Group VIA phospholipase A2 deficiency in mice chronically fed with high-fat-diet attenuates hepatic steatosis by correcting a defect of phospholipid remodeling

A defect of hepatic remodeling of phospholipids (PL) is seen in non-alcoholic fatty liver disease and steatohepatitis (NASH) indicating pivotal role of PL metabolism in this disease. The deletion of group VIA calcium-independent phospholipase A2 (iPla2β) protects ob/ob mice from hepatic steatosis (BBAlip 1861, 2016, 440–461), however its role in high-fat diet (HFD)-induced NASH is still elusive. Here, wild-type and iPla2β-null mice were subjected to chronic feeding with HFD for 6 months. We showed that protection was observed in iPla2β-null mice with an attenuation of diet-induced body and liver-weight gains, liver enzymes, serum free fatty acids as well as hepatic TG and steatosis scores. iPla2β deficiency under HFD attenuated the levels of 1-stearoyl lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI) as well as elevation of hepatic arachidonate, arachidonate-containing cholesterol esters and prostaglandin E₂. More importantly, this deficiency rescued a defect in PL remodeling and attenuated the ratio of saturated and unsaturated PL. The protection by iPla2β deficiency was not observed during short-term HFD feeding of 3 or 5 weeks which showed no PL remodeling defect. In addition to PC/PE, this deficiency reversed the suppression of PC/PI and PE/PI among monounsaturated PL. However, this deficiency did not modulate hepatic PL contents and PL ratios in ER fractions, ER stress, fibrosis, and inflammation markers. Hence, iPla2β inactivation protected mice against hepatic steatosis and obesity during chronic dietary NASH by correcting PL remodeling defect and PI composition relative to PC and PE.     

Preconditioning limits mitochondrial Ca(2+) during ischemia in rat hearts: role of K(ATP) channels

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca(2+)]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca(2+)]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca(2+)]m during ischemia-reperfusion and that this effect was mediated by opening mitochondrial K(ATP) channels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial K(ATP) channel opening (diazoxide, 100 microM) and blockade [5-hydroxydecanoic acid (5-HD), 100 microM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca(2+)]i and [Ca(2+)]m were measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca(2+)]m during ischemia-reperfusion. In addition, PC lowered [Ca(2+)]i during reperfusion, whereas diazoxide paradoxically resulted in increased [Ca(2+)]i during reperfusion. There was an inverse linear relationship between [Ca(2+)]m and developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca(2+)]i, an effect mediated by opening mitochondrial K(ATP) channels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.     

Studies on the origin of biliary phospholipid. Effect of dehydrocholic acid and cholic acid infusions on hepatic and biliary phospholipids.

The correlation between the secretion of biliary phospholipid (PL) and bile acid suggests a regulatory effect of bile acid on PL secretion. Bile acids may influence PL synthesis and/or the mobilization of a preformed PL pool. The objective of this study was to determine the contribution of these two sources to biliary PL, by using an experimental protocol in which dehydrocholic acid (DHCA) and cholic acid (CA) were infused to manipulate biliary PL secretion. In control rats, there was a steady state in bile flow. PL secretion and the biliary secretion of newly synthesized phosphatidylcholine (PC). The specific radioactivity of PC in bile was significantly higher than in plasma, microsomes and canalicular membranes. DHCA infusion decreased biliary PC secretion rate by 80%, and secretion returned to normal values at the transport maximum of CA. The specific radioactivity of biliary PC was decreased by 30% by DHCA infusion and reached normal values during CA infusion. There were no significant changes in the specific radioactivity of PC in plasma or cellular organelles during infusion of bile acids. These data indicate that: (1) newly synthesized PC contributes a small percentage to biliary PC; thus a preformed pool (microsomal and extrahepatic) is a major source of biliary PL; (2) the contribution of the extrahepatic pool to the biliary PL may be more important than the microsomal pool.     

Quantification of surfactant phospholipids in the dog lung

We quantified total phospholipid (PL), total and disaturated phosphatidylcholine (PC and DSPC), phosphatidylglycerol (PG), and total protein in alveolar washings and lung tissue in 22 dog lungs. Quantitative recovery of alveolar material and assessment of its possible contamination by blood lipids were important determinants of methodology. To remove blood, the vessels of half the lungs were perfused with a fluorocarbon emulsion before lavage. The volume of blood removed by perfusion and the quantity and fatty acid patterns of its whole blood and plasma PL and PC were determined. Washings of unperfused lungs contained means of 21% more PL and 24% more PC than those of perfused lungs. Although this excess could be accounted for by the PL and PC in pulmonary blood, the hemoglobin and total protein content of washings and their PC fatty acid patterns indicated that blood lipids were not a major source of the excess lipid in washings of unperfused lungs. Using more recent morphometric estimates rather than the indirect ones previously used by others, the quantity of alveolar DSPC (1 mg/g lung) is calculated to be 1.8 times the amount necessary to form a packed monolayer on the internal surface of the lung at functional residual capacity.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ?C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1? cells grown on synthetic media at both 30 ?C and 37 ?C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1?gpd1? double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.     

Phospholipid and Fatty Acid Composition of Phosphatidylcholine and Phosphatidylethanolamine in the Black Plaice <Emphasis Type="Italic">Pleuronectes obscura</Emphasis> during Thermoadaptation

The phospholipid (PL) and fatty acid (FA) composition of major membrane lipid constituents, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as the cholesterol/phospholipid (CL/PL) ratio were assayed in the muscles, gills and liver of the black plaice Pleuronectes (Liopsetta) obscura at different ambient temperatures (18, 9 and 0°C). PL and CL were shown to be actively involved in adaptation of the fish to changes in the seawater temperature. As temperature declines, the monounsaturated FA (MUFA) level increases while the polyunsaturated FA (PUFA) fraction in gills and liver PC and PE, on the contrary, decreases, resulting in diminished functional activity of the fish. However, in muscles this correlation is lacking. The PC and PE composition was shown to be organ- and ambient temperature-dependent. Major PC forms are saturated FA (SFA)/PUFA and MUFA/PUFA composed of a relatively small number of major molecular species. A temperature drop results in an increased SFA/PUFA level and decreased MUFA/PUFA and PUFA/PUFA levels in muscles and gills, and this may promote a drop in the viscosity of the outer lipid monolayer of membranes and in their functional activity. In contrast to PC, the PE composition in all organs tested is characterized by a decrease in the SFA/ PUFA level and an increase in MUFA/PUFA and PUFA/PUFA levels. Such changes promote the retention of functional activity of the inner lipid monolayer of membranes and are not synchronized with rearrangements in their outer monolayer. Due to intermolecular transfer of acyl radicals at a constancy of their composition, functional rearrangement of the lipid matrix appears to be achieved through changes in the membrane viscosity. Our data support the idea that different adaptation strategies in fish are driven by certain sets of PL molecular species.     

Effect of GRP75/mthsp70/PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells

Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonged to the heat shock protein (HSP) family. To evaluate the effect of GRP75 overexpression on PC12 cells under glucose deprivation, cell viability and mitochondrial function of GRP75-overexpressing PC12 cells and the vector transfected control PC12 cells were monitored during glucose deprivation. Upon exposure to glucose deprivation, GRP75-overexpressing PC12 cells exhibited more moderate cell damage than control PC12 cells. Both of the two groups of cells showed a decreased ATP level following an early increase in the condition of glucose deprivation, and the mitochondrial potential were also reduced in the similar manner in the two groups of cells. Control PC12 cells showed an immediate and rapid increase in ROS accumulation after the onset of GD treatment, and this accumulation was slowed and reduced in GRP75-overexpressing PC12 cells. These findings suggested that GRP75 could inhibit the ROS accumulation, and it may be associated with the cytoprotective effect of GRP75 overexpression upon glucose deprivation. (Mol Cell Biochem 268: 45–51, 2005)     

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N₂-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

ESI–MS and matrix-assisted laser desorption ionization–mass spectrometry imaging reveal alterations in Medicago truncatula nodules membrane lipid composition and spatial distribution in phosphorus deficiency.     

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.     

Intracellular oxygen tension and energy metabolism in the cat brain cortex during haemorrhagic shock.

The changes in intracellular oxygen tension and energy metabolism of the cat brain cortex were studied by surface fluororeflectometry during haemorrhagic shock. The results may be summarized as follows. (a) Intracellular oxygen tension, i.e. the maximum cortical NAD reduction obtained during nitrogen gas inhalation decreased gradually during the hypovolaemic phase of shock and finally, the brain cortex became ischaemic. (b) Partial uncoupling of the cerebrocortical mitochondrial respiration and oxidative phosphorylation appeared in the very early period of bleeding, as indicated by the overshot of the cortical NAD/NADH redox state towards oxidation subsequent to the cessation of nitrogen gas inhalation. Partial uncoupling of mitochondrial respiration and oxidative phosphorylation became more pronounced during the later phases of bleeding, finally, the mitochondrial electron transport stopped. In line with these changes the frequency and the amplitude of ECoG decreased gradually and markedly during the hypovolaemic phase of shock. (c) Microcirculation and energy metabolism of the cat brain cortex were severely and irreversibly damaged during the hypovolaemic phase of shock. This was clearly shown by the fact that in the majority of experiments the nitrogen anoxia after reinfusion failed to bring about changes in the cortical NAD/NADH redox state and the ECoG changes occurred during bleeding did not improve after reinfusion. It is concluded that the early disturbances of cerebrocortical energy metabolism play an important role in the development of neural and vascular lesions of the brain that occur during haemorrhagic shock.     

The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

Pulmonary surfactant is a lipoprotein complex essential for lung function, and insufficiency or altered surfactant composition is associated with major lung diseases, such as acute respiratory distress syndromes, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Pulmonary surfactant is primarily composed of phosphatidylcholine (PC) in complex with specialized surfactant proteins and secreted by alveolar type 2 (AT2) cells. Surfactant homeostasis on the alveolar surface is balanced by the rates of synthesis and secretion with reuptake and recycling by AT2 cells, with some degradation by pulmonary macrophages and loss up the bronchial tree. However, whether phospholipid (PL) transporters exist in AT2 cells to mediate reuptake of surfactant PL remains to be identified. Here, we demonstrate that major facilitator superfamily domain containing 2a (Mfsd2a), a sodium-dependent lysophosphatidylcholine (LPC) transporter, is expressed at the apical surface of AT2 cells. A mouse model with inducible AT2 cell–specific deficiency of Mfsd2a exhibited AT2 cell hypertrophy with reduced total surfactant PL levels because of reductions in the most abundant surfactants, PC containing dipalmitic acid, and PC species containing the omega-3 fatty acid docosahexaenoic acid. These changes in surfactant levels and composition were mirrored by similar changes in the AT2 cell lipidome. Mechanistically, direct tracheal instillation of fluorescent LPC and PC probes indicated that Mfsd2a mediates the uptake of LPC generated by pulmonary phospholipase activity in the alveolar space. These studies reveal that Mfsd2a-mediated LPC uptake is quantitatively important in maintaining surfactant homeostasis and identify this lipid transporter as a physiological component of surfactant recycling.     

Dipalmitoylphosphatidylcholine is not the major surfactant phospholipid species in all mammals

Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0/14:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal.     

Differential effects of n-3 fatty acid deficiency on phospholipid molecular species composition in the rat hippocampus

In this study, we have examined the effects of n-3 fatty acid deficient diets on the phospholipids (PL) molecular species composition in the hippocampus. Female rats were raised for two generations on diets containing linoleic acid (18:2n-6), with or without supplementation of alpha-linolenic acid (18:3n-3) or 18:3n-3 plus docosahexaenoic acid (22:6n-3). At 84 days of age, the hippocampal phospholipids were analyzed by reversed phase HPLC-electrospray ionization mass spectrometry. Depleting n-3 fatty acids from the diet led to a reduction of 22:6n-3 molecular species in phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-plasmalogens (PLE), and phosphatidylserine (PS) by 70-80%. In general, 22:6n-3 was replaced with 22:5n-6 but the replacement at the molecular species level did not always occur in a reciprocal manner, especially in PC and PLE. In PC, the 16:0,22:6n-3 species was replaced by 16:0,22:5n-6 and 18:0,22:5n-6. In PLE, substantial increases of both 22:5n-6 and 22:4n-6 species compensated for the decreases in 22:6n-3 species in n-3 fatty acid deficient groups. While the total PL content was not affected by n-3 deficiency, the relative distribution of PS decreased by 28% with a concomitant increase in PC.The observed decrease of 22:6n-3 species along with PS reduction may represent key biochemical changes underlying losses in brain-hippocampal function associated with n-3 deficiency.     

The value of immunohistochemical research on PCNA, p53 and heat shock proteins in prostate cancer management: a review

This review addresses the significance of the expression of proliferating cell nuclear antigen (PCNA), p53 and some heat shock proteins (Hsps) in prostate carcinoma (PC). In fact, PCNA and p53 are two widely discussed tools in PC diagnosis, mainly because of the controversy regarding the significance of their expression during prostate cancer development and progression. At the same time, only few studies have shown the potential role of Hsps in carcinogenesis and their overexpression in pre-neoplastic and neoplastic lesions of the prostate. We briefly describe the physiological roles of Hsps in normal cells, and the significance of their immunohistochemical detection in PC as well as in pre-cancerous lesions of the prostate. We will also discuss the possible functional interactions of these molecules in both dysplastic and neoplastic cells.     

Influence of Saturated and Polyunsaturated Egg Yolk Phospholipids on Hyperlipidemia in Rats

The supplementation of egg yolk phospholipid (PL) containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to a cholesterol-free purified diet causes a reduction in the serum cholesterol level in rats [J. Nutr., 112,1805 (1982)]. The present study was carried out to determine if dietary egg yolk PL also exerts this hypocholesterolemic action in rats given a high cholesterol diet and if this action is influenced by the constituent fatty acids. Egg yolk PL suppressed the elevation of serum cholesterol irrespective of its fatty acid composition, while purified PC had no effect, suggesting that the ethanolamine portion is responsible for this hypocholesterolemic effect. Egg yolk PL and PC containing longer-chain polyunsaturated fatty acids (arachidonic and docosahexaenoic acids) lowered the serum triglyceride level, while their hydrogenated forms did not. The present results, therefore, indicate that the hypolidemic effect of dietary egg yolk PL can be modulated by the combination of the constituent fatty acids as well as the base moieties. This hypolipidemic effect, however, appeared not to be related to the activities of adipocyte lipoprotein lipase and serum lecithin: cholesterol acyltransferase.     

Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry--a cautionary note

In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution.     

Lipid composition of the main fractions of rabbit semen

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 microg/10(9) but was only 21-23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp(p)) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE+PC ratios than in the germinal cells. Some physiological implications are discussed.     

Postnatal development of phospholipids and their fatty acid profile in rat heart

The aim of this study was to determine the concentration of phospholipids (PL), plasmalogen components of choline (PC) and ethanolamine (PE) phosphoglycerides (PLPC, PLPE) and fatty acid profile of PL and triacylglycerols (TAG) in developing rat left ventricular myocardium between postnatal day (d) 2 and 100. The steepest increase of total PL (TPL) concentration occurs between d2 and d5, followed by a further slower increase between d20 and d40. Similar developmental changes were observed in PC and PE. The PLPE concentration rises by d10, whereas PLPC does not change during the whole period investigated, except for the transient decline on d5. The concentration of diphosphatidylglycerol (DPG) increases by d60; the steepest rise occurs between d20 and d40. Phosphatidylinositol (PI) concentration rises only by d5. The concentration of phosphatidylserine (PS) decreases between d5 and d10 and then it does not change. Sphingomyelin (SM) concentration is maintained till d10, it declines on d20 and does not change thereafter. The proportion of saturated fatty acids (SFA) increases by d5 in PC, PE, PS and TAG, and by d10 in DPG and PI. After d20 the SFA proportion gradually decline in all lipids. Monounsaturated FA (MUFA) proportion decreases in PC, PE, PI and PS from d2 till d10, and in the weaning period it tends to rise again. In contrast, in DPG and TAG the proportion of MUFA declines during the whole postnatal period. N-6 polyunsaturated FA (PUFA) decrease in all PL by d20 and rise again thereafter; in TAG they decline between d2 and d10 and return to the initial level by d100. N-3 PUFA increase in all PL during the suckling period and decline after weaning; in TAG they increase only by d5 and then they decline. This remodeling of myocardial PL and TAG composition during postnatal development may affect membrane properties and contribute to developmental changes in the function of membrane proteins and cell signaling.     

Deleterious action of FA metabolites on ATP synthesis: possible link between lipotoxicity, mitochondrial dysfunction, and insulin resistance

Insulin resistance is a characteristic feature of type 2 diabetes and obesity. Insulin-resistant individuals manifest multiple disturbances in free fatty acid (FFA) metabolism and have excessive lipid accumulation in insulin target tissues. Although much evidence supports a causal role for altered FFA metabolism in the development of insulin resistance, i.e., "lipotoxicity", the intracellular mechanisms by which elevated plasma FFA levels cause insulin resistance have yet to be completely elucidated. Recent studies have implicated a possible role for mitochondrial dysfunction in the pathogenesis of insulin resistance in skeletal muscle. We examined the effect of FFA metabolites [palmitoyl carnitine (PC), palmitoyl-coenzyme A (CoA), and oleoyl-CoA] on ATP synthesis in mitochondria isolated from mouse and human skeletal muscle. At concentrations ranging from 0.5 to 2 microM, these FFA metabolites stimulated ATP synthesis; however, above 5 microM, there was a dose-response inhibition of ATP synthesis. Furthermore, 10 microM PC inhibits ATP synthesis from pyruvate. Elevated PC concentrations (> or =10 microM) inhibit electron transport chain activity and decrease the mitochondrial inner membrane potential. These acquired mitochondrial defects, caused by a physiological increase in the concentration of FFA metabolites, provide a mechanistic link between lipotoxicity, mitochondrial dysfunction, and muscle insulin resistance.