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• W3023804959 abstract "The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis. The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis. acid sphingomyelinase calreticulin lysosomal acid sphingomyelinase mitochondria-associated membrane mitochondrial fraction mitochondrial outer membrane permeability neutral sphingomyelinase pure mitochondria secreted acid sphingomyelinase staurosporine Lipids are present as structural components of membranes but are also, through their interactions with proteins, instrumental in many cellular functions under both normal and pathophysiological situations (1Drin G. Topological regulation of lipid balance in cells.Annu. Rev. Biochem. 2014; 83: 51-77Crossref PubMed Scopus (60) Google Scholar). In addition to global composition, distribution of lipids among intracellular membranes is essential for cell growth, function, and survival, as it participates directly and indirectly in the cellular compartmentalization of major signaling pathways (1Drin G. Topological regulation of lipid balance in cells.Annu. Rev. Biochem. 2014; 83: 51-77Crossref PubMed Scopus (60) Google Scholar). This is particularly true for sphingolipids, which are present in plasma and intracellular organelle membranes where they are involved in their structures as well as in signal transduction. Sphingolipids are divided into several species that include sphingosine, ceramides, and SM, which are metabolically and structurally related but exhibit different biological properties (2Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar). In particular, the hydrophobic properties of the sphingolipids, which are determined by the high number of carbons in the fatty acid chains linked to the sphingoid backbone, impact on the biological responses. Alterations in sphingolipid metabolites are related to aging and common pathologies such as diabetes and cancers, and neurodegenerative, cardiovascular, and respiratory diseases (3Huang X. Withers B.R. Dickson R.C. Sphingolipids and lifespan regulation.Biochim. Biophys. Acta. 2014; 1841: 657-664Crossref PubMed Scopus (69) Google Scholar). Sphingolipids are involved in the process of apoptosis either through clustering of lipid microdomains or as second messengers by binding specific proteins and regulating their phosphorylation (2Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar). Ceramides with various lengths in fatty acid chains are formed in different cell compartments or membranes by a variety of mechanisms, which enable specific, rapid, and transient production following a given stimulus (4Castro B.M. Prieto M. Silva L.C. Ceramide: a simple sphingolipid with unique biophysical properties.Prog. Lipid Res. 2014; 54: 53-67Crossref PubMed Scopus (232) Google Scholar, 5Pewzner-Jung Y. Park H. Laviad E.L. Silva L.C. Lahiri S. Stiban J. Erez-Roman R. Brugger B. Sachsenheimer T. Wieland T. et al.A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways.J. Biol. Chem. 2010; 285: 10902-10910Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). During apoptosis, sphingolipids can affect mitochondria through different mechanisms such as ceramide-formed channels, which could participate in mitochondrial outer membrane permeability (MOMP), kinase activation, and inhibition of respiration (1Drin G. Topological regulation of lipid balance in cells.Annu. Rev. Biochem. 2014; 83: 51-77Crossref PubMed Scopus (60) Google Scholar, 2Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar, 3Huang X. Withers B.R. Dickson R.C. Sphingolipids and lifespan regulation.Biochim. Biophys. Acta. 2014; 1841: 657-664Crossref PubMed Scopus (69) Google Scholar, 4Castro B.M. Prieto M. Silva L.C. Ceramide: a simple sphingolipid with unique biophysical properties.Prog. Lipid Res. 2014; 54: 53-67Crossref PubMed Scopus (232) Google Scholar, 5Pewzner-Jung Y. Park H. Laviad E.L. Silva L.C. Lahiri S. Stiban J. Erez-Roman R. Brugger B. Sachsenheimer T. Wieland T. et al.A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways.J. Biol. Chem. 2010; 285: 10902-10910Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 6Stiban J. Perera M. Very long chain ceramides interfere with C16-ceramide-induced channel formation: a plausible mechanism for regulating the initiation of intrinsic apoptosis.Biochim. Biophys. Acta. 2015; 1848: 561-567Crossref PubMed Scopus (72) Google Scholar). Of note, ceramide metabolites can have opposite effects: for example, sphingosine-1-phosphate (S1P) signaling inhibits ceramide-mediated apoptosis in endothelial cells (7Bonnaud S. Niaudet C. Pottier G. Gaugler M.H. Millour J. Barbet J. Sabatier L. Paris F. Sphingosine-1-phosphate protects proliferating endothelial cells from ceramide-induced apoptosis but not from DNA damage-induced mitotic death.Cancer Res. 2007; 67: 1803-1811Crossref PubMed Scopus (55) Google Scholar). Other results highlight the importance of compartment-specific lipid-mediated cell death and the many partners implicated in this process (8Mignard V. Lalier L. Paris F. Vallette F.M. Bioactive lipids and the control of Bax pro-apoptotic activity.Cell Death Dis. 2014; 5: e1266Crossref PubMed Scopus (42) Google Scholar). It has been shown that ceramide induces cell death specifically when generated in mitochondria (9Birbes H. El Bawab S. Hannun Y.A. Obeid L.M. Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis.FASEB J. 2001; 15: 2669-2679Crossref PubMed Scopus (221) Google Scholar). Recently, a direct implication of sphingolipids in MOMP has been further highlighted by a study showing the cooperation of microsomal sphingolipids (namely S1P and its metabolite hexadecenal) with pro-apoptotic members of the BCL-2 family, respectively BAK and BAX, to promote cytochrome c release (10Chipuk J.E. McStay G.P. Bharti A. Kuwana T. Clarke C.J. Siskind L.J. Obeid L.M. Green D.R. Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.Cell. 2012; 148: 988-1000Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). These data suggest that nonmitochondrial membranes are necessary to provide an appropriate sphingolipid environment for the executive phase of apoptosis. Indeed, ceramide has been shown to be present in the so-called mitochondria-associated membranes (MAMs), an elusive structure that connects the ER to mitochondria (11Ardail D. Popa I. Bodennec J. Louisot P. Schmitt D. Portoukalian J. The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases.Biochem. J. 2003; 371: 1013-1019Crossref PubMed Scopus (84) Google Scholar, 12Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?.Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (202) Google Scholar). Primitively, MAM functions have been assigned to lipid transfer from the ER to mitochondria and to Ca2+ exchange between the two organelles (13van Vliet A.R. Verfaillie T. Agostinis P. New functions of mitochondria associated membranes in cellular signaling.Biochim. Biophys. Acta. 2014; 1843: 2253-2262Crossref PubMed Scopus (265) Google Scholar). The protein composition of MAMs appears to be tissue-specific with a corpus of proteins mainly involved in mitochondrial functions (14Poston C.N. Krishnan S.C. Bazemore-Walker C.R. In-depth proteomic analysis of mammalian mitochondria-associated membranes (MAM).J. Proteomics. 2013; 79: 219-230Crossref PubMed Scopus (155) Google Scholar). Recently, new roles have been attributed to MAMs, including the control of autophagy/mitophagy, mitochondrial fusion/fission, and apoptosis (13van Vliet A.R. Verfaillie T. Agostinis P. New functions of mitochondria associated membranes in cellular signaling.Biochim. Biophys. Acta. 2014; 1843: 2253-2262Crossref PubMed Scopus (265) Google Scholar). This structure has also been implicated in the basic mechanisms of neurodegenerative diseases and diabetes (15Paillard M. Tubbs E. Thiebaut P.A. Gomez L. Fauconnier J. Da Silva C.C. Teixeira G. Mewton N. Belaidi E. Durand A. et al.Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.Circulation. 2013; 128: 1555-1565Crossref PubMed Scopus (172) Google Scholar, 16Schon E.A. Area-Gomez E. Mitochondria-associated ER membranes in Alzheimer disease.Mol. Cell. Neurosci. 2013; 55: 26-36Crossref PubMed Scopus (170) Google Scholar, 17Tubbs E. Theurey P. Vial G. Bendridi N. Bravard A. Chauvin M.A. Li-Cao J. Zoulim F. Bartosch B. Ovize M. et al.Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance.Diabetes. 2014; 63: 3279-3294Crossref PubMed Scopus (272) Google Scholar). However, the precise molecular mechanisms and actors implicated in MAM function in such an essential program as apoptosis remain poorly characterized. Because many MAM functions appear to be similar to those implicating sphingolipids (18Vance J.E. MAM (mitochondria-associated membranes) in mammalian cells: lipids and beyond.Biochim. Biophys. Acta. 2014; 1841: 595-609Crossref PubMed Scopus (413) Google Scholar), we analyzed the composition of the main subspecies of sphingolipids and related enzymes in vivo and in vitro under resting and apoptotic conditions. MS allows analysis and quantification with reproductive accuracy of a large number of lipid species and subspecies in a very small amount of biological samples of diverse origins (cell, cell medium, tissue, blood, etc.). In this work, we used these properties to attempt to define the distribution of a large number of sphingolipids and their different subspecies between ER, MAM, and mitochondria by LC-MS/MS. The U251 cell line was grown in DMEM (4.5 g/l glucose) supplemented with 10% FCS, antibiotics (penicillin, streptomycin), and glutamine (Life Technologies, Carlsbad, CA) in 5% CO2 at 37°C. Apoptosis was induced in cultures at 70% confluency with 0.5 μg/ml staurosporine (STS; Santa Cruz Biotechnology, Heidelberg, Germany). Livers from C57BL6 mice (about 10 g) or U251 cells from 40 petri dishes (representing about 16 × 107 cells) were homogenized and subcellular fractionation prepared by differential centrifugation as previously described (19Wieckowski M.R. Giorgi C. Lebiedzinska M. Duszynsk J. Pinton P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.Nat. Protoc. 2009; 4: 1582-1590Crossref PubMed Scopus (582) Google Scholar). Briefly, after homogenization, unbroken cells and nuclei were removed, and then an intermediate pellet was isolated from a fraction later centrifuged for ER and cytosol isolation and finally separated by ultracentrifugation (20,000 g for 30 min and then 100,000 g for 1 h). The crude mitochondrial fraction (MF) was obtained by centrifugation of the previous resuspended pellet at 10,000 g for 10 min. Crude MF was then layered on top of 30% Percoll medium and centrifuged at 95,000 g for 30 min. After this centrifugation, a dense band of pure mitochondria (PM) was observed at the bottom of the tube and a diffused white band of MAM at the top of the tube. These fractions were collected with a Pasteur pipette, diluted, and then pelleted respectively at 6,300 g for 10 min and 100,000 g for 1 h. The different fractions were diluted in different volumes as described in supplemental Fig. S1. For Western blot analysis, 10 μg total protein (cells) or 30 μg (liver) from each fraction (supplemental Fig. S1) were loaded onto 8% or 12% SDS-PAGE and transferred onto Immobillion-P transfer membrane (Millipore, Darmstadt, Germany) for immunoblotting. The primary antibodies used were anti-FACL-4 (ACSL-4) (sc-365230; Santa Cruz Biotechnology), anti-calreticulin (CR) (c1036; US Biological, Salem, MA), anti-VDAC (V2139; Sigma-Aldrich, St Louis, MO), anti-cytochrome c (7H82C12; R&D Systems, Minneapolis, MN), anti-TOM20 (sc-11415; Santa Cruz Biotechnology), anti-RTN3 (sc-374599; Santa Cruz Biotechnology), and anti-LAMP2 (PA5-85327; Thermo Fisher Scientific). Transmission electron microscopy was done on cells fixed with 4% glutaraldehyde in PBS (pH 7.4) followed by a postfixation with 2% OsO4. After dehydration in a graded series of ethanol, adherent cells were embedded in epoxy resin, and thin sections (60 to 70 nm) were cut on a Reichert Ultracut E microtome and stained with uranyl acetate and lead citrate for observation at 80 KV under a JEM-1010 transmission electron microscope (JEOL). For microscopic analyses, cells were grown on gelatin-coated coverslips. The cells were incubated with MitoTracker Red (Life Technologies) for 30 min at 37°C, washed two times with PBS, and then fixed in 4% paraformaldehyde for 30 min. The cells were washed with PBS and then mounted with Prolong antifade (Life Technologies) polymerizing solution and observed under a microscope with a 63× objective (Zeiss with apotome). Cells treated or not treated with STS were harvested after 2, 4, 6, or 8 h, permeabilized in PBS/0.002% digitonin, and fixed for 15 min with 2% paraformaldehyde. Cytosolic cytochrome c is lost during mild digitonin-induced membrane permeabilization, apoptotic cells thus appear negative for cytochrome c staining. Staining for cytochrome c (Clone 6H2.B4; BD Biosciences, San Jose, CA) and active Bax (Clone 6A7; BD Biosciences) was performed overnight at 4°C. The percent of positive cells was determined compared with isotype. For cell viability assessment, cells were harvested after STS treatment and the number of dead PI positive cells was determined after 5 min incubation with propidium iodide (Sigma). The internal standard mixture composed of C17 ceramide, C17 SM, C17 sphingosine-1-phosphate, C12 ceramide-1-phosphate, C17 sphinganine, C12 lactosyl-ceramide, and C12 glucosyl-ceramide was added to each biological sample. Lipid extraction was carried out in two steps. First, 1.5 ml of hexane/propan-2-ol (60:40, v/v) containing 0.6% formic acid were added, samples were centrifuged at 1,000 g for 5 min (4°C), and the upper phase was removed into a glass tube. Then, 1.5 ml of methanol containing 0.6% formic acid were added to the lower phase. After centrifugation at 7, 000 g for 5 min (4°C), the two upper phases were combined and dried under nitrogen at room temperature. The total lipid extract was suspended in 150 μl of hexane/propan-2-ol (60:40 v/v) (0.6% formic acid). Targeted lipid purification was carried out using NH2 SPE cartridges (100 mg). Total extract was deposited on cartridges preconditioned with 2 ml of hexane. Different classes of lipids were separated following the method described in (20Bodennec J. Koul O. Aguado I. Brichon G. Zwingelstein G. Portoukalian J. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges.J. Lipid Res. 2000; 41: 1524-1531Abstract Full Text Full Text PDF PubMed Google Scholar). The four recovered fractions (F2, F4, F5, and F6) were dried down under nitrogen and removed in optimized volume of UPLC eluent A. The sphingolipids were quantified by LC-ESI-MS/MS. The purified sphingolipids were analyzed on an Acquity H-Class UPLC system (Waters Corporation, Milford, MA) combined with a Waters Xevo TQD triple quadrupole mass spectrometer. All analyses were performed using ESI in the positive ion mode (ESI+) with multiple reaction monitoring. All sphingolipid classes were analyzed on a Waters C18 BEH column (2.1 × 50 mm) with 1.8 μm particle size equipped with a 0.5 μM prefilter. The column heater was set at 43°C for all compounds except ceramide-1-phosphate and sphingosine-1-phosphate where column temperature was set at 60°C. The autosampler temperature was maintained at 10°C. The mobile phases consisted of methanol (eluent A) or water (eluent B) containing 1% formic acid and 5 mM of ammonium formate. Purified sphingolipid fractions (sample and calibration) were eluted according to the elution step program. Measurement and data analysis were collected by Mass Lynx software version 4.1 (Waters, Manchester, UK). Integration and quantification were performed using the TargetLinks™ software (Waters). Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) enzymatic activities were determined using the fluorescent substrate BODIPY-FL-C12-SM (Invitrogen-Life Technologies; D-7711). ASMase reaction (21Mühle C. Huttne H.B. Walte S. Reichel M. Canneva F. Lewczuk P. Gulbins E. Kornhuber J. Characterization of acid sphingomyelinase activity in human cerebrospinal fluid.PLoS One. 2013; 8: e62912Crossref PubMed Scopus (27) Google Scholar) was initiated by the addition of 1 μg protein sample in a volume of 100 μl containing 73 pmol substrate in 12.5 mM sucrose buffer (pH 5.0) with 1% Triton X-100 and 0.1 mM of ZnCl2 or 5 mM of EDTA activation buffer. EDTA and ZnCl2 buffers were used respectively for lysosomal ASMase (L-ASMase) and total ASMase activity quantification. NSMase activity was magnesium dependent (22Rao B.G. Spence M.W. Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0.J. Lipid Res. 1976; 17: 506-515Abstract Full Text PDF PubMed Google Scholar). The reaction was initiated by 1 μg of protein sample in a volume of 100 μl containing 10 pmol of substrate in 200 mM of HEPES buffer (pH 7.4) with 20 mM of MgCl2. The reaction was processed at 37°C over 24 h and stopped by adding chloroform:methanol (1:1, v/v). Samples were centrifuged at 1,000 g for 5 min. The organic phase was harvested, evaporated under N2, and resuspended in chloroform/methanol (2:1, v/v). Five microliters were spotted on silica gel 60 thin-layer chromatography plates (Merck-Millipore; 105626). Ceramide and uncleaved SM were separated using a mixture of chloroform:methanol (95:5, v/v) as a solvent and were quantified on a ChemiDoc MP imaging system (Bio-Rad) with the ImageLab software (Bio-Rad). Enzymatic activity is presented as the hydrolysis rate of SM (picomoles) per time (hours) and per protein concentration (micrograms). Two-way ANOVA and, as posttest, Tukey's multiple comparison test were used in this study. Research involving animals were performed in agreement with the French National Ethics Committee. Investigations with animals were approved by the local institutional review committee of the animal house facilities. We first analyzed the mitochondrial and ER proteins present in the different submembrane fractions from mouse liver by immunoblots (see the Materials and Methods). As illustrated in Fig. 1A, we found a repartition of these different proteins from ER to MAM to MF similar to that described in previous studies: CR (23Arnaudeau S. Frieden M. Nakamura K. Castelbou C. Michalak M. Demaurex N. Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria.J. Biol. Chem. 2002; 277: 46696-46705Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and the long-chain fatty acid-CoA ligase 4 (FACL-4) (24Lewin T.M. Kim J.H. Granger D.A. Vance J.E. Coleman R.A. Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently.J. Biol. Chem. 2001; 276: 24674-24679Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) were found in ER and MAM, while VDAC1 and cytochrome c were predominantly found in mitochondria. Similar but not identical results were found in mouse brain (supplemental Fig. S2). Next, we analyzed and quantified the sphingolipid content and, in particular, precursors and metabolites of ceramides in the different liver fractions by LC-ESI-MS/MS. Ceramides and SM were present mostly in MAM and the ER (Fig. 1B). On the other hand, sphinganine was essentially present in MAM and PM fractions, while sphingosine was found mostly in PM (Fig. 1C). Statistical data for lipid measurements are shown in supplemental Fig. S3. Glycosylated forms of ceramides are biologically active and versatile derivatives of ceramides. These glucosyl- and lactosyl-ceramides are thought be synthesized in the late ER compartments. although there is some evidence that they are present in MAM (11Ardail D. Popa I. Bodennec J. Louisot P. Schmitt D. Portoukalian J. The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases.Biochem. J. 2003; 371: 1013-1019Crossref PubMed Scopus (84) Google Scholar). We confirmed the latter observation and found that, in mouse liver, glucosyl-ceramide is essentially present in MAM, while lactosyl-ceramide is located both in PM and MAM (Fig. 1D). Analysis by the chain lengths of the sphingolipids shows some differences between the different fractions: MAM and PM fractions were enriched in C18 compared with ER, while ER and PM fractions were enriched in C24 and C24:1 compared with MAM (Table 1). However, major and minor chain lengths in the different fractions appear to be roughly constant and no chain length is specific from any organelle (except from C18 ceramide, which is the only ceramide found in cytosol) (Table 1).TABLE 1Subcellular fractionation performed from mouse liversHMFPMMAMERCytoMeanSEMMeanSEMMeanSEMMeanSEMMeanSEMMeanSEMCeramide C14:04.871.617.111.259.103.964.721.4910.472.070.510.37 C16:11.250.581.980.283.721.102.570.601.430.410.850.42 C1638.7612.6378.4110.11112.0034.9258.6317.8297.2413.903.510.52 C18:10.930.312.030.273.841.070.290.291.130.290.190.19 C1832.377.77137.4528.70144.1722.86204.3456.8354.828.11152.2034.72 C20:10.000.000.000.000.000.000.000.000.000.000.000.00 C201.640.604.090.648.864.072.810.546.051.340.250.25 C22:11.090.382.270.584.081.881.350.294.240.490.000.00 C227.592.5218.142.1035.7211.3712.844.1725.244.061.300.05 C24:1123.5541.27258.8429.87445.91131.01173.1148.49358.6836.2511.392.48 C2440.5314.5084.537.12131.5035.3557.6813.29120.3613.376.171.17SM C14:0317.9368.991,464.30106.82593.72131.791608.6664.32993.28135.6121.6110.82 C16:1376.2281.261,708.5697.20672.38123.041,852.9228.601,135.45158.0436.653.90 C164,653.71880.0322,298.322,219.499,495.851,969.8824,336.04714.9913,682.741,602.26906.61389.91 C18:1112.3725.73470.8045.54192.9024.54513.9923.53307.0424.206.503.58 C18422.0079.762,019.3688.37981.44183.302,030.7631.161,152.21137.07119.1873.24 C20:118.765.5974.4310.0735.553.0165.038.9051.858.200.000.00 C2081.0919.07406.0835.36157.1022.46382.6139.81223.8932.9657.2554.12 C22:1116.0318.77529.3434.78224.4228.59543.2431.37315.9146.5519.8514.53 C22204.0140.391,108.36132.84475.9196.141,014.94141.00551.2378.4022.8413.53 C24:11,656.56257.778,205.661,135.303,683.24932.798,478.13442.034,878.83917.29385.63170.81 C24371.8653.232,049.34333.96898.11235.671,839.27232.731,096.05221.38368.50323.24Sphingosine C18:121.664.01113.3518.81213.2527.40128.5071.5923.565.1061.7918.04Sphinganine C182.310.5612.181.8634.561.965.132.242.320.351.530.48Glucosyl-ceramide C14:00.000.000.010.010.000.000.010.010.010.010.000.00 C16:10.510.132.860.271.730.311.990.031.760.040.000.00 C1650.6211.05252.433.77199.7918.40197.4012.31127.859.444.150.40C18:10.140.080.640.330.720.360.310.310.250.120.000.00 C183.981.0321.571.2317.762.2118.602.389.040.810.120.12 C20:10.070.070.000.000.300.190.070.070.110.090.000.00 C201.120.186.380.265.730.505.740.392.140.070.040.04 C22:15.861.2932.692.4828.521.7724.072.4512.701.290.170.17 C220.250.171.280.661.370.690.890.490.450.230.010.01 C24:144.098.69255.2310.99242.0113.46166.9016.7299.798.433.530.53 C2430.637.12154.0911.89126.754.81111.9610.4284.627.462.020.38Lactosyl-ceramide C14:01.130.554.831.511.720.514.310.903.510.510.000.00 C16:10.060.060.280.280.070.070.500.290.250.120.000.00 C1655.5725.53240.4963.68101.1822.27263.9054.47176.6033.993.991.49 C18:10.640.351.870.640.920.141.190.751.170.220.000.00 C186.213.0428.889.8912.973.1536.2310.4116.443.910.540.54 C20:10.010.010.100.100.010.010.070.070.040.040.000.00 C200.310.141.610.420.940.152.680.380.870.100.000.00 C22:10.310.121.340.490.350.351.470.740.660.200.000.00 C223.081.1717.183.598.341.9525.204.288.621.520.240.24 C24:134.7211.85203.0054.79115.1630.89320.8478.76103.8421.415.832.08 C2419.117.1689.5414.3142.226.60118.8416.1857.395.011.800.55Lipid extraction and purification were performed on 50 μl of each fraction. The sphingolipid composition of the fractions was then analyzed by UPLC-MS. The results are expressed in picomoles of lipids per milligram of proteins for each chain length of ceramide and SM (mean ± SEM). Cyto, cytosol; H, homogenate. Open table in a new tab Lipid extraction and purification were performed on 50 μl of each fraction. The sphingolipid composition of the fractions was then analyzed by UPLC-MS. The results are expressed in picomoles of lipids per milligram of proteins for each chain length of ceramide and SM (mean ± SEM). Cyto, cytosol; H, homogenate. Our data support previous observations on the presence of sphingolipids in MAM. However, sphingolipids present in MAM are quantitatively and qualitatively different from those found in ER and mitochondria. Sphingolipids have been implicated in apoptosis, but little is known about their intracellular distribution between ER, MAM, and mitochondria during the induction of this cell death program (8Mignard V. Lalier L. Paris F. Vallette F.M. Bioactive lipids and the control of Bax pro-apoptotic activity.Cell Death Dis. 2014; 5: e1266Crossref PubMed Scopus (42) Google Scholar). Induction of apoptosis in liver is a multimodal and complex phenomenon that involves different processes. In order to obtain a more homogeneous population, we investigated the sphingolipid composition in resting and apoptotic human glioma U251 cells. This cell line has been extensively studied as a model for apoptosis (25Gratas C. Sery Q. Rabe M. Oliver L. Vallette F.M. Bak and Mcl-1 are essential for Temozolomide induced cell death in human glioma.Oncotarget. 2014; 5: 2428-2435Crossref PubMed Scopus (43) Google Scholar). As illustrated in Fig. 2A, transmission electron microscopy studies reveal that U251 cells exhibited ER/mitochondria contacts that resemble classical MAM morphology. Resting U251 cells were subjected to su" @default.
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• W3023804959 title "Sphingolipid distribution at mitochondria-associated membranes (MAMs) upon induction of apoptosis" @default.
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