus muscle can adapt metabolism from resting to different stages of exercise. The aim of this study was to compare the morphological features of these three muscles and the expression of genes that are important for energy metabolism. Therefore, histochemical studies were performed for determination of muscle fibre type composition. Oxidative and glycolytic capacity was assessed by measuring isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities. The mRNA expression of glucose transporter 4 (GLUT 4), growth hormone receptor (GHR) and AMP-activated kinase (AMPK) α₁ and α₂ subunits was studied by semiquantitative Northern blotting. Heart, and to a slightly lesser extent diaphragm were highly oxidative muscles characterised by high expression of oxidative muscle fibres and ICDH activity. Longissimus muscle exhibited the highest percentage of glycolytic fibres and LDH activity. GLUT 4 mRNA was lowest in heart reflecting the dependency of heart muscle on fatty acids as major energy source. Higher expression of GLUT 4 in diaphragm indicated that glucose is an important energy substrate in this oxidative muscle. Highest GLUT 4 expression in longissimus should be essential for the refilling of glycogen stores after exercise. AMPK subunits, which are important stimulators of GLUT 4 protein insertion into the sarcolemma, are also highest expressed in longissimus muscle indicating the strong capacity to adapt energy metabolism to large changes in energy demand. Interestingly, AMPK α₁ subunit expression on protein level is strongly restricted to muscle fibres containing type I myosin in this muscle. GHR mRNA expression was also highest in longissimus muscle indicating that an enhanced effect of growth hormone, which is described to be diabetogenic, could be involved in the lower insulin sensitivity of glycolytic muscles.     

Relationship between muscle fibre composition, glucose transporter protein 4 and exercise training: possible consequences in non-insulin-dependent diabetes mellitus.

Skeletal muscle is composed of different fibre types, which differ in contractile as well as in metabolic properties. The myosin molecule, which exists in several different isoforms, is of major importance in determining the contractile properties of the muscle cell. The plasticity of skeletal muscle is reflected in this tissue's adaptability to changes in the functional demand. In both rats and humans, a decrease in activity level will in most cases change the muscle fibre composition towards faster myosin isoforms and an increase in activity level (such as seen with exercise training) will induce an increase in slower myosin isoforms. The glucose transporter protein 4 (GLUT4), which is the major insulin regulatable glucose transporter in mammalian skeletal muscle, is found in larger amounts in slow muscle fibres compared with fast muscle fibres. An increase in activity level will increase the GLUT4 protein expression and a decrease in activity level will in most cases decrease GLUT4. Thus, there seems to be some kind of relationship between the muscle fibre type and GLUT4. However, the main factor regulating both the GLUT4 protein expression and the muscle fibre composition seems to be the activity level of the muscle fibre. Patients suffering from non-insulin-dependent diabetes mellitus (NIDDM) are insulin resistant in their skeletal muscles but are generally normal when it comes to skeletal muscle fibre composition and the GLUT4 protein expression. There is good evidence that exercise training beneficially impacts on insulin sensitivity in healthy individuals and in patients with type II diabetes. An increase in the GLUT4 protein expression in skeletal muscle may at least partly explain this effect of training.     

GLUT4 expression in human muscle fibres is not correlated with intracellular triglyceride (TG) content. Is TG a maker or a marker of insulin resistance?

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.     

AMPK调节骨骼肌细胞GLUT4基因表达的机制研究

腺苷酸活化蛋白激酶(AMPK)能调节运动/肌肉收缩所引起的骨骼肌细胞葡萄糖转运蛋白4(GLUT4)基因的表达,但至今它的调节机制不清.研究显示在非运动刺激引起的细胞信号事件中由组蛋白去乙酰化酶(HDACs)以及组蛋白乙酰化酶(HATs)控制的组蛋白乙酰化状态是调节基因表达的重要机制,所以我们假设AMPK信号途径是通过征用HDACs中的HDAC5(在骨骼肌细胞内高表达)来实现对运动/肌肉收缩引起的GLUT4基因表达控制.细胞分为正常浓度葡萄糖对照组(NGLU组)、正常浓度AICAR组(NGLU AICAR组)、高浓度对照组(HGLU组)、高浓度AICAR组(HGLU AICAR组).用5 mmol/L和20 mmol/L葡萄糖浓度培养骨骼肌细胞后,NGLU AICAR组和HGLU AICAR组与肌肉收缩模拟信号刺激5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)孵育.AICAR能激活NGLU组骨骼肌细胞AMPKα2、减少骨骼肌细胞核HDAC5蛋白、促使HDAC5与骨骼肌细胞加强因子(MEF2)蛋白分离和上调GLUT4基因的表达;相反,高浓度葡萄糖延迟由AICAR引起的AMPKα2磷酸化、AMPKα2向细胞核转入、HDAC5向细胞核转出和GLUT4基因的表达.实验结果说明在不同葡萄糖浓度下的骨骼肌细胞GLUT4基因表达变化都对应着上游AMPK蛋白和下游HDAC5蛋白的变化,AMPK可能是征用转录抑制子HDAC5来调节MEF2的活性而达到控制肌肉收缩所引起的GLUT4基因表达.     

Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise

The pattern of muscle glycogen synthesis following its depletion by exercise is biphasic. Initially, there is a rapid, insulin independent increase in the muscle glycogen stores. This is then followed by a slower insulin dependent rate of synthesis. Contributing to the rapid phase of glycogen synthesis is an increase in muscle cell membrane permeability to glucose, which serves to increase the intracellular concentration of glucose-6-phosphate (G6P) and activate glycogen synthase. Stimulation of glucose transport by muscle contraction as well as insulin is largely mediated by translocation of the glucose transporter isoform GLUT4 from intracellular sites to the plasma membrane. Thus, the increase in membrane permeability to glucose following exercise most likely reflects an increase in GLUT4 protein associated with the plasma membrane. This insulin-like effect on muscle glucose transport induced by muscle contraction, however, reverses rapidly after exercise is stopped. As this direct effect on transport is lost, it is replaced by a marked increase in the sensitivity of muscle glucose transport and glycogen synthesis to insulin. Thus, the second phase of glycogen synthesis appears to be related to an increased muscle insulin sensitivity. Although the cellular modifications responsible for the increase in insulin sensitivity are unknown, it apparently helps maintain an increased number of GLUT4 transporters associated with the plasma membrane once the contraction-stimulated effect on translocation has reversed. It is also possible that an increase in GLUT4 protein expression plays a role during the insulin dependent phase.     

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinization

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinizationAims : To examine invasive head and neck squamous carcinomas for expression of GLUT1, a glucose transporter and marker of increased glucose uptake, glycolytic metabolism and response to tissue hypoxia; p63, a p53 homologue that is a marker of the undifferentiated proliferative basaloid phenotype; and phospho-histone H1, a marker of activation of the cell cycle-promoting cyclin-dependent kinases 1 and 2. Methods : Routinely processed slides from 34 invasive squamous carcinomas, including 25 with intraepithelial components, were immunostained with anti-GLUT1 (Chemicon), anti-p63 (4A4, Santa Cruz), and antiphospho-histone H1 (monoclonal 12D11). Results : In keratinizing carcinomas, all three markers were most commonly immunodetected peripherally, with loss of expression in central keratinized zones. In contrast, in non-keratinizing carcinomas, p63 and phospho-histone H1 expression was most commonly observed throughout tumour nests and anti-GLUT1 stained in a pattern suggestive of hypoxia-induced expression ('antistromal' staining), in which cells at the tumour-stromal interface were GLUT1- and cells in central, perinecrotic zones showed progressive induction of GLUT1. Intraepithelial components also displayed basal and 'antibasal' GLUT1 staining patterns, homologous to the pro- and antistromal patterns in invasive carcinoma; basal patterns in intraepithelial lesions appeared to be more predictive of keratinizing invasive carcinoma and antibasal intraepithelial staining more predictive of non-keratinizing poorly differentiated carcinomas. Conclusions : Keratinizing and non-keratinizing squamous carcinomas differ in expression patterns of GLUT1, p63 and phospho-histone H1. In the former, all three markers were typically suppressed in conjunction with keratinization; in the latter, GLUT1 expression was more likely to occur in a hypoxia-inducible pattern and expression of p63 and phospho-histone H1 was unsuppressed. GLUT1 expression patterns in intraepithelial lesions may be predictive of the differentiation status of the associated invasive carcinoma.