Branched chain fatty acid synthesis drives tissue-specific innate immune response and infection dynamics of Staphylococcus aureus.

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.

Lipid Profile, Lipoprotein Subfractions, and Fluidity of Membranes in Children and Adolescents with Depressive Disorder: Effect of Omega-3 Fatty Acids in a Double-Blind Randomized Controlled Study.

Depressive disorder (DD) is a psychiatric disorder whose molecular basis is not fully understood. It is assumed that reduced consumption of fish and omega-3 fatty acids (FA) is associated with DD. Other lipids such as total cholesterol (TCH), LDL-, and HDL-cholesterols (LDL-CH, HDL-CH) also play a role in depression. The primary endpoint of the study was the effect of omega-3 FA on the severity of depression in children and adolescents. This study aimed to investigate the secondary endpoint, relationship between depressive disorder symptoms and lipid profile, LDL- and HDL-cholesterol subfractions, Paraoxonase 1 (PON1) activities, and erythrocyte membrane fluidity in 58 depressed children and adolescents (calculated by the statistical program on the effect size), as well as the effect of omega-3 FA on the monitored parameters. Depressive symptoms were assessed by the Children's Depression Inventory (CDI), lipid profile by standard biochemical procedures, and LDL- and HDL-subfractions by the Lipoprint system. Basic biochemical parameters including lipid profile were compared with levels in 20 healthy children and were in the physiological range. Improvement of symptoms in the group supplemented with a fish oil emulsion rich in omega-3 FA in contrast to omega-6 FA (emulsion of sunflower oil) has been observed. We are the first to report that omega-3 FAs, but not omega-6 FA, increase large HDL subfractions (anti-atherogenic) after 12 weeks of supplementation and decrease small HDL subfractions (proatherogenic) in depressed children. We found a negative correlation between CDI score and HDL-CH and the large HDL subfraction, but not LDL-CH subfractions. CDI score was not associated with erythrocyte membrane fluidity. Our results suggest that HDL-CH and its subfractions, but not LDL-CH may play a role in the pathophysiology of depressive disorder. The study was registered under ISRCTN81655012.

Partitioning of caffeine in lipid bilayers reduces membrane fluidity and increases membrane thickness.

Caffeine is a small amphiphilic molecule, which is widely consumed as a stimulant to prevent fatigue, but is also used as a common drug adjuvant in modern medicine. Here, we show that caffeine interacts with unsaturated lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By combining X-ray diffraction and molecular dynamics simulations, we present evidence that caffeine partitions in lipid membranes and locates at the head group-tail group interface of the bilayers. By attracting water molecules from neighboring lipid molecules, it leads to the formation of "water pockets", i.e., a local increase of water density at this interface. Through this mechanism, caffeine leads to an overall decrease of the gauche defect density in the membranes and an increase of membrane thickness, indicating a loss of membrane fluidity. These non-specific membrane interactions may increase the efficacy of analgesic drugs through changes in the bioavailability and rate of metabolism of these drugs.

Membrane fluidity does not explain how solvents act on the middle-ear reflex.

Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesia's depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.

A Model of Electrostimulation Based on the Membrane Capacitance as Electromechanical Transducer for Pore Gating.

BACKGROUND: Electrostimulation has gained enormous importance in modern medicine, for example, in implantable pacemakers and defibrillators, pain stimulators, and cochlear implants. Most electrostimulation macromodels use the electrical current as the primary parameter to describe the conventional strength-duration relationship of the output of a generator. These models normally assume that the stimulation pulse charges up the passive cell membrane capacitance, and then the increased (less-negative) transmembrane potential activates voltage-gated sodium channels. However, this model has mechanistic and accuracy limitations. NOVEL CONCEPT: Our model assumes that the membrane capacitance is an electromechanical transducer and that the membrane is compressed by the endogenous electric field. The pressure is quadratically correlated with the transmembrane voltage. If the pressure is reduced by an exogenous field, the compression is released and, thus, opening the pores for Na(+) influx initiates excitation. RESULTS: The exogenous electric field must always be equal to or greater than the rheobase field strength (rheobase condition). This concept yields a final result that the voltage-pulse-content produced by the exogenous field between the two ends of a cell is a linear function of the pulse duration at threshold level. Thus, the model yields mathematical formulations that can describe and explain the characteristic features of electrostimulation. CONCLUSIONS: Our model of electrostimulation can describe and explain electrostimulation at cellular level. The model's predictions are consistent with published experimental studies. Practical applications in cardiology are discussed in the light of this model of electrostimulation.

Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5).

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential.

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.

Inhibition of phospholipase A2 in rat brain modifies different membrane fluidity parameters in opposite ways.

Fluidity is an important neuronal membrane property and it is influenced by the concentration of polyunsaturated fatty acids (PUFAs) in membrane phospholipids. Phospholipase A(2) (PLA(2)) is a key enzyme in membrane phospholipid metabolism, generating free PUFAs. In Alzheimer disease (AD), reduced PLA(2) activity, specifically of calcium-dependent cytosolic PLA(2) (cPLA(2)) and calcium-independent intracellular PLA(2) (iPLA(2)), and phospholipid metabolism was reported in the frontal cortex and hippocampus. This study investigated the effects of in vivo infusion of the dual cPLA(2) and iPLA(2) inhibitor MAFP into rat brain on PLA(2) activity and membrane fluidity parameters in the postmortem frontal cortex and dorsal hippocampus. PLA(2) activity was measured by radioenzymatic assay and membrane fluidity was determined by fluorescence anisotropy technique using three different probes: DPH, TMA-DPH, and pyrene. MAFP significantly inhibited PLA(2) activity, reduced the flexibility of fatty acyl chains (indicated by increased DPH anisotropy), increased the fluidity in the lipid-water interface (indicated by decreased TMA-DPH anisotropy), and increased the lipid lateral diffusion in the hydrocarbon core (represented by pyrene excimer formation) of membranes in both brain areas. The findings suggest that reduced cPLA(2) and iPLA(2) activities in AD brain might contribute to the cognitive impairment, in part, through alterations in membrane fluidity parameters.

Relationship between barotolerance and fatty acids constitution of brain cell membrane.

OBJECTIVE: It is suggested that one of the mechanisms for high pressure nervous syndrome (HPNS) is related to nervous cell membranous fluidity. Both pressure and fatty acid components of cell membranes would influence membrane fluidity. The present research probed into the relationship between different fatty acid components of brain cell membrane and individuals' degree of HPNS. METHODS: Four groups of mice were compressed to 4.1 MPa with an He-O2 mixture over a period of two hours. These animals had been fed with different diets for a period of months prior to the procedure. We recorded interpeak latency of Wave 1 to Wave 4 (IPL1-4) of brainstem auditory-evoked potential (BAEP) at different stages of compression. Animals were sacrificed immediately after surfacing. Both polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) of brain cell membranes were analyzed by HPLC. RESULTS: Upon arriving at 4 MPa, the IPL1-4 readings of the four groups were prolonged 0.294 +/- 0.400 milliseconds (ms), 0.156 +/- 0.200 ms, 0.009 +/- 0.182 ms and 0.025 +/- 0.137 ms separately; each corresponded to its own PUFA-percent constitution of 16.2 +/- 4.5%, 24.8 +/- 4.3%, 33.5 +/- 8.8% and 32.3 +/- 2.9% respectively on the basis of total fatty acids. DISCUSSION AND CONCLUSION: Varying fractions of PUFAs, implying different membrane fluidity, interfered with disturbance of synaptic transmission during hyperbaric exposure. In other words, the higher the ratio of PUFAs/SFAs to the brain cell membrane, the stronger the ability for animals to antagonize the pressure effect.

Arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats.

Previous studies indicate that long-term dietary supplementation with arachidonic acid (AA) in 20-month-old rats (OA) effectively restores performance in a memory task and the induction of long-term potentiation in the hippocampus to the level of young control animals (YC). The present study examined protein mobility using the live cell imaging technique "Fluorescent Recovery After Photobleaching (FRAP)" in YC, old control (OC) and OA neurons in hippocampal slice preparations. Three measures; mobile fraction (M(f)), diffusion constant (D) and time constant (tau), were estimated among YC, OC and OA. Each of these parameters was significantly different between OC and YC, suggesting that membrane fluidity is lower in OC than in YC. In contrast, D and tau were comparable in OA and YC, indicating that hippocampal neuronal membranes supplemented with AA were more fluid than those in OC, whereas the fraction of diffusible protein in the bleached region remained smaller than in YC. Long-term administration of AA to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity.

The effects of dietary N-3 and antioxidant supplementation on erythrocyte membrane fatty acid composition and fluidity in exercising horses.

REASONS FOR PERFORMING STUDY: Fatty acid supplementation could modulate erythrocyte membrane fluidity in horses at rest and during exercise, but information is lacking on the effect of exercise. OBJECTIVES: To assess the effect of exercise with, and without, an oral antioxidant supplementation enriched with n-3 fatty acids on erythrocyte membrane fluidity (EMF) and fatty acid composition in eventing horses. METHODS: Twelve healthy and regularly trained horses were divided randomly into 2 groups: group S received an oral antioxidant cocktail enriched in n-3 fatty acid (alphatocopherol, eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) whereas group P was placebo-treated. At the end of 4 weeks, all horses performed a standardised exercise test (ET) under field conditions. Venous blood was sampled before starting treatment (TO), immediately before (T1) as well as 15 min (T2) and 24 h (T3) after ET. Spin labelled (16-DOXYL-stearic acid) red blood cell membranes were characterised using the relaxation correlation time (Tc in inverse proportion to EMF). Fatty acid composition (%) of the membrane was determined by gas-liquid chromatography. RESULTS: Supplementation did not induce changes in EMF (T1 vs. TO) but significant changes in membrane composition were observed and there were increases in n-3 polyunsaturated fatty acid PUFA, n-3/n-6 ratio, and total n-3 fatty acids. Exercise (T2 vs. T1) induced a significant decrease of EMF in group P (Tc: +19%, P<0.05) and nonsignificant decrease in group S (Tc: +5%), whereas membrane fatty acid composition did not change in either group. During the recovery period (T3 vs. T2), EMF decreased significantly in group S (Tc: +29%, P<0.05) and nonsignificantly in group P (Tc: +18%) without any significant changes in fatty acid composition. CONCLUSION AND POTENTIAL RELEVANCE: An enriched oral antioxidant supplementation induced changes in membrane composition, which modulated the decrease in EMF induced by exercise. Long chain n-3 fatty acid supplementation might therefore be beneficial.

Omega-3 fatty acid treatment and T(2) whole brain relaxation times in bipolar disorder.

OBJECTIVE: The authors hypothesized that changes in brain membrane composition resulting from omega-3 fatty acid administration in patients with bipolar disorder would result in greater membrane fluidity, as detected by reductions in T(2) values. METHOD: Women with bipolar disorder (N=12) received omega-3 fatty acids for 4 weeks. A cohort of bipolar subjects (N=9) and a group without bipolar disorder (N=12) did not receive omega-3 fatty acids. T(2) values were acquired at baseline and after 4 weeks. RESULTS: Bipolar subjects who received omega-3 fatty acids had significant decreases in T(2). There was a dose-dependent effect when the bipolar omega-3 fatty acid group was subdivided into high- and low-dose cohorts. CONCLUSIONS: Omega-3 fatty acids lowered T(2) values, consistent with the hypothesis that the fluidity of cell membranes was altered. Further studies are needed to clarify the significance of alterations in brain physiology induced by omega-3 fatty acids, as reflected in T(2) values.

The effect of Pycnogenol on the erythrocyte membrane fluidity.

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.

[Effects of spirulina on serum lipids, erythrocyte membrane fluidity and vascular endothelial cells in tail-suspended rats].

OBJECTIVE: To study the changes of erythrocyte membrane fluidity, serum lipid and vascular endothelial cell caused by simulated weightlessness in rats and the beneficial effect of spirulina. METHOD: Thirty male SD rats were divided into 3 groups: free control group (group A) and two simulated weightlessness groups (groups B, C). Rats in group A and B were fed with normal forage, and the rats in group C were fed with normal forage supplemented with 5% (W/W) spirulina. Water was taken ad libitum. RESULT: Levels of serum CHO, HDL, TG, HDL-C/CHO and erythrocyte membrane fluidity decreased significantly, and number of vascular endothelial cells in plasma increased markedly in group B as compared with those in group A; The ratio of LDL-C/HDL-C, and atherosclerosis index (AI) decreased, number of vascular endothelial cells significantly lowered; level of CHO, HDL-C and value of the IDmax of plasma as well as erythrocyte membrane fluidity remarkedly increased in group C compared with those in group B. CONCLUSION: Spirulina can improve the physiological conditions of erythrocyte membrane fluidity, serum lipid and vascular endothelial cell caused by simulated weightlessness in rats.

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics.

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.

Lizards, lipids, and dietary links to animal function.

Our experiments were designed to test the hypotheses that dietary lipids can affect whole-animal physiological processes in a manner concordant with changes in the fluidity of cell membranes. We measured (1) the lipid composition of five tissues, (2) body temperatures selected in a thermal gradient (T(sel)), (3) the body temperature at which the righting reflex was lost (critical thermal minimal [CTMin]), and (4) resting metabolic rate (RMR) at three body temperatures in desert iguanas (Dipsosaurus dorsalis) fed diets enriched with either saturated or unsaturated fatty acids. The composition of lipids in tissues of the lizards generally reflected the lipids in their diets, but the particular classes and ratios of fatty acids varied among sampled organs, indicating the conservative nature of some tissues (e.g., brain) relative to others (e.g., depot fat). Lizards fed the diet enriched with saturated fatty acids selected warmer nighttime body temperatures than did lizards fed a diet enriched with unsaturated fatty acids. This difference is concordant with the hypothesis that the composition of dietary fats influences membrane fluidity and that ectotherms may compensate for such changes in fluidity by selecting different body temperatures. The CTMin of the two treatment groups was indistinguishable. This may reflect the conservatism of some tissues (e.g., brain) irrespective of diet treatment. The RMR of the saturated treatment group nearly doubled between 30 degrees and 40 degrees C. Here, some discrete membrane domains in the lizards fed the saturated diet may have been in a more-ordered phase at 30 degrees C and then transformed to a less-ordered phase at 40 degrees C. In contrast, the RMR of the unsaturated treatment group exhibited temperature independence in metabolic rate from 30 degrees to 40 degrees C. Perhaps the unsaturated diet resulted in membranes that developed a higher degree of disorder (i.e., a certain phase) at a lower temperature than were membranes of lizards fed the saturated diet. Our study demonstrates links between dietary fats and whole-animal physiology; however, the mechanistic basis of these links, and the general knowledge of lipid metabolism in squamate reptiles, remain poorly understood and warrant further study.

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency.

BACKGROUND: Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. METHODS: K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. RESULTS: K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V(max) of brush-border membrane (BBM) Na/Pi cotransport activity (1943 +/- 95 in control vs. 1184 +/- 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 1152 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. CONCLUSION: K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Membrane fluidity and fatty acid metabolism in kidney cells from rats fed purified eicosapentaenoic acid or purified docosahexaenoic acid.

Rats were given a supplement (1.5 ml/day) of purified eicosapentaenoic acid (EPA, 20:5,n-3), purified docosahexaenoic acid (DHA, 22:6,n-3)), or corn oil for 10 days. Membrane fluidity, measured as the steady-state fluorescence polarization of diphenylhexatriene (DPH), was approximately 20% lower in kidney cells from rats fed purified EPA than in cells from the DHA-fed or corn-oil fed animals. The level of 20:5(n-3) in kidney phospholipids was 18 times higher in rats fed EPA, and four times higher in those fed DHA as compared to the corn-oil group. The level of arachidonic acid (20:4,n-6) was concomitantly decreased, while linoleic acid (18:2,n-6) was increased in kidney-phospholipids in the n-3 fatty acid fed rats. The proportion of 22:6(n-3) in kidney phospholipids was not affected by EPA supplementation, while the DHA diet slightly increased the level of this fatty acid. The distribution of phospholipid subclasses was significantly altered in that phosphatidylcholine was increased and phosphatidylethanolamine was concomitantly decreased. It is suggested that the decrease in 20:4(n-6) is relatively more important in the regulation of fluidity than a concomitant increase in 20:5(n-3). It is also suggested that the compensatory modifications of the phospholipid subclass distribution as a response to decreased 20:4(n-6)/20:5(n-3) ratio was not sufficient to maintain fluidity when the ratio was as low as in the present study. The incorporation of labelled linolenic acid (18:3,n-3) in phospholipids was decreased in cells from the n-3 supplemented rats. Since endogenous 22:5(n-3) in phospholipids was only increased in the EPA group, 22:6(n-3) only in the DHA group, and 20:5(n-3) in both, it is suggested that the decreased incorporation of labelled 18:3(n-3) into phospholipids of the DHA-fed rats in particular is correlated to the increased level of 22:6(n-3) in the membrane phospholipids. The incorporation of fatty acids into phopholipids may thus show substrate specificity, in that 22:6(n-3) is less exchangable with labelled 18:3(n-3) than is 20:5(n-3). These results demonstrate that increasing levels of n-3 fatty acids in membranes affect the uptake and intracellular metabolism of fatty acids as well as membrane fluidity in the kidney.

Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes.

The fluorescent probe diI was used to study the lateral mobility of lipids in in vitro strains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 microM all-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement of D among the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lower D values. The other FRAP parameters (mobile fraction and bleach depth) were larger in the "slow" group than in the "fast" group. This trend of negative correlation between these parameters and D was also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.