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Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

O diabetes melito gestacional (DMG) está relacionado ao crescimento fetal exagerado. Entender a influência de fatores relacionados ao crescimento fetal auxilia na identificação dos fetos com maior risco de desvios da normalidade. Objetivo: comparar fatores clínicos, laboratoriais e a expressão placentária de transportadores de glicose segundo o crescimento fetal em pacientes com DMG. Método: Para análise dos fatores clínicos e laboratoriais foi realizado um estudo retrospectivo com 425 gestantes com DMG do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC FM-USP) no período de janeiro de 2003 a novembro de 2009. Para a análise da expressão placentária dos transportadores de glicose dos tipos 1 (GLUT1), 3 (GLUT3) e 4 (GLUT4) foram selecionados todos os casos de recém-nascidos grandes para idade gestacional (RNGIG) pareados com um caso controle de recém-nascido adequado para idade gestacional (RNAIG). Foram incluídas apenas gestações únicas e com DMG diagnosticado pelo teste de tolerância à glicose oral de 100 gramas, sem malformações fetais e com idade gestacional definida e confiável. Todas as gestantes realizaram dieta para diabetes, controle glicêmico diário e uso de insulina quando necessário. Os critérios de seguimento e tratamento seguiram rigorosamente as normas do Protocolo de Condutas do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do HC-FMUSP. As gestantes foram divididas para análise dos dados em dois grupos: Fatores clínicos e laboratoriais com: 376 RNAIG e 49 RNGIG num total de 425 DMG. Expressão Placentária dos Transportadores de Glicose: 50 RNAIG e 44 RNGIG. Foram realizados testes de associação e médias das variáveis e relacionadas com os grupos de RNAIG e RNGIG. Resultados: Na análise univariada, dos fatores clínicos e laboratoriais, não houve diferenças entre os grupos quanto a: idade materna, antecedente familiar de diabetes, antecedente pessoal de hipertensão arterial, número de gestações, valores de glicemia de jejum e 1 hora no TTGO-100g, idade gestacional no parto, sexo do RN, tipo de parto e índice de Apgar no 1º e 5º minutos. Houve diferenças estatisticamente significativas entre os grupos quanto a: índice de massa corpórea pré-gestacional (p < 0,02); uso de insulina (p < 0,041); macrossomia anterior (p < 0,001); idade gestacional do diagnóstico do DMG (p < 0,001); glicemias de duas e três horas no TTGO-100g respectivamente com (p < 0,003) e (p < 0,026). Na análise de regressão logística foram considerados preditores independentes da ocorrência de RNGIG: o índice de massa corpórea pré - gestacional, a macrossomia anterior, aidade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas. Em relação a expressão dos transportadores de glicose não diferiram entre os grupos em relação a expressão de GLUT1 na decídua, GLUT3 na decídua e vilosidades e GLUT4 na decídua e vilosidades. Houve diferença entre os grupos quanto à: a expressão do GLUT1 nas vilosidades. Conclusões: O índice de massa corpórea pré - gestacional, a macrossomia anterior, a idade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas foram preditores da ocorrência de RNGIG. A expressão de GLUT1 nas vilosidades coriônicas teve relação com a ocorrência de RNGIG / Gestational diabetes mellitus (GDM) is related to excessive fetal growth. Knowing the influence of factors related to fetal growth assists in the identification of fetuses at high risk of deviations from normality. Objective: To compare clinical and laboratory tests and the placental expression of glucose transporters according to fetal growth in patients with GDM. Method: A retrospective study of clinical and laboratory factors related with large for gestational age newborns, included 425 pregnant women with GDM was carried out at Sector Endocrine Clinic of Obstetrics Hospital of the School of Medicine, University of São Paulo (HC-FMUSP), between January 2003 to November 2009. For the analysis of placental expression of glucose transporters types 1 (GLUT1), 3 (GLUT3) and 4 (GLUT4) were selected all cases of newborns large for gestational age (LGA) paired with a case control newly born appropriate for gestational age (AGA). We included only patients with singleton pregnancies and GDM diagnosed by OGTT-100g, with newborns without malformations and birth weight classified as adequate or large for gestational age. All pregnant women received diet for diabetes, daily glycemic control and insulin when necessary. The criteria for monitoring and treatment followed strictly the standards of Conduct Protocol Endocrine Obstetric Clinic of the Clinic Hospital, School of Medicine, University of São Paulo. The pregnancies were divided for analysis into two groups: 376 cases of newborns AGA and 49 cases of newborns LGA. Data were analyzed and considered the probability value p <0.05. Results: In the univariate analysis of clinical and laboratory factors, there were no differences between the groups regarding maternal age, family history of diabetes, personal history of hypertension, number of pregnancies, blood fasting glucose and 1 hour in- OGTT 100g, gestational age at delivery, gender of the newborn, type of delivery, Apgar score at 1st and 5th minutes. There were statistically significant differences between the groups regarding: body mass index before pregnancy (p <0.02), insulin (p <0.041), previous macrosomia (p <0.001), gestational age at diagnosis of GDM (p <0.001), blood glucose levels two and three hours at 100 g OGTT, respectively, with (p <0.003) (p <0.026). In logistic regression analysis were considered independent predictors of the occurrence of LGA: body mass index before pregnancy, previous macrosomia gestational age at diagnosis of GDM and two hours after glucose overload 100 grams. Regarding the expression of glucose transporters, the groups did not differ regarding the expression of GLUT1 in the decidua, GLUT3 in the decidua and villi and GLUT4 in the decidua and villi. There were differences between the groups regarding the expression of GLUT1 in the villi. Conclusions: The body mass index before pregnancy, previous macrosomia, gestational age of diagnosis of GDM and two hours after glucose overload 100 grams were predictors of the occurrence of LGA. The expression of GLUT1 in chorionic villi was related to the occurrence of LGA newborn

O diabetes melito gestacional (DMG) está relacionado ao crescimento fetal exagerado. Entender a influência de fatores relacionados ao crescimento fetal auxilia na identificação dos fetos com maior risco de desvios da normalidade. Objetivo: comparar fatores clínicos, laboratoriais e a expressão placentária de transportadores de glicose segundo o crescimento fetal em pacientes com DMG. Método: Para análise dos fatores clínicos e laboratoriais foi realizado um estudo retrospectivo com 425 gestantes com DMG do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC FM-USP) no período de janeiro de 2003 a novembro de 2009. Para a análise da expressão placentária dos transportadores de glicose dos tipos 1 (GLUT1), 3 (GLUT3) e 4 (GLUT4) foram selecionados todos os casos de recém-nascidos grandes para idade gestacional (RNGIG) pareados com um caso controle de recém-nascido adequado para idade gestacional (RNAIG). Foram incluídas apenas gestações únicas e com DMG diagnosticado pelo teste de tolerância à glicose oral de 100 gramas, sem malformações fetais e com idade gestacional definida e confiável. Todas as gestantes realizaram dieta para diabetes, controle glicêmico diário e uso de insulina quando necessário. Os critérios de seguimento e tratamento seguiram rigorosamente as normas do Protocolo de Condutas do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do HC-FMUSP. As gestantes foram divididas para análise dos dados em dois grupos: Fatores clínicos e laboratoriais com: 376 RNAIG e 49 RNGIG num total de 425 DMG. Expressão Placentária dos Transportadores de Glicose: 50 RNAIG e 44 RNGIG. Foram realizados testes de associação e médias das variáveis e relacionadas com os grupos de RNAIG e RNGIG. Resultados: Na análise univariada, dos fatores clínicos e laboratoriais, não houve diferenças entre os grupos quanto a: idade materna, antecedente familiar de diabetes, antecedente pessoal de hipertensão arterial, número de gestações, valores de glicemia de jejum e 1 hora no TTGO-100g, idade gestacional no parto, sexo do RN, tipo de parto e índice de Apgar no 1º e 5º minutos. Houve diferenças estatisticamente significativas entre os grupos quanto a: índice de massa corpórea pré-gestacional (p < 0,02); uso de insulina (p < 0,041); macrossomia anterior (p < 0,001); idade gestacional do diagnóstico do DMG (p < 0,001); glicemias de duas e três horas no TTGO-100g respectivamente com (p < 0,003) e (p < 0,026). Na análise de regressão logística foram considerados preditores independentes da ocorrência de RNGIG: o índice de massa corpórea pré - gestacional, a macrossomia anterior, aidade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas. Em relação a expressão dos transportadores de glicose não diferiram entre os grupos em relação a expressão de GLUT1 na decídua, GLUT3 na decídua e vilosidades e GLUT4 na decídua e vilosidades. Houve diferença entre os grupos quanto à: a expressão do GLUT1 nas vilosidades. Conclusões: O índice de massa corpórea pré - gestacional, a macrossomia anterior, a idade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas foram preditores da ocorrência de RNGIG. A expressão de GLUT1 nas vilosidades coriônicas teve relação com a ocorrência de RNGIG / Gestational diabetes mellitus (GDM) is related to excessive fetal growth. Knowing the influence of factors related to fetal growth assists in the identification of fetuses at high risk of deviations from normality. Objective: To compare clinical and laboratory tests and the placental expression of glucose transporters according to fetal growth in patients with GDM. Method: A retrospective study of clinical and laboratory factors related with large for gestational age newborns, included 425 pregnant women with GDM was carried out at Sector Endocrine Clinic of Obstetrics Hospital of the School of Medicine, University of São Paulo (HC-FMUSP), between January 2003 to November 2009. For the analysis of placental expression of glucose transporters types 1 (GLUT1), 3 (GLUT3) and 4 (GLUT4) were selected all cases of newborns large for gestational age (LGA) paired with a case control newly born appropriate for gestational age (AGA). We included only patients with singleton pregnancies and GDM diagnosed by OGTT-100g, with newborns without malformations and birth weight classified as adequate or large for gestational age. All pregnant women received diet for diabetes, daily glycemic control and insulin when necessary. The criteria for monitoring and treatment followed strictly the standards of Conduct Protocol Endocrine Obstetric Clinic of the Clinic Hospital, School of Medicine, University of São Paulo. The pregnancies were divided for analysis into two groups: 376 cases of newborns AGA and 49 cases of newborns LGA. Data were analyzed and considered the probability value p <0.05. Results: In the univariate analysis of clinical and laboratory factors, there were no differences between the groups regarding maternal age, family history of diabetes, personal history of hypertension, number of pregnancies, blood fasting glucose and 1 hour in- OGTT 100g, gestational age at delivery, gender of the newborn, type of delivery, Apgar score at 1st and 5th minutes. There were statistically significant differences between the groups regarding: body mass index before pregnancy (p <0.02), insulin (p <0.041), previous macrosomia (p <0.001), gestational age at diagnosis of GDM (p <0.001), blood glucose levels two and three hours at 100 g OGTT, respectively, with (p <0.003) (p <0.026). In logistic regression analysis were considered independent predictors of the occurrence of LGA: body mass index before pregnancy, previous macrosomia gestational age at diagnosis of GDM and two hours after glucose overload 100 grams. Regarding the expression of glucose transporters, the groups did not differ regarding the expression of GLUT1 in the decidua, GLUT3 in the decidua and villi and GLUT4 in the decidua and villi. There were differences between the groups regarding the expression of GLUT1 in the villi. Conclusions: The body mass index before pregnancy, previous macrosomia, gestational age of diagnosis of GDM and two hours after glucose overload 100 grams were predictors of the occurrence of LGA. The expression of GLUT1 in chorionic villi was related to the occurrence of LGA newborn