Several studies have demonstrated through the activation of its central nervous system (CNS) receptor, that insulin has a key role in important functions related to neuronal plasticity, thus modulat ing the cascade activat ion of intracellular signaling. In addit ion, research f rom our laboratory demonstrates that insulin regulates lipid signal transduction pathways in the SNC, which are altered in the process of normal aging. The purpose of the present research is to study a lipid signaling pathway associated to the CNS insulin signaling transduct ion mechanism and its role in normal aging. Particular emphasis is put on the action of insulin as activator of the diacylglycerol kinase (DAGK), a key enzyme in the regulation of two important lipid messengers, namely phosphat idic acid (PA) and diacylglycerol (DAG), both of which are essential molecules in the regulation of cellular responses. The f irst stage of this research focused on the character izat ion of DAGK activity in cerebral cortex synaptosomes of adult rats in order to evaluate its activity in endogenous and exogenous DAGs by means of the use of detergents such as the carrier of lipid substrate DAG. The kinetic parameters of DAGK activity f rom endogenous DAG and exogenous DAG with dif ferent acyl composition were also studied. We analyzed dipalmitoylglycerol (DPG), 1-stearoyl, 2-arachidonoylglycerol (SAG) and dioleoylglycerol (DOG) as exogenous lipid substrates. Results show that the enzymatic activity preferentially uses DAG containing arachydonic acid in position 2 of the chain of glycerol (SAG) as substrate, thus indicating the presence of épsilon type of DAGK (DAGKe) in CC synaptosomes, which is the only type with substrate preferences. The analysis of DAGK act ivat ion by insulin revealed that hormonal action stimulates DAGK act ivity by means of two mechanisms: 1) by increasing the levels of DAG in the plasmatic membrane f rom the stimulus of PI-PLC and PC-PLD/PAP2 pathways, thus producing an increase of DAGK action; and 2) by stimulat ing DAGK act ivity in a direct and independent form of DAG generation in the membrane. It was also found that the stimulus that exerts insulin independently of DAG generation is associated to an epsilon DAGK act ivity which is possibly regulated by a decrease in phosphoinosit ide levels and which could be associated to polyphosphoinositides resynthesis. In CC synaptosomal membranes of senile animals, an increase in DAG content was observed without a signif icant modif ication in acyl composition. This can be correlated with the simultaneous f inding of a decreased DAGK activity in endogenous DAGs. Whereas the use of saturated exogenous DAG (DPG) showed no modif icat ions by aging, an important decrease in DAGK ability to transform SAG into PA was observed. A similar result was observed in synaptosomes f rom hippocampus. It was also observed that insulin act ing in CC synapt ic terminals exerted a minor st imulatory ef fect with respect to adult animals in DPG transformation, whereas it showed the same ef fectiveness to increase SAG transformation. Interestingly, insulin strongly st imulated DAGK activity in SAG in hippocampus synaptosomes of senile rats with respect to adults. As to the recovery of a PA with the potential ability to be phosphoinositide precursor, our f indings suggest that this hormonal action is a compensatory mechanism in the regulation of the synthesis of these phospholipids. This partly explains the hormone role as neuroprotector agent.

El receptor de acetilcolina nicotínico (AChR) es un canal iónico activado por ligandos, presente en el sistema nervioso central y en la unión neuromuscular. Su estabilidad en la membrana celular es de suma importancia, ya que determina el nivel de respuesta colinérgica, que puede ser alterada ante diversas situaciones fisiológicas y patológicas. En esta Tesis utilizamos como modelo experimental a las células CHO-K1/A5, una línea celular que expresa de forma estable al AChR muscular adulto. En el primer capítulo nos centramos en el estudio de la posible acción de los diacilgliceroles (DAG) sobre la expresión/distribución del AChR. Realizamos los experimentos con el dioctanoilglicerol (DOG), y definimos dos tiempos de tratamiento, a los que denominamos tiempos cortos (3-4 hs) y largos (18 hs) de incubación. A tiempos cortos, el DOG aumenta los niveles de AChR presentes en la superficie celular, mientras que a tiempos largos los disminuye con el concomitante aumento de los AChR intracelulares. A su vez, el incremento de los DAG endógenos como consecuencia de la alteración de diversas vías metabólicas, afecta de igual manera al AChR a tiempos cortos. Como el tratamiento con un éster de forbol, molécula análoga al DAG por su capacidad de interaccionar con los dominios C1 de diversas proteínas, resultó en los mismos cambios sobre la distribución del AChR, concluimos que los efectos del DOG eran debidos a su papel como segundo mensajero lipídico. Mediante el empleo de inhibidores enzimáticos y de anticuerpos específicos para las proteínas quinasas C (PKC) clásicas, las PKC noveles y las PKD, determinamos que los DAG a tiempos cortos de incubación favorecerían la inhibición de las PKCc al favorecer su desfosforilación, y esto disminuiría la internalización del AChR. Por otra parte, el estímulo con DOG a tiempos largos de las distintas PKC estudiadas, provocaría el efecto opuesto. El segundo capítulo se centró en el estudio de la distribución de los clústeres de AChR, generados por la unión de anticuerpos, en dominios lipídicos con diferentes valores de polarización generalizada (GP). Esta medición se realizó gracias a la tinción de las células con di-4-ANEPPDHQ, una sonda fluorescente capaz de poner en evidencia los cambios en la polaridad de la membrana, relacionados a un orden de membrana determinado. La depleción del colesterol (Col) disminuye los valores promedio de la GP de la membrana, aumentando en consecuencia la asociación de los clústeres de AChR a dominios de membrana más desordenados. Por otra parte, la despolimerización del citoesqueleto de actina con Latrunculina A, produce el efecto contrario al aumentar la asociación de los acúmulos de AChR con dominios de membrana más ordenados. Estos resultados indican que la distribución del AChR en la superficie celular depende del contenido de Col y de la integridad del citoesqueleto de actina. Por otra parte, el AChR se internaliza en pequeñas vesículas con valores bajos de la GP y bajo contenido de Col, situación que se mantiene frente a la depleción del Col producida por el tratamiento con metil-β-ciclodextrina, demostrando entonces que la endocitosis del AChR requiere de la disociación previa del receptor con dominios ricos en Col presentes en la membrana. / The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel, present at the neuromuscular junction and central nervous synapses. Its stability at the cell membrane is important in determining cholinergic responses and can be altered under physiological and pathological conditions. In this thesis, we used CHO-K1/A5 cells, a cell line that expresses the adult muscle AChR in a stable manner. In the first chapter, we focus on the study of the possible effects of diacylglycerols (DAGs) on expression/distribution of AChR. We performed experiments with dioctanoylglycerol (DOG), and define two treatment times, which we name short (3-4 h) and long (18 h) incubation times. DOG increases AChR levels on the cell surface at short incubation times. At long treatment times however, surface AChRs decrease with the concomitant increase of intracellular AChRs. Besides, endogenous DAGs increase due to the alteration of various metabolic pathways, equally affecting AChR at short times. Since treatment with a phorbol ester, a DAG analogous because of its ability to interact with the C1 domain of different proteins, resulted in similar changes on AChR distribution, we conclude that DOG effects were due to its role as a lipid second messenger. By using specific enzyme inhibitors and antibodies to classical and novel protein kinases C (PKC) and PKD, we determined that DAGs inhibit PKCc by promoting its dephosphorylation at shorter incubation times, and thus the internalization of AChR decreases. Moreover, DOG stimulation of different PKCs at long treatment times, causes the opposite effect. The second chapter is focused on the study of AChR clusters distribution, generated by antibody binding, at lipid domains with different generalized polarization (GP) values. This measurement was performed by staining cells with di-4-ANEPPDHQ, a fluorescent probe that senses changes in the polarity of the membrane, related to membrane order. Cholesterol depletion diminishes the mean cell membrane di-4- ANEPPDHQ GP and concomitantly the number of AChR clusters associated with more disordered membrane domains increases. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A has the opposite effect, with more AChR clusters associated with more ordered domains. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane depends on cholesterol content and cytoskeleton integrity. AChR internalizes via small vesicles having lower GP and lower cholesterol content than cell surface. This also occurs upon cholesterol depletion by methyl-β-cyclodextrin. Thus, AChR endocytosis is primed by receptor dissociation from Chol-rich plasmalemma domains.

El diacilglicerol (DAG) es un importante intermediario en la síntesis de muchas clases de lípidos, y es, además, un segundo mensajero producido en respuesta a diversos estímulos extracelulares, capaz de modular la actividad de numerosas enzimas. Una de las vías de metabolización del DAG es la fosforilación llevada a cabo por las DAGK, para dar ácido fosfatídico (PA). El ácido fosfatídico es también un importante segundo mensajero lipídico involucrado en una gran variedad de respuestas celulares. Dada la importancia fisiológica del DAG y del PA como segundos mensajeros lipídicos, y, teniendo en cuenta los numerosos resultados que indican la presencia de las vías de señalización responsables de la síntesis y degradación de estos segundos mensajeros a nivel nuclear, iniciamos nuestros estudios analizando la actividad de las DAGK en los núcleos de la retina bovina, proyectando la investigación hacia la interpretación de su participación en las funciones esenciales de la retina, la recepción y transmisión de la luz. Además, analizamos a nivel nuclear, en las células fotorreceptoras de la retina, los efectos de la insulina, un reconocido protector del sistema nervioso central (SNC) y modulador de la actividad DAGK en otros tipos celulares del SNC. Mediante un protocolo diseñado en nuestro laboratorio, obtuvimos a partir de la retina entera una fracción nuclear enriquecida en los núcleos de las células fotorreceptoras (FNF). En esta fracción nuclear, se pudo detectar actividad DAGK, transformadora del DAG endógeno y exógeno. Se determinó linealidad en función del tiempo de ensayo, el contenido proteico de la fracción nuclear y se analizaron parámetros cinéticos aparentes (Km y Vmax para cada sustrato). Los resultados con detergentes y sustratos diferentes permitieron sugerir la coexistencia de varios tipos de DAGK. Esto fue confirmado por Western Blot (WB) y se detectaron, además, efectos significativos en el contenido nuclear de estas isoformas por efecto de la luz (aumento de la DAGKδ y disminución de las DAGKe, ß y B). Nuestros hallazgos se reforzaron con ensayos enzimáticos en los que se utilizaron condiciones selectivas (preferenciales) para medir la actividad de las isoformas δ y e.En ellos se observó, por efecto de la exposición de la retinas a la luz, una fuerte correlación entre los cambios en el contenido y la actividad de ambas isoformas en la FNF. Se demostró asimismo que el aumento de la actividad DAGK nuclear en respuesta a la luz es dependiente de la actividad PIP2-PLC, y se observó que dicho incremento en la condición lumínica no es debido exclusivamente a un aumento del sustrato de la reacción enzimática, DAG, sino que también participa en la activación el PIP2. También demostramos la presencia de la PKCα en la FNF, PKC de tipo convencional que es activada en presencia de Ca2+ y DAG. La exposición de las retinas a la luz produjo un aumento del estado de fosforilación de la PKCα. Dado que la fosforilación de PKC puede ser mediada por la activación de PDK1, que es dependiente de la activación previa de PI3K, enzima participante de la vía de señalización de insulina, el siguiente objetivo fue determinar la presencia de los principales componentes de las vías de señalización de insulina en la FNF. Nuestros resultados demostraron la presencia de Akt total y en su estado fosforilado (proteína quinasa de la via de la PI3K), y de ERK1/2, pERK1/2 y p38 fosforilada (quinasas correspondientes a la vía de las MAPK). Nuestros estudios demostraron, además, que la exposición de las retinas bovinas a la luz produce un aumento en el estado de fosforilación de Akt, y que induce la translocación de ERK1/2 activada a la FNF. Por otro lado, al analizar los efectos directos e indirectos de la insulina sobre la actividad DAGK nuclear en la FNF de retinas expuestas a la luz o a la oscuridad, fue posible demostrar que la insulina es capaz de modular la actividad DAGK nuclear tanto de forma directa (incubación de los núcleos aislados con insulina) como de manera indirecta (incubación de las retinas bovinas con insulina y posterior análisis de la actividad DAGK en la FNF). Además, se observó que la insulina cumple un rol en los cambios de la actividad DAGK nuclear en respuesta a la luz, y que los efectos de la insulina sobre la actividad DAGK son dependientes de su concentración (los efectos se incrementan con un aumento concomitante de la concentración de insulina empleada en el ensayo enzimático). Teniendo en cuenta los efectos directos de la insulina sobre la actividad DAGK nuclear, analizamos la presencia del receptor de insulina en la FNF por WB e inmunofluorescencia (IF) y demostramos la presencia del mismo en los núcleos de las células fotorreceptoras. Un hallazgo de particular interés fue el de haber demostrado que el contenido del RI aumenta en la FNF cuando las retinas bovinas son expuestas a la luz, lo cual sugiere que la luz puede ser un estímulo capaz de promover la translocación del RI al núcleo de las células fotorreceptoras. Por último, analizamos si la insulina es capaz de participar en la translocación del receptor de insulina al núcleo de las células fotorreceptoras y de mediar la activación de las vías de señalización activadas por la misma a nivel nuclear. Nuestros resultados indicaron que la insulina produce un aumento en el contenido del receptor de insulina nuclear con respecto a la condición luz en ausencia de hormona. Además, la insulina produjo un aumento de ERK1/2 activado en la FNF. En conclusión, nuestros resultados demostraron por primera vez que la exposición de las retinas bovinas a su estímulo natural, la luz, paralelamente a la activación de la típica vía de la fototransducción que se inicia en los segmentos externos, induce a nivel nuclear, la activación de distintas vías de señalización responsables de funciones críticas para la célula, y que a nivel nuclear, pueden intervenir en la transcripción de genes. Nuestros resultados demostraron también que la luz y la insulina son capaces de intervenir en la translocación del receptor de insulina desde la membrana plasmática hacia el núcleo de las células fotorreceptoras, y que la insulina promueve la activación de vías de señalización tanto de forma indirecta, actuando sobre la retina entera, como directa a nivel nuclear, sugiriendo para este último caso que mediaría sus efectos a través de la población nuclear de dicho receptor. / Diacylglycerol (DAG) is an important intermediate in the synthesis of several types of lipids, and it is also a second messenger produced in response to various extracellular stimuli, with the ability to modulate the activity of numerous enzymes. One route of metabolism of DAG is phosphorylation by DAGK to yield phosphatidic acid (PA). PA is also an important lipid second messenger that has been involved in a variety of cellular responses. Given the physiological importance of DAG and PA as lipid second messengers and taking into account the many results that indicate the presence of the signaling pathways responsible for the synthesis and degradation of these second messengers at the nuclear level, we initiated our studies assessing the activity of DAGK in the nuclei of bovine retina, projecting them to the interpretation of their participation in the essential functions of the retina, the receipt and transmission of light. We also analyzed at the nuclear level in retina photoreceptor cells, the effects of insulin, a known protector of the central nervous system (CNS) and DAGK activity modulator in other cell types of the CNS. Using a protocol designed in our laboratory, we obtained from the entire retina a nuclear fraction enriched in photoreceptor cell nuclei (PNF). In the nuclear fraction, DAGK activity could be detected, which is responsible for the transformation of endogenous and exogenous DAG. A linear response was obtained as a function of protein content of the nuclear fraction and as a function of time. The apparent kinetic parameters (Vmax and Km for each substrate) were also determined. The results derived from the different substrates and detergents used suggest the coexistence of various types of DAGK. This was confirmed by Western Blot (WB), and significant effects were detected in the nuclear content of these isoforms by the effect of light (increased of DAGKδ and decreased of DAGK E, ß and B). Our findings were strengthened by further enzyme assays using selective conditions to measure the activity of E and δ isoforms in which, due to the exposure of retina to light, strong correlation was observed between changes in the content and the activity of both isoforms in the PNF. It was also demonstrated that the increase in nuclear DAGK activity in response to light is dependent on PIP2-PLC activity and that this increase in light condition is not due only to an increase of the substrate of the enzymatic reaction, DAG, but also to the fact that PIP2 participates in DAGK activation. We also showed the presence of PKCα in FNF, conventional PKC which is activated in the presence of Ca2+ and DAG. Retina light exposure produced an increase in the phosphorylation status of PKCα. In addition, because phosphorylation of PKC could be mediated by the activation of PDK1, which is dependent on the prior activation of PI3K, an enzyme participant of insulin signaling pathway, our second main objective in the present study was to determine the presence of the main components of the insulin signaling pathway in PNF. Our results showed the presence of total Akt and its phosphorylated state (protein kinase of PI3K pathway) and ERK1/2, pERK1/2 and phospho-p38 (components of the MAP kinases pathway). Our results also showed that light exposure to bovine retinas causes an increase in the phosphorylation status of Akt and induces the translocation of ERK1/2 activated to PNF. In assessing the direct and indirect effects of insulin on nuclear DAGK activity in the FNF from retinas exposed either to light or darkness, it was demonstrated that insulin can modulate DAGK activity nuclear both directly (the incubation of isolated nuclei with insulin), and indirectly (the incubation of bovine retinas with insulin and the subsequent analysis of the DAGK activity in the FNF). We also observed that insulin has a role in the changes of nuclear DAGK activity in response to light, and that these effects on DAGK activity are dependent on its concentration (the effects are increased with a concomitant increase in the insulin concentration used in the enzyme assay). Taking into account the direct effects of insulin on nuclear DAGK activity, we analyzed the presence of the insulin receptor in the FNF by WB and IF and we could interest was to have shown that the contents of RI increase in FNF when bovine retinas are exposed to light, thus suggesting that light can be a stimulus capable of promoting the translocation of RI to the nucleus of the photoreceptor cells. Finally, we examined whether or not insulin is able to participate in insulin receptor translocation to the nucleus of the photoreceptor cells and to mediate the activation of signaling pathways related with insulin at the nuclear level. Our results indicated that insulin causes an increase in the content of the nuclear insulin receptor with respect to light condition in the absence of the hormone. Furthermore, incubation of PNF with insulin resulted in an increase of nuclear, activated ERK1/2. Summing up, our results demonstrate for the first time that light exposure of bovine retinas, its natural stimulus, in parallel to the typical activation of phototransduction pathways which starts in the outer segments, induces, at the nuclear level, the activation of different signaling pathways known to be responsible for critical functions to the cell, as gene transcription. Our results also reveal that light and insulin are involved in the translocation of the insulin receptor from the plasma membrane to the nucleus of the photoreceptor cells. Insulin also promotes the activation of cell signaling pathways, thus indirectly acting on the retina, or directly in the nucleus, suggesting that it mediates their effects through a nuclear population of the insulin receptor.

Made available in DSpace on 2017-07-10T18:08:17Z (GMT). No. of bitstreams: 1 Jamal Abd Awadallak.pdf: 7067148 bytes, checksum: 8e321c5aa3d2b9b3eeaafb2fd17b9d47 (MD5) Previous issue date: 2012-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diacylglycerol rich oils have its organoleptic characteristics very similar to those of conventional edible oils, but these oils do not tend to accumulate in the body, even when consumed in high quantities, making them a great resource in the fight against obesity. Palm oil ranks first the world production of edible oils mainly due to its low cost. This work aimed to propose a new technology for enzyme production using diacylglycerol lipase Lipozyme RM IM and ultrasound to promote water in oil emulsions, which increases the interfacial area of the system leading to higher reaction rates compared to conventional enzymatic processes. . The reactions were carried out at 55 °C with two different methods. First, the reaction system was exposed to ultrasonic waves for the whole reaction time, which led to enzymatic inactivation and water evaporation. Ultrasound was then used to promote emulsification of the water/oil system before the hydrolysis reaction, avoiding contact between the probe and the enzymes. Achieved conversions were superior to the conventional method further hydrolysis rate when the ultrasound is employed for emulsion formation was significantly greater. For 12 hours of reaction the conversion was 85% higher than the conventional method and 15% higher for a period of 24 hours of reaction. . An experimental design was used to optimize the ultrasound-related parameters and maximize the hydrolysis rate, and in these conditions, with a change in equilibrium, DAG production was evaluated.Better reaction conditions were achieved for the second method: 11.20 wt% (water+oil mass) water content, 1.36 wt% (water+oil mass) enzyme load, 12 h of reaction time, 1.2 min and 200 W of exposure to ultrasound. In these conditions diacylglycerol yield was 37.69 wt%. / Óleos enriquecidos com diacilglicerol possuem características organolépticas muito semelhantes às dos óleos comestíveis convencionais, porém, estes óleos não tendem a se acumular no organismo, mesmo quando consumidos em altas quantidades, tornando-os um grande recurso no combate à obesidade. O óleo de palma está no topo da produção mundial de óleos comestíveis principalmente devido ao seu baixo custo. Este trabalho teve como objetivo propor uma nova tecnologia para a produção enzimática de diacilglicerol empregando a lipase Lipozyme RM IM e utilizando ultrassom como gerador de emulsões de água em óleo, o que aumenta a área interfacial do sistema conduzindo a maiores taxas de reação em relação aos processos enzimáticos convencionais. A hidrólise parcial do óleo de palma foi realizada em meio livre de solventes a 55 °C em duas etapas distintas e comparadas com reações em condições semelhantes sem o uso do ultrassom. Primeiramente o sistema reacional foi exposto às ondas ultrassônicas, o que levou a taxas iniciais de reação elevadas, porém, as conversões obtidas foram baixas, em função da desativação enzimática e da evaporação de água, pelo longo período de exposição ao ultrassom. Posteriormente, utilizou-se o ultrassom para gerar emulsões antes a etapa reacional, não permitindo seu contato com o sistema contendo a enzima. As conversões obtidas foram superiores ao método convencional, além disso, taxa de hidrólise quando se empregou o ultrassom para a formação de emulsões foi significativamente maior. Para 12 horas de reação a conversão foi 85% superior ao método convencional e de 15% superior para um período de 24 horas de reação. Foi desenvolvido um planejamento fatorial, o delineamento central composto rotacional para avaliar os efeitos das variáveis tempo de exposição ao ultrassom, potência do ultrassom e razão água/óleo na conversão em ácidos graxos livres da reação, sendo que a razão água/óleo e o produto tempo x potência apresentaram os maiores efeitos. Nas melhores condições, foi produzido um óleo concentrado com 37,69% de DAG em de 12h de reação, exposto ao ultrassom por 1,2min à 200W.