RESUMO
The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.
Assuntos
Descoberta de Drogas , Transportador de Glucose Tipo 3/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 2/química , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 5/antagonistas & inibidores , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/genética , Transportador de Glucose Tipo 5/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Amongst a wide range of biological macromolecules, saccharides exhibit the potential to be specifically recognized by cell-surface receptors and hence can be utilized as ligands in targeted drug delivery. The current study aims to use saccharides viz. Galactose, Pectin and Chitosan to improve targeting of Atenolol by oxalyl chloride mediated grafting. Conjugates were engineered by grafting Atenolol, a cardiovascular agent with the modified saccharide units. The conjugates were characterized by FTIR, DSC and 1H NMR study. Drug release analysis and cellular uptake study was carried out using H9c2 cell lines which represent that concentration of drug in cells treated with all atenolol-saccharide conjugates is enhanced by almost two-folds in comparison with cells treated with atenolol solution. Thus cell line study confers the evidence of selective cardiac delivery. No significant cytotoxicity was observed in case of all synthesized conjugates in the Brine shrimp lethality bioassay. Possible binding of the developed conjugates with the GLUT-4 receptors was assessed by in silico analysis using homology model developed by Swiss Model server. Hence it was concluded that the application of these conjugates with saccharides in selective cardiovascular drug delivery can be a promising approach to increase bioavailability, minimize drug loss by degradation and prevent harmful side effects by increasing specific cell targeting.
Assuntos
Atenolol/química , Atenolol/farmacologia , Portadores de Fármacos/química , Antagonistas de Receptores Adrenérgicos beta 1/química , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Artemia/efeitos dos fármacos , Atenolol/metabolismo , Atenolol/toxicidade , Linhagem Celular , Quitosana/química , Simulação por Computador , Portadores de Fármacos/farmacologia , Portadores de Fármacos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Galactose/química , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Dose Letal Mediana , Espectroscopia de Ressonância Magnética , Miocárdio/citologia , Pectinas/química , Ratos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P2 regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P2 metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P2 regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P2 is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P2 on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P2 levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P2 in controlling of vesicle docking process. Local depletion of PI(4,5)P2 at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P2 plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P2 not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.
Assuntos
Adipócitos/citologia , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Exocitose/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Simulação de Acoplamento Molecular , Optogenética , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Defective translocation of glucose transporter 4 (GLUT4) to the cell surface is a key feature of insulin resistance in type 2 diabetes. Therefore, elucidating the mechanism of GLUT4 translocation is of primary importance. The mammalian Bag6/Bat3 gene has been suggested to be linked with potential obesity- and diabetes-associated loci, while its function in the control of glucose incorporation into the cytoplasm has not been investigated. In this study, we established a series of cell lines that stably expressed GLUT4 with three tandem repeats of the antigenic peptide inserted into its 1st extracellular loop. With these cell lines, we found that the depletion of endogenous BAG6 downregulated the cell surface expression of GLUT4, concomitant with the reduced incorporation of a glucose analog into the cells. Defective intracellular translocation of GLUT4 in BAG6-depleted cells is similar to the case observed for the depletion of Rab8a, an essential regulator of insulin-stimulated GLUT4 translocation. In addition, we observed that the assembly of syntaxin 6 into the endoplasmic reticulum membrane was slightly disturbed under BAG6 depletion. Given that Rab8a and syntaxin 6 are critical for GLUT4 translocation, we suggest that BAG6 may play multiple roles in the trafficking of glucose transporters to the cell surface.This article has an associated First Person interview with the first author of the paper.
Assuntos
Membrana Celular/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Camundongos , Modelos Moleculares , Chaperonas Moleculares/genética , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoRESUMO
Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose-dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform-selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.
Assuntos
Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 4/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Glucose/antagonistas & inibidores , Glucose/metabolismo , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 2/química , Transportador de Glucose Tipo 3/química , Transportador de Glucose Tipo 4/química , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
In situ detection of the expression level of cell-surface receptors has become a hotspot study in recent years. We propose in this manuscript a novel strategy for sensitive electrochemiluminescence (ECL) detection of glucose transporter 4 (GLUT4) on human skeletal muscle cells (HSMCs). Graphene hydrogel (GH) was selected to fabricate a permeable electrode with the purpose of overcoming the steric hindrance of cells on electrode, which leads to errors in the detection of cell-surface receptors. GLUT4 was labeled with carbon dots (CDs), which generate ECL emission at the interface between GH and cells, so about half the amount of GLUT4 expressed at the cell surface could be determined, which provided an accurate GLUT4 expression quantification. The prepared cytosensor exhibited good analytical performance for HSMC cells, ranging from 500 to 1.0 × 106 cells·mL-1, with a detection limit of 200 cells·mL-1. The average amount of GLUT4 per HSMC cell was calculated to be 1.88 × 105. Furthermore, GLUT4 on HSMC surface had a 2.3-fold increase under the action of insulin. This strategy is capable of evaluating the receptors on the cell surface, which may push the application of ECL for disease diagnosis.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Transportador de Glucose Tipo 4/biossíntese , Medições Luminescentes , Células-Tronco Mesenquimais/química , Músculo Esquelético/química , Eletrodos , Transportador de Glucose Tipo 4/química , Grafite/química , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Músculo Esquelético/citologia , Imagem Óptica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Transportador de Glucose Tipo 4/imunologia , Transportador de Glucose Tipo 4/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Galinhas , Mapeamento de Epitopos , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Células HEK293 , Humanos , Vírus da Leucemia Murina/genética , Modelos Moleculares , Domínios Proteicos , Vacinas de Partículas Semelhantes a Vírus/químicaRESUMO
Tankyrase 1 and 2, members of the poly(ADP-ribose) polymerase family, have previously been shown to play a role in insulin-mediated glucose uptake in adipocytes. However, their precise mechanism of action, and their role in insulin action in other cell types, such as myocytes, remains elusive. Treatment of differentiated L6 myotubes with the small molecule tankyrase inhibitor XAV939 resulted in insulin resistance as determined by impaired insulin-stimulated glucose uptake. Proteomic analysis of XAV939-treated myotubes identified down-regulation of several glucose transporter GLUT4 storage vesicle (GSV) proteins including RAB10, VAMP8, SORT1, and GLUT4. A similar effect was observed following knockdown of tankyrase 1 in L6 myotubes. Inhibition of the proteasome using MG132 rescued GSV protein levels as well as insulin-stimulated glucose uptake in XAV939-treated L6 myotubes. These studies reveal an important role for tankyrase in maintaining the stability of key GLUT4 regulatory proteins that in turn plays a role in regulating cellular insulin sensitivity.
Assuntos
Transportador de Glucose Tipo 4/química , Resistência à Insulina , Insulina/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Tanquirases/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Estabilidade Proteica , Proteômica , RatosRESUMO
We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids employing an easy and short synthetic pathway. The compounds were tested in vitro against a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40 (GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPARγ) and solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound 1 displayed an EC50 value of 0.075 µM against GPR40 and was an AKR1B1 inhibitor, showing IC50 = 7.4 µM. Compounds 2 and 3 act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase of 2 to 4-times in the mRNA expression of PPARγ, as well as the GLUT-4 levels. Docking studies were conducted in order to explain the polypharmacological mode of action and the interaction binding mode of the most active molecules on these targets, showing several coincidences with co-crystal ligands. Compounds 1-3 were tested in vivo at an explorative 100 mg/kg dose, being 2 and 3 orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model. Compounds 2 and 3 displayed robust in vitro potency and in vivo efficacy, and could be considered as promising multitarget antidiabetic candidates. This is the first report of a single molecule with these four polypharmacological target action.
Assuntos
Desenho de Fármacos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Aldeído Redutase/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Técnicas de Química Sintética , Transportador de Glucose Tipo 4/agonistas , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Humanos , Hipoglicemiantes/síntese química , Ligantes , Camundongos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Terapia de Alvo Molecular , PPAR gama/antagonistas & inibidores , PPAR gama/química , Fenilpropionatos/síntese química , Ligação Proteica , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/químicaRESUMO
Presently, many studies have focused on exploring in silico approaches in the identification and development of alternative therapy for the treatment and management of cancer. Solute carrier family-2-member-4-gene (Slc2a4) which encodes glucose transporter 4 protein (GLUT4), has been identified as a promising therapeutic target for cancer. Though Slc2a4 is known to play a major regulatory role in the pathophysiology of type 2 diabetes, emerging evidence suggests that successful pharmacological inhibition of this protein may lead to the development of a novel drug candidate for the treatment of cancer. In this study, Slc2a4 protein sequence was retrieved and analysed using in silico approaches, and we identified seven putative antimicrobial peptides (AMPs; RAB1-RAB7) as anti-cancer. The structures of the protein and AMPs were modelled using I-TASSER server, and the overall quality of the Slc2a4 model was validated using PROCHECK. Subsequently, the probable motifs and active site of the protein were forecasted. Also, the molecular interaction between the AMPs and Slc2a4 was ascertained using PatchDock. The result revealed that, all the AMPs are good Slc2a4 inhibitors with RAB1 having the highest binding affinity of 12,392 and binding energy of -39.13 kcal/mol. Hence, this study reveals that all the generated AMPs can serve as therapeutic drug in treating cancer by inhibiting Slc2a4 which is responsible for the production of energy for cancer cells during angiogenesis. This is the first report on AMPs as inhibitors of Slc2a4 for the treatment of cancer.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Transportador de Glucose Tipo 4/química , Simulação de Acoplamento Molecular , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Sítios de Ligação , Transportador de Glucose Tipo 4/metabolismo , Humanos , Ligação ProteicaRESUMO
Camels have exceptional carbohydrate metabolism as their plasma glucose level is high and have low whole body insulin sensitivity, similar to that observed in type 2 diabetes patients. We aimed at studing an important component of insulin signalling pathway, the GLUT4, in camel. Camelus dromedarius GLUT4 (CdGLUT4) CDS is 1530 nucleotide in length that encodes for a 55KDa protein. CdGLUT4 has 23 amino acid substitutions and 3N-glycosylation sites, compared to 2 in Human GLUT4. 3 D structures of CdGLUT4 and HsGLUT4 generated by homology modelling revealed conservation of characteristic signature motifs. CdGLUT4 was cloned and expressed optimally in C43(DE3)pLysS strain and maximum detergent solubility was observed in n-Dodecyl-ß-d-maltopyranoside. These preliminary data provide information on residual differences between CdGLUT4 and HsGLUT4 that may be responsible for camel's unique glucose metabolism. These differences are postulated to assist in designing and development of efficacious GLUT4 that might help in management of diabetic patients.
Assuntos
Escherichia coli/metabolismo , Transportador de Glucose Tipo 4/isolamento & purificação , Transportador de Glucose Tipo 4/metabolismo , Fígado/metabolismo , Sequência de Aminoácidos , Animais , Camelus , Clonagem Molecular , Simulação por Computador , Escherichia coli/genética , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Glicosilação , Modelos Moleculares , Fosforilação , Filogenia , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F5QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F5QQI (F5QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F5QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance.
Assuntos
Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Microscopia/métodos , Células 3T3-L1 , Adipócitos/metabolismo , Motivos de Aminoácidos , Animais , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/genética , Insulina/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Mutação/genética , Processos EstocásticosRESUMO
BACKGROUND: The Rana dybowskii distribute in northeast region of China which have seasonally cold climates. During winter they survival freezing by biosynthesizing carbohydrate cryoprotectants such as high concentrations glucose into blood and all tissues. The essential role of glucose transporter 4 is a high-affinity glucose transporter, which can increase glucose uptake in cells when it stimulated by insulin. OBJECTIVE: In this study, we analysis the full-length GLUT4 mRNA detect the gene levels of GLUT4 in R. dybowskii main tissues by qPCR during low temperature. RESULTS: We found in heart, fat body, skeletal muscle and skin four tissues all express GLUT4, and the levels of GLUT4 decreased on initial cold exposure stage, 8~12 hours, followed 24 hours it recovered. CONCLUSION: This study we firstly indentified and characterized GLUT4 in amphibious, and provide a novel insight into the role of GLUT4 in cryoprotectant synthesis and cell protection in cold hardiness amphibians.
Assuntos
Clonagem Molecular/métodos , Temperatura Baixa , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/genética , Ranidae/genética , Sequência de Aminoácidos , Animais , China , Criopreservação , Evolução Molecular , Perfilação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Masculino , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de AminoácidosRESUMO
WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 â« GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.
Assuntos
3-O-Metilglucose/metabolismo , Eritrócitos/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Hidroxibenzoatos/farmacologia , Animais , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Citocalasina B/metabolismo , Eritrócitos/efeitos dos fármacos , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 3/química , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Humanos , Cinética , Camundongos , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.
Assuntos
Corantes Fluorescentes/análise , Transportador de Glucose Tipo 4/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Transportador de Glucose Tipo 4/química , Glicosilação , Células HeLa , Humanos , Estrutura Molecular , Transporte ProteicoRESUMO
BACKGROUND: Rosmarinic acid (RA) is a natural substance that may be useful for treating diabetes mellitus. The present study investigated the effects of RA on glucose homeostasis and insulin regulation in rats with streptozocin (STZ)-induced type 1 diabetes or high-fat diet (HFD)-induced type 2 diabetes. METHODS: Glucose homeostasis was determined using oral glucose tolerance tests and postprandial glucose tests, and insulin activity was evaluated using insulin tolerance tests and the homeostatic model assessment for insulin resistance. Additionally, the protein expression levels of PEPCK and GLUT4 were determined using Western blot analysis. RESULTS: RA administration exerted a marked hypoglycemic effect on STZ-induced diabetic rats and enhanced glucose utilization and insulin sensitivity in HFD-fed diabetic rats. These effects of RA were dose-dependent. Meanwhile, RA administration reversed the STZ- and HFD-induced increase in PEPCK expression in the liver and the STZ- and HFD-induced decrease in GLUT4 expression in skeletal muscle. CONCLUSION: RA reduces hyperglycemia and ameliorates insulin sensitivity by decreasing PEPCK expression and increasing GLUT4 expression.
Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Estreptozocina/farmacologia , Animais , Cinamatos/metabolismo , Depsídeos/metabolismo , Dieta Hiperlipídica , Transportador de Glucose Tipo 4/química , Resistência à Insulina , Músculo Esquelético/química , Ratos , Estreptozocina/química , Ácido RosmarínicoRESUMO
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 kcat of transport without changing the Km 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.
Assuntos
Transportador de Glucose Tipo 3 , Transportador de Glucose Tipo 4 , Fosfolipídeos , Transporte Biológico Ativo/fisiologia , Transportador de Glucose Tipo 3/química , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismoRESUMO
Glucose transporter GLUT4 (also known as SLC2A4) plays a major role in glucose homeostasis and is efficiently retained intracellularly in adipocytes and myocytes. To simplify the analysis of its retention, here, various intracellular GLUT4 domains were fused individually to reporter molecules. Of the four short cytoplasmic loops of GLUT4, only the first nine-residue-long loop conferred intracellular retention of truncated forms of the transferrin receptor and CD4 in adipocytes. In contrast, the same loop of GLUT1 was without effect. The reporter molecules to which the first loop of GLUT4 was fused localized, unlike GLUT4, to the trans-Golgi network (TGN), possibly explaining why these molecules did not respond to insulin. The retention induced by the GLUT4 loop was specific to adipocytes as it did not induce retention in preadipocytes. Of the SQWLGRKRA sequence that constitutes this loop, mutation of either the tryptophan or lysine residue abrogated reporter retention. Mutation of these residues individually into alanine residues in the full-length GLUT4 molecule resulted in a decreased retention for GLUT4-W105A. We conclude that the first intracellular loop of GLUT4 contains the retention motif WLGRK, in which W105 plays a prominent role.
Assuntos
Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Motivos de Aminoácidos , Animais , Antígenos CD4/metabolismo , Análise Mutacional de DNA , Genes Reporter , Insulina/farmacologia , Espaço Intracelular/metabolismo , Camundongos , Mutação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.
Assuntos
Cromatografia em Gel/métodos , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/isolamento & purificação , Glucose/metabolismo , Proteolipídeos/metabolismo , Animais , Clonagem Molecular , Transportador de Glucose Tipo 4/química , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Humanos , Micelas , Modelos Moleculares , Polímeros/química , Propilaminas/química , Ligação Proteica , Ratos , Células Sf9 , Spodoptera , TemperaturaRESUMO
Recently, we identified Glut4 as a palmitoylated protein in adipocytes. To understand the role of Glut4 palmitoylation in Glut4 membrane trafficking, a process that is essential for maintenance of whole body glucose homeostasis, we have characterized Glut4 palmitoylation. We found that Glut4 is palmitoylated at Cys223 and Glut4 palmitoylation at Cys223 is essential for insulin dependent Glut4 membrane translocation as substitution of Cys223 with a serine residue in Glut4 (C223S Glut4) diminished Glut4 responsiveness to insulin in membrane translocation in both adipocytes and CHO-IR cells. We have examined C223S Glut4 subcellular localization and observed that it was absence from tubular-vesicle structure, where insulin responsive Glut4 vesicles were presented. Together, our studies uncover a novel mechanism under which Glut4 palmitoylation regulates Glut4 sorting to insulin responsive vesicles, thereby insulin-dependent Glut4 membrane translocation.