Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.     

急、慢性运动对2型糖尿病大鼠脂肪PI3K/AKT/GLUT4通路的影响

目的:探讨急性和慢性运动对2型糖尿病(T2DM)大鼠脂肪组织明磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/葡萄糖运载体4(GLUT4)信号通路的影响。方法:15月龄SD雄性大鼠52只随机分为正常对照组(n=13)和高脂组(n=39),分别喂养普通和高脂饲料。8周后,高脂组体重＞正常对照组20％,注射小剂量STZ后,血糖＞16.7 mmol/l,造模成功。将糖尿病模型组随机分为糖尿病对照组(DC,n=13),糖尿病慢性运动组(DCE,n=13),糖尿病急性运动组(DAE,n=13)。DCE组进行8周的游泳运动,DAE组进行一次性游泳运动。测定血脂,血糖和血清胰岛素,Western blot法测定脂肪PI3K、AKT和GLUT4蛋白含量。结果:糖尿病组体重、血脂、血糖、胰岛素显著高于正常对照组(P均＜0.01);高密度脂蛋白胆固醇(HDL-C)水平降低(P＜0.05),脂肪组织中PI3K、AKT和GLUT4蛋白表达下降(P均＜0.01)。糖尿病慢性运动组体重、血脂、血糖、胰岛素均出现显著性下降(P均＜0.01);HDL-C升高(P＜0.05),脂肪PI3K、AKT和GLUT4蛋白表达上升(P＜0.01)。糖尿病急性运动组血脂、血糖、胰岛素下降(P均＜0.05);HDL-C升高(P＜0.05),脂肪PI3K、AKT和GLUT4含量显著上升(P均＜0.05)。结论:①高脂饮食结合小剂量STZ诱导的T2DM大鼠脂肪组织PI3K/AKT通路受损,降低了胰岛素的敏感性。②急性、慢性有氧运动,均可以通过PI3K/AKT通路,改善糖脂代谢紊乱,慢性运动略优于急性运动。     

Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

Glucose Transporter 8 (GLUT8) Mediates Fructose-induced de Novo Lipogenesis and Macrosteatosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.     

GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment

Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 - sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL[LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.     

Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression

In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.     

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Growth factors, mitogens, oncogenes and the regulation of glucose transport

The erythrocyte (or HepG2/brain) type glucose transporter (GLUT 1) was the first of the family of facilitative glucose transporter proteins to be cloned [M. Mueckler et al., Science 229, 941–945, 1985]. GLUT 1 is expressed in most tissue types, all cells lines, transformed cells and tumour cells. It is thought to be responsible for ‘housekeeping’ levels of glucose transport, i.e. the uptake of glucose required for oxidative phosphorylation. The rate of glucose transport via GLUT 1 can be regulated under conditions in which the metabolic rate must be adjusted such as cell division (mitosis and meiosis), differentiation, transformation and nutrient starvation. Here we review the recent literature on the control of glucose transport of mitogens, growth factors and oncogenes, and discuss some of the implications for the integration of cellular signalling pathways and cell growth.     

氯化钴增强培养海马神经元葡萄糖转运活性参与抗缺氧的作用

本文用培养新生大鼠海马神经元观察了氯化钴对葡萄糖转运活性的影响及其在神经元抗缺氧中的作用。结果表明,用CoCl2处理的培养海马神经元,24h后其2-脱氧-D-[1-^3H]葡萄糖摄取率和葡萄糖转运体GLUT1和GLUT3mRNA表达明显高于对照组,并且其在缺氧6或8h后的损伤也明显减轻,氯化钴对神经元缺氧损伤的保护作用被葡萄糖转运体抑制剂细胞松弛素B大部分消除,结果提示,氯化钴能够增强神经元GLUT1和GLUT3mRNA的表达和葡萄糖转运活性,CoCl2的这一作用可能是其增强神经元抗缺氧的重要机制。     

Metabolic and therapeutic lessons from genetic manipulation of GLUT4

This review focuses on the effects of varying levels of GLUT4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. Three mouse models are discussed including MLC-GLUT4 mice which overexpress GLUT4 specifically in skeletal muscle, GLUT4 null mice which express no GLUT4, and the MLC-GLUT4 null mice which express GLUT4 only in skeletal muscle. Overexpressing GLUT4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT4 mice. In contrast, the GLUT4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT4 expression in skeletal muscle in the MLC-GLUT4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.     

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α₁-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α₁-AR stimulation protected against increased lactate dehydrogenase release or Annexin V⁺ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α₁-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α₁-AR stimulation increased ³H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α₁-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α₁-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α₁-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α_1A-AR but not α_1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α_1A-AR but not α_1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α₁-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α_1A-AR subtype.     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.     

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb     

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.     

2-Deoxyglucose conjugated platinum (II) complexes for targeted therapy: design,synthesis, and antitumor activity

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. To target the Warburg effect, we designed a new series of 2-deoxyglucose (2-DG) conjugated platinum (II) complexes for glucose transporter 1 (GLUT1)-mediated anticancer drug delivery. The potential GLUT1 transportability of the complexes was investigated through a comparative molecular docking analysis utilizing the latest GLUT1 protein crystal structure. The key binding site for 2-DG as GLUT1’s substrate was identified with molecular dynamics simulation, and the docking study demonstrated that the 2-DG conjugated platinum (II) complexes can be recognized by the same binding site as potential GLUT1 substrate. The conjugates were synthesized and evaluated for in vitro cytotoxicity study with seven human cancer cell lines. The results of this study revealed that 2-DG conjugated platinum (II) complexes are GLUT1 transportable substrates and exhibit improved cytotoxicities in cancer cell lines that over express GLUT1 when compared to the clinical drug, Oxaliplatin. The correlation between GLUT1 expression and antitumor effects are also confirmed. The study provides fundamental information supporting the potential of the 2-DG conjugated platinum (II) complexes as lead compounds for further pharmaceutical R&D.     

Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids

The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the k_cat of transport without changing the K_m values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.     

自噬对葡萄糖转运蛋白4动力学影响分析

葡萄糖转运蛋白4(GLUT4)与胰岛素抵抗有着紧密联系,抑制自噬能减缓胰岛素抵抗.为了探讨自噬对胰岛素抵抗方面的作用,现以GLUT4囊泡为动力学模型,通过全内反射荧光显微镜实时观测3T3-L1成熟脂肪细胞中GLUT4囊泡的运动,并采用高斯拟合及相应的搜索算法,从TIRFM时间序列中提取运动轨迹、速度等信息进行统计分析.结果显示:自噬对GLUT4的运动具有一定的影响.抑制自噬后,GLUT4囊泡运动的胰岛素响应程度增强,长距离运动囊泡增多,平均运动速度加快.