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

Mitochondrial Glycerol Phosphate Acyltransferase Directs the Incorporation of Exogenous Fatty Acids into Triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [14C]oleate (∼3 μM), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [14C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 μM oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.Instituto de Investigaciones Bioquímicas de La Plat

Mitochondrial Glycerol Phosphate Acyltransferase Directs the Incorporation of Exogenous Fatty Acids into Triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [14C]oleate (∼3 μM), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [14C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 μM oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.Instituto de Investigaciones Bioquímicas de La Plat

Regulation of Triglyceride Metabolism II. Function of mitochondrial GPAT1 in the regulation of triacylglycerol biosynthesis and insulin action

GPAT1, one of four known glycerol-3-phosphate acyltransferase isoforms, is located on the mitochondrial outer membrane, allowing reciprocal regulation with carnitine palmitoyltransferase-1. GPAT1 is upregulated transcriptionally by insulin and SREBP-1c and downregulated acutely by AMP-activated protein kinase, consistent with a role in triacylglycerol synthesis. Knockout and overexpression studies suggest that GPAT1 is critical for the development of hepatic steatosis and that steatosis initiated by overexpression of GPAT1 causes hepatic, and perhaps also peripheral, insulin resistance. Future questions include the function of GPAT1 in relation to the other GPAT isoforms and whether the lipid intermediates synthesized by GPAT and downstream enzymes in the pathway of glycerolipid biosynthesis participate in intracellular signaling pathways

Mitochondrial Glycerol Phosphate Acyltransferase Directs the Incorporation of Exogenous Fatty Acids into Triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [14C]oleate (∼3 μM), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [14C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 μM oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.Instituto de Investigaciones Bioquímicas de La Plat

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV)

Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER

Magnetic proximity effect features in antiferromagnetic/ferrimagnetic core-shell nanoparticles

A study of "inverted" core-shell, MnO/γ-Mn2O3, nanoparticles is presented. Crystal and magnetic structures and characteristic sizes have been determined by neutron diffraction for the antiferromagnetic core (MnO) and the ferrimagnetic shell (γ-Mn2O3). Remarkably, while the MnO core is found to have a TN not far from its bulk value, the magnetic order of the γ-Mn2O3 shell is stable far above TC, exhibiting two characteristic temperatures, at T~ 40  K [TC(γ-Mn2O3)] and at T~120  K [~ TN(MnO)]. Magnetization measurements are consistent with these results. The stabilization of the shell moment up to TN of the core can be tentatively attributed to core-shell exchange interactions, hinting at a possible magnetic proximity effect

Spontaneous formation of spiral-like patterns with distinct periodic physical properties by confined electrodeposition of Co-In disks

Spatio-temporal patterns are ubiquitous in different areas of materials science and biological systems. However, typically the motifs in these types of systems present a random distribution with many possible different structures. Herein, we demonstrate that controlled spatio-temporal patterns, with reproducible spiral-like shapes, can be obtained by electrodeposition of Co-In alloys inside a confined circular geometry (i.e., in disks commensurate with the typical size of the spatio-temporal features). These patterns are mainly of compositional nature, i.e., with virtually no topographic features. Interestingly, the local changes in composition lead to a periodic modulation of the physical (electric, magnetic and mechanical) properties. Namely, the Co-rich areas show higher saturation magnetization and electrical conductivity and are mechanically harder than the In-rich ones. Thus, this work reveals that confined electrodeposition of this binary system constitutes an effective procedure to attain template-free magnetic, electric and mechanical surface patterning with specific and reproducible shapes

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89 kDa band in liver and testis from GPAT1 null mice and both 89 and 80 kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis

Spontaneous formation of spiral-like patterns with distinct periodic physical properties by confined electrodeposition of Co-In disks

Golvano-Escobal, Irati et al.Spatio-temporal patterns are ubiquitous in different areas of materials science and biological systems. However, typically the motifs in these types of systems present a random distribution with many possible different structures. Herein, we demonstrate that controlled spatio-temporal patterns, with reproducible spiral-like shapes, can be obtained by electrodeposition of Co-In alloys inside a confined circular geometry (i.e., in disks that are commensurate with the typical size of the spatio-temporal features). These patterns are mainly of compositional nature, i.e., with virtually no topographic features. Interestingly, the local changes in composition lead to a periodic modulation of the physical (electric, magnetic and mechanical) properties. Namely, the Co-rich areas show higher saturation magnetization and electrical conductivity and are mechanically harder than the In-rich ones. Thus, this work reveals that confined electrodeposition of this binary system constitutes an effective procedure to attain template-free magnetic, electric and mechanical surface patterning with specific and reproducible shapes.This work has been partially funded by the 2014-SGR-1015, 2014-SGR-1216 and 2014-SGR-753 projects from the Generalitat de Catalunya, the MAT2014-57960-C3-1-R, MAT2014-51778-C2-1-R and FIS2013-48668-C2-1-P from the Spanish Ministerio de Economía y Competitividad (MINECO) and the ‘Fondo Europeo de Desarrollo Regional’ (FEDER). Dr. Eva Pellicer and Dr. Neus Domingo are also grateful to MINECO for the “Ramon y Cajal” contracts (RYC-2012-10839 and RYC-2010-06365 respectively). ICN2 and ICMAB acknowledge support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295 and Grant SEV-2015-0496, respectively). The PEEM experiments were performed at CIRCE beamline at ALBA Synchrotron with the collaboration of ALBA staff.Peer reviewe