Ethanol alters lipid profiles and phosphorylation status of AMP-activated protein kinase in the neonatal mouse brain

Previously, we have shown that ethanol-induced apoptosis in cultured neurons is accompanied by changes in cellular lipid profiles. In the present study, the effects of ethanol on brain lipid metabolism were studied using 7-day-old C57BL/6ByJ mice, which display apoptotic neurodegeneration upon exposure to ethanol. The brain lipids were extracted 4-24 h after the ethanol or saline treatment, and analyzed by TLC. We found that the levels of triglyceride, cholesterol ester, ceramide, and N-acylphosphatidylethanolamine increased significantly in the brains of ethanol-treated mice compared to those of saline-treated mice. Concomitantly, ethanol reduced Thr172 phosphorylation of AMP-activated protein kinase (AMPK) alpha subunits. Ethanol also reduced phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK and a lipogenic enzyme known to be activated by dephosphorylation. In contrast, lipid profiles of 19-day-old mouse brains, which scarcely manifested neurodegeneration upon ethanol exposure, were not significantly affected by ethanol. Also, the basal levels of Thr172-phosphorylated AMPK alpha were lower in these brains than in 7-day-old mouse brains, and no detectable changes in the phosphorylation status were observed by ethanol treatment. Our findings indicate that the ethanol-induced apoptotic neurodegeneration observed in mice during restricted developmental periods is accompanied by alterations in both the lipid content and the activity of AMPK in the brain.     

Evaluation of sphingolipids changes in brain tissues of rats with pentylenetetrazol-induced kindled seizures using MALDI-TOF-MS

Abnormal lipid metabolism has been implicated in the pathogenesis of many neural system diseases, including epilepsy. Pentylenetetrazol (PTZ)-induced kindling in rodents is considered a model of human absence epilepsy and myoclonic, generalized tonic-clonic seizure. In an effort to further understand the mechanism for PTZ-induced seizure, we analyzed crude lipids and sphingolipids in the cortex, hippocampus, and brain stem of normal and PTZ-rats using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). It was found that phosphatidylcholines dominated the crude lipids in different tissues and there were no obvious differences in crude lipid profiles of different tissues between normal and PTZ-rats. However, ceramide, sphingomyelins, and ceramide-monohexoside were differently distributed in normal and PTZ-rats. Using the reference mass spectra method established in our laboratory, it was shown that sphingomyelins and ceramide-monohexoside levels were elevated in the brain tissues of PTZ-rats. Ceramide levels were found to be higher in brain stem than in cortex and hippocampus of normal rats, and PTZ caused a general decrease in ceramide levels. These data suggest that changes in sphingolipid metabolism contribute to PTZ-induced seizure.     

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.     

A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.     

Ceramide pathways modulate ethanol-induced cell death in astrocytes

We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.     

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite,Caloric Intake and Fat Storage in Drosophila

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.     

Maternal Alcohol Consumption Increases Sphingosine Levels in the Brains of Progeny Mice

The effect of ‘binge’ alcohol upon sphingolipid metabolism in the fetal alcohol syndrome (FAS) was examined in pregnant mice (C57BL/6J) by administering a single dose of alcohol during the third trimester (gestational day 15–16). The control mice were administered a sucrose solution of equal caloric value. Brains from progeny at postnatal days 5, 15, 21 and 30 were dissected into three regions, and sphingolipid concentrations of the brain regions were determined including assay of monoglycosylceramide, ceramide, sphingosine and sphingomyelin. We found that a single dose of ethanol induces an elevation of sphingosine (2–3.5-fold) in the brain of progeny. The level of brain ceramide at a dose of 1.5 g/kg was significantly higher than control. Alcohol consumption during pregnancy induces neuronal loss in progeny brains. Our result suggests that the elevation of sphingosine in progeny brain induced by maternal alcohol consumption may be responsible for observed neuronal loss in FAS. Special issue in honor of Naren Banik.     

Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp^−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.     

Jasmonates modulate sphingolipid metabolism and accelerate cell death in the ceramide kinase mutant acd5

Sphingolipids are structural components of the lipid bilayer that acts as signaling molecules in many cellular processes, including cell death. Ceramides, key intermediates in sphingolipid metabolism, are phosphorylated by the ceramide kinase ACCELERATED CELL DEATH5 (ACD5). The loss of ACD5 function leads to ceramide accumulation and spontaneous cell death. Here, we report that the jasmonate (JA) pathway is activated in the Arabidopsis (Arabidopsis thaliana) acd5 mutant and that methyl JA treatment accelerates ceramide accumulation and cell death in acd5. Moreover, the double mutants of acd5 with jasmonate resistant1-1 and coronatine insensitive1-2 exhibited delayed cell death, suggesting that the JA pathway is involved in acd5-mediated cell death. Quantitative sphingolipid profiling of plants treated with methyl JA indicated that JAs influence sphingolipid metabolism by increasing the levels of ceramides and hydroxyceramides, but this pathway is dramatically attenuated by mutations affecting JA pathway proteins. Furthermore, we showed that JAs regulate the expression of genes encoding enzymes in ceramide metabolism. Together, our findings show that JAs accelerate cell death in acd5 mutants, possibly by modulating sphingolipid metabolism and increasing ceramide levels.     

Pulsatilla chinensis saponins cause liver injury through interfering ceramide/sphingomyelin balance that promotes lipid metabolism dysregulation and apoptosis

Background: P. chinensis saponins (PRS) are pentacyclic triterpenoid bioactive constituents from Pulsatilla chinensis (Bunge) Regel. In our previous study, PRS caused chronic liver injury (CLI) with the significant changes of lipid metabolites including sphingomyelin (SM) in serum after long-term administration. The SM in the hepatocytes membrane plays an indispensable role in maintaining cell membrane stability and regulating the extracellular and intracellular signal transduction. However, it is still unknown the pathway related to SM and the mechanism of CLI on hepatocyte.Purpose: The purpose of this study was to explore the hepatotoxicity mechanism of PRS in vivo and in vitro, to reveal the action of mechanism of SM and the pathway related to liver injury.Methods: SD rats were orally administered with PRS for 240 days and liver injury was evaluated by histological examinations. Metabolomics analysis was used to explore the liver metabolic pathway affected by PRS, and the expressions of related proteins were evaluated by western blots. To discover and elucidate the underlying mechanisms of metabolites changes induced by PRS at the cellular level, cellular morphology, MTT assays, western blots and cell membrane potential measurements were carried out using LO2 cells. Furthermore, the roles of SM and cholesterol (Chol) in hepatocyte injury were investigated individually in overload Chol and SM groups. Sphingolipid metabolic pathway related with ceramide/sphingomyelin (Cer/SM) balance was explored using cellular lipidomics and RT-PCR.Results: PRS gradually damaged the rat's liver in a time-dependent manner. The analysis of liver metabolism profiles showed that lipids metabolites were changed, including sphingolipid, bile acid, linoleic acid and fatty acid. We found that PRS induced apoptosis by interfering with bile acid-mediated sphingolipid metabolic pathway and Cer/SM balance in CLI. In in vitro experiments, PRS led to the increase of LDH leakage, depolarized cell membrane potential and caused cell membrane toxicity. Furthermore, PRS inducedG0/G1 phase cell cycle arrest in LO2 cells, simultaneously activated cellular extrinsic and intrinsic apoptosis pathways. PRS acted on SM and interfered with Cer/SM balance, which promote lipid metabolism dysregulation and apoptosis.Conclusion: PRS acted on SM to interfere Cer/SM balance on LO2 cell. Both in vivo and in vitro, PRS induced Cer/SM imbalance which promoted lipid metabolism disorder and apoptosis. Apoptosis and lipids changes gradually damaged the rats liver, and ultimately developed into CLI.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Effects of chronic consumption of ethanol and low-thiamin, low-protein diets on the lipid composition of rat whole brain and brain membranes

Four groups of rats were used in a nutritionally-controlled study of effects of chronic ethanol consumption on brain membrane lipid composition. Rats chronically consuming ethanol were fed high-nutrient or low-thiamin, low-protein diets. After 4 months, lipid analyses were performed on brains, brain microsomes and myelin from each group and from pair-fed, non-ethanol controls. Among the effects of ethanol was an increase of the relative proportion of cholesterol in microsomal lipids while there was decrease of it in myelin. Ethanol also increased plasmenylethanolamine while decreasing phosphatidylethanolamine proportions in myelin and in whole brain lipids, decreased the total lipid phosphorus of whole brain, and elevated the proportion of phosphatidylserine in microsomal and whole brain lipids. Effects of poor diet generally did not interfere with ethanol effects except in the case of microsomal lipids, where it apparently prevented an ethanol-induced increase in proportion of cholesterol. These changes may be adaptive responses to the fluidizing effect of ethanol on membranes.     

Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate

Ceramide is an important lipid signaling molecule and a key intermediate in sphingolipid biosynthesis. Recent studies have implied a previously unappreciated role for the ceramide N-acyl chain length, inasmuch as ceramides containing specific fatty acids appear to play defined roles in cell physiology. The discovery of a family of mammalian ceramide synthases (CerS), each of which utilizes a restricted subset of acyl-CoAs for ceramide synthesis, strengthens this notion. We now report the characterization of mammalian CerS2. qPCR analysis reveals that CerS2 mRNA is found at the highest level of all CerS and has the broadest tissue distribution. CerS2 has a remarkable acyl-CoA specificity, showing no activity using C16:0-CoA and very low activity using C18:0, rather utilizing longer acyl-chain CoAs (C20-C26) for ceramide synthesis. There is a good correlation between CerS2 mRNA levels and levels of ceramide and sphingomyelin containing long acyl chains, at least in tissues where CerS2 mRNA is expressed at high levels. Interestingly, the activity of CerS2 can be regulated by another bioactive sphingolipid, sphingosine 1-phosphate (S1P), via interaction of S1P with two residues that are part of an S1P receptor-like motif found only in CerS2. These findings provide insight into the biochemical basis for the ceramide N-acyl chain composition of cells, and also reveal a novel and potentially important interplay between two bioactive sphingolipids that could be relevant to the regulation of sphingolipid metabolism and the opposing functions that these lipids play in signaling pathways.     

Ceramide kinase regulates TNFα-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNFα exposure dramatically increased Mg(2+)-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNFα exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNFα. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation.     

Lipid Integration in Neurodegeneration: An Overview of Alzheimer’s Disease

Various types of lipids and their metabolic products associated with the biological membrane play a crucial role in signal transduction, modulation, and activation of receptors and as precursors of bioactive lipid mediators. Dysfunction in the lipid homeostasis in the brain could be a risk factor for the many types of neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. These neurodegenerative disorders are marked by extensive neuronal apoptosis, gliosis, and alteration in the differentiation, proliferation, and development of neurons. Sphingomyelin, a constituent of plasma membrane, as well as its primary metabolite ceramide acts as a potential lipid second messenger molecule linked with the modulation of various cellular signaling pathways. Excessive production of reactive oxygen species associated with enhanced oxidative stress has been implicated with these molecules and involved in the regulation of a variety of different neurodegenerative and neuroinflammatory disorders. Studies have shown that alterations in the levels of plasma lipid/cholesterol concentration may result to neurodegenerative diseases. Alteration in the levels of inflammatory cytokines and mediators in the brain has also been found to be implicated in the pathophysiology of neurodegenerative diseases. Although several mechanisms involved in neuronal apoptosis have been described, the molecular mechanisms underlying the correlation between lipid metabolism and the neurological deficits are not clearly understood. In the present review, an attempt has been made to provide detailed information about the association of lipids in neurodegeneration especially in Alzheimer’s disease.     

Exogeneous and endogenous CCK inhibit ethanol ingestion in Sardinian alcohol-preferring rats

Ethanol ingestion, like food ingestion, stimulates release of the signaling molecule cholecystokinin (CCK) from the small intestine. Here, we investigated the possibility that ethanol-induced CCK release might be a negative-feedback control of ethanol ingestion, similar to its function as part of the mechanism by which ingested food produces meal-ending satiation. We used Sardinian alcohol-preferring (sP) and Marchesian Sardinian (msP) alcohol-preferring rats, two apparently identical substrains that spontaneously ingest pharmacologically relevant amounts of ethanol, as well as their background strain, Wistar (W) rats. We demonstrated that: (1) intraperitoneal (IP), but not intracerebroventricular, injections of 0.5-4 microg/kg CCK-8 produced transient, dose-related reductions in 10% ethanol ingestion; (2) this inhibitory effect of CCK-8 on ethanol intake appeared behaviorally similar to its inhibitory action on ingestion of sucrose solutions; (3) the inhibitory effect of IP CCK-8 on ethanol ingestion occurred without evidence of tolerance when tests were repeated on consecutive days; (4) IP CCK-8 reduced ethanol intake despite simultaneously reducing blood ethanol levels (BALs); and (5) antagonism of CCK1 receptors with devazepide increased ethanol intake, indicating that endogenous CCK normally limits the size of bouts of ethanol ingestion. These results implicate peripheral CCK in the control of ethanol ingestion in sP and msP alcohol-preferring rats.     

Cancer and sphingolipid storage disease therapy using novel synthetic analogs of sphingolipids

Sphingolipid metabolites have become recognized for their participation in cell functions and signaling events that control a wide array of cellular activities. Two main sphingolipids, ceramide and sphingosine-1-phosphate, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, angiogenesis, stress responses, protein synthesis, carbohydrate metabolism, and intracellular trafficking. Ceramide and S1P often exert opposing effects on cell survival, ceramide being pro-apoptotic and S1P generally promoting cell survival. Therefore, the conversion of one of these metabolites to the other by sphingolipid enzymes provides a vast network of regulation and provides a useful therapeutic target. Here we provide a survey of the current knowledge of the roles of sphingolipid metabolites in cancer and in lipid storage disease. We review our attempts to interfere with this network of regulation and so provide new treatments for a range of diseases. We synthesized novel analogs of sphingolipids which inhibit the hydrolysis of ceramide or its conversion to more complex sphingolipids. These analogs caused elevation of ceramide levels, leading to apoptosis of a variety of cancer cells. Administration of a synthetic analog to tumor-bearing mice resulted in reduction and even disappearance of the tumors. Therapies for sphingolipid storage diseases, such as Niemann-Pick and Gaucher diseases were achieved by two different strategies: inhibition of the biosynthesis of the substrate (substrate reduction therapy) and protection of the mutated enzyme (chaperone therapy). Sphingolipid metabolism was monitored by the use of novel fluorescent sphingolipid analogs. The results described in this review indicate that our synthetic analogs could be developed both as anticancer drugs and for the treatment of sphingolipid storage diseases.     

Sphingolipid Turnover in the Hippocampus and Cognitive Dysfunction in Alcoholized Rats: Correction with the Help of Alimentary <Emphasis Type="Italic">n</Emphasis>-3 Fatty Acids

We studied the effect of long-lasting (during 60 days) consumption of 15% ethanol solution by 2- to 4-month-old rats on the turnover of sphingolipids in the hippocampus and cognitive functions in these animals. It was found that chronic action of ethanol led to a decrease in the content of newly synthesized sphingomyelin and to an increase in the level of ceramide and the ceramide/sphingomyelin ratio in the hippocampus; this was accompanied by significant worsening of conditioned-reflex activity in experimental rats (testing of active avoidance in a shuttle chamber). The addition of fish oil containing a considerable amount of n-3 fatty acids to the diet of animals consuming ethanol prevented, to a considerable extent, the development of disorders of sphingolipid turnover and cognitive functions.     

Decreased caveolae in AGPAT2 lacking adipocytes is independent of changes in cholesterol or sphingolipid levels: A whole cell and plasma membrane lipidomic analysis of adipogenesis

BackgroundAdipocytes from lipodystrophic Agpat2^?/? mice have impaired adipogenesis and fewer caveolae. Herein, we examined whether these defects are associated with changes in lipid composition or abnormal levels of caveolae-associated proteins. Lipidome changes were quantified in differentiated Agpat2^?/? adipocytes to identify lipids with potential adipogenic roles.MethodsAgpat2^?/? and wild type brown preadipocytes were differentiated in vitro. Plasma membrane was purified by ultracentrifugation. Number of caveolae and caveolae-associated proteins, as well as sterol, sphingolipid, and phospholipid lipidome were determined across differentiation.ResultsDifferentiated Agpat2^?/? adipocytes had decreased caveolae number but conserved insulin signaling. Caveolin-1 and cavin-1 levels were equivalent between Agpat2^?/? and wild type adipocytes. No differences in PM cholesterol and sphingolipids abundance were detected between genotypes. Levels of phosphatidylserine at day 10 of differentiation were increased in Agpat2^?/? adipocytes. Wild type adipocytes had increased whole cell triglyceride, diacylglycerol, phosphatidylglycerol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, and trihexosyl ceramide, and decreased 24,25-dihydrolanosterol and sitosterol, as a result of adipogenic differentiation. By contrast, adipogenesis did not modify whole cell neutral lipids but increased lysophosphatidylcholine, sphingomyelin, and trihexosyl ceramide levels in Agpat2^?/? adipocytes. Unexpectedly, adipogenesis decreased PM levels of main phospholipids in both genotypes.ConclusionIn Agpat2^?/? adipocytes, decreased caveolae is not associated with changes in PM cholesterol nor sphingolipid levels; however, increased PM phosphatidylserine content may be implicated. Abnormal lipid composition is associated with the adipogenic abnormalities of Agpat2 ?/? adipocytes but does not prevent insulin signaling.