The relationship between human erythrocyte glucose transporter (GLUT1) oligomeric structure and function was studied. GLUT1 was purified from human erythrocytes in the absence (GLUT1-DTT) or the presence (GLUT1+DTT) of dithiothreitol. Chemical cross-linking studies of lipid bilayer-resident purified GLUT1 and hydrodynamic studies of cholate-solubilized GLUT1 support the view that GLUT1-DTT is a homotetramer and GLUT1+DTT is a homodimer. Parallel studies on human erythrocyte, and studies employing conformation-specific antibodies (anti-GLUT1-DTT antibodies, ∂-IgGs), indicate that erythrocyte-resident GLUT1 resembles GLUT1-DTT (a homotetramer). While the D-glucose binding capacities of GLUT1-DTT and GLUT1+DTT are indistinguishable, GLUT1-DTT presents at least two population of binding sites to D-glucose whereas GLUT1+DTT presents only one population of sugar binding sites. The cytochalasin B (CCB) binding capacity of GLUT1-DTT (0.4 sites/monomer) is one half of that of GLUT1+DTT. GLUT1-DTT and GLUT1+DTT contain 2 and 6 free sulfhydryls per monomer respectively. The subunits (monomers) of tetrameric and dimeric GLUT1 are not linked by disulfide bridges. Erythrocyte resident GLUT1 presents at least two binding sites to D-glucose and binds CCB with a molar stoichiometry of 0.55 sites per GLUT1 monomer. Following treatment with high pH plus dithiothreitol, the sugar binding capacity of erythrocyte membrane resident transporter is unaltered but the transporter now presents only one population of binding sites to D-glucose, binds CCB with molar stoichiometry of 1.3 sites per GLUT1 monomer and displays significantly reduced affinity for ∂-IgGs. These findings demonstrate that erythrocyte resident glucose transporter is GLUT1-DTT (a GLUT1 tetramer) and that GLUT1 oligomeric structure determines GLUT1 functional properties. A model which rationalizes these findings is proposed.

Since the initial characterization of the human glucose transporter GLUT1, it has been observed that the presence of intracellular sugar stimulates the unidirectional rate of sugar uptake by a kinetic phenomenon known as trans-acceleration. Both GLUTs 1 and 3 catalyze transacceleration, while both GLUTs 2 and 4 do not. Although the basis for trans-acceleration is unknown, potential explanations include the requirement of a modulating cofactor, cellular context, or that the behavior is an artifact of imperfect transport measurements. This thesis examines whether trans-acceleration is a sequence-specific property intrinsic to the transporter. A method for detecting trans-acceleration in mammalian cells at physiologic temperature was developed through transport of two different glucose analogs. Homology-scanning mutagenesis was employed to exchange transmembrane domains (TMs) of GLUTs 1 and 4, and thereby test for accelerated-exchange loss- or gain-of-function. This approach was extended to GLUTs 2 and 3. The catalytic rates of these chimeric proteins were determined through transport measurements and expression measured by cell-surface biotinylation. These studies show that the sequence of putative scaffolding domain TM6 is both necessary and sufficient for trans-acceleration in scaffolds of GLUT1, GLUT2, and GLUT4. The substitution of TM6 sequence between these transporters has no effect on the turnover under exchange conditions, yet profoundly modifies turnover in the absence of intracellular sugar. We propose that the sequence-specific interaction of TM6 with other TMs structurally restrains relaxation of the empty carrier in GLUTs which catalyze trans-acceleration, and that binding of intracellular sugar affects these interactions to reduce the overall duration of the transport cycle. In addition, our model suggests that the substrate binding constant and rate of carrier relaxation are inter-dependent. In this model, the dissociation constant determined by substrate binding and dissociation rates at the endofacial sugar binding site must be larger than the equivalent constant at the exofacial site in order for trans-acceleration to occur.

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.

Protein-mediated sugar transport is nominally absent in normoxic (adequately oxygenated) pigeon erythrocytes. Following exposure to metabolic inhibitors (cyanide or carbonylcyanide-p-trifluoromethoxyphenylhydrazone), pigeon red cells transport sugars by a saturable, stereoselective pathway that is inhibited by cytochalasin B or forskolin. The sugar transport capacity of fully poisoned cells is consistent with a transporter density of approximately 30 carriers per erythrocyte. Immunoblot analyses and competition ELISA indicate that pigeon red cells contain approximately 200 copies of an integral plasma membrane protein immunologically related to the glucose transporter isoform GLUT1. GLUT1 is quantitatively restricted to the plasma membrane at all times. Pigeon red cells and brain lack proteins immunologically related to the sugar transporter isoforms GLUT3 and GLUT4. Specific immunodepletion of red cell GLUT1 content results in the subsequent loss of reconstitutable protein-mediated sugar transport. These findings demonstrate that avian erythrocyte sugar transport is mediated by a GLUT1-like sugar transport protein and that sugar transport stimulation by metabolic inhibitors results from derepression of cell surface sugar transport proteins. Lysis-resealing experiments suggest that derepression is a glutathione (OSH) dependent phenomenon. This mechanism of transport regulation contrasts with insulin stimulation of sugar transport in muscle and adipose tissue which is believed to result from recruitment of intracellular sugar transporters to the plasma membrane.

Zhan Siyan. / "November 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 133-152) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Human erythrocyte glucose sugar transport displays a complexity that is not explained by available models. Sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions (intracellular [sugar] = extracellular [sugar]). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional) or chemical (anomerization) barrier to equilibrate with cell water. The anomerization model was rejected through several lines of direct experimental investigation. 1) The sizes of the fast and slow phases of sugar transport do not correlate with the equilibrium anomer distributions of all GLUT1 sugar substrates. 2) Increasing the rate of anomerization by addition of exogenous intracellular mutarotase has no effect on biphasic transport kinetics. 3) Direct measurement of initial rates of sugar uptake or exchange demonstrates that GLUT1 shows no anomer preference. The physical barrier model was further refined by the use of the counterflow condition (intracellular [sugar] >> extracellular [sugar]). The presence of a physical barrier alone was unable to explain the complex counterflow time courses observed. As a result, the model was modified to include the action of a specific sugar export that is compartmentalized from rapidly equilibrating, GLUT1-mediated uptake and exit.

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C. The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.

A nefropatia diabética (ND) decorre da hiperglicemia crônica, de fatores de risco como a hipertensão arterial e a dislipidemia e de uma susceptibilidade genética já evidenciada em inúmeros estudos clínicos. Uma das características histológicas da ND é o acúmulo de proteínas de matriz extracelular no mesângio, para o qual contribuem várias vias bioquímicas. O GLUT-1, codificado pelo gene SLC2A1, é o principal transportador de glucose da célula mesangial e sua expressão está aumentada no glomérulo de animais diabéticos, o que constitui uma alça de feedback positivo pela qual a glicose extracelular aumentada estimula ainda mais sua própria captação, piorando a lesão mesangial. O GLUT-2, codificado pelo gene SLC2A2, é expresso nas células tubulares e nos podócitos e sua expressão também está aumentada na ND. A expressão deste transportador de glicose é regulada pelo fator de transcrição HNF-1. Participa, ainda, da lesão renal induzida pela hiperglicemia o fator de crescimento transformante - (TGF-), que exerce vários efeitos deletérios, tais como diminuir a atividade de metaloproteinases de matriz e promover fibrose renal. Esse fator de crescimento determina a ativação transcricional de genes-alvo, mas necessita de outros ativadores e co-ativadores da transcrição, tais como a proteína SMIF, codificada pelo gene DCP1A. Tendo em vista a participação das proteínas mencionadas acima na patogênese da ND, o presente estudo teve o objetivo de avaliar a associação de polimorfismos de um único nucleotídeo (SNPs) nos genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A com a doença renal em portadores de diabetes mellitus tipo 1 (DM1). Um total de 449 pacientes (56,4% do sexo feminino, idade média de 36,0±11,0 anos) com mais de 10 anos de doença foram incluídos e classificados de acordo com o estágio de ND: (1) Ausência de ND: excreção urinária de albumina (EUA) normal (< 30 mg/24h ou < 20 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo; (2) ND incipiente: microalbuminúria (EUA de 30 299 mg/24h ou 20 199 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo e (3) ND Franca: macroalbuminúria (EUA > 300 mg/24h ou > 200 g/min) ou proteinúria ou tratamento para reposição renal. Também foram avaliadas as associações dos SNPs com o ritmo de filtração glomerular estimado (RFGe). Os SNPs foram genotipados pela metodologia de reação em cadeia da polimerase em tempo real, com o uso de sondas fluorescentes. As associações dos SNPs com a ND foram avaliadas por análise de regressão logística e os odds ratios (OR) e respectivos intervalos de confiança (IC) de 95% foram calculados após ajuste para possíveis confundidores, que foram incluídos como co-variáveis no modelo de regressão. Valores de P < 0.05 (bicaudal) foram considerados estatisticamente significantes. As seguintes associações foram observadas: (1) gene SLC2A1: genótipos CT+TT do SNP rs841848 conferiram risco para a ND incipiente na população global (OR 1,88; CI95% 1,06-3,34; P= 0,03) e nos pacientes do sexo masculino (OR 2,67; CI95% 1,13-6,35; P=0,0247) e para a ND franca (OR 2,70; CI95% 1,18-6,31; e P= 0,0197) apenas nos pacientes do sexo masculino; genótipos GA+AA do SNP rs1385129 conferiram risco para a ND franca na população do sexo masculino (OR 3,09; CI95% 1,34-7,25; P=0,0085); genótipos AT + TT do SNP rs3820589, conferiram proteção contra a ND incipiente na população global (OR 0,36; CI95% 0,16-0,78; P=0,0132) e na população do sexo feminino (OR 0,14; CI95% 0,02-0,52; P=0,0122). (2) gene SLC2A2: genótipos GA+GG do SNP rs5396 conferiram proteção contra ND franca nos pacientes do sexo masculino (OR 0,29; CI95% 0,12-0,69; P=0,0052); os genótipos AG+GG do SNP rs6800180 conferiram proteção contra a ND franca nos pacientes do sexo masculino (OR 0,16; CI95% 0,14-0,90; P=0,0324). (3) gene HNF1A: genótipos AC + CC do SNP rs1169288 conferiram risco para ND franca na população global (OR 2,23; CI95% 1,16-4,38; P=0,0175); genótipos CG+GG do SNP rs1169289 conferiram risco para ND franca na população global (OR 3,43; CI95% 1,61-7,73; P=0,002); (4) Gene TGFB1: genótipos CT + TT do SNP 1800468 conferiram risco para ND incipiente na população total (OR 2,99; CI95% 1,26-7,02; P 0,0116) e o alelo polimórfico T do SNP rs1800469 conferiu risco para um menor RFGe (p=0,0271). (5) gene DCP1A: o alelo polimórfico A do SNP rs11925433 também se associou com um menor RFGe (p=0,0075). Em conclusão, SNPs em genes que codificam as proteínas envolvidas na patogênese da ND GLUT-1, GLUT-2, HNF-1, TGF- e SMIF conferem susceptibilidade para essa complicação crônica nos portadores de DM1 avaliados no presente estudo / Diabetic nephropathy (DN) results from chronic hyperglycemia, risk factors such as hypertension and dyslipidemia as well as from genetic susceptibility, already demonstrated in numerous clinical studies. A histological feature of DN is the accumulation of extracellular matrix proteins in the mesangium after activation of multiple biochemical pathways. GLUT-1, encoded by gene SLC2A1, is the major glucose transporter in mesangial cell and its expression is increased in the glomeruli of diabetic animals, comprising a positive feedback loop whereby high extracellular glucose stimulates its own uptake and worsening mesangial injury. GLUT-2, encoded by SLC2A2 gene, is expressed in podocytes and tubular cells and its expression is also increased in DN. The expression of this glucose transporter is regulated by the transcription factor HNF-1. Transforming growth factor - (TGF-) also participates in renal injury induced by hyperglycemia, exerting several deleterious effects, such as to decrease the activity of matrix metalloproteinases and to promote renal fibrosis. This growth factor determines the transcriptional activation of target genes, but needs other activators and co-activators, such as the protein named SMIF, encoded by the gene DCP1A. Given the involvement of the aforementioned proteins in the pathogenesis of DN, the present study aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A with renal disease in patients with type 1 diabetes mellitus (T1DM). A total of 449 patients (56.4% female, mean age 36.0±11.0 years) with disease duration > 10 years were included and grouped according to DN stages: (1) absence of DN: normal urinary albumin excretion (UAE) (< 30 mg/24h or < 20 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment; (2) incipient DN: microalbuminuria (UAE 30 299 mg/24h or 20 199 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment and (3) overt DN: macroalbuminúria (UAE > 300 mg/24h or > 200 g/min) or proteinuria or renal replacement therapy. Associations of SNPs with estimated glomerular filtration rate (eGFR) were also evaluated. All SNPs were genotyped by real time polymerase chain reaction using fluorescent-labelled probes. Associations of the SNPs with DN were assessed by logistic regression analyses and odds ratios (OR) were calculated after adjustments for possible confounders included as covariables in the regressive model. P values <0.05 (two-tails) were considered significant. The following associations were observed: (1) SLC2A1: genotypes CT+TT from rs841848 conferred risk to incipient DN in the overall population (OR 1.88; 95%IC 1.06-3.34; P= 0.03) and in the male patients (OR 2.67; CI95% 1.13-6.35; P=0.0247) and to overt DN (OR 2.70; CI95% 1.18-6.31; e P= 0.0197) only in the male patients; genotypes GA+AA from rs1385129 conferred risk to overt DN in the male population (OR 3.09; CI95% 1.34-7.25; P=0.0085); genotypes AT + TT from rs3820589 conferred protection against incipient DN in the overall population (OR 0.36; CI95% 0.16-0.78; P=0.0132) and in the female population (OR 0.14; CI95% 0.02-0.52; P=0.0122). (2) SLC2A2: genotypes GA+GG from rs5396 conferred protection against overt DN in the male patients (OR 0.29; CI95% 0.12-0.69; P=0.0052); genotypes AG+GG from rs6800180 conferred protection against overt DN in the male patients (OR 0.16; CI95% 0.14-0.90; P=0.0324). (3) HNF1A: genotypes AC + CC from rs1169288 conferred risk to overt DN in the overall population (OR 2.23; CI95% 1.16-4.38; P=0.0175); genotypes CG+GG from rs1169289 conferred risk to overt DN in the overall population (OR 3.43; CI95% 1.61-7.73; P=0.002); (4) TGFB1: genotypes CT + TT from 1800468 conferred risk to incipient DN in the overall population (OR 2.99; CI95% 1.26-7.02; P=0.0116) and the polymorphic allele T from SNP rs1800469 conferred risk to a lower eGFR (p=0.0271). (5) DCP1A: the polymorphic allele A from SNP rs11925433 was also associated with a lower eGFR (p=0.0075). In conclusion, SNPs in the genes encoding proteins GLUT-1, GLUT-2, HNF-1, TGF- e SMIF, all involved in the pathogenesis of DN, conferred susceptibility to this chronic complication in the T1DM patients evaluated in the present study