Maria R. Gonzalez-Baro

Lipid characterization and distribution among tissues of the freshwater crustacean Macrobrachium borellii during an annual cycle

Abstract 1. 1. Lipid and fatty acid seasonal distribution from tissues of the freshwater shrimp Macrobrachium borelli was studied. 2. 2. Female gonads and hepatopancreas were the organs with the highest lipid percentages. They were mainly composed of triacylglycerols, with oleic acid as the major fatty acid. 3. 3. Total lipids and triacylglycerols from ovarian tissue increased during sexual maturation and they presented a peak during winter in hepatopancreas. 4. 4. Phospholipids were the dominant lipids in muscle. They were mainly constituted by eicosapentaenoic, oleic and arachidonic acids. 5. 5. The w3:w6 acid ratio was more than 1, which is very high compared to other freshwater invertebrates.

Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol

The topography of mitochondrial glycerol-3-phosphate acyltransferase (GPAT) was determined using rat liver mitochondria and mutagenized recombinant rat GPAT (828 aa (amino acids)) expressed in CHO cells. Hydrophobicity analysis of GPAT predicts two transmembrane domains (TMDs), residues 472–493 and 576–592. Residues 224–323 correspond to the active site of the enzyme, which is believed to lie on the cytosolic face of the outer mitochondrial membrane. Protease treatment of rat liver mitochondria revealed that GPAT has a membrane-protected segment of 14 kDa that could correspond to the mass of the two predicted TMDs plus a loop between aa 494 and 575. Recombinant GPAT constructs containing tagged epitopes were transiently expressed in Chinese hamster ovary cells and immunolocalized. Both the C and N termini epitope tags could be detected after selective permeabilization of only the plasma membrane, indicating that both termini face the cytosol. A 6–8-fold increase in GPAT-specific activity in the transfected cells confirmed correct protein folding and orientation. When the C terminus and loop-tagged GPAT construct was immunoassayed, the epitope at the C terminus could be detected when the plasma membrane was permeabilized, but loop-epitope accessibility required disruption of the outer mitochondrial membrane. Similar results were observed when GPAT was truncated before the second TMD, again consistent with an orientation in which the loop faces the mitochondrial intermembrane space. Although protease digestion of the HA-tagged loop resulted in preservation of a 14-kDa fragment, consistent with a membrane protected loop domain, neither the truncated nor loop-tagged enzymes conferred GPAT activity when overexpressed, suggesting that the loop plays a critical structural or regulatory role for GPAT function. Based on these data, we propose a GPAT topography model with two transmembrane domains in which both the N (aa 1–471) and C (aa 593–end) termini face the cytosol and a single loop (aa 494–575) faces the intermembrane space.

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, contributing 131 and 60 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 45.0% (calculated as percentage of vitelline energy transformed into embryonic tissues). Lipids were the most important energy reserve (CE 39.3%), followed by proteins (CE 57.1%), both being simultaneously utilized during development while carbohydrates were synthesized de novo (CE 587.5%). Variation in the lipid class composition 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 pigment 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.

FATTY ACID METABOLISM OF MACROBRACHIUM BORELLII : DIETARY ORIGIN OF ARACHIDONIC AND EICOSAPENTAENOIC ACIDS

Abstract To determine the origin of arachidonic (20:4n-6) and eicosapentaenoic (20:5n-3) acids in the freshwater shrimp Macrobrachium borellii , the capacity of bioconversion of radioactive α -linolenic (18:3n-3) and dihomogammalinolenic (20:3n-6) acids was assayed in vivo and in vitro . No conversion of these two fatty acids into 20:4n-6 and 20:5n-3 was observed. Both 20:4n-6 and 20:5n-3 acids were detected in the stomach contents of wild animals. When we analyzed the natural diet of M. borellii , these fatty acids were found in the associated periphyton and detritus. When shrimps were fed artificial diets rich in either linoleic (18:2n-6) or α -linolenic acids, a sharp decrease of 20:4n-6 and 20:5n-3 was observed in vitamin- and mineral-deficient treatment groups. When the animals were fed diets with added minerals and vitamins, the same effect was observed after longer treatment times. Under our experimental conditions, it can be concluded that shrimps are unable to synthesize either 20:4n-6 or 20:5n-3 from shorter chain fatty acids, and these fatty acids must be supplied by the diet.

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

Crustancean 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.

Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the breast cancer MDA-MB-231 cell line.

The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2′ deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.

Effect of fenitrothion on the physical properties of crustacean lipoproteins.

The effect of the liposoluble organophosphorus insecticide fenitrothion (FS) on lipid packing and rotation of two crustacean plasma HDL was investigated. These lipoproteins, HDL-1 and HDL-2, differed in their lipid composition, but their lipid/protein ratios were similar. The rotational behavior of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 3-(p-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (DPH-PA) was used to obtain information about the lipid dynamics in the outer and inner regions, respectively, of the lipid phase of the lipoproteins. Fluorescent steady-state anisotropy (rs), lifetime (τ), rotational correlation time (τr), and the limiting anisotropy (r∞) of these probes were measured in the lipoproteins exposed to different concentrations of FS in vitro. The results showed the penetration of FS into both plasma lipoproteins, altering the lipid dynamics of the inner as well as the outer regions. The overall effect of the insecticide was to induce an increase in the lipid order in a concentration-dependent fashion. DPH and DPH-PA fluorescence-lifetime shortening indicated that FS increased the polarity of the probe environment, suggesting an enhanced water penetration into the lipoprotein lipid phase, may be due to the induction of failures in the lipid packing. Even in the absence of FS, a higher ordering of the lipid phase was found in HDL-2 compared to HDL-1, a fact that might be attributed to a higher percentage of sphingomyelin in HDL-2.

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 GPAT2s 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.

Fenitrothion-induced structural and functional perturbations in the yolk lipoproteins of the shrimp Macrobrachium borellii

Two lipovitellin (LV) forms containing the same apoproteins but differing in their lipid composition were isolated from Macrobrachium borelii eggs at early (LVe) and late (LVI) embryogenic stages and characterized. These two forms of LV, as well as liposomes prepared with lipids extracted from them, were used as simpler models to study the effect of the pesticide fenitrothion (FS) on their structures and functions. Rotational diffusion and fluorescence lifetime of two fluorescent probes [1,6-diphenyl-1,3,5-hexatriene (DPH) and 3-(p(6-phenyl)-1,3,5-hexatrienal)phenylpropionic acid (DPH-PA)] were used to obtain information on structural changes induced by FS in the inner and outer regions of the LV, respectively. Comparison of the rotational behavior of these probes in native LV and liposomes (LP) from extracted LV lipids suggests that apoprotein-lipid interactions result in an ordered neutral lipid core. FS increased the lipid phase polarity of both LV and LP forms. The rotation of these probes in LP was not affected, suggesting a dependence of FS action on lipid-protein interactions. DPH-PA steady-state anisotropy showed that, unlike the LVe form, the LVI form was sensitive to extremely low FS concentrations. The ability of both LV to transfer palmitic acid to albumin was increased, but in a dissimilar manner, by the presence of FS. Such differences in the sensitivity of the LV at different steps of embryogenesis to FS influence the toxic action of this insecticide.

Lipid transport in snails. Partial characterization of a high-density lipoprotein isolated from Ampullaria canaliculata plasma

1. 1. The mechanism of cholesterol transport in snail hemolymph was studied in Ampullaria canaliculata. Plasma and hemocytes were obtained 7 hr after injecting labelled cholesterol into the foot muscle. 2. 2. The radioactive tracer circulated in the free form mainly in plasma (90%) and to a lesser extent, associated with hematic cells. 3. 3. Labelled plasma was centrifuged in density gradients and fractionated. It was possible to detect a high-density lipoprotein (HDL), with a hydrated density in the range 1.10–1.15 g/ml. 4. 4. HDL was isolated from non-labelled plasma and partially characterized. It presents a protein-to-lipid ratio of about 1:9. Its lipid moiety is mainly composed of phospholipids, moderate amounts of triacylglycerols, free cholesterol and free fatty acids, and minor concentrations of hydrocarbons.