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

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases −3 and −4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition

Background and aims: The transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Methods: We used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). Results: We showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4−/− BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. Conclusions: Our results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.Fil: Quiroga, Ivana Yoseli. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Gonzalez, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Coleman, Rosalind A.. University of North Carolina; Estados UnidosFil: Gonzalez Baro, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Problematization about articulation between community programs, teaching activities and research at University of La Plata and its impact on higher university education

Este trabajo se focaliza en la problematización acerca de la articulación entre las distintas funciones de la Universidad y fue realizado en el contexto de la acreditación del seminario “Políticas de articulación, docencia, investigación, extensión, y transferencia”, de la Especialización en Docencia Universitaria de la Universidad Nacional de La Plata. Mediante el debate y la discusión grupal hemos identificado distintas problemáticas, que dificultan la formación integral de los y las estudiantes, como la desinformación de la planta docente en cuanto a la estructuración y gestión de la extensión universitaria, la falta de reconocimiento de la actividad extensionista y su escasa incorporación en los currículos de las carreras, la formación profesional descontextualizada, el uso no democrático de los espacios de trabajo en las distintas unidades académicas o las escasas iniciativas para promover el aprendizaje desde la práctica. Para cada uno de los problemas encontrados se proponen estrategias para su abordaje, con el propósito de pensar la formación y la educación como una construcción social del conocimiento. Concluimos que, resulta necesario promover el conocimiento a partir de la vinculación entre la extensión, la investigación y la docencia, tres funciones de la Universidad estrechamente vinculadas, pero no reconocidas todavía de igual modo.This work focuses on the problematization about the articulation between the different functions of the University, and was carried out in the context of the accreditation of the seminar “Policies of articulation, teaching, research, community programs and knowledge translation”, of the “Specialization in teaching activities at the National University of La Plata”. Through debate and group discussion, we have identified problems that hinder the comprehensive training of students, such as the misinformation of the teaching staff regarding the structuring and management of community programs, the lack of recognition of the community programs activity and its scarce incorporation into career curricula, decontextualized professional training, undemocratic use of work spaces in different academic units or few initiatives to promote learning from practice. For each of the problems found, strategies are proposed for their approach, with the purpose of thinking of training and education as social construction of knowledge. We conclude that it is necessary to promote knowledge from the link between community programs, research and teaching activities, three roles of the University closely linked, but not yet recognized in the same way.Fil: Fusé, Santiago Héctor. Universidad Nacional de La Plata; ArgentinaFil: Henning, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Karpenko Wilman, Ingrid Denise. Universidad Nacional de La Plata; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo 2 -lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3 −/− and Gpat4 −/− BMDM was impaired. Additionally, inhibiting fatty acid β-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4 −/− BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.Fil: Quiroga, Ivana Yoseli. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Suchanek, Amanda L.. University of North Carolina; Estados UnidosFil: Grandinetti, Rosalinda. University of North Carolina; Estados UnidosFil: Gonzalez Baro, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases −3 and −4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition

Background and aims: The transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Methods: We used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). Results: We showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4−/− BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. Conclusions: Our results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.Fil: Quiroga, Ivana Yoseli. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Gonzalez, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Coleman, Rosalind A.. University of North Carolina; Estados UnidosFil: Gonzalez Baro, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentin

Identification of signaling pathways modulated by RHBDD2 in breast cancer cells: a link to the unfolded protein response

Rhomboid domain containing 2 (RHBDD2) was previously observed overexpressed and amplified in breast cancer samples. In order to identify biological pathways modulated by RHBDD2, gene expression profiles of RHBDD2 silenced breast cancer cells were analyzed using whole genome human microarray. Among the statistically significant overrepresented biological processes, we found protein metabolism—with the associated ontological terms folding, ubiquitination, and proteosomal degradation—cell death, cell cycle, and oxidative phosphorylation. In addition, we performed an in silico analysis searching for RHBDD2 co-expressed genes in several human tissues. Interestingly, the functional analysis of these genes showed similar results to those obtained with the microarray data, with negative regulation of protein metabolism and oxidative phosphorylation as the most enriched gene ontology terms. These data led us to hypothesize that RHBDD2 might be involved in endoplasmic reticulum (ER) stress response. Thus, we specifically analyzed the unfolding protein response (UPR) of the ER stress process. We used a lentivirus-based approach for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line, and we examined the transcriptional consequences on UPR genes as well as the phenotypic effects on migration and proliferation processes. By employing dithiothreitol as an UPR inducer, we observed that cells with silenced RHBDD2 showed increased expression of ATF6, IRE1, PERK, CRT, BiP, ATF4, and CHOP (p < 0.01). We also observed that RHBDD2 silencing inhibited colony formation and decreased cell migration. Based on these studies, we hypothesize that RHBDD2 overexpression in breast cancer could represent an adaptive phenotype to the stressful tumor microenvironment by modulating the ER stress response.Fil: Lacunza, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Rabassa, Martín Enrique. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Canzoneri, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Croce, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; ArgentinaFil: Aldaz, Claudio Marcelo. University of Texas; Estados UnidosFil: Abba, Martín Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentin

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or DK107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with DK107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (DK226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by DK107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/ sphyngomyelin distribution produced by wild-type apoA-I is not observed with either DK107 or DK226.Fil: Toledo, Juan Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Garda, Horacio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Cabaleiro, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Cuellar, Angela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Pellon Maison, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Gonzalez Baro, Maria del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Gonzalez, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

GPAT2 overexpression increased TAG storage in CHO-K1 cells.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (*<i>p</i><0.05).</p

GPAT2 overexpression increased phosphatidic acid synthesis.

<p>A) The reaction products of GPAT reaction measured in control (C), GPAT1 (G1) and GPAT2 (G2)-overexpressing CHO-K1 cells with [¹⁴C]glycerol-3-phosphate and palmitoyl-CoA (16∶0-CoA) or arachidonoyl-CoA (20∶4-CoA) were visualized by a Storm radioactivity scanner. DAG, diacylglycerol, PA, phosphatidic acid, LPA, lysophosphatidic acid. B) AGPAT activity was measured with oleoyl-CoA or arachidonoyl-CoA and [¹⁴C]oleoyl-lysophosphatidic acid. GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate (*<i>p</i><0.05, **<i>p</i><0.01).</p

GPAT2 overexpression increased arachidonoyl-CoA esterification.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs. A) NEM-sensitive GPAT activity was measured with [³H]glycerol-3-phosphate and the acyl-CoA esters substrates palmitoyl-CoA (16∶0-CoA), oleoyl-CoA (18∶1-CoA), linoleoyl-CoA (18∶2-CoA), arachidonoyl-CoA (20∶4-CoA), eicosapentaenoyl-CoA (20∶5-CoA) and docosahexanoyl-CoA (22∶6-CoA) in CHO-K1 cells. GPAT2 overexpression significantly increased GPAT activity only when arachidonoyl-CoA was used as a substrate. B) GPAT activity was measured in both control and GPAT2-overexpressing CHO-K1 and Vero cells with [³H]glycerol-3-phosphate and arachidonoyl-CoA in the absence (Total GPAT activity) and presence (NEM-resistant, NEM+) of 2 mM NEM. NEM-sensitive GPAT activity (NEM−) was calculated by difference of the other two activity values. C) NEM-resistant GPAT activity was measured in control and GPAT1-overexpressing CHO-K1 cells with the substrates palmitoyl-CoA (16∶0-CoA) and arachidonoyl-CoA (20∶4-CoA). D) The ratio between NEM-sensitive GPAT activity in control and GPAT2-overexpressing CHO-K1 cells with the same fatty acyl-CoA substrates as in A) and Vero cells with palmitoyl-CoA and arachidonoyl-CoA was calculated. Bars represent the mean ± SD of three independent experiments (**<i>p</i><0.01).</p