Um dos principais fatores patogênicos do quadro de resistência à insulina é a deficiência na expressão de Slc2a4/GLUT4 que, ao longo prazo, causa perda da homeostasia glicêmica. Além disso, a hiperglicemia constante leva a uma glicotoxicidade que também contribui para a patogênese desse quadro. O aumento da glicose circulante leva à formação de produtos finais de glicação avançada (AGEs) que, ao interagem com seu receptor RAGE, desencadeiam processos inflamatórios e estresse oxidativo e de retículo na célula, culminando na ativação da via do NFKB, fator transcricional conhecido por ser repressor da expressão do gene Slc2a4. Nesse sentido, o objetivo do presente estudo visou investigar os efeitos dos AGEs na expressão de Slc2a4/GLUT4 e se esse efeito é mediado pela ativação da via do NFKB. Para isso, utilizamos adipócitos da linhagem 3T3-L1 tratados com albumina sérica bovina sem modificações ou conjugada com glicolaldeído e verificamos a expressão dos genes Slc2a4, Nfkb1 e Rela, a abundância das proteínas GLUT4, p65 e p50, o grau de fosforilação das proteínas IKK alfa e IKK beta e a atividade de ligação da subunidade p65 no promotor do gene Slc2a4. Os resultados indicaram que a albumina glicada é capaz de modular a expressão de Slc2a4/GLUT4 de maneira hormética, isto é, em concentração baixa (0,4 mg/mL) e tempo curto (24 horas), houve um aumento na expressão do gene e da proteína, porém, há uma redução da expressão do gene e da proteína quando em concentração alta (5,4 mg/mL) e tempo prolongado (72 horas). Além disso, a albumina glicada induz atividade pró-inflamatória no adipócito e aumento da quantidade de p65 e p50 no núcleo em concentração alta e tempo prolongado. No entanto, a ativação da via do NFKB não se dá pela via canônica, pois não foi observado ativação de IKK. Por fim, apesar de ser um dado preliminar, a albumina glicada induz aumento de ligação do NFKB na região promotora do gene Slc2a4. Em suma, o presente estudo demonstra que os AGEs participam não só da gênese das complicações degenerativas presentes no diabetes mellitus, como também, contribui ativamente para a perda da homeostasia glicêmica. / One of the main pathogenic factors of the insulin resistance is the deficiency in the expression of Slc2a4/GLUT4, which in the long term causes impaired glucose homeostasis. In addition, constant hyperglycemia leads to a glycotoxicity that also contributes to the pathogenesis of this condition. Increased circulating glucose leads to the formation of advanced glycated end products (AGEs) which, when interacting with RAGE receptor, trigger inflammatory processes and oxidative and reticulum stress in the cell, culminating in the activation of the NFKB pathway. This transcriptional factor is a known repressor of the Slc2a4 gene. In this sense, the objective of the present study was to investigate the effects of AGEs on the expression of Slc2a4/GLUT4 and whether this effect is mediated by the activation of the NFKB pathway. For this, we used 3T3-L1 adipocytes treated with bovine serum albumin without modification or conjugated with glycolaldehyde and verified the expression of the genes Slc2a4, Nfkb1 and Rela, abundance of GLUT4, p65 and p50 proteins, the degree of phosphorylation of IKK alpha and IKK beta proteins and p65 subunit binding activity in the Slc2a4 gene promoter. The results indicated that glycated albumin is able to modulate the expression of Slc2a4/GLUT4 in a hormetic manner, i.e. at low concentration (0.4 mg/mL) and short time (24 hours), there was an increase in gene expression and protein. However, there is a reduction of the gene and the protein expression when in high concentration (5.4 mg/mL) and prolonged time (72 hours). In addition, glycated albumin induces proinflammatory activity in the adipocyte and increases the amount of p65 and p50 in the nucleus in high concentration and prolonged time. Nonetheless, the activation of the NFKB pathway does not occur through canonical pathway, since no IKK activation was observed. Finally, despite being a preliminary finding, glycated albumin induces increased NFKB binding in the promoter region of the Slc2a4 gene. In summary, the present study demonstrates that AGEs participate not only in the genesis of the degenerative complications present in diabetes mellitus, but also contributes actively to the loss of glycemic homeostasis.

O estudo investigou a participação do sistema nervoso simpático ß-adrenérgico sobre a regulação da expressão gene do GLUT4 em músculo vermelho (sóleo) e branco (EDL) no jejum. Foram estudados ratos alimentados livremente, jejuados por 48 horas e sob bloqueio ß1/ß2 ou bloqueio ß1/ß2/ß3 ou sob estímulo ß1/ß2/ß3-adrenérgicos. Os músculos sóleo e EDL foram analisados por Northern e Western Blotting. O jejum diminuiu o peso dos animais, a glicemia, a insulinemia, e aumentou concentração de ácidos graxos livres (AGL). O bloqueio ß1/ß2 aumentou a insulinemia, o bloqueio ß1/ß2/ß3 reduziu a glicemia e o estímulo ß1/ß2/ß3 aumentou AGL. O jejum aumentou mRNA do GLUT4 no sóleo e EDL, os bloqueios ß-adrenérgicos reduziram o mRNA no sóleo e EDL, e o estímulo ß aumentou o mRNA somente no sóleo. O jejum aumentou a proteína GLUT4 no sóleo e nos tratamentos somente reduziu com o bloqueio triplo, no EDL. O jejum aumentou a expressão do gene do GLUT4 em músculo esquelético. A atividade simpática ß-adrenérgica é fundamental para aumentar a expressão do GLUT4 no sóleo e manter no EDL. / The study investigated the participation of ß-adrenergic sympathetic nervous system in the regulation of GLUT4 gene expression in red (soleus) and white (EDL) muscles during fasting. Rats were fasted for 48hours and treated with ß1/ß2-adrenoceptors antagonist or ß1/ß2/ß3-adrenoceptors antagonist or ß1/ß2/ß3-adrenoceptors agonist. Soleus and EDL muscles were analyzed by Northern and Western Blotting. Fasting decreased body weight, glycemia, insulinemia, and increased non esterified free fatty acids (NEFA). The insulinemia increased with ß1/ß2 blockade, glycemia decreased with triple blockade, NEFA increased with ß stimulus. The GLUT4 mRNA increased in soleus and EDL after fasting, and it decreased in soleus and EDL after ß1/ß2 blockade and triple blockade, and increased in soleus after ß stimulus. GLUT4 protein increased in soleus of fasted rats, and decreased with triple blockade in EDL. Fasting increased GLUT4 gene expresion in skeletal muscle. The ß-adrenergic sympathetic activity is essencial to increase GLUT4 expression in soleus and to preserve it in EDL.

O estudo investigou a participação do sistema nervoso simpático ß-adrenérgico sobre a regulação da expressão gene do GLUT4 em músculo vermelho (sóleo) e branco (EDL) no jejum. Foram estudados ratos alimentados livremente, jejuados por 48 horas e sob bloqueio ß1/ß2 ou bloqueio ß1/ß2/ß3 ou sob estímulo ß1/ß2/ß3-adrenérgicos. Os músculos sóleo e EDL foram analisados por Northern e Western Blotting. O jejum diminuiu o peso dos animais, a glicemia, a insulinemia, e aumentou concentração de ácidos graxos livres (AGL). O bloqueio ß1/ß2 aumentou a insulinemia, o bloqueio ß1/ß2/ß3 reduziu a glicemia e o estímulo ß1/ß2/ß3 aumentou AGL. O jejum aumentou mRNA do GLUT4 no sóleo e EDL, os bloqueios ß-adrenérgicos reduziram o mRNA no sóleo e EDL, e o estímulo ß aumentou o mRNA somente no sóleo. O jejum aumentou a proteína GLUT4 no sóleo e nos tratamentos somente reduziu com o bloqueio triplo, no EDL. O jejum aumentou a expressão do gene do GLUT4 em músculo esquelético. A atividade simpática ß-adrenérgica é fundamental para aumentar a expressão do GLUT4 no sóleo e manter no EDL. / The study investigated the participation of ß-adrenergic sympathetic nervous system in the regulation of GLUT4 gene expression in red (soleus) and white (EDL) muscles during fasting. Rats were fasted for 48hours and treated with ß1/ß2-adrenoceptors antagonist or ß1/ß2/ß3-adrenoceptors antagonist or ß1/ß2/ß3-adrenoceptors agonist. Soleus and EDL muscles were analyzed by Northern and Western Blotting. Fasting decreased body weight, glycemia, insulinemia, and increased non esterified free fatty acids (NEFA). The insulinemia increased with ß1/ß2 blockade, glycemia decreased with triple blockade, NEFA increased with ß stimulus. The GLUT4 mRNA increased in soleus and EDL after fasting, and it decreased in soleus and EDL after ß1/ß2 blockade and triple blockade, and increased in soleus after ß stimulus. GLUT4 protein increased in soleus of fasted rats, and decreased with triple blockade in EDL. Fasting increased GLUT4 gene expresion in skeletal muscle. The ß-adrenergic sympathetic activity is essencial to increase GLUT4 expression in soleus and to preserve it in EDL.

GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.

Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation. PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P`s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase a (PIP4K2a) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although PtdIns5P is converted by PIP4K2a to PtdIns(4,5)P2 which is a precursor of the potent PI PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2a, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach. The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2a. Quantifying the level of PIs suggested that PIP4K2a expression increases the removal of PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2a. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.

Ad libitum high-fat diet (HFD)-induced obesity leads to insulin resistance in skeletal muscle, altered gene expression, and altered growth signaling, all of which contributes to pathological changes in metabolism. Liver kinase B1 (LKB1) is an important metabolism regulator. The purpose of this dissertation was to understand how knocking out LKB1 influences HFD induced adaptations in mouse skeletal muscle. To do so, control and skeletal muscle LKB1 knock-out (LKB1-KO) mice were put on either standard diet (STD) or HFD for 1 week or 14 weeks, or put on the HFD for 14 weeks and then switched to STD for 1 week (switched diet). The major differences in adaptation in the LKB1-KO mice include: 1) lower fasting blood glucose levels but impaired glucose tolerance compared to WT mice (although conflicting results are generated if the data is not normalized to fasting blood glucose levels), 2) altered expression of 16 HFD-induced genes, and 3) decreased muscle weight. The lower fasting blood glucose in LKB1-KO mice was likely due to elevated serum insulin levels, and the impaired glucose tolerance was associated with decreased phosphorylation of TBC1D1, an important regulator of insulin stimulated glucose uptake. 16 potential important target genes (metabolism, mitochondrial, cytoskeleton, cell cycle, cell-cell interactions, enzyme, ion channel) were identified in the context of HFD feeding and LKB1-KO. These genes were quantified by RT-PCR and grouped according to changes in their patterns of expression among the different groups. Among several other interesting changes in gene expression, the muscle growth-related protein, Ky was not affected by short-term HFD, but increased after long-term HFD, and did not decrease after switched diet, showing that its expression may be an important long-term adaptation to HFD. LKB1-KO promoted anabolic signaling through increasing t-eIF2α and eIF4E expression, and promoted protein degradation through increasing protein ubiquitination. Because the degradation is the main effect and lead to muscle weight decrease. The effect of HFD and/or LKB1-KO on the LKB1-AMPK system was also determined. The results showed that knocking-out LKB1 decreased AMPK activity, decreased nuclear distribution for AMPK α2 and increased AMPK α1 expression. Long-term HFD increased t-AMPK expression in LKB1-KO mice, decreased the cytoplasm p-AMPK and nuclear p/t-AMPK ratio in CON mice. Together the findings of this dissertation demonstrated HFD induced glucose/insulin tolerance, while LKB1-KO had a controversial effect on glucose/insulin sensitivity. Both HFD and LKB1-KO affect AMPK expression and cellular location, while LKB1-KO also affects AMPK activity. LKB1-KO promoted protein degradation through ubiquitination in skeletal muscle.

GLUT4 is the principal insulin-responsive glucose transporter in skeletal muscle. Insulin stimulation leads to exocytosis of intracellular GLUT4-containing vesicles to the cell surface, thereby increasing glucose uptake. Muscle contraction also elevates cell surface GLUT4 by a less understood mechanism. Once at the cell surface, GLUT4 may be subject to additional regulation, such as by modulation of its internalization rate or its intrinsic activity. The objective of this thesis was to identify the mechanism of GLUT4 internalization in muscle cells and to determine whether it is regulated by insulin treatment or by the signals elicited by muscle contraction. Skeletal muscle cells in culture stably expressing myc-tagged GLUT4 were used. We found that GLUT4 internalizes simultaneously through a clathrin-dependent and a clathrin- and caveolae-independent and cholesterol- and dynamin-dependent pathway. Insulin did not regulate GLUT4 internalization. In contrast, mitochondrial uncoupling, which may mimic the heightened energy demand that occurs during muscle contraction, retarded GLUT4 internalization by inhibiting the clathrin-independent route. Activation of both AMP-dependent kinase (AMPK) and protein kinase C (PKC) was necessary and sufficient for this response. We further hypothesized that the intrinsic activity of GLUT4 may be regulated under some conditions, based on a discrepancy between the amount of cell surface transporters and the rate of glucose uptake. In particular, inhibitors of p38 mitogen-activated protein kinase (p38MAPK) lowered insulin-dependent glucose uptake without reducing the number of GLUT4 units at the surface. We found that p38MAPK is activated by insulin through TAB1-dependent autophosphorylation, yet p38MAPK was dispensable for insulin-stimulated glucose uptake. Mechanisms other than p38MAPK must be involved in the regulation of GLUT4 intrinsic activity. In conclusion, in addition to its exocytosis, the activity and endocytosis of GLUT4 are regulated by stimuli that increase the rate of glucose uptake into muscle.

GLUT4 is the principal insulin-responsive glucose transporter in skeletal muscle. Insulin stimulation leads to exocytosis of intracellular GLUT4-containing vesicles to the cell surface, thereby increasing glucose uptake. Muscle contraction also elevates cell surface GLUT4 by a less understood mechanism. Once at the cell surface, GLUT4 may be subject to additional regulation, such as by modulation of its internalization rate or its intrinsic activity. The objective of this thesis was to identify the mechanism of GLUT4 internalization in muscle cells and to determine whether it is regulated by insulin treatment or by the signals elicited by muscle contraction. Skeletal muscle cells in culture stably expressing myc-tagged GLUT4 were used. We found that GLUT4 internalizes simultaneously through a clathrin-dependent and a clathrin- and caveolae-independent and cholesterol- and dynamin-dependent pathway. Insulin did not regulate GLUT4 internalization. In contrast, mitochondrial uncoupling, which may mimic the heightened energy demand that occurs during muscle contraction, retarded GLUT4 internalization by inhibiting the clathrin-independent route. Activation of both AMP-dependent kinase (AMPK) and protein kinase C (PKC) was necessary and sufficient for this response. We further hypothesized that the intrinsic activity of GLUT4 may be regulated under some conditions, based on a discrepancy between the amount of cell surface transporters and the rate of glucose uptake. In particular, inhibitors of p38 mitogen-activated protein kinase (p38MAPK) lowered insulin-dependent glucose uptake without reducing the number of GLUT4 units at the surface. We found that p38MAPK is activated by insulin through TAB1-dependent autophosphorylation, yet p38MAPK was dispensable for insulin-stimulated glucose uptake. Mechanisms other than p38MAPK must be involved in the regulation of GLUT4 intrinsic activity. In conclusion, in addition to its exocytosis, the activity and endocytosis of GLUT4 are regulated by stimuli that increase the rate of glucose uptake into muscle.

La función de las caveolas y de la caveolina in vivo ha sido motivo de controversia, de hecho se han implicado en procesos de endocitosis y transcitosis, transporte de colesterol y ácidos grasos, regulación de procesos de transducción de señales y tumorigénesis (Liu et al., 2002). Sin embargo, la generación de los ratones KO de caveolina-1 (Drab et al., 2001; Razani et al., 2001a) mostraba que estos ratones eran perfectamente viables y fértiles, aunque estudios posteriores relacionaban a caveolina-1 con la homeostasis lipídica (Razani et al., 2002a).A partir de estos antecedentes, el objetivo general de esta tesis ha sido: · Definir la función de las caveolas/ caveolina-1 en la fisiología del adipocitoNos planteamos así, nuestro primer objetivo:1- Obtención de líneas 3T3L1 deficientes en caveolina-1 mediante silenciamiento génico posttranscripcional.2- Analizar la presencia de caveolas y la distribución de la caveolina-1 remanente en las membranas de los adipocitos y estudiar la estructura y composición de los rafts lipídicos/caveolas en estos adipocitos deficientes en caveolina-1.3- Analizar la diferenciación adipocitaria de los preadipocitos 3T3L1 deficientes en caveolina-1. 4- Analizar la acción de la insulina en los adipocitos deficientes en caveolina-1, 5- Estudiar el papel de caveolina-1 en el metabolismo lipídico del adipocito.A partir de los objetivos planteados conseguimor obtener las siguientes conclusiones en el desarrollo de esta tesis:1. Se ha conseguido la transducción eficiente de adipocitos 3T3L1 maduros usando estas partículas lentivirales que expresan transgenes, aunque no se ha logrado transducir los adipocitos maduros eficazmente en el caso de los lentivirus que expresan moléculas de siRNA.2. Los preadipocitos 3T3L1 son eficientemente transducidos usando las partículas lentivirales que permiten la expresión tanto de transgenes como de moléculas de siRNA. Mediante esta técnica hemos generado líneas estables 3T3L1 que expresan moléculas de siRNA de caveolina-1 y que presentan una reducción en la expresión de la proteína caveolina-1 de más del 93%.3. La disminución de la proteína caveolina-1 en más de un 93% en adipocitos 3T3L1 provoca una disminución equivalente en el número de caveolas en la membrana plasmática.4. La deficiencia en caveolina-1 en los adipocitos 3T3L1 no afecta a la localización de los marcadores proteicos y lipídicos característicos de los rafts lipídicos, que continúan recuperándose en las fracciones ligeras de los extractos celulares durante el aislamiento bioquímico de los rafts lipídicos. 5. La deficiencia en caveolina-1 y caveolas no afecta a la diferenciación de los preadipocitos 3T3L1 a adipocitos maduros, ni tampoco a las características morfológicas y el contenido lipídico (triacilglicéridos y colesterol) de los adipocitos maduros. 6. Los adipocitos 3T3L1 con una disminución del 93% en la expresión de caveolina-1, presentan una reducción del 50% en la expresión de las proteínas GLUT4 y del 40% en el caso del receptor de insulina, sin que este hecho responda a una menor cantidad de sus RNA mensajeros, sino a una disminución en la vida media de estas proteínas. Este dato sugiere que caveolina-1 participe en la estabilidad de estas proteínas.7. El déficit de caveolina-1 en los adipocitos 3T3L1 no afecta a las vías de señalización de la insulina (dependiente de PI3K y dependiente de Cbl-TC10) ni tampoco a la translocación de GLUT4 a la membrana plasmática inducida por la insulina ni a la estimulación del transporte de glucosa. 8. La presencia de caveolas/caveolina-1 no es necesaria para la correcta internalización de GLUT4 durante la reversión de la acción de la insulina.9. Las caveolas/caveolina-1 tampoco son necesarias para la acción de la insulina inhibiendo o activando la lipólisis y la lipogénesis en los adipocitos 3T3L1, respectivamente.10. La deficiencia de caveolina-1 y caveolas no altera el transporte de ácido palmítico a través de la membrana plasmática de adipocitos 3T3L1.Esta deficiencia tampoco altera la activación lipolítica por los efectores beta-adrenérgicos: Isoproterenol 10mu-M e IBMX 0,5mM en los adipocitos 3T3L1.PALABRAS CLAVE: Cavelonia-1, Caveola, Adipocito, Insulina, GLUT4, Receptor de insulina / Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin protein. Caveolae are particularly enriched in adipocytes. Here we analyzed the effects of caveolin-1 knockdown and caveolae ablation on adipocyte function. To this end, we obtained several multiclonal mouse 3T3-L1 cell lines with a reduced expression of caveolin-1 (95% reduction) by a small interfering RNA approach using lentiviral vectors. Control cell lines were obtained by lentiviral infection with lentiviral vectors encoding appropriate scrambled RNAs. Caveolin-1 knockdown adipocytes showed a drastic reduction in the number of caveolae (95% decrease) and cholera toxin-labeling was reorganized in dynamic plasma membrane microdomains. Caveolin-1 depletion caused a specific decrease in GLUT4 glucose transporter and insulin receptor protein levels. This reduction was not the result of a generalized defect in adipocyte differentiation or altered gene expression but was explained by faster degradation of these proteins. Caveolin-1 knockdown adipocytes showed reductions in insulin-stimulated glucose transport, insulin-triggered GLUT4 recruitment to the cell surface and insulin receptor activation. In all, our data indicate that caveolin-1 loss-of-function reduces maximal insulin response through lowered stability and diminished expression of insulin receptors and GLUT4. We propose that caveolin-1/caveolae control insulin action in adipose cells.

Physical activity can prevent cardiovascular disease, obesity, type II diabetes and some types of cancer. With only 3.5% of adults meeting the recommended physical activity guidelines, research has focused on the regulatory factors that influence physical activity level. Genetic influence accounts for the majority of physical activity regulation. However, there is limited information on the mechanisms that affect physical activity, in part, because of a lack of reliable methods to silence genes in vivo. The purpose of this dissertation was to identify mechanisms in skeletal muscle that influence physical activity. The methods used to accomplish the purpose of this dissertation were the evaluation of Vivo-morpholinos as a gene silencing tool in skeletal muscle and brain, identification of proteins in skeletal muscle associated with increased physical activity level, and the use Vivo-morpholinos to transiently knockdown the identified skeletal muscle proteins as a means to elucidate mechanisms for the peripheral regulation of physical activity. Overall, this study showed that Vivo-morpholinos effectively silenced genes in skeletal muscle yet required the use of a pharmacological aid to achieve gene silencing in the brain. Additionally proteins associated with calcium regulation (Annexin A6 and Calsequestrin 1) and the Kreb’s (TCA) cycle were found to be over expressed in the high active animals. The knockdown of Annexin A6 and Calsequestrin 1 resulted in a significant decrease in physical activity, thus showing that calcium regulation could influence the physical activity response. While these results provide a potential mechanism for the peripheral regulation of physical activity, a side effect observed was that Vivo-morpholinos can hybridize resulting in increased mortality rates of the treatment animals. Therefore, we developed methods to alleviate the toxic effects of Vivo-morpholinos. Thus, this dissertation refined a technique for determining a gene’s effect in an in vivo model and identified two candidate proteins (Annexin A6 and Calsequestrin 1) that play a role in regulating daily physical activity.