Effect of fenitrothion on dipalmitoyl and 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

The effects of the organophosphorous insecticide fenitrothion (phosphorothioic acid, O,O-dimethyl O-(3-methyl-4-nitrophenyl) ester; FS) on the physical state of pure dipalmitoyl (DPPC) and 1-palmitoyl-2- oleoylphosphatidylcholine (POPC) membranes were investigated. FS lowers the phase transition temperature of DPPC. It has no large effects on the DPPC gel phase, but it increases the order of the liquid-crystalline state of DPPC and POPC. FS also decreases 1,6-diphenyl-1,3,5-hexatriene (DPH) lifetime (τ) in the DPPC and POPC liquid-crystalline states. Since a direct quenching of DPH emission by FS was ruled out, τ shortening is assigned to an increased water penetration in the bilayer. The effect of FS is different from most perturbing agents for which an increased order is accompanied by a higher τ. Furthermore, quenching of DPH by KI was increased by FS in POPC liposomes indicating an increased accessibility of the quencher to the hydrophobic core where DPH distributes. The effect of FS on dipole relaxation at the hydrophilic-hydrophobic interface of POPC bilayers was studied with 2- dimethylamino-6-lauroylnaphthalene (Laurdan). FS produces a decrease in Laurdan τ and a narrowing of its emission band. FS significantly increases the generalized polarization values at both emission band ends. These results indicate that FS may allow the coexistence of microdomains that have different physical properties.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Lipid and fatty acid composition and energy partitioning during embryo development in the shrimp Macrobrachium borellii

Energy partitioning, composition of lipids and fatty acids, and their utilization by embryos were determined in the lecithotrophic shrimp Macrobrachium borellii during seven development stages. The biochemical composition at stage I is represented by lipids, proteins, and carbohydrates, with 29.3, 28.7, and 0.2% dry weight, respectively. The former two were identified as the major energy-providing components, contribut- ing 131 and 60 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 45.0% (calculated as per- centage of vitelline energy transformed into embryonic tissues). Lipids were the most important energy reserve (CE 39.3%), fol- lowed by proteins (CE 57.1%), both being simultaneously uti- lized during development while carbohydrates were synthe- sized de novo (CE 587.5%). Variation in the lipid class compo- sition of embryos and vitellus showed an accumulation of triacylglycerols (TAG) and phospholipids (PL) up to stage IV, a more active accumulation and selective utilization phase (stages V and VI), and a consumption and de novo synthesis period until hatching. Structural lipids (PL and cholesterol) and pig- ment astaxanthin were selectively conserved in embryos, but TAG, hydrocarbons, and esterified sterols were preferentially depleted. Monounsaturated fatty acids (FA) were the major group in TAG, whereas polyunsaturated FA (PUFA) were the major group in PL after organogenesis. Certain PUFA such as 22:6n-3 and 20:5n-3 were selectively accumulated in PL.Instituto de Investigaciones Bioquímicas de La Plat

Effect of fenitrothion on dipalmitoyl and 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

The effects of the organophosphorous insecticide fenitrothion (phosphorothioic acid, O,O-dimethyl O-(3-methyl-4-nitrophenyl) ester; FS) on the physical state of pure dipalmitoyl (DPPC) and 1-palmitoyl-2- oleoylphosphatidylcholine (POPC) membranes were investigated. FS lowers the phase transition temperature of DPPC. It has no large effects on the DPPC gel phase, but it increases the order of the liquid-crystalline state of DPPC and POPC. FS also decreases 1,6-diphenyl-1,3,5-hexatriene (DPH) lifetime (τ) in the DPPC and POPC liquid-crystalline states. Since a direct quenching of DPH emission by FS was ruled out, τ shortening is assigned to an increased water penetration in the bilayer. The effect of FS is different from most perturbing agents for which an increased order is accompanied by a higher τ. Furthermore, quenching of DPH by KI was increased by FS in POPC liposomes indicating an increased accessibility of the quencher to the hydrophobic core where DPH distributes. The effect of FS on dipole relaxation at the hydrophilic-hydrophobic interface of POPC bilayers was studied with 2- dimethylamino-6-lauroylnaphthalene (Laurdan). FS produces a decrease in Laurdan τ and a narrowing of its emission band. FS significantly increases the generalized polarization values at both emission band ends. These results indicate that FS may allow the coexistence of microdomains that have different physical properties.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Transfer of lipids between hemolymph and hepatopancreas in the shrimp Macrobrachium borellii

Crustacean lipids are transported in the hemolymph by an HDL. The hepatopancreas is the most important and active organ regarding lipid metabolism, so we studied the interchange of FA and acylglycerols between both components of the hepatopancreas-hemolymph system in the decapod crustacean Macrobrachium borellii. The hepatopancreas and a sole plasma lipoprotein were labeled by in vivo incubations with 14C palmitic acid injected into the hemolymph. Then they were incubated in vitro with unlabeled hepatopancreas and hemolymph, and the transfer of lipids between them was measured by radiochromatographic techniques. It was determined in vivo that more than 80% of the circulating palmitic acid was taken up by the hepatopancreas and incorporated into PC and TAG. Both classes of lipids, but mainly PC, were transferred back from tissues to the hemolymph. Lipid transfer was also demonstrated in vitro. The transfer of PC (30% of labeling) as well as that of FFA (48% of labeling) from hemolymph to hepatopancreas was determined. On the other hand, FFA were released more efficiently than the acylglycerols from intact hepatopancreas to hemolymph, and they were the only lipid transferred when the hepatopancreas had been previously washed.Instituto de Investigaciones Bioquímicas de La Plat

Função da isoforma 2 da glicerol-3-fosfatoaciltransferase no metabolismo lipídico testicular

El primer paso en la síntesis de novo de glicerolípidos es la acilación del glicerol-3-fosfato, la cual es catalizada por la enzima glicero-3-fosfato aciltransferasa (GPAT). En mamíferos han sido descriptas hasta el momento cuatro isoformas de GPAT, las cuales pueden diferenciarse por su localización tisular y subcelular, y por su sensibilidad a reactivos que reaccionan con los grupos sulfhidrilos. El objetivo de este trabajo fue estudiar la localización y función de GPAT2, una isoforma mitocondrial que se expresa principalmente en testículo utilizando como modelo células CHO-K1 que sobreexpresan GPAT2. Se observó que esta isoforma utiliza como sustrato específicamente al ácido araquidónico, estimula la síntesis de triacilgliceroles (TAG) con alto contenido de ácido araquidónico, y favorece la incorporación de [1-14C] ácido araquidónico en los TAG. Utilizando modelos animales se observó por un lado que el ARNm de GPAT2 se expresa sólo en los espermatocitos primarios, mientras que la expresión de la proteína se mantiene a lo largo de la espermatogénesis; y por otro lado que la expresión de GPAT2 varía durante el desarrollo sexual, alcanzando un máximo de expresión y actividad a los 30 días de edad en la rata. A esta edad se observó también un máximo en el contenido de TAG y de AA esterificado en los TAG del testículo. Se concluye que los resultados sugieren que GPAT2 sería la enzima responsable de esterificar el ácido araquidónico en los TAG de las células de la línea espermática.De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. The objective of the present work was to study localization and function of GPAT2, a mitochondrial isoform that is mainly expressed in testis, using as a model CHO-K1 cells that overexpress GPAT2. Incubation of GPAT2-transfected CHO-K1 cells with arachidonoyl-CoA increased GPAT activity 2-fold and the incorporation of [1-14C] arachidonate into TAG. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2’s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.Este es un resumen de la tesis doctoral dela autora, dirigida por el Dr R. Brenner, la cual fue aprobada el 10 de agosto de 2011 en la Facultad de Ciencias Exactas de la Universidad Nacional de La Plata.Instituto de Investigaciones Bioquímicas de La Plat

La síntesis de novo de glicerolípidos aumenta durante la diferenciación de macrófagos en concordancia con el incremento de la expresión de GPAT3

La diabetes tipo 2 y la obesidad se caracterizan por un desbalance en el metabolismo energético que ocasiona un exceso en los depósitos de triacilgliceroles (TAG) en el hígado. En este sistema, dos enzimas mitocondriales poseen un papel fundamental: la glicerol-3-fosfato aciltransferasa (GPAT), que cataliza el primer paso de la síntesis de novo de glicerolípidos celulares, y la carnitina aciltransferasa 1a (CPT1a), cuya función es ingresar acil-CoAs a la mitocondria para su posterior oxidación. Ambas enzimas representan los pasos limitantes de cada vía y están regulados en forma recíproca. Con el objetivo de evaluar si estas enzimas mitocondriales también juegan un papel importante en otro modelo de acumulación de TAG, evaluamos sus niveles de expresión en dos líneas celulares: las RAW 264 derivadas de macrófagos de ratón y las THP1 de monocitos humanos.Facultad de Ciencias Médica

La síntesis de novo de glicerolípidos aumenta durante la diferenciación de macrófagos en concordancia con el incremento de la expresión de GPAT3

La diabetes tipo 2 y la obesidad se caracterizan por un desbalance en el metabolismo energético que ocasiona un exceso en los depósitos de triacilgliceroles (TAG) en el hígado. En este sistema, dos enzimas mitocondriales poseen un papel fundamental: la glicerol-3-fosfato aciltransferasa (GPAT), que cataliza el primer paso de la síntesis de novo de glicerolípidos celulares, y la carnitina aciltransferasa 1a (CPT1a), cuya función es ingresar acil-CoAs a la mitocondria para su posterior oxidación. Ambas enzimas representan los pasos limitantes de cada vía y están regulados en forma recíproca. Con el objetivo de evaluar si estas enzimas mitocondriales también juegan un papel importante en otro modelo de acumulación de TAG, evaluamos sus niveles de expresión en dos líneas celulares: las RAW 264 derivadas de macrófagos de ratón y las THP1 de monocitos humanos.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

Glicerol-3-fosfato aciltransferasa 2: su relación con la espermatogénesis y la proliferación celular

La enzima glicerol-3-fosfato aciltransferasa (GPAT) cataliza el primer paso y limitante en la síntesis de novo de glicerolípidos celulares. En mamíferos han sido descriptas al menos cuatro isoformas con diferentes localizaciones celulares y parámetros cinéticos. La isoforma mitocondrial 1 (GPAT1) ha sido extensamente estudiada y se ha demostrado que participa en la síntesis de triacilglicéridos (TAG) en hígado de rata. La isoforma mitocondrial 2 (GPAT2) presenta homología de secuencia con GPAT1, pero se expresa preferencialmente en testículo de rata y no tiene preferencia por los ácidos grasos saturados, por lo cual hipotetizamos que estaría involucrada en la síntesis de TAG ricos en ácidos grasos insaturados, moléculas que actúan como reservorios de estos ácidos grasos necesarios para la espermatogénesis. Nuestro objetivo es determinar si la función de GPAT2 (isoforma mitocondrial 2) está asociada a la síntesis celular de TAG (triacilglicéridos), y si su expresión se correlaciona con el inicio de la espermatogénesis.Facultad de Ciencias Médica

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols

Background: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. Methods and Results: Incubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. Conclusions: These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.Facultad de Ciencias Médica