Regulación de las enzimas desaturantes en la diabetes mellitus experimental

Durante el desarrollo del presente trabajo de tesis se utilizaron diferentes modelos de Diabetes Mellitus de rata, de tipo I inducida y de tipo II, inducida o genética, teniendo como objetivos principales: a) Estudiar el rol regulador que tienen sobre el metabolismo de la biosíntesis de ácidos grasos, a través de las desaturasas hepáticas, las vías de activación dependientes de: - Insulina/SREBP-1c - PPAR-α - LXR-α b) Realizar un estudio pormenorizado de las interacciones entre las distintas vías de regulación antes mencionadas, y del resultado de estas interacciones sobre el metabolismo de las desaturasas hepáticas y sobre las composiciones de ácidos grasos de membrana de hígado. c) Realizar un aporte a la elucidación de la compleja red reguladora de la biosíntesis de ácidos grasos en el hígado de mamíferos y de las alteraciones que pueden ocurrir como consecuencia de la patología diabética de tipo I o II. d) Determinar las influencias diferenciales de cada tipo de diabetes (I versus II) sobre: - el nivel de expresión de SCD1, Δ6 y Δ5 desaturasas de ácidos grasos hepáticas, en rata. - la actividad enzimática de las desaturasas hepáticas. - las composiciones de ácidos grasos de membranas celulares, microsomales hepáticas o de las fracciones que resulten de interés en el experimento en particular, resultantes de las alteraciones producidas en los puntos anteriores.Facultad de Ciencias Exacta

Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis

Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as a glycerol-3-phospate acyltransferase by sequence homology to Gpat1 , GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs, a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analyzed by qPCR, in situ hybridization, immunohistochemistry and Western blot. Gpat2 mRNA content and protein expression were maximal at 15 dpp and restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.Fil: Garcia Fabiani, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Montanaro, Mauro Aldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Lacunza, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Cattaneo, Elizabeth Renee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Coleman, Rosalind A.. University of North Carolina; Estados UnidosFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Gonzalez-Baró; MR. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

Role of Liver X Receptor, Insulin and Peroxisome Proliferator Activated Receptor α on in Vivo Desaturase Modulation of Unsaturated Fatty Acid Biosynthesis

We examined the in vivo contribution of insulin, T090137 (T09), agonist of liver X receptor (LXR), fenofibrate, agonist of peroxisome proliferator activated receptor (PPAR-α) and sterol regulatory element binding protein-1c (SREBP-1c) on the unsaturated fatty acid synthesis controlled by Δ6 and Δ5 desaturases, compared with the effects on stearoylcoenzyme A desaturase-1. When possible they were checked at three levels: messenger RNA (mRNA), desaturase protein and enzymatic activity. In control rats, only fenofibrate increased the insulinemia that was maintained by the simultaneous administration of T09, but this increase has no specific effect on desaturase activity. T09 enhanced SREBP-1 in control animals and the mRNAs and activity of the three desaturases in control and type-1 diabetic rats, demonstrating a LXR/SREBP-1-mediated activation independent of insulin. However, simultaneous administration of insulin and T09 to diabetic rats led to a several-fold increase of the mRNAs of the desaturases, suggesting a strong synergic effect between insulin and LXR/retinoic X receptor (RXR). Moreover, this demonstrates the existence of an interaction between unsaturated fatty acids and cholesterol metabolism performed by the insulin/SREBP-1c system and LXR/RXR. PPAR-α also increased the expression and activity of the three desaturases independently of the insulinemia since it was equivalently evoked in streptozotocin diabetic rats. Besides, PPAR-α increased the palmitoylcoenzyme A elongase, evidencing a dual regulation in the fatty acid biosynthesis at the level of desaturases and elongases. The simultaneous administration of fenofibrate and T09 did not show additive effects on the mRNA expression and activity of the desaturases. Therefore, the results indicate a necessary sophisticated interaction of all these factors to produce the physiological effects.Instituto de Investigaciones Bioquímicas de La Plat

El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

Glycerol-3-phosphate acyltransferase 2 expression modulates cell roughness and membrane permeability: An atomic force microscopy study

In mammalian cells, de novo glycerolipid synthesis begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions, and overexpressed in several types of cancers and cancer-derived human cell lines where its expression contributes to the tumor phenotype. Using gene silencing and atomic force microscopy, we studied the correlation between GPAT2 expression and cell surface topography, roughness and membrane permeability in MDA-MB-231 cells. In addition, we analyzed the glycerolipid composition by gas-liquid chromatography. GPAT2 expression altered the arachidonic acid content in glycerolipids, and the lack of GPAT2 seems to be partially compensated by the overexpression of another arachidonic-acid-metabolizing enzyme, AGPAT11. GPAT2 expressing cells exhibited a rougher topography and less membrane damage than GPAT2 silenced cells. Pore-like structures were present only in GPAT2 subexpressing cells, correlating with higher membrane damage evidenced by lactate dehydrogenase release. These GPAT2-induced changes are consistent with its proposed function as a tumor-promoting gene, and might be used as a phenotypic differentiation marker. AFM provides the basis for the identification and quantification of those changes, and demonstrates the utility of this technique in the study of cancer cell biology.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Investigaciones Fisicoquímicas Teóricas y AplicadasFacultad de Ciencias ExactasFacultad de Ciencias Médica

Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Methylation of the <i>Gpat2</i> promoter regulates transient expression during mouse spermatogenesis

Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as a glycerol-3-phospate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs, a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analyzed by qPCR, in situ hybridization, immunohistochemistry and Western blot. Gpat2 mRNA content and protein expression were maximal at 15 dpp and restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La PlataCentro de Investigaciones Inmunológicas Básicas y Aplicada

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada