Odile Colard

Seipin deficiency alters fatty acid Δ9 desaturation and lipid droplet formation in Berardinelli-Seip congenital lipodystrophy

Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue and severe insulin resistance. In most cases, BSCL is due to loss-of-function mutations in the genes encoding either seipin of unknown function or 1-acyl-glycerol-3-phosphate O-acyltransferase 2 (AGPAT2) which catalyses the formation of phosphatidic acid from lysophosphatidic acid. We studied the lipid profile of lymphoblastoid cell-lines from 20 BSCL patients with null mutations in the genes encoding either seipin (n=12) or AGPAT2 (n=8) in comparison to nine control cell-lines. In seipin deficient cells, we observed alterations in the pattern of lipid droplets which were decreased in size and increased in number as compared to control cells. We also observed alterations in the triglycerides content as well as in the fatty acid composition from triglycerides and phosphatidylethanolamine, with an increased proportion of saturated fatty acids at the expense of the corresponding monounsaturated fatty acids, reflecting a defect in Delta9-desaturase activity. In AGPAT2 deficient cells, no specific alterations in lipid droplet pattern nor in fatty acid composition was observed but the cellular level of lysophosphatidic acid was increased as compared to that of control and seipin deficient cells. These results indicate that seipin like AGPAT2 is involved in lipid metabolism but exerts a different function. Seipin intervenes at a proximal step in triglycerides and phospholipids biosynthesis being involved in the pathway that links fatty acid Delta9 desaturation to lipid droplet formation. These findings provide new insights into how seipin deficiency causes severe lipodystrophy.

Rafts Promote Assembly and Atypical Targeting of a Nonenveloped Virus, Rotavirus, in Caco-2 Cells

ABSTRACT Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.

Induction by lysophospholipids of CoA-dependent arachidonyl transfer between phospholipids in rat platelet homogenates

Rat platelet homogenates are able to catalyze CoA-mediated, ATP-independent transfer of arachidonic acid from platelet phospholipids to added lysophospholipids. Homogenates of platelets prelabelled with radioactive arachidonic or oleic acid were incubated in the presence of CoA and various lysophospholipids. Transfer observed with arachidonic acid-labelled platelets was dependent on the lysophospholipid added. When 1-alkenyl- or 1-acyllysophosphatidylethanolamine was used, there was a more efficient arachidonyl transfer from phosphatidylcholine than from phosphatidylinositol to the phosphatidylethanolamine fraction. Lysophosphatidylserine also accepted arachidonyl from phosphatidylcholine. Addition of lysophosphatidylcholine resulted in a decrease in the labelling of phosphatidylinositol and to a lesser extent of phosphatidylethanolamine with concomitant transfer to phosphatidylcholine. Lysophosphatidylinositol and lysophosphatic acid did not act as substrate for this transfer reaction. Free, non-radioactive arachidonic acid did not compete for the labelled arachidonic acid transfer. This pathway may play a major role in the synthesis of arachidonyl species of phosphatidylethanolamine and phosphatidylserine and for the arachidonyl transfer to the phosphatidylethanolamine plasmologen in stimulated platelets.

Acylation of endogenous phospholipids and added lysoderivatives by rat liver plasma membranes

Phospholipid acyltransferase activities of plasma membranes have been investigated with various acyl-CoA thioesters (palmitoyl, stearoyl, oleoyl, linoleoyl and arachidonoyl) with and without added lysoderivatives. Different patterns of incorporation were observed for each acyl-CoA into endogenous phosphatidylcholine and phosphatidylethanolamine. The turnover rates calculated with tracer amounts of 10 microM acyl-CoA thioesters were five times faster for the polyunsaturated than for the saturated acyl moieties of phosphatidylethanolamine and phosphatidylcholine. Arachidonoyl-CoA was the best acyl donor at low concentrations and the maximal turnover rate was observed at about 25 microM. No saturation appeared at up to 100 microM linoleoyl-CoA. Linoleoyl-CoA transacylase acylated the lyso-compounds in the following order: lysophosphatidylcholine greater than lysophosphatidylserine and lysophosphatidylinositol, while lysophosphatidylethanolamine inhibited linoleate incorporation into the phosphatidylethanolamine itself. Linoleoyl-CoA transacylation was not affected by the fatty acyl moiety at the 1-position of the lysophosphatidylcholine. The results support the view that the plasma membrane acyltransferase activity might contribute to the formation of bile phosphatidylcholines.

Changes induced by PAF-acether in diacyl and ether phospholipids from guinea-pig alveolar macrophages

The release and the mobilization of arachidonic acid from guinea-pig alveolar macrophages labeled with [1-14C]arachidonic acid for short (1 h) and long (18 h) periods and stimulated with PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied. After short labeling periods arachidonic acid was primarily incorporated into alkylacyl- and diacylglycerophosphocholine (alkylacylGPC, diacylGPC) and glycerophosphoinositol (GPI), whereas after long labeling periods arachidonic acid was mainly incorporated into alkenylacylglycerophosphoethanolamine (alkenylacylGPE). In macrophages labeled for 1 h, PAF-acether (1 microM) induced a significant decrease in the amount of arachidonic acid esterified into diacyl- and alkylacylGPC and GPI, as well as a significant increase of arachidonate transferred into alkenylacylGPE. No significant decrease in arachidonate esterified in GPC fractions and in GPI was induced by PAF-acether in macrophages labeled for 18 h, whereas the increased transfer of the fatty acid into alkenylacylGPE was still measurable. This study shows that PAF-acether induces the release and the mobilization of newly incorporated arachidonic acid in alveolar macrophages. When cells are labeled for long periods and the majority of arachidonic acid is retained in ether-linked phospholipids, no PAF-acether-induced release of arachidonate was obtained, whereas its transfer was maintained.

Spike Protein VP4 Assembly with Maturing Rotavirus Requires a Postendoplasmic Reticulum Event in Polarized Caco-2 Cells

ABSTRACT Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.

Oxidant-induced arachidonic acid release and impairment of fatty acid acylation in vascular smooth muscle cells

Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with A[Formula: see text], a process not involving phospholipase A2(PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2or A[Formula: see text] as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.Oxidative damage, which plays a major role in the early stages of atherosclerosis, is associated with arachidonic acid (AA) release in vascular smooth muscle cells (VSMC) as in other cell types. In this study, H2O2 was used to investigate mechanisms of AA release from VSMC on oxidative stress. Cell treatment with H2O2 inhibited AA incorporation in an inverse relationship to prolonged H2O2-induced AA release. Identical kinetics of inhibition of AA incorporation and AA release were observed after cell treatment with AlF4-, a process not involving phospholipase A2 (PLA2) activation as recently described (A. Cane, M. Breton, G. Béréziat, and O. Colard. Biochem. Pharmacol. 53: 327-337, 1997). AA release was not specific, since oleic acid also increased in the extracellular medium of cells treated with H2O2 or AlF4- as measured by gas chromatography-mass spectrometry. In contrast, AA and oleic acid cell content decreased after cell treatment. Oleoyl and arachidonoyl acyl-CoA synthases and acyltransferases, assayed using a cell-free system, were not significantly modified. In contrast, a good correlation was observed between decreases in AA acylation and cell ATP content. The decrease in ATP content is only partially accounted for by mitochondrial damage as assayed by rhodamine 123 assay. We conclude that oxidant-induced arachidonate release results from impairment of fatty acid esterification and that ATP availability is probably responsible for free AA accumulation on oxidative stress by preventing its reesterification and/or transmembrane transport.

Biosynthesis of paf-acether XVII. Regulation by the CoA-independent transacylase in human neutrophils

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1–10 ng/ml) induced paf‐acether (pat) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA‐independent lyso paf : transacylase as assayed in a cell‐free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA‐treated intact PMN. When higher concentrations of PMA (40–100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA‐independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA‐activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA‐independent transacylase in controling the level of lyso paf availability for paf formation.

Hydrolysis of endogenous phospholipids by rat platelet phospholipase A2: ether or acyl bond and polar head group selectivity

Substrate specificity of platelet phospholipase A2 was investigated following Ca2+-dependent hydrolysis by endogenous enzyme of linoleate- or arachidonate-labelled platelet phospholipids. Alkylacyl, alkenylacyl and diacyl classes of ethanolamine and choline glycerophospholipids (GPE and GPC) were separated after their diacylglycerol derivation, and molecular species of diacyl-GPE were analyzed by HPLC. Hydrolysis of platelet ethanolamine and choline glycerophospholipids was dependent on Ca2+ and was maximal at neutral pH. In the presence of 0.2 mM Ca2+ the hydrolysis rate for [14C]arachidonate-labelled phospholipids was in the order diacyl-GPE greater than alkylacyl-GPE = diacyl-GPC = alkenylacyl-GPE greater than alkylacyl-GPC. In addition to being the best substrate at high Ca2+ concentration, diacyl-GPE could be degraded with Ca2+ concentrations in the micromolar range, concentrations which are unable to induce any degradation of diacyl-GPC. As a function of Ca2+ concentration, the hydrolysis rate of [14C]linoleate- and [14C]arachidonate-labelled diacyl-GPE or diacyl-GPC was identical. The five main molecular species of diacyl-GPE labelled with arachidonate or with linoleate were hydrolyzed at the same rate in the presence of 50 microM Ca2+. This study shows that platelet phospholipase A2 is specific for endogenous diacyl-GPE and is independent of fatty chain composition. These results are discussed in relation to the Ca2+ concentration observed in stimulated platelets and in relation to the lysophospholipid-induced specific transfer of arachidonate. They suggest that diacyl-GPE hydrolysis by phospholipase A2 could play a key role in stimulated platelets.

Heterogeneity of Raft-Type Membrane Microdomains Associated with VP4, the Rotavirus Spike Protein, in Caco-2 and MA 104 Cells

ABSTRACT Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-β-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.