Tau Is Truncated in Five Regions of the Normal Adult Human Brain.

The truncation of Tau is thought to be important in promoting aggregation, with this feature characterising the pathology of dementias such as Alzheimer disease. Antibodies to the C-terminal and N-terminal regions of Tau were employed to examine Tau cleavage in five human brain regions: the entorhinal cortex, prefrontal cortex, motor cortex, hippocampus, and cerebellum. These were obtained from normal subjects ranging in age from 18 to 104 years. Tau fragments of approximately 40 kDa and 45 kDa with an intact N-terminus retained were found in soluble and insoluble brain fractions. In addition, smaller C-terminal Tau fragments ranging in mass from 17 kDa to 25 kDa were also detected. These findings are consistent with significant Tau cleavage taking place in brain regions from 18 years onwards. It appears that site-specific cleavage of Tau is widespread in the normal human brain, and that large Tau fragments that contain the N-terminus, as well as shorter C-terminal Tau fragments, are present in brain cells across the age range.

Honey bee caste lipidomics in relation to life-history stage and the long life of the queen.

Honey bees have evolved a system in which fertilised eggs transit through the same developmental stages but can become either workers or queens. This difference is determined by their diet through development. Whereas workers live for weeks (normally 2-6 weeks), queens can live for years. Unfertilised eggs also develop through the same stages but result in a short-lived male caste (drones). Workers and drones are fed pollen throughout their late larval and adult life stages, while queens are fed exclusively on royal jelly and do not eat pollen. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has a negligible amount of PUFA. To investigate the role of dietary PUFA lipids and their oxidation in the longevity difference of honey bees, membrane fatty acid composition of the three castes was characterised at six different life-history stages (larva, pupa, emergent and different adult stages) through mass spectrometry. All castes were found to share a similar membrane phospholipid composition during early larval development. However, at pupation, drones and workers increased their level of PUFA, whilst queens increased their level of monounsaturated fatty acids. After emergence, worker bees further increased their level of PUFA by 5-fold across most phospholipid classes. In contrast, the membrane phospholipids of adult queens remained highly monounsaturated throughout their adult life. We postulate that this diet-induced increase in membrane PUFA results in more oxidative damage and is potentially responsible for the much shorter lifespan of worker bees compared with long-lived queens.

An antioxidant-like action for non-peroxidisable phospholipids using ferrous iron as a peroxidation initiator.

The degradation of phospholipids containing polyunsaturated fatty acids, termed peroxidation, poses a constant challenge to membranes lipid composition and function. Phospholipids with saturated (e.g. PC 16:0/16:0) and monounsaturated fatty acids (e.g. PC 16:0/18:1) are some of the most common phospholipids found in membranes and are generally not peroxidisable. The present experiments show that these non-peroxidisable phospholipids, when present in liposomes with peroxidisable phospholipids (i.e. those containing polyunsaturated fatty acids) such as PC 16:0/18:2 and Soy PC, produce an inhibitory effect on rates of peroxidation induced by ferrous-iron. This inhibitory effect acts to extend the duration of the lag phase by several-fold. If present in natural systems, this action could enhance the capacity of conventional antioxidant mechanisms in membranes. The results of this preliminary work suggest that non-peroxidisable phospholipids may exert an antioxidant-like action in membranes.

Docosahexaenoic and arachidonic acid peroxidation: It's a within molecule cascade.

Peroxidation is a well-known natural phenomenon associated with both health and disease. We compared the peroxidation kinetics of phosphatidylcholine (PC) molecules with different fatty acid compositions (i.e. 18:0, 18:1n-9, 18:2n-6, 20:4n-6 and 22:6n-3 at the sn-2 and 16:0 at sn-1 position) either as molecules free in solution or formed into liposomes. Fatty acid levels, oxygen consumption plus lipid hydroperoxide and malondialdehyde production were measured from the same incubations, at the same time during maximal elicitable peroxidation. PCs with highly peroxidizable fatty acids (i.e. 20:4n-6 and 22:6n-3) in the same incubation were found to be either fully peroxidized or intact. Rates of peroxidation of PCs with multiple bisallylic groups (i.e. 20:4n-6 and 22:6n-3) peroxidized at 2-3 times the rate per bisallylic bond than the same phospholipid with 18:2n-6. The results suggest that propagation of peroxidation (H-atom transfer) is firstly an intramolecular process that is several-fold faster than intermolecular peroxidation. PCs in solution peroxidized twice as fast as those in liposomes suggesting that only half of the phospholipids in liposomes were available to peroxidize i.e. the outer leaflet. Experiments on liposomes suggest that even after heavy peroxidation of the outer leaflet the inner leaflet is unaffected, indicating how cells may protect themselves from external peroxidation and maintain control over internal peroxidation. Intramolecular peroxidation may produce highly concentrated, localized sites of peroxidation product that together with internal control of peroxidation of the inner leaflet of membranes provide new insights into how cells control peroxidation at the membrane level.

The thermal dependence of Na+ flux in isolated liver cells from ectotherms and endotherms.

The thermal dependence (0-40°C) of Na(+) flux in isolated liver cells of three endotherms (mice, rat and rabbit) was compared with that of ectotherms in the form of a thermally tolerant amphibian (cane toad), a cold-water fish (rainbow trout) and a thermophilic reptile (lizard). Mammals were found to share similar high rates of Na(+) flux (3.0-3.7 nmol Na(+) mg(-1) protein min(-1)) at their normal body temperatures (36-39°C). These Na(+) flux rates were significantly greater (P<0.0004-0.0001) than those of the ectotherms, which shared similar low rates of Na(+) flux (0.7-1.3 nmol Na(+) mg(-1) protein min(-1)) at their very different normal acclimated body temperatures (15°C for trout, 25°C for toad and 37°C for the lizard species). Trout, which possess highly unsaturated membranes (similar to those of mammals), showed a Na(+) flux with high thermal sensitivity at low temperatures similar to that found in mammals at higher temperatures. The thermal sensitivity of toad Na(+) flux was significantly less (P<0.05-0.01) than that of rat and rabbit. Trout Na(+) flux did not increase with increasing temperature much above 20°C, whereas all other species measured increased their Na(+) flux with increasing temperature up to 40°C. In conclusion, at normal operating body temperatures, the rate of Na(+) flux is much lower in ectotherms.

Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

Dinosaur lactation?

Lactation is a process associated with mammals, yet a number of birds feed their newly hatched young on secretions analogous to the milk of mammals. These secretions are produced from various sections (crop organ, oesophageal lining and proventriculus) of the upper digestive tract and possess similar levels of fat and protein, as well as added carotenoids, antibodies and, in the case of pigeons and doves, epidermal growth factor. Parental care in avian species has been proposed to originate from dinosaurs. This study examines the possibility that some dinosaurs used secretory feeding to increase the rate of growth of their young, estimated to be similar to that of present day birds and mammals. Dinosaur 'lactation' could also have facilitated immune responses as well as extending parental protection as a result of feeding newly hatched young in nest environments. While the arguments for dinosaur lactation are somewhat generic, a case study for lactation in herbivorous site-nesting dinosaurs is presented. It is proposes that secretory feeding could have been used to bridge the gap between hatching and establishment of the normal diet in some dinosaurs.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease.

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.

Dietary docosahexaenoic Acid (22:6) incorporates into cardiolipin at the expense of linoleic Acid (18:2): analysis and potential implications.

Cardiolipin is a signature phospholipid of major functional significance in mitochondria. In heart mitochondria the fatty acid composition of cardiolipin is commonly viewed as highly regulated due to its high levels of linoleic acid (18:2n - 6) and the dominant presence of a 4×18:2 molecular species. However, analysis of data from a comprehensive compilation of studies reporting changes in fatty acid composition of cardiolipin in heart and liver mitochondria in response to dietary fat shows that, in heart the accrual of 18:2 into cardiolipin conforms strongly to its dietary availability at up to 20% of total dietary fatty acid and thereafter is regulated. In liver, no dietary conformer trend is apparent for 18:2 with regulated lower levels across the dietary range for 18:2. When 18:2 and docosahexaenoic acid (22:6n - 3) are present in the same diet, 22:6 is incorporated into cardiolipin of heart and liver at the expense of 18:2 when 22:6 is up to ~20% and 10% of total dietary fatty acid respectively. Changes in fatty acid composition in response to dietary fat are also compared for the two other main mitochondrial phospholipids, phosphatidylcholine and phosphatidylethanolamine, and the potential consequences of replacement of 18:2 with 22:6 in cardiolipin are discussed.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole−linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18−104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Mammals to membranes: A reductionist story.

This is the story of a series of reductionist studies that started with an attempt to explain what underpins the high-level of aerobic metabolism in mammals (i.e. associated with the evolution of endothermy) and almost forty years later had led to investigations into the role of membrane lipids in determining metabolism. Initial studies showed that the increase in aerobic metabolism in mammals was driven by a combination of increases in mitochondrial volume and membrane densities, organ size and changes in the molecular activity of enzymes. The increase in the capacity to produce energy was matched by an increase in energy use, notably driven by increases in H+, Na+ and K+ fluxes. In the case of increased Na+ flux, it was found this was matched by increases in Na+-dependent metabolism at the tissue level and increases in enzyme activity at a cellular level but not by an increase in the number of sodium pumps. To maintain Na+ gradient across cell membranes, increased Na+ flux is not controlled by an increase in sodium pump number but rather by an increase in sodium pump molecular activity (i.e. an increase the substrate turnover rate of each sodium pump) in tissues of endotherms. This increase in molecular activity is coupled to an increase in the level of highly unsaturated polyunsaturated fatty acids (PUFA) in membranes, a mechanism similar to that used by ectotherms to ameliorate decreasing activities of metabolic processes in the cold. Determination of how changes in membrane fatty acid composition can change the activities of proteins in membranes will be the next step in this story.

Fingertip Whole Blood as an Indicator of Omega-3 Long-Chain Polyunsaturated Fatty Acid Changes during Dose-Response Supplementation in Women: Comparison with Plasma and Erythrocyte Fatty Acids.

The sensitivity of fingertip whole blood to reflect habitual dietary and dose-dependent supplemental omega-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) intake in premenopausal women was compared to that of venous erythrocytes and plasma fatty acids. Samples were obtained from women in a randomised, double-blind, placebo-controlled trial in which premenopausal women (n = 53) were supplemented with DHA-rich tuna oil capsules and/or placebo (Sunola oil) capsules (6 capsules per day) for 8 weeks to achieve doses of either 0, 0.35, 0.7 or 1.05 g/day n-3 LCPUFA. All blood biomarkers were very similar in their ability to reflect dietary n-3 LCPUFA intake (r = 0.38-0.46 for EPA and DHA intake), and in their dose-dependent increases in n-3 LCPUFA levels after supplementation (R2 = 0.41-0.51 for dose effect on biomarker EPA and DHA levels (mol %)). Fingertip whole blood is an effective alternative to erythrocytes and plasma as a biomarker n-3 LCPUFA intake in premenopausal women.

Effect of liraglutide on neural and peripheral markers of metabolic function during antipsychotic treatment in rats.

BACKGROUND: Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist that prevents metabolic side effects of the antipsychotic drugs (APDs) olanzapine and clozapine through unknown mechanisms. AIM: This study aimed to investigate the effect of chronic APD and liraglutide co-treatment on key neural and peripheral metabolic signals, and acute liraglutide co-treatment on clozapine-induced hyperglycaemia. METHODS: In study 1, rats were administered olanzapine (2 mg/kg), clozapine (12 mg/kg), liraglutide (0.2 mg/kg), olanzapine + liraglutide co-treatment, clozapine + liraglutide co-treatment or vehicle for six weeks. Feeding efficiency was examined weekly. Examination of brain tissue (dorsal vagal complex (DVC) and mediobasal hypothalamus (MBH)), plasma metabolic hormones and peripheral (liver and kidney) cellular metabolism and oxidative stress was conducted. In study 2, rats were administered a single dose of clozapine (12 mg/kg), liraglutide (0.4 mg/kg), clozapine + liraglutide co-treatment or vehicle. Glucose tolerance and plasma hormone levels were assessed. RESULTS: Liraglutide co-treatment prevented the time-dependent increase in feeding efficiency caused by olanzapine, which plateaued by six weeks. There was no effect of chronic treatment on melanocortinergic, GABAergic, glutamatergic or endocannabionoid markers in the MBH or DVC. Peripheral hormones and cellular metabolic markers were unaltered by chronic APD treatment. Acute liraglutide co-treatment was unable to prevent clozapine-induced hyperglycaemia, but it did alter catecholamine levels. CONCLUSION: The unexpected lack of change to central and peripheral markers following chronic treatment, despite the presence of weight gain, may reflect adaptive mechanisms. Further studies examining alterations across different time points are required to continue to elucidate the mechanisms underlying the benefits of liraglutide on APD-induced metabolic side effects.

Cardiac glycosides ouabain and digoxin interfere with the regulation of glutamate transporter GLAST in astrocytes cultured from neonatal rat brain.

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na(+), K(+))-dependent ATPase (Na(+)/K(+)-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na(+)/K(+)-ATPase. We now report that Na(+)/K(+)-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 µM). Therefore, while we may not have established a direct link between GLAST regulation and Na(+)/K(+)-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic.

Membrane fatty acid composition of rat skeletal muscle is most responsive to the balance of dietary n-3 and n-6 PUFA.

The present study quantifies the relationships between diet fatty acid profile and fatty acid composition of rat skeletal muscle phospholipids. Young adult male Sprague-Dawley rats were fed, for 8 weeks, on one of twelve moderate-fat diets (25 % of total energy) differing only in fatty acid profile. SFA content ranged from 8-88 % of total fatty acids, MUFA 6-65 %, total PUFA 4-81 %, n-6 PUFA 3-70 % and n-3 PUFA 1-70 %. Diet PUFA included only essential fatty acids 18 : 2n-6 and 18 : 3n-3. The balance between n-3 and n-6 PUFA (PUFA balance) in the diet ranged from 1 : 99 to 86 : 14 % n-3 PUFA:n-6 PUFA. The slope of muscle phospholipid composition plotted against diet composition quantifies the response of muscle membrane composition to dietary fat (0, no response; 1, complete conformity with diet). The resulting slopes were 0.02 (SFA), 0.10 (PUFA), 0.11 (MUFA), 0.14 (n-3 PUFA) and 0.23 (n-6 PUFA). The response to PUFA balance was biphasic with a slope of 0.98 below 10 % diet PUFA balance and 0.16 above 10 %. Thus, low diet PUFA balance has greater influence on muscle composition than 18-carbon n-3 or n-6 PUFA individually. Equations provided may allow prediction of muscle composition for other diet studies. Diet PUFA balance dramatically affects muscle 20 : 4n-6 and 22 : 6n-3. This may have significant implications for some disease states in human subjects.

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet.

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of ~30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.

The highly unnatural fatty acid profile of cells in culture.

The fatty acid profile of cells in culture are unlike those of natural cells with twice the monounsaturated (MUFA) and half the polyunsaturated fatty acids (PUFA) level (Mol%). This is not due to cell lines primarily being derived from cancers but is due to limited access to lipid and an inability to make PUFA de novo as vertebrate cells. Classic culture methods use media with 10% serum (the only exogenous source of lipid). Fetal bovine serum (FBS), the serum of choice has a low level of lipid and cholesterol compared to other sera and at 10% of media provides 2-3% of the fatty acid and cholesterol, 1% of the PUFA and 0.3% of the essential fatty acid linoleic acid (18:2n-6) available to cells in the body. Since vertebrate cell lines cannot make PUFA they synthesise MUFA, offsetting their PUFA deficit and reducing their fatty acid diversity. Stem and primary cells in culture appear to be similarly affected, with a rapid loss of their natural fatty acid compositions. The unnatural lipid composition of cells in culture has substantial implications for examining natural stems cell in culture, and for investigations of cellular mechanisms using cell lines based on the pervasive influence of fats.

Postnatal development in the rat: Changes in Na+ flux, sodium pump molecular activity and membrane lipid composition.

The cellular mechanisms underpinning changes in metabolism during postnatal development in young mammals have not been extensively examined. This study examines changes in sodium pump capacity (Na+, K+-ATPase activity), number and molecular activity, as well as, Na+ flux, cholesterol level and fatty acid composition in a number of major organs during postnatal development in the rat. In liver, Na+ flux was highest (2.6 times) in the youngest rats (3-day old) and decreased with increasing age, whereas Na+, K+-ATPase activity increased with age (up to 9-28 days) in liver, kidney and brain, but not in heart. Increases in Na+, K+-ATPase activity where primarily driven by increases in molecular activity, 4-fold in brain and 7-fold in kidney, rather than by increases in sodium pump number. Membrane polyunsaturation increased in both kidney and brain during development, with kidney becoming increasingly dominated by omega-6 (18:2n-6 and 20:4n-6) and brain by omega-3 (22:6n-3) fatty acids. Membrane reconstitution experiments support the concept that changes in membrane composition might underpin higher sodium molecular activities in the adult. In conclusion, at birth rats possess high Na+ flux but a lower sodium pump capacity that increases with age being driven by increases in molecular activities associate with changes in membrane lipid composition.

High Variability in Erythrocyte, Plasma and Whole Blood EPA and DHA Levels in Response to Supplementation.

(1) Aim: the aim of this secondary analysis was to report the variability in response to n-3 long chain polyunsaturated fatty acids (LCPUFA) supplementation in erythrocytes, plasma and whole blood of a previously published dose response study. (2) Methods: a randomized, double-blind, placebo-controlled trial of parallel design was conducted, whereby pre-menopausal women were randomly assigned to consume 0, 0.35, 0.7 or 1 g/day of supplemental eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Fasted blood samples were taken at baseline and after eight weeks intervention. Erythrocyte, plasma and whole blood fatty acids were extracted using the method of Lepage and Roy and analysed using gas chromatography. (3) Results: There were significant increases in EPA plus DHA levels in the 0.7 g and 1 g dose groups, with the highest increase with the 1 g dose notably: in erythrocytes (from 5.69% to 7.59%), plasma (from 2.94% to 5.48%) and in whole blood (from 3.81% to 6.03%). There was high variability in response to the supplement in erythrocytes, plasma and whole blood across the different doses. (4) Conclusion: there is high individual variability in n-3 LCPUFA levels in response to n-3 LCPUFA supplementation, which should be taken into account in clinical trials using n-3 LCPUFA supplements.

Rottlerin inhibits (Na+, K+)-ATPase activity in brain tissue and alters D-aspartate dependent redistribution of glutamate transporter GLAST in cultured astrocytes.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-delta) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-delta mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na+, K+)-dependent ATPase (Na+, K+-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation ("metabolic poison"). Rottlerin also inhibited Na+, K+-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na+, K+-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na+, K+-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-delta-catalysed phosphorylation in the process.