The glucose transporter 4 (GLUT4) is the major insulin-responsive glucose transporter in adipose and muscle tissues. Although the early steps in the insulin signaling pathway governing translocation of GLUT4 to the plasma membrane are well understood, the final steps in this pathway are not. TUG is a protein which has been shown to affect trafficking of GLUT4 both in the basal state and in response to insulin. One protein-protein interaction between TUG and the large cytosolic loop of GLUT4 has previously been identified. Based on reports of the requirement of the GLUT4 N-terminal domain for its proper targeting to the plasma membrane, we postulated that an interaction might also exist between TUG and the N-terminal domain of GLUT4, and we tested this hypothesis using two sets of pull-down experiments. In the first set, using the N-terminal domain of GLUT4 fused with glutathione S-transferase (GST), we were able to pull TUG down from the lysates of TUG-transfected HEK 293 cells. TUG was also pulled down by the GLUT4 cytosolic loop and, to a much lesser extent, its C-terminal domain. However, there was no specific interaction between these fusion proteins and the lysates of cells transfected with a truncated form of TUG lacking its own N-terminal domain. In the second set of experiments, using a biotinylated synthetic GLUT4 N-terminal peptide, we pulled down a protein detected by an anti-TUG antibody and running at ~64 kDa, a slightly higher molecular weight than wild-type TUG. We believe that this band represents modified full-length TUG. This interaction was not seen using synthetic GLUT4 N-terminal peptide mutated at 4 amino acids previously identified as necessary for proper GLUT4 retention and insulin-responsive trafficking. We conclude that TUG interacts not only with the large cytosolic loop of GLUT4, but also with the N-terminal domain of GLUT4, and that this latter interaction can be disrupted by mutations in GLUT4 that cause defective trafficking, suggesting that this interaction is critical for GLUT4 intracellular retention and insulin-responsive GLUT4 trafficking.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 164-175).

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

Orientador: Doris Hissako Matsushita / Banca: Maria Aparecida Visconti / Banca: Flávia Lombardi Lopes / Banca: Joel Claudio Heimann / Banca: Fernando Yamamoto Chiba / Resumo: Atualmente, está bem estabelecido que o ambiente fetal está ligado à saúde materna, e estímulos ou agressões anormais durante a vida intra-uterina podem resultar em mudanças na fisiologia e metabolismo da prole, aumentando o risco de doenças na vida adulta, este fenômeno é conhecido como programação fetal. Alterações na metilação do DNA e expressão gênica são consideradas mecanismos moleculares responsáveis por esta programação. Estudos anteriores demonstraram que a doença periodontal (DP) materna promove resistência insulínica, aumento nas concentrações plasmáticas de citocinas, redução do conteúdo de GLUT4 e do seu índice de translocação para membrana plasmática em sua prole adulta. E citocinas, como por exemplo, o TNF-α, têm sido relacionadas com a redução da expressão de GLUT4 por meio da ativação do fator de transcrição nuclear κappa B (NF-κB). Além disso, esta citocina pode estimular algumas serinas quinases, incluindo IκB quinase (IKK), c-Jun amino-terminal kinase (JNK) e quinases reguladas por sinais extracelulares (ERKs) que estão envolvidas na resistência insulínica. Tais achados evidenciam a necessidade de realizar mais estudos para verificar os mecanismos envolvidos nestas alterações. Portanto, os objetivos do presente estudo foram avaliar em ratos adultos, proles de ratas com DP: 1) glicemia e insulinemia; 2) expressão do RNAm da proteína transportadora de glicose GLUT4 e do IRS1 em muscular esquelético gastrocnêmio (MG); 3) o grau de metilação do DNA na região p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is well establishedthat the fetal environment is linked to maternal health, and abnormal stimuli or aggressions during intrauterine life can result in changes in the physiology and metabolism of offspring, increasing the risk of disease in adult life, this phenomenon is known as fetal programming. Changes in DNA methylation and gene expression are considered molecular mechanisms responsible for this programming. Previous studies have demonstrated that maternal periodontaldisease (PD) promotes insulin resistance, increased plasma concentrations of cytokines, reduced GLUT4 content and its plasma membrane translocation index in its adult offspring. And cytokines, such as TNF-α, have been linked to reduced GLUT4 expressionthrough the activation of nuclear transcription factor kappa B (NF-κB). In addition, this cytokine can stimulate some serine kinases including IκB kinase (IKK), c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs)that are involved in insulin resistance. These findings evidenced the need for further studies to verify the mechanisms involved in these changes. Therefore, the objectives of the present study were to evaluate in adult rats, offspring of rats with PD: 1) birth weight and during the75 days of age;2) glycemia and insulinemia; 3) GLUT4 and IRS1mRNA expression in skeletal muscle gastrocnemius (MG); 4) the degree of DNA methylation in the promoter region of the GLUT4 gene in MG; 5) phosphorylation of JNK, IKKα/β, ERK 1/2, N... (Complete abstract click electronic access below) / Doutor

O diabetes mellitus tipo 2 (DM2) é caracterizado pela presença de resistência à insulina, a qual pode ser modulada pelo estrógeno, tanto em fêmeas como em machos. Nesse processo, o transportador de glicose GLUT4 (gene Slc2a4, solute carrier family 2 member 4) desempenha papel importante, pois aumento da expressão do GLUT4 melhora o controle glicêmico. Estradiol (E2) regula a expressão do Slc2a4 por meio do balanço dos efeitos contrários de seus receptores (ERs): ER-alfa estimula e ER-beta inibe a expressão. Efeitos transcricionais dos ERs envolvem a participação de co-reguladores, destacadamente o SP1 (transcription factor Sp1), potente estimulador do Slc2a4. Entretanto, o papel do SP1 na regulação do Slc2a4 mediada pelos ERs é desconhecido; e este foi o objetivo do presente estudo. Investigou-se adipócitos maduros 3T3-L1, tratados por 24 horas com E2, agonista de ER-alfa (PPT) ou agonista de ER-beta (DPN). Avaliou-se: a expressão gênica (RT-qPCR) de Slc2a4 e Sp1; o conteúdo (Western blotting) total de GLUT4 e o nuclear de ER-alfa/beta e SP1; a atividade de ligação do SP1 no Slc2a4 (ensaio de mobilidade eletroforética); e a formação de complexos SP1/ER-alfa (imunoprecipitação). Os resultados confirmaram que E2 aumenta a expressão de Slc2a4/GLUT4 pela ação preponderante do ER-alfa. O agonista PPT aumentou: o conteúdo nuclear de SP1, a interação SP1/ER-alfa e a atividade de ligação do SP1 no Slc2a4. O agonista DPN indicou que a ação repressora do ER-beta não envolve o SP1. Conclui-se que o efeito estimulador do ER-alfa na expressão do Slc2a4 envolve mecanismo de transativação gênica via SP1. Essas observações colocam a cooperação ER-alfa/SP1 como um novo alvo para o desenvolvimento de medidas terapêuticas para resistência à insulina e diabetes mellitus tipo 2 / Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, which can be modulated by estrogen in both females and males. In this process, the glucose transporter GLUT4 (solute carrier family 2 member 4 gene - Slc2a4) plays an important role, since increasing GLUT4 expression improves glycemic control. Estradiol (E2) regulates the expression of Slc2a4, by a mechanism in which estrogen receptors (ERs) play opposite effects: ER-alpha stimulates, whereas ER-beta inhibits the expression. Transcriptional effects of ERs involve co-regulators, notably the transcription factor SP1, a powerful enhancer of Slc2a4. However, the role of SP1 in the ERs-mediated regulation of Slc2a4 is unknown; and that was the aim of the present study. Differentiated adipocytes 3T3-L1 were treated (24 hours) with E2, ER-alpha agonist (PPT) or ER-beta agonist (DPN). It was analyzed: gene expression (RT-qPCR) of Slc2a4 and Sp1; total content o GLUT4 and nuclear content of ER-alpha/beta and SP1 (Western blotting); binding activity of SP1 into Slc2a4 promoter (electrophoretic mobility shift assay); and content of nuclear SP1/ER-alpha complexes (immunoprecipitation). Results confirmed that E2 increases the expression of Slc2a4/GLUT4, by the dominant effect of ER-alpha. The ER-alpha agonist PPT increased the nuclear content of SP1, the interaction of SP1/ER-alpha, and the binding activity of SP1 into the Slc2a4. The agonist DPN evinced that ER-beta activity does not involve the SP1. In conclusion, the enhancer effect of ER-alpha upon Slc2a4 gene expression involves a transactivation mechanism via SP1. This observation point outs the cooperation of ER-alpha/SP1 as a new target for the development of approaches to treat insulin resistance and T2DM

O diabetes mellitus tipo 2 (DM2) é caracterizado pela presença de resistência à insulina, a qual pode ser modulada pelo estrógeno, tanto em fêmeas como em machos. Nesse processo, o transportador de glicose GLUT4 (gene Slc2a4, solute carrier family 2 member 4) desempenha papel importante, pois aumento da expressão do GLUT4 melhora o controle glicêmico. Estradiol (E2) regula a expressão do Slc2a4 por meio do balanço dos efeitos contrários de seus receptores (ERs): ER-alfa estimula e ER-beta inibe a expressão. Efeitos transcricionais dos ERs envolvem a participação de co-reguladores, destacadamente o SP1 (transcription factor Sp1), potente estimulador do Slc2a4. Entretanto, o papel do SP1 na regulação do Slc2a4 mediada pelos ERs é desconhecido; e este foi o objetivo do presente estudo. Investigou-se adipócitos maduros 3T3-L1, tratados por 24 horas com E2, agonista de ER-alfa (PPT) ou agonista de ER-beta (DPN). Avaliou-se: a expressão gênica (RT-qPCR) de Slc2a4 e Sp1; o conteúdo (Western blotting) total de GLUT4 e o nuclear de ER-alfa/beta e SP1; a atividade de ligação do SP1 no Slc2a4 (ensaio de mobilidade eletroforética); e a formação de complexos SP1/ER-alfa (imunoprecipitação). Os resultados confirmaram que E2 aumenta a expressão de Slc2a4/GLUT4 pela ação preponderante do ER-alfa. O agonista PPT aumentou: o conteúdo nuclear de SP1, a interação SP1/ER-alfa e a atividade de ligação do SP1 no Slc2a4. O agonista DPN indicou que a ação repressora do ER-beta não envolve o SP1. Conclui-se que o efeito estimulador do ER-alfa na expressão do Slc2a4 envolve mecanismo de transativação gênica via SP1. Essas observações colocam a cooperação ER-alfa/SP1 como um novo alvo para o desenvolvimento de medidas terapêuticas para resistência à insulina e diabetes mellitus tipo 2 / Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, which can be modulated by estrogen in both females and males. In this process, the glucose transporter GLUT4 (solute carrier family 2 member 4 gene - Slc2a4) plays an important role, since increasing GLUT4 expression improves glycemic control. Estradiol (E2) regulates the expression of Slc2a4, by a mechanism in which estrogen receptors (ERs) play opposite effects: ER-alpha stimulates, whereas ER-beta inhibits the expression. Transcriptional effects of ERs involve co-regulators, notably the transcription factor SP1, a powerful enhancer of Slc2a4. However, the role of SP1 in the ERs-mediated regulation of Slc2a4 is unknown; and that was the aim of the present study. Differentiated adipocytes 3T3-L1 were treated (24 hours) with E2, ER-alpha agonist (PPT) or ER-beta agonist (DPN). It was analyzed: gene expression (RT-qPCR) of Slc2a4 and Sp1; total content o GLUT4 and nuclear content of ER-alpha/beta and SP1 (Western blotting); binding activity of SP1 into Slc2a4 promoter (electrophoretic mobility shift assay); and content of nuclear SP1/ER-alpha complexes (immunoprecipitation). Results confirmed that E2 increases the expression of Slc2a4/GLUT4, by the dominant effect of ER-alpha. The ER-alpha agonist PPT increased the nuclear content of SP1, the interaction of SP1/ER-alpha, and the binding activity of SP1 into the Slc2a4. The agonist DPN evinced that ER-beta activity does not involve the SP1. In conclusion, the enhancer effect of ER-alpha upon Slc2a4 gene expression involves a transactivation mechanism via SP1. This observation point outs the cooperation of ER-alpha/SP1 as a new target for the development of approaches to treat insulin resistance and T2DM

Energy homeostasis is an important physiological mechanism essential for balancingenergy flow through the living systems by managing overall metabolism in the body. Thus, energy homeostasis is under a tight control by means of extremely well-regulated energy metabolism. One of the most common metabolic disorders that occur following disruption in energy homeostasis mechanisms is obesity. Obese individuals develop insulin resistance in the peripheral tissues (fat and muscle) and may also include non-alcoholic fatty liver disease. Insulin resistance is the primary factor responsible for the development of type 2 diabetes mellitus (T2D). Towards control and management of T2D condition, insulin, drugs that regulate the insulin sensitivity and drugs that regulate glucose metabolism are widely used. Repeated insulin administration is painful, expensive and requires constant glucose monitoring while other drugs have various limitations and side effects. Therefore, there is wide scope development of new anti-diabetic molecules for effective management of T2D. Studies related to energy metabolism are necessary to understand the cause of such disorders and improve existing methods to manage metabolic abnormalities. Animal models to understand such metabolic disorders have been developed by chemical treatments and genetic modifications, but diet-induced obese (DIO) animal models appear to be the better among all the models reported. DIO animal models are known to most closely mimic the physiological situation. Apart from the experimental model system studies have been conducted under physiological conditions to gain knowledge on possible mechanisms behind energy balance maintained and established during extreme situations such as pregnancy and lactation. To support fetal growth and milk synthesis several metabolic adjustments occur during pregnancy and lactation without the major disruption in the maternal energy homeostasis. In the present study, to gain knowledge on the mother’s body glucose, lipid management and insulin responses throughout the gestation and lactation periods analyses were carried out during at different stages of pregnancy and lactation in rats. It was observed that during pregnancy, the dam developed insulin resistance in peripheral tissues with decreased activation of insulin pathway and reduced glucose utilization while the liver remained unaffected. Although, as soon as the lactation began, peripheral tissue such as muscle developed increased insulin sensitivity associated with increased expression of glucose transporter gene and higher glucose metabolism. The reversal of insulin response in the muscle tissue observed during lactation appears to be a suitable model system for understanding the process by which the body undergoes a transition from insulin resistant state to sensitive state under a physiological condition. Interestingly, early lactation period is known to have much lower levels of insulin available to act upon peripheral tissues. Factors involved in this transition could be potential therapeutic agents for control of T2D, since during early stages of T2D muscle appears to be the first metabolic organ to exhibit resistance to insulin. The undercarboxylated osteocalcin (UNOC) has been reported to function as anti-diabetic molecule. UNOC is released from skeletal system during bone turnover, especially due to resorption process. Experiments were carried out to examine the role of UNOC during the transition from insulin resistant state of pregnancy to sensitive state of lactation period. It was observed that UNOC levels were lower during pregnancy, but increased during early lactation (day 3 to 6 of lactation). The increased UNOC levels seen during early lactation was higher than the levels observed in non-pregnant, non-lactating (NPNL) rats and the UNOC levels decreased following removal of pups immediately after parturition. It was noted that altering UNOC levels during early lactation altered the insulin response of the whole body and muscle transporter-4 expression (glut4) of lactating rats. A significant increase in bone turnover was also observed during lactation compared to NPNL and pregnant rats. The data suggest that increased bone turnover leads to increased UNOC levels in blood during lactation. Estrogen is known as bone protector molecule which acts via its receptors, estrogen receptor α and β (ERα and β). It was reported that ERβ is a dominant regulator of estrogen signaling when both the receptors of estrogen i.e. ERα and ERβ coexist in the target tissue and estrogen levels are relatively higher. Compared to NPNL rats estrogen levels have shown to be higher during late pregnancy and lower during early lactation. It was observed that liver and adipose tissues largely express ERα, but the muscle showed expression of both the receptors in NPNL rats indicating that muscle is the metabolic tissue that may be modulated by both the receptors. It has been reported that ERβ suppresses ERα action on glut4 transcription in the myocytes. It is possible that the altered ERs ratio modulates glut4 expression during late pregnancy and early lactation. The receptor expression ratio data indicated that muscle is an ERβ dominant during late pregnancy, while it is ERα dominant during early lactation. Further, alteration in UNOC levels during early lactation changed ERs ratio but not sufficient enough to alter the ER dominance, indicating lack of effect of UNOC on ER dominance during early lactation. Experiments were conducted to alter insulin sensitivity during early lactation to extrapolate physiological findings to a pathological condition of the DIO model by feeding rats with high-fat diet (HFD). During early lactation, HFD dams had lower insulin response, lower circulatory UNOC level and lower UNOC receptor (GPRC6A) expression in the muscle. Gene expression of muscle glut4 was lower in HFD rats and the tissue remained ERα dominant indicating no role of HFD on ERs ratio in muscle during early lactation. UNOC has been found to have negative effect on lipid accumulation. During pregnancy, lipid accumulation is one of the first events essential for proper fetal development. Since UNOC levels were suppressed during pregnancy, experiments were carried out to examine relevance of UNOC suppression on lipid accumulation during early pregnancy. For this purpose, pharmacological approaches were utilized to alter UNOC levels during early pregnancy. It was observed that the transient elevation of UNOC levels caused decrease in maternal fat depots without changing circulatory triacylglyceride (TAG) levels. In experiments that decreased UNOC levels in NPNL state to mimic lower levels of UNOC present during early pregnancy, it was found fat storage was higher and TG was found to be lowered in the circulation. These results indicate that UNOC can cause a reduction in fat accumulation and TG levels but UNOC effects on TG levels, was not observed during pregnancy. The data taken together suggest that suppression of UNOC is required for better fat deposition in the mother’s body. Although, some studies have indicated an insulin response transition occurring during pregnancy to lactation, but the factors involved in this transition have not been reported. This report discusses about the factors such as UNOC and ERs and their involvement in the transition process. UNOC role has been studied in genetically modified models and in metabolic disorders such as obesity model system and evidence for physiological role of UNOC would further support its candidature as anti-diabetic molecule. The present research work is the first report to detail relevance of UNOC in physiological conditions such as pregnancy and lactation for glucose and lipid management.