Omega-3 triglycerides modify blood clearance and tissue targeting pathways of lipid emulsions

Omega-3-rich (n-3) triglycerides (TG) are increasingly recognized as having modulating roles in many physiological and pathological conditions. We questioned whether the catabolism of lipid emulsions would be changed after enrichment with fish oil (n-3) TG as compared to enrichment with omega-6-rich soy oil (n-6) TG. Phospholipid-stabilized emulsions of n-3 TG and n-6 TG were labeled with [(3)H]cholesteryl oleoyl ether and administered by bolus injection to wild-type (WT) mice, mice lacking the low-density lipoprotein receptor (LDL-R) (LDL-R -/-), and apolipoprotein E (apoE) knockout mice (apoE -/-). The effects of exogenous apoE, heparin, Triton WR 1339, and lactoferrin on catabolism of emulsions were also assayed. n-3 TG emulsions were cleared faster from blood and had different extrahepatic tissue targeting compared to n-6 TG emulsions. In apoE -/- and LDL-R -/- mice, blood clearance of n-6 TG emulsions slowed with decreased liver uptake, but no changes were observed in n-3 TG emulsion clearance and tissue uptake compared to WT mice. In WT mice, addition of exogenous apoE to the emulsion increased liver uptake of n-6 TG emulsions but had no impact on n-3 TG emulsions. Pre-injection of heparin increased and Triton WR 1339 and lactoferrin decreased blood clearance of n-6 TG emulsions with little or no effect on n-3 TG emulsions. Liver uptake of n-6 TG emulsions increased after heparin injection and decreased after Triton WR 1339 injection, but uptake of n-3 TG emulsions was not changed. These data show that the catabolism of n-3 TG emulsions and the catabolism of n-6 TG emulsions occur via very different mechanisms. Removal of chylomicron-sized n-6 TG emulsions is modulated by lipoprotein lipase (LPL), apoE, LDL-R, and lactoferrin-sensitive pathways. In contrast, clearance of chylomicron-sized n-3 TG emulsions relies on LPL to a very minor extent and is independent of apoE, LDL-R, and lactoferrin-sensitive pathways.     

Acute Administration of n-3 Rich Triglyceride Emulsions Provides Cardioprotection in Murine Models after Ischemia-Reperfusion

Dietary n-3 fatty acids (FAs) may reduce cardiovascular disease risk. We questioned whether acute administration of n-3 rich triglyceride (TG) emulsions could preserve cardiac function and decrease injury after ischemia/reperfusion (I/R) insult. We used two different experimental models: in vivo, C57BL/6 mice were exposed to acute occlusion of the left anterior descending coronary artery (LAD), and ex-vivo, C57BL/6 murine hearts were perfused using Langendorff technique (LT). In the LAD model, mice treated with n-3 TG emulsion (1.5g/kg body weight), immediately after ischemia and 1h later during reperfusion, significantly reduced infarct size and maintained cardiac function (p<0.05). In the LT model, administration of n-3 TG emulsion (300mgTG/100ml) during reperfusion significantly improved functional recovery (p<0.05). In both models, lactate dehydrogenase (LDH) levels, as a marker of injury, were significantly reduced by n-3 TG emulsion. To investigate the mechanisms by which n-3 FAs protects hearts from I/R injury, we investigated changes in key pathways linked to cardioprotection. In the ex-vivo model, we showed that n-3 FAs increased phosphorylation of AKT and GSK3β proteins (p<0.05). Acute n-3 TG emulsion treatment also increased Bcl-2 protein level and reduced an autophagy marker, Beclin-1 (p<0.05). Additionally, cardioprotection by n-3 TG emulsion was linked to changes in PPARγ protein expression (p<0.05). Rosiglitazone and p-AKT inhibitor counteracted the positive effect of n-3 TG; GSK3β inhibitor plus n-3 TG significantly inhibited LDH release. We conclude that acute n-3 TG injection during reperfusion provides cardioprotection. This may prove to be a novel acute adjunctive reperfusion therapy after treating patients with myocardial infarction.     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.     

Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids

Male rat pups (21 days old) were placed on a diet deficient in n-3 polyunsaturated fatty acids (PUFAs) or on an n-3 PUFA adequate diet containing alpha-linolenic acid (alpha-LNA; 18 : 3n-3). After 15 weeks on a diet, [4,5-3H]docosahexaenoic acid (DHA; 22 : 6n-3) was injected into the right lateral cerebral ventricle, and the rats were killed at fixed times over a period of 60 days. Compared with the adequate diet, 15 weeks of n-3 PUFA deprivation reduced plasma DHA by 89% and brain DHA by 37%; these DHA concentrations did not change thereafter. In the n-3 PUFA adequate rats, DHA loss half-lives, calculated by plotting log10 (DHA radioactivity) against time after tracer injection, equaled 33 days in total brain phospholipid, 23 days in phosphatidylcholine, 32 days in phosphatidylethanolamine, 24 days in phosphatidylinositol and 58 days in phosphatidylserine; all had a decay slope significantly greater than 0 (p < 0.05). In the n-3 PUFA deprived rats, these half-lives were prolonged twofold or greater, and calculated rates of DHA loss from brain, Jout, were reduced. Mechanisms must exist in the adult rat brain to minimize DHA metabolic loss, and to do so even more effectively in the face of reduced n-3 PUFA availability for only 15 weeks.     

n-3 Deficient and docosahexaenoic acid-enriched diets during critical periods of the developing prenatal rat brain.

The last period of the intrauterine life in the rat (embryonic day 17 to 21, ED17-ED21) is demarcated by an increase in brain and body weight and active neuronogenesis. During this period, a rapid accumulation of DHA (22:6 n-3), unparalleled to other fatty acids, takes place. The details of DHA rapid acquisition in the fetal brain were investigated after imposing a diet deficient in n-3 fatty acids (FA) as of ED1 and subsequently examining the distribution of DHA in major brain phospholipid (PL) classes on ED20, having added on ED15 a triglyceride (TG) mixture enriched up to 43% with DHA. The n-3 deficiency maintained for 19 days resulted at ED20 in more than 30% reduction of DHA in PL, which was counterbalanced by an increase of docosapentaenoic acid (DPA, 22:5 n-6). No effect on body weight, nor major changes in PL composition or other FA in fetal brain PL were observed. Feeding dams a DHA-TG diet on ED15 induced an immediate increase of DHA in maternal liver PL, followed by a subsequent increase of DHA in fetal liver PL, as well as in fetal brain PL.Thus the content of fetal brain DHA in n-3 deficient embryos could be restored within 48 hours. Dietary manipulation of fetal tissues is a rapid phenomenon and can be used to enrich DHA at critical periods of development in utero.     

Autophagic neuron death in neonatal brain ischemia/hypoxia

Hypoxia/ischemia (H/I) brain injury at birth is an important cause of cerebral palsy, mental retardation, and epilepsy. The H/I insult also causes energy failure, oxidative stress, and unbalanced ion fluxes, leading to high induction of autopahgy in brain neurons. Since the mice unable to execute autophagy (due to brain-specific deletion of Atg7 or Atg5) die by massive loss of cerebral and cerebellar neurons with accumulation of ubiquitin aggregates, induction of neuronal autophagy after H/I injury is generally considered neuroprotective by maintaining cellular homeostasis. However, our recent results show that hippocampal pyramidal neurons undergoing caspase-dependent or -independent death following neonatal H/I injury possess abundant LC3-positive granules, and such H/I neuronal death is largely prevented by Atg7 deficiency. In the present review we discuss the roles of autophagy and other forms of programmed cell death in the neonatal H/I brain insult.     

Autophagic neuron death in neonatal brain ischemia/hypoxia

Hypoxia/ischemia (H/I) brain injury at birth is an important cause of cerebral palsy, mental retardation, and epilepsy. The H/I insult also causes energy failure, oxidative stress, and unbalanced ion fluxes, leading to high induction of autopahgy in brain neurons. Since the mice unable to execute autophagy (due to brain-specific deletion of Atg7 or Atg5) die by massive loss of cerebral and cerebellar neurons with accumulation of ubiquitin aggregates, induction of neuronal autophagy after H/I injury is generally considered neuroprotective by maintaining cellular homeostasis. However, our recent results show that hippocampal pyramidal neurons undergoing caspase-dependent or -independent death following neonatal H/I injury possess abundant LC3-positive granules, and such H/I neuronal death is largely prevented by Atg7 deficiency. In the present review we discuss the roles of autophagy and other forms of programmed cell death in the neonatal H/I brain insult.     

Altered essential fatty acid metabolism and composition in rat liver, plasma, heart and brain after microalgal DHA addition to the diet

To investigate the effect of docosahexaenoic acid (DHA) without other highly unsaturated fatty acids (HUFA) on n-3 and n-6 essential fatty acid (EFA) metabolism and fatty acid composition in mammals, a stable isotope tracer technique was used in adult rats fed diets with or without 1.3% of algal DHA in a base diet containing 15% of linoleic acid and 3% of alpha-linolenic acid over 8 weeks. The rats were administered orally a mixed oil containing 48 mg/kg body weight of deuterated linoleic and alpha-linolenic acids and euthanized at 4, 8, 24, 96, 168, 240, 360 and 600 h after administration of the isotopes. Fatty acid compositions and the concentrations of deuterated precursors and their respective metabolites were determined in rat liver, plasma, heart and brain as a function of time. DHA, docosapentaenoic acid and eicosapentaenoic acid in the n-3 EFA family were significantly increased in all organs tested in the DHA-fed group, ranging from 5% to 200% greater in comparison with the control group. The accumulation of the metabolites, deuterated-DHA and deuterated-docosapentaenoic acid n-6 was greatly decreased by 1.5- to 2.5-fold in the dietary DHA group. In summary, feeding preformed DHA led to a marked increase in n-3 HUFA content of rat organs at the expense of n-6 HUFA and also prevented the accumulation of newly synthesized deuterated end products. This is the first study which has isolated the effects of DHA on the de novo metabolism on both the n-6 and n-3 EFA pathways.     

Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet

We quantified incorporation rates of plasma-derived alpha-linolenic acid (alpha-LNA, 18:3n-3) into "stable" liver lipids and the conversion rate of alpha-LNA to docosahexaenoic acid (DHA, 22:6n-3) in male rats fed, after weaning, an n-3 PUFA-adequate diet (4.6% alpha-LNA, no DHA) or an n-3 PUFA-deficient diet (0.2% alpha-LNA, no DHA) for 15 weeks. Unanesthetized rats were infused intravenously with [1-14C]alpha-LNA, and arterial plasma was sampled until the liver was microwaved at 5 min. Unlabeled alpha-LNA and DHA concentrations in arterial plasma and liver were reduced >90% by deprivation, whereas unlabeled arachidonic acid (20:4n-6) and docosapentaenoic acid (22:5n-6) concentrations were increased. Deprivation did not change alpha-LNA incorporation coefficients into stable liver lipids but increased synthesis-incorporation coefficients of DHA from alpha-LNA by 6.6-, 8.4-, and 2.3-fold in triacylglycerol, phospholipid, and cholesteryl ester, respectively. Assuming that synthesized-incorporated DHA eventually would be secreted within lipoproteins, calculated liver DHA secretion rates equaled 2.19 and 0.82 micromol/day in the n-3 PUFA-adequate and -deprived rats, respectively. These rates exceed the published rates of brain DHA consumption by 6- and 10-fold, respectively, and should be sufficient to maintain normal and reduced brain DHA concentrations, respectively, in the two dietary conditions.     

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1β and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.     

Regulation of intracellular calcium levels by polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, in astrocytes: possible involvement of phospholipase A2

Pathological conditions in the brain, such as ischemia, trauma and seizure are accompanied by increased levels of free n-6 and n-3 polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). A neuroprotective role has been suggested for PUFA. For investigation of the potential molecular mechanisms involved in neuroprotection by PUFA, we studied the regulation of the concentration of intracellular Ca2+ ([Ca2+]i) in rat brain astrocytes. We evaluated the presence of extracellular PUFA and the release of intracellular PUFA. Interestingly, only the constitutive brain PUFA AA and DHA, but not eicosapentaenoic acid (EPA) had prominent effects on intracellular Ca2+. AA and DHA suppressed [Ca2+]i oscillation, inhibited store-operated Ca2+ entry, and reduced the amplitudes of Ca2+ responses evoked by agonists of G protein-coupled receptors. Moreover, prolonged exposure of astrocytes to AA and DHA brought the cells to a new steady state of a moderately elevated [Ca2+]i level, where the cells became virtually insensitive to external stimuli. This new steady state can be considered as a mechanism of self-protection. It isolates disturbed parts of the brain, because AA and DHA reduce pathological overstimulation in the tissue surrounding the damaged area. In inflammation-related events, frequently AA and DHA exhibit opposite effects. However, in astrocytes AA and DHA exerted comparable effects on [Ca2+]i. Extracellularly added AA and DHA, but not EPA, were also able to induce the release of [3H]AA from prelabeled astrocytes. Therefore, we also suggest the involvement of phospholipase A2 activation and lysophospholipid generation in the regulation of intracellular Ca2+ in astrocytes.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.     

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.     

Hypobaric Hypoxia Postconditioning Reduces Brain Damage and Improves Antioxidative Defense in the Model of Birth Asphyxia in 7-Day-Old Rats

Perinatal brain insult mostly resulting from hypoxia–ischemia (H–I) often brings lifelong permanent disability, which has a major impact on the life of individuals and their families. The lack of progress in clinically—applicable neuroprotective strategies for birth asphyxia has led to an increasing interest in alternative methods of therapy, including induction of brain tolerance by pre- and particularly postconditioning. Hypoxic postconditioning represents a promising strategy for preventing ischemic brain damage. The aim of this study was to investigate the potential neuroprotective effect of hypobaric hypoxia (HH) postconditioning applied to 7-day old rats after H–I insult. The mild hypobaric conditions (0.47 atm) used in this study imitate an altitude of 5,000 m. We show that application of mild hypobaric hypoxia at relatively short time intervals (1–6 h) after H–I, repeated for two following days leads to significant neuroprotection, manifested by a reduction in weight loss of the ipsilateral hemisphere observed 14 days after H–I. HH postconditioning results in decrease in reactive oxygen species level observed in all experimental groups. The increase in superoxide dismutase activity observed after H–I is additionally enhanced by HH postconditioning applied 1 h after H–I. The increase observed 3 and 6 h after H–I was not statistically significant. Postconditioning with HH suppresses the glutathione concentration decrease evoked by H–I and increased glutathione peroxidase activity and this effect is not dependent on the time of postconditioning initiation. HH postconditioning had no effect on catalase activity. We show for the first time that HH postconditioning reduces brain damage resulting from H–I in immature rats and that the mechanism potentially involved in this effect is related to antioxidant defense mechanisms of immature brain.     

Reversibility of n-3 fatty acid deficiency-induced alterations of learning behavior in the rat: level of n-6 fatty acids as another critical factor

Rats fed a semipurified diet supplemented with 3% (w/w) safflower oil [Saf, n-3 fatty acid deficient, high linoleic acid (18:2n-6)] through two generations exhibit decreased correct response ratios in a brightness-discrimination learning test compared with rats fed 3% perilla oil [Per, high alpha-linolenic acid (18:3n-3)]. This is associated with a decreased DHA (22:6n-3)-to-arachidonic acid (20:4n-6) ratio in brain lipids. In the first set of experiments, dietary oil was shifted from Saf to a mixture of 2.4% safflower oil plus 0.6% DHA after weaning (Saf-DHA), but all parameters measured in the learning test were essentially unchanged. Brain 22:6n-3 content of the Saf-DHA group reached that of the Per group but the levels of 20:4n-6 and docosatetraenoic acid (22:4n-6) did not decrease to those of the Per group at the start of the test. In the second set of experiments, dietary oil was shifted to a mixture of 0.6% safflower oil plus 1.2% oleic acid (OA) plus 1.2% DHA (Saf-OA-DHA group) with 18:2n-6 content comparable to that of the Per group. The Saf-OA-DHA group exhibited a learning performance similar to that of the Per group; brain 22:6n-3, 20:4n-6, and 22:4n-6 contents were also comparable to those of the Per group. These results indicate that the altered learning behavior associated with a long-term n-3 fatty acid deficiency is reversed by supplementing 22:6n-3 after weaning, when the levels of competing n-6 fatty acids in the diet and brain lipids are limited.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Transgenic conversion of omega-6 into omega-3 fatty acids in a mouse model of Parkinson's disease

We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.