耐力运动对大鼠葡萄糖运载体基因表达及转位的影响

目的　研究耐力运动对大鼠骨骼肌细胞葡萄糖运载体 4(glucosetransporter 4,GLUT4)基因表达及转位机制的影响。方法　将SD大鼠随机分为两组 :对照组和耐力运动组。耐力运动组大鼠进行 6周游泳训练。用Western印迹法检测大鼠骨骼肌细胞内膜和外膜的GLUT4蛋白含量 ,用Northern杂交法和斑点印迹法检测大鼠骨骼肌细胞内GLUT4mRNA含量。实验前后检测大鼠血清胰岛素和血糖浓度。结果　运动组大鼠经过 6周游泳训练后 ,与对照组大鼠相比 ,骨骼肌细胞内膜GLUT4含量增加 16.0 %(P <0 .0 1) ,细胞外膜GLUT4含量增加 71.9% (P <0 .0 1) ,骨骼肌细胞内GLUT4mRNA含量增加2 5 .6% (P <0 .0 1)。结论　耐力运动可增加骨骼肌细胞内GLUT4基因表达水平 ,促进骨骼肌细胞内的GLUT4从内膜向细胞外膜转位 ,从而提高骨骼肌细胞对葡萄糖的摄取和利用。     

Effect of diabetes on glucoregulation. From glucose transporters to glucose metabolism in vivo.

Peripheral resistance to insulin is a prominent feature of both insulin-dependent and non-insulin-dependent diabetes. Skeletal muscle is the primary site responsible for decreased insulin-induced glucose utilization in diabetic subjects. Glucose transport is the rate-limiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. The transport of glucose across the muscle cell plasma membrane is mediated by glucose transporter proteins, and two isoforms (GLUT1 and GLUT4) are expressed in muscle. Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT4 transporter (but not GLUT1) from an intracellular pool to the plasma membrane. In skeletal muscles of streptozocin-induced diabetic rats, there is a decreased GLUT4 protein content in intracellular and plasma membranes. In these rats, insulin induced the mobilization of GLUT4 from the internal pool, but the incorporation of the transporter protein into the plasma membrane is diminished. Conversely, the content of the GLUT1 transporter increases in the plasma membrane of these diabetic rats. Normalization of glycemia with phlorizin fully restores the amount of GLUT1 and GLUT4 proteins to normal levels in the plasma membrane without altering insulin levels. This suggests that glycemia regulates the number of glucose transporters at the cell surface, GLUT1 varying directly and GLUT4 inversely, to glycemia. The regulatory role of glycemia also can be seen in diabetic dogs in vivo, where correction of hyperglycemia with phlorizin restores, at least in part, the defective metabolic clearance rate of glucose seen in these animals. In addition to acutely stimulating glucose transport in muscle, insulin controls exercise- and possibly stress-mediated glucose uptake in vivo, by preventing hyperglycemia and by restraining the effects of catecholamines on lipolysis and/or muscle glycogenolysis. Finally, we postulated a neural pathway that requires the permissive effect of insulin to increase glucose uptake by the muscle. Thus, insulin, glucose, and neural pathways regulate muscle glucose utilization in vivo and are, therefore, important determinants of glucoregulation in diabetes.     

Insulin resistance and glucose transporter

Insulin stimulates glucose transport in muscle and adipose tissue by promoting the appearance of GLUT4, the main glucose transporter isoform in these tissues, on the cell surface. Insulin resistance is instrumental in pathogenesis of type 2 diabetes mellitus and involves decreased glucose transport activity in these tissues. No significant differences are observed between the diabetic and non-diabetic subjects in muscle GLUT4 levels. Polymorphism in the GLUT4 gene, which is very rare, has the same prevalence between subjects with type 2 diabetes mellitus and the non-diabetic subjects. The most likely explanation for the insulin resistance is a defect in insulin signaling pathways or GLUT4 intracellular trafficking pathways.     

MUNC-ing around with insulin action

Defective uptake of glucose into muscle and fat cells, or insulin resistance, is a central feature of obesity and type 2 diabetes. As we brace ourselves for the diabetes epidemic, it is reassuring to know that real progress is being made in defining the molecular biology of how insulin stimulates glucose uptake and what goes awry in obesity and type 2 diabetes. An understanding of the molecular determinants of insulin-stimulated glucose transport has been one of the holy grails of hormone action research. A major breakthrough was the discovery that insulin stimulates the translocation of a specific glucose transport protein, GLUT4, from intracellular vesicles to the cell surface. Elucidating how this process is regulated has remained a challenge because it represents a convergence of 2 disparate and complex fields of research--namely, vesicle transport and signal transduction. A study reported in this issue of the JCI using mice lacking Munc18c, one of the vesicle-trafficking proteins involved in GLUT4 translocation, has provided new insights into the signaling/trafficking intersection that controls insulin-stimulated GLUT4 movement.     

Glycemic improvement in diabetic db/db mice by overexpression of the human insulin-regulatable glucose transporter (GLUT4).

The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter) expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and 360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice (approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in the basal state, which markedly increased after an insulin/glucose injection. In contrast, nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein in the basal state with a relatively small increase after an insulin/glucose challenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the glucose transport defect in db/db mice is at the level of glucose transporter translocation. Together, these data demonstrate that GLUT4 upregulation overcomes the glucose transporter translocation defect and alleviates insulin resistance in genetically diabetic mice, thus resulting in markedly improved glycemic control.     

运动对胰岛素抵抗大鼠脂肪细胞葡萄糖转运蛋白-4的影响

目的研究运动对高糖高脂饮食诱导的胰岛素抵抗大鼠脂肪细胞葡萄糖转运蛋白4(GLUT4)的影响。方法将实验大鼠随机分为正常组、模型组和运动组,每组各10只。各组以基础饲料适应性喂养1周后,模型组和运动组改用高糖、高脂饲料喂养,第5周时运动组进行为期6周的游泳训练。各组于实验第1,5和11周检测大鼠的体重、血甘油三酯、血胆固醇、空腹血糖和血浆胰岛素水平,计算胰岛素敏感指数。采用Westernblot法检测大鼠脂肪细胞内、外膜GLUT4含量。结果模型组大鼠与正常组相比,脂肪细胞内、外膜GLUT4含量均显著降低,差异均有统计学意义(P<0.05)。与模型组相比,经过6周游泳训练的运动组大鼠脂肪细胞内膜GLUT4含量无明显变化,而细胞外膜GLUT4含量显著增加,差异有统计学意义(P<0.05)。结论运动可提高胰岛素抵抗大鼠脂肪细胞外膜GLUT4含量,促进葡萄糖的摄取和利用。     

运动对大鼠骨骼肌胰岛素信号转导蛋白表达和活性的影响

目的：观察运动对大鼠骨骼肌中胰岛素信号转导蛋白表达和活性的影响，探讨运动调节骨骼肌葡萄糖转运的细胞内机制。方法：将20只SD大鼠随机分为运动组和对照组，运动组按Ploug方法进行8w的游泳训练。结果：运动组大鼠游泳8w后骨骼肌中蛋白激酶B蛋白表达和磷酸化程度均增加，细胞外信号调节激酶蛋白表达有上升趋势．磷酸化程度显著增加。结论：运动可激活正常骨骼肌中胰岛素信号传递两大途径中的关键蛋白，这可能是运动增强骨骼肌的胰岛素敏感性，促进葡萄糖转运的机制之一。     

Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle of diabetic rats.

This study was designed to determine whether altered glucose transporter expression is essential for the in vivo insulin-resistant glucose uptake characteristic of streptozocin-induced diabetes. Immunofluorescence in rat skeletal muscle colocalizes GLUT4 with dystrophin, both intrinsic to muscle fibers. In contrast, GLUT1 is extrinsic to muscle fibers, probably in perineurial sheath. Immunoblotting shows that levels of GLUT1 and GLUT4 protein per DNA in hindlimb muscle are unaltered from control levels at 7 d of diabetes but decrease to approximately 20% of control at 14 d of diabetes. This decrease is prevented by insulin treatment. In adipose cells of 7 d diabetic rats, GLUT4 levels are depressed. Thus, GLUT4 undergoes tissue-specific regulation in response to diabetes. GLUT4 and GLUT1 mRNA levels in muscle are decreased 62-70% at both 7 and 14 d of diabetes and are restored by insulin treatment. At 7 d of diabetes, when GLUT4 protein levels in muscle are unaltered, in vivo insulin-stimulated glucose uptake measured by euglycemic clamp is 54% of control. This reflects impairment in both glycogen synthesis and glycolysis and the substrate common to these two pathways, glucose-6-phosphate, is decreased approximately 30% in muscle of diabetic rats. These findings suggest a defect early in the pathway of glucose utilization, probably at the step of glucose transport. Because GLUT1 and GLUT4 levels are unaltered at 7 d of diabetes, reduced glucose uptake in muscle probably reflects impaired glucose transporter translocation or intrinsic activity. Later, at 14 d of diabetes, GLUT1 and GLUT4 protein levels are reduced, suggesting that sequential defects may contribute to the insulin-resistant glucose transport characteristic of diabetes.     

Glucose transport and NIDDM.

Three major metabolic abnormalities contribute to hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) including defective glucose-induced insulin secretion, elevated rates of hepatic glucose output, and insulin's impaired ability to stimulate glucose uptake in peripheral target tissues (insulin resistance). These functions involve cellular glucose transport in beta-cells, liver, adipose tissue, and skeletal muscle; and, in some instances, abnormalities in glucose transporter isoforms (GLUT) specifically expressed in these tissues may constitute key biochemical lesions underlying defective glucose homeostasis. In animal models of NIDDM, suppression of GLUT2 in beta-cells is correlated with loss of high-Km glucose transport and glucose-sensitive insulin secretion. Although there are no data on humans with NIDDM, GLUT2 loss would constitute an attractive mechanism for defective glucose sensing in beta-cells if it can be shown that transport then becomes rate limiting for glucose metabolism. In the liver, however, hepatocyte glucose transport via GLUT2 probably plays only a permissive role in sustaining increased glucose efflux. Peripheral insulin resistance is associated with decreased glucose transport activity, the likely rate-limiting step for glucose uptake in fat and muscle. Accordingly, the insulin-responsive GLUT4 isoform expressed exclusively in insulin target tissues has been studied intensively in NIDDM. In these studies, pretranslational suppression of GLUT4 appears to be the key mechanism of insulin resistance in adipocytes. However, levels of GLUT4 protein and mRNA are normal in vastus lateralis and rectus abdominis, inferring that defects in GLUT4 functional activity or insulin-mediated translocation cause insulin resistance in muscle. Thus, the intensified study of glucose transport has provided important new insights into NIDDM pathogenesis over the past 5 yr and has presented investigators with additional intriguing hypotheses.     

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance.

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.     

Glucose transport in cultured human skeletal muscle cells. Regulation by insulin and glucose in nondiabetic and non-insulin-dependent diabetes mellitus subjects.

A primary human skeletal muscle culture (HSMC) system, which retains cellular integrity and insulin responsiveness for glucose transport was employed to evaluate glucose transport regulation. As previously reported, cells cultured from non-insulin-dependent diabetic (NIDDM) subjects displayed significant reductions in both basal and acute insulin-stimulated transport compared to nondiabetic controls (NC). Fusion/differentiation of NC and NIDDM HSMC in elevated media insulin (from 22 pM to 30 microM) resulted in increased basal transport activities but reduced insulin-stimulated transport, so that cells were no longer insulin responsive. After fusion under hyperinsulinemic conditions, GLUT1 protein expression was elevated in both groups while GLUT4 protein level was unaltered. Fusion of HSMC under hyperglycemic conditions (10 and 20 mM) decreased glucose transport in NC cells only when combined with hyperinsulinemia. Hyperglycemia alone down-regulated transport in HSMC of NIDDM, while the combination of hyperglycemia and hyperinsulinemia had greater effects. In summary: (a) insulin resistance of glucose transport can be induced in HSMC of both NC and NIDDM by hyperinsulinemia and is accompanied by unaltered GLUT4 but increased GLUT1 levels; and (b) HSMC from NIDDM subjects demonstrate an increased sensitivity to impairment of glucose transport by hyperglycemia. These results indicate that insulin resistance in skeletal muscle can be acquired in NC and NIDDM from hyperinsulinemia alone but that NIDDM is uniquely sensitive to the additional influence of hyperglycemia.     

Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene.

Glucose transporter type 4 (GLUT4) is insulin responsive and is expressed in striated muscle and adipose tissue. To investigate the impact of a partial deficiency in the level of GLUT4 on in vivo insulin action, we examined glucose disposal and hepatic glucose production (HGP) during hyperinsulinemic clamp studies in 4-5-mo-old conscious mice with one disrupted GLUT4 allele [GLUT4 (+/-)], compared with wild-type control mice [WT (+/+)]. GLUT4 (+/-) mice were studied before the onset of hyperglycemia and had normal plasma glucose levels and a 50% increase in the fasting (6 h) plasma insulin concentrations. GLUT4 protein in muscle was approximately 45% less in GLUT4 (+/-) than in WT (+/+). Euglycemic hyperinsulinemic clamp studies were performed in combination with [3-3H]glucose to measure the rate of appearance of glucose and HGP, with [U-14C]-2-deoxyglucose to estimate muscle glucose transport in vivo, and with [U-14C]lactate to assess hepatic glucose fluxes. During the clamp studies, the rates of glucose infusion, glucose disappearance, glycolysis, glycogen synthesis, and muscle glucose uptake were approximately 55% decreased in GLUT4 (+/-), compared with WT (+/+) mice. The decreased rate of in vivo glycogen synthesis was due to decreased stimulation of glucose transport since insulin's activation of muscle glycogen synthase was similar in GLUT4 (+/-) and in WT (+/+) mice. By contrast, the ability of hyperinsulinemia to inhibit HGP was unaffected in GLUT4 (+/-). The normal regulation of hepatic glucose metabolism in GLUT4 (+/-) mice was further supported by the similar intrahepatic distribution of liver glucose fluxes through glucose cycling, gluconeogenesis, and glycogenolysis. We conclude that the disruption of one allele of the GLUT4 gene leads to severe peripheral but not hepatic insulin resistance. Thus, varying levels of GLUT4 protein in striated muscle and adipose tissue can markedly alter whole body glucose disposal. These differences most likely account for the interindividual variations in peripheral insulin action.     

Pretranslational suppression of a glucose transporter protein causes insulin resistance in adipocytes from patients with non-insulin-dependent diabetes mellitus and obesity

A major portion of insulin-mediated glucose uptake occurs via the translocation of GLUT 4 glucose transporter proteins from an intracellular depot to the plasma membrane. We have examined gene expression for the GLUT 4 transporter isoform in subcutaneous adipocytes, a classic insulin target cell, to better understand molecular mechanisms causing insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM) and obesity. In subgroups of lean (body mass index [BMI] = 24 +/- 1) and obese (BMI = 32 +/- 2) controls and in obese NIDDM (BMI = 35 +/- 2) patients, the number of GLUT 4 glucose transporters was measured in total postnuclear and subcellular membrane fractions using specific antibodies on Western blots. Relative to lean controls, the cellular content of GLUT 4 was decreased 40% in obesity and 85% in NIDDM in total cellular membranes. In obesity, cellular depletion of GLUT 4 primarily involved low density microsomes (LDM), leaving fewer transporters available for insulin-mediated recruitment to the plasma membrane (PM). In NIDDM, loss of GLUT 4 was profound in all membrane subfractions, PM, LDM, as well as high density microsomes. These observations corresponded with decrements in maximally stimulated glucose transport rates in intact cells. To assess mechanisms responsible for depletion of GLUT 4, we quantitated levels of mRNA specifically hybridizing with human GLUT 4 cDNA on Northern blots. In obesity, GLUT 4 mRNA was decreased 36% compared with lean controls, and the level was well correlated (r = + 0.77) with the cellular content of GLUT 4 protein over a wide spectrum of body weight. GLUT 4 mRNA in adipocytes from NIDDM patients was profoundly reduced by 86% compared with lean controls and by 78% relative to their weight-matched nondiabetic counterparts (whether expressed per RNA, per cell, or for the amount of CHO-B mRNA). Interestingly, GLUT 4 mRNA levels in patients with impaired glucose tolerance (BMI = 34 +/- 4) were decreased to the same level as in overt NIDDM. We conclude that, in obesity, insulin resistance in adipocytes is due to depletion of GLUT 4 glucose transporters, and that the cellular content of GLUT 4 is determined by the level of encoding mRNA over a wide range of body weight. In NIDDM, more profound insulin resistance is caused by a further reduction in GLUT 4 mRNA and protein than is attributable to obesity per se. Suppression of GLUT 4 mRNA is observed in patients with impaired glucose tolerance, and therefore, may occur early in the evolution of diabetes. Thus, pretranslational suppression of GLUT 4 transporter gene expression may be an important mechanism that produces and maintains cellular insulin resistance in NIDDM.     

Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss. Effect on muscle glucose transport and glucose transporter GLUT4.

A major defect contributing to impaired insulin action in human obesity is reduced glucose transport activity in skeletal muscle. This study was designed to determine whether the improvement in whole body glucose disposal associated with weight reduction is related to a change in skeletal muscle glucose transport activity and levels of the glucose transporter protein GLUT4. Seven morbidly obese (body mass index = 45.8 +/- 2.5, mean +/- SE) patients, including four with non-insulin-dependent diabetes mellitus (NIDDM), underwent gastric bypass surgery for treatment of their obesity. In vivo glucose disposal during a euglycemic clamp at an insulin infusion rate of 40 mU/m2 per min was reduced to 27% of nonobese controls (P less than 0.01) and improved to 78% of normal after weight loss of 43.1 +/- 3.1 kg (P less than 0.01). Maximal insulin-stimulated glucose transport activity in incubated muscle fibers was reduced by approximately 50% in obese patients at the time of gastric bypass surgery but increased twofold (P less than 0.01) to 88% of normal in five separate patients after similar weight reduction. Muscle biopsies obtained from vastus lateralis before and after weight loss revealed no significant change in levels of GLUT4 glucose transporter protein. These data demonstrate conclusively that insulin resistance in skeletal muscle of mobidly obese patients with and without NIDDM cannot be causally related to the cellular content of GLUT4 protein. The results further suggest that morbid obesity contributes to whole body insulin resistance through a reversible defect in skeletal muscle glucose transport activity. The mechanism for this improvement may involve enhanced transporter translocation and/or activation.     

不同强度的耐力运动对糖尿病大鼠骨骼肌 GLUT4 mRNA表达的影响

目的探讨不同强度的耐力运动对糖尿病大鼠骨骼肌GLUT4 mRNA表达的影响.方法雄性SD大鼠,其中36只大鼠经尾静脉注射链脲霉素,建立糖尿病模型.然后随机分为低强度运动组(EL)、高强度运动组(EH)、低强度运动加胰岛素治疗组(LI)、高强度运动加胰岛素治疗组(HI)、胰岛素治疗非运动组(DI)和非胰岛素治疗非运动组(DM).6只SD大鼠为非运动正常血糖组(CN).耐力训练采用活动平板,胰岛素采用皮下注射,共8周.运用RT-PCR法测定骨骼肌GLUT4 mRNA.结果DM组骨骼肌GLUT4 mRNA表达水平显著低于其它各组(P＜0.05).LI组骨骼肌GLUT4 mRNA表达水平明显增高接近CN组,并且显著高于DI组.DI组GLUT4 mRNA含量与EL、EH、HI各组相当,差异无显著性意义.结论运动可以促进GLUT4 mRNA的表达,而运动强度对GLUT4 mRNA表达量无显著影响;低强度运动加胰岛素所具有最佳的GLUT4 mRNA表达水平是其它单独干预措施所无法替代的.     

Differential regulation of adipose tissue glucose transporters in genetic obesity (fatty rat). Selective increase in the adipose cell/muscle glucose transporter (GLUT 4) expression.

Adipocytes from young obese Zucker rats exhibit a hyperresponsive insulin-mediated glucose transport, together with a marked increase in cytochalasin B binding as compared with lean rat adipocytes. Here, we examined in these cells the expression of two isoforms of glucose transporter, the erythroid (GLUT 1) and the adipose cell/muscle (GLUT 4) types, in rats aged 16 or 30 d, i.e., before and after the emergence of hyperinsulinemia. GLUT 1 protein and mRNA levels were identical in the two genotypes at both ages. In contrast, the levels of GLUT 4 protein in obese rat adipocytes were 2.4- and 4.5-fold those of lean littermates at 16 and 30 d of age, respectively, in perfect agreement with the genotype effect on insulin-stimulated glucose transport activity. The levels of GLUT 4 mRNA per fat pad were increased 2.3- and 6.2-fold in obese vs. lean rats 16- and 30-d-old, indicating a pretranslational level of regulation. The obese phenotype was not associated with overexpression of GLUT 4 mRNA in gastrocnemius muscle. This work indicates that the fa gene exerts a differential control on the expression of GLUT 1 and GLUT 4 in adipose tissue and provides evidence that independent of hyperinsulinemia, genotype is a major regulatory factor of GLUT 4 expression in this tissue.     

GLUT4 glucose transporter deficiency increases hepatic lipid production and peripheral lipid utilization

A critical defect in type 2 diabetes is impaired insulin-stimulated glucose transport and metabolism in muscle and adipocytes. To understand the metabolic adaptations this elicits, we generated mice with targeted disruption of the GLUT4 glucose transporter in both adipocytes and muscle (AMG4KO). In contrast to total body GLUT4-null mice, AMG4KO mice exhibit normal growth, development, adipose mass, and longevity. They develop fasting hyperglycemia and glucose intolerance and are at risk for greater insulin resistance than mice lacking GLUT4 in only one tissue. Hyperinsulinemic-euglycemic clamp studies showed a 75% decrease in glucose infusion rate and markedly reduced 2-deoxyglucose uptake into skeletal muscle (85-90%) and white adipose tissue (65%). However, AMG4KO mice adapt by preferentially utilizing lipid fuels, as evidenced by a lower respiratory quotient and increased clearance of lipids from serum after oral lipid gavage. While insulin action on hepatic glucose production and gluconeogenic enzymes is impaired, hepatic glucokinase expression, incorporation of 14C-glucose into lipids, and hepatic VLDL-triglyceride release are increased. The lipogenic activity may be mediated by increased hepatic expression of SREBP-1c and acetyl-CoA carboxylase. Thus, inter-tissue communication results in adaptations to impaired glucose transport in muscle and adipocytes that involve increased hepatic glucose uptake and lipid synthesis, while muscle adapts by preferentially utilizing lipid fuels. Genetic determinants limiting this "metabolic flexibility" may contribute to insulin resistance and type 2 diabetes in humans.     

Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozocin diabetic rats.

Chronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier-mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats.     

Muscle-specific transgenic complementation of GLUT4-deficient mice. Effects on glucose but not lipid metabolism.

We have taken the approach of introducing the muscle-specific myosin light chain (MLC)-GLUT4 transgene into the GLUT4-null background to assess the relative role of muscle and adipose tissue GLUT4 in the etiology of the GLUT4-null phenotype. The resulting MLC-GLUT4-null mice express GLUT4 predominantly in the fast-twitch extensor digitorum longus (EDL) muscle. GLUT4 is nearly absent in female white adipose tissue (WAT) and slow-twitch soleus muscle of both sexes of MLC-GLUT4-null mice. GLUT4 content in male MLC-GLUT4-null WAT is 20% of that in control mice. In transgenically complemented EDL muscle, 2-deoxyglucose (2-DOG) uptake was restored to normal (male) or above normal (female) levels. In contrast, 2-DOG uptake in slow-twitch soleus muscle of MLC-GLUT4-null mice was not normalized. With the normalization of glucose uptake in fast-twitch skeletal muscle, whole body insulin action was restored in MLC-GLUT4-null mice, as shown by the results of the insulin tolerance test. These results demonstrate that skeletal muscle GLUT4 is a major regulator of skeletal muscle and whole body glucose metabolism. Despite normal skeletal muscle glucose uptake and insulin action, the MLC-GLUT4-null mice exhibited decreased adipose tissue deposits, adipocyte size, and fed plasma FFA levels that are characteristic of GLUT4-null mice. Together these results indicate that the defects in skeletal muscle and whole body glucose metabolism and adipose tissue in GLUT4-null mice arise independently.     

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.