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1.  Endogenous Signaling by Omega-3 Docosahexaenoic Acid-derived Mediators Sustains Homeostatic Synaptic and Circuitry Integrity 
Molecular Neurobiology  2011;44(2):216-222.
The harmony and function of the complex brain circuits and synapses are sustained mainly by excitatory and inhibitory neurotransmission, neurotrophins, gene regulation, and factors, many of which are incompletely understood. A common feature of brain circuit components, such as dendrites, synaptic membranes, and other membranes of the nervous system, is that they are richly endowed in docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family. DHA is avidly retained and concentrated in the nervous system and known to play a role in neuroprotection, memory, and vision. Only recently has it become apparent why the surprisingly rapid increases in free (unesterified) DHA pool size take place at the onset of seizures or brain injury. This phenomenon began to be clarified by the discovery of neuroprotectin D1 (NPD1), the first-uncovered bioactive docosanoid formed from free DHA through 15-lipoxygenase-1 (15-LOX-1). NPD1 synthesis includes, as agonists, oxidative stress and neurotrophins. The evolving concept is that DHA-derived docosanoids set in motion endogenous signaling to sustain homeostatic synaptic and circuit integrity. NPD1 is anti-inflammatory, displays inflammatory resolving activities, and induces cell survival, which is in contrast to the pro-inflammatory actions of the many of omega-6 fatty acid family members. We highlight here studies relevant to the ability of DHA to sustain neuronal function and protect synapses and circuits in the context of DHA signalolipidomics. DHA signalolipidomics comprises the integration of the cellular/tissue mechanism of DHA uptake, its distribution among cellular compartments, the organization and function of membrane domains containing DHA phospholipids, and the precise cellular and molecular events revealed by the uncovering of signaling pathways regulated by docosanoids endowed with prohomeostatic and cell survival bioactivity. Therefore, this approach offers emerging targets for prevention, pharmaceutical intervention, and clinical translation involving DHA-mediated signaling.
doi:10.1007/s12035-011-8200-6
PMCID: PMC3180614  PMID: 21918832
Epilepsy; Neuroprotectin D1; Photoreceptors; Retinal pigment epithelial cells; Liver
2.  Phosphatidylserine-dependent neuroprotective signaling promoted by docosahexaenoic acid 
Enrichment of polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA, 22:6n–3), in the brain is known to be critical for optimal brain development and function. Mechanisms for DHA’s beneficial effects in the nervous system are not clearly understood at present. DHA is incorporated into the phospholipids in neuronal membranes, which in turn can influence not only the membrane chemical and physical properties but also the cell signaling involved in neuronal survival, proliferation and differentiation. Our studies have indicated that DHA supplementation promotes phosphatidylserine (PS) accumulation and inhibits neuronal cell death under challenged conditions, supporting a notion that DHA is an important neuroprotective agent. This article summarizes our findings on the DHA-mediated membrane-related signaling mechanisms that might explain some of the beneficial effects of DHA, particularly on neuronal survival.
doi:10.1016/j.plefa.2010.02.025
PMCID: PMC3383770  PMID: 20207120
3.  Docosahexaenoic Acid Signaling Modulates Cell Survival in Experimental Ischemic Stroke Penumbra and Initiates Long-Term Repair in Young and Aged Rats 
PLoS ONE  2012;7(10):e46151.
Background
Docosahexaenoic acid, a major omega-3 essential fatty acid family member, improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia. We hypothesize that DHA elicits neuroprotection by inducing AKT/p70S6K phosphorylation, which in turn leads to cell survival and protects against ischemic stroke in young and aged rats.
Methods and Results
Rats underwent 2 h of middle cerebral artery occlusion (MCAo). DHA, neuroprotectin D1 (NPD1) or vehicle (saline) was administered 3 h after onset of stroke. Neurological function was evaluated on days 1, 2, 3, and 7. DHA treatment improved functional recovery and reduced cortical, subcortical and total infarct volumes 7 days after stroke. DHA also reduced microglia infiltration and increased the number of astrocytes and neurons when compared to vehicle on days 1 and 7. Increases in p473 AKT and p308 AKT phosphorylation/activation were observed in animals treated with DHA 4 h after MCAo. Activation of other members of the AKT signaling pathway were also observed in DHA treated animals including increases in pS6 at 4 h and pGSK at 24 h. DHA or NPD1 remarkably reduced total and cortical infarct in aged rats. Moreover, we show that in young and aged rats DHA treatment after MCAo potentiates NPD1 biosynthesis. The phosphorylation of p308 AKT or pGSK was not different between groups in aged rats. However, pS6 expression was increased with DHA or NPD1 treatment when compared to vehicle.
Conclusions
We suggest that DHA induces cell survival, modulates the neuroinflammatory response and triggers long term restoration of synaptic circuits. Both DHA and NPD1 elicited remarkable protection in aged animals. Accordingly, activation of DHA signaling might provide benefits in the management of ischemic stroke both acutely as well as long term to limit ensuing disabilities.
doi:10.1371/journal.pone.0046151
PMCID: PMC3484151  PMID: 23118851
4.  Depletion of Brain Docosahexaenoic Acid Impairs Recovery from Traumatic Brain Injury 
PLoS ONE  2014;9(1):e86472.
Omega-3 fatty acids are crucial for proper development and function of the brain where docosahexaenoic acid (DHA), the primary omega-3 fatty acid in the brain, is retained avidly by the neuronal membranes. We investigated the effect of DHA depletion in the brain on the outcome of traumatic brain injury (TBI). Pregnant mice were put on an omega-3 fatty acid adequate or deficient diet from gestation day 14 and the pups were raised on the respective diets. Continuation of this dietary regime for three generations resulted in approximately 70% loss of DHA in the brain. Controlled cortical impact was delivered to both groups of mice to produce severe TBI and the functional recovery was compared. Compared to the omega-3 adequate mice, the DHA depleted mice exhibited significantly slower recovery from motor deficits evaluated by the rotarod and the beam walk tests. Furthermore, the DHA deficient mice showed greater anxiety-like behavior tested in the open field test as well as cognitive deficits evaluated by the novel object recognition test. The level of alpha spectrin II breakdown products, the markers of TBI, was significantly elevated in the deficient mouse cortices, indicating that the injury is greater in the deficient brains. This observation was further supported by the reduction of NeuN positive cells around the site of injury in the deficient mice, indicating exacerbated neuronal death after injury. These results suggest an important influence of the brain DHA status on TBI outcome.
doi:10.1371/journal.pone.0086472
PMCID: PMC3903526  PMID: 24475126
5.  Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and 16O/18O labeling 
Journal of proteome research  2011;10(12):5472-5480.
Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC/ESI-MS/MS and 16O/18O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC/ESI-MS/MS after SDS-PAGE, in-gel digestion and differential O18/O16 labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.
doi:10.1021/pr2007285
PMCID: PMC3458425  PMID: 22003853
Synaptic plasma membrane (SPM); synaptic proteins; docosahexaenoic acid (DHA); 18O labeling; nano-LC/ESI-MS/MS; brain
6.  The Salutary Effects of DHA Dietary Supplementation on Cognition, Neuroplasticity, and Membrane Homeostasis after Brain Trauma 
Journal of Neurotrauma  2011;28(10):2113-2122.
Abstract
The pathology of traumatic brain injury (TBI) is characterized by the decreased capacity of neurons to metabolize energy and sustain synaptic function, likely resulting in cognitive and emotional disorders. Based on the broad nature of the pathology, we have assessed the potential of the omega-3 fatty acid docosahexaenoic acid (DHA) to counteract the effects of concussive injury on important aspects of neuronal function and cognition. Fluid percussion injury (FPI) or sham injury was performed, and rats were then maintained on a diet high in DHA (1.2% DHA) for 12 days. We found that DHA supplementation, which elevates brain DHA content, normalized levels of brain-derived neurotrophic factor (BDNF), synapsin I (Syn-1), cAMP-responsive element-binding protein (CREB), and calcium/calmodulin-dependent kinase II (CaMKII), and improved learning ability in FPI rats. It is known that BDNF facilitates synaptic transmission and learning ability by modulating Syn-I, CREB, and CaMKII signaling. The DHA diet also counteracted the FPI-reduced manganese superoxide dismutase (SOD) and Sir2 (a NAD+-dependent deacetylase). Given the involvement of SOD and Sir2 in promoting metabolic homeostasis, DHA may help the injured brain by providing resistance to oxidative stress. Furthermore, DHA normalized levels of calcium-independent phospholipase A2 (iPLA2) and syntaxin-3, which may help preserve membrane homeostasis and function after FPI. The overall results emphasize the potential of dietary DHA to counteract broad and fundamental aspects of TBI pathology that may translate into preserved cognitive capacity.
doi:10.1089/neu.2011.1872
PMCID: PMC3191367  PMID: 21851229
brain-derived neurotrophic factor; plasticity; Sir2; superoxide dismutase; traumatic brain injury
7.  Oral Supplementation with Docosahexaenoic Acid and Uridine-5’-Monophosphate Increases Dendritic Spine Density in Adult Gerbil Hippocampus 
Brain research  2007;1182:50-59.
Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer’s disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA’s behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension, and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5’-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (> 30%) is accompanied by parallel increases in membrane phosphatides, and in pre- and post-synaptic proteins within the hippocampus. Hence oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.
doi:10.1016/j.brainres.2007.08.089
PMCID: PMC2140951  PMID: 17950710
docosahexaenoic acid; uridine; membrane synthesis; spine formation; synaptogenesis; phosphatides
8.  Differential Cerebral Cortex Transcriptomes of Baboon Neonates Consuming Moderate and High Docosahexaenoic Acid Formulas 
PLoS ONE  2007;2(4):e370.
Background
Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels.
Methods and Findings
Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; “L”, LCPUFA, with 0.33%DHA-0.67%ARA; “L3”, LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner.
Conclusions
These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.
doi:10.1371/journal.pone.0000370
PMCID: PMC1847718  PMID: 17426818
9.  N-Docosahexaenoylethanolamide promotes development of hippocampal neurons 
The Biochemical journal  2011;435(2):327-336.
DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n−3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
doi:10.1042/BJ20102118
PMCID: PMC3169088  PMID: 21281269
docosahexaenoic acid (DHA); N-docosahexaenoylethanolamide (DEA); hippocampus; neurite growth; neuron; synaptogenesis
10.  Omega-3 Fatty Acid Docosahexaenoic Acid Increases SorLA/LR11, a Sorting Protein with Reduced Expression in Sporadic Alzheimer’s Disease (AD): Relevance to AD Prevention 
Environmental and genetic factors, notably ApoE4, contribute to the etiology of late-onset Alzheimer’s disease (LOAD). Reduced mRNA and protein for an apolipoprotein E (ApoE) receptor family member, SorLA (LR11) has been found in LOAD but not early-onset AD, suggesting that LR11 loss is not secondary to pathology. LR11 is a neuronal sorting protein that reduces amyloid precursor protein (APP) trafficking to secretases that generate β-amyloid (Aβ). Genetic polymorphisms that reduce LR11 expression are associated with increased AD risk. However these polymorphisms account for only a fraction of cases with LR11 deficits, suggesting involvement of environmental factors. Because lipoprotein receptors are typically lipid-regulated, we postulated that LR11 is regulated by docosahexaenoic acid (DHA), an essential ω-3 fatty acid related to reduced AD risk and reduced Aβ accumulation. In this study, we report that DHA significantly increases LR11 in multiple systems, including primary rat neurons, aged non-Tg mice and an aged DHA-depleted APPsw AD mouse model. DHA also increased LR11 in a human neuronal line. In vivo elevation of LR11 was also observed with dietary fish oil in young rats with insulin resistance, a model for type II diabetes, another AD risk factor. These data argue that DHA induction of LR11 does not require DHA-depleting diets and is not age dependent. Because reduced LR11 is known to increase Aβ production and may be a significant genetic cause of LOAD, our results indicate that DHA increases in SorLA/LR11 levels may play an important role in preventing LOAD.
doi:10.1523/JNEUROSCI.3593-07.2007
PMCID: PMC2628584  PMID: 18160637
SorLA; LR11; Alzheimer; docosahexaenoic acid; diet; omega-3 fatty acid; amyloid
11.  DHA does not protect ELOVL4 transgenic mice from retinal degeneration 
Molecular Vision  2009;15:1185-1193.
Purpose
Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3.
Methods
Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1–/TG2+, Fat1+/TG2–, and Fat1–/TG2–. All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels.
Results
Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene.
Conclusions
We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.
PMCID: PMC2697457  PMID: 19536303
12.  Improved Mitochondrial Function with Diet-Induced Increase in Either Docosahexaenoic Acid or Arachidonic Acid in Membrane Phospholipids 
PLoS ONE  2012;7(3):e34402.
Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.
doi:10.1371/journal.pone.0034402
PMCID: PMC3316678  PMID: 22479624
13.  The Effect of varying ratios of docosahexaenoic Acid and arachidonic acid in the prevention and reversal of biochemical essential fatty acid deficiency in a murine model 
Objective
Essential fatty acids (EFA) are necessary for growth, development, and biological function, and must be acquired through the diet. While linoleic acid (LA) and alpha-linolenic acid (ALA) have been considered the true EFAs, we previously demonstrated that docosahexaenoic acid (DHA) and arachidonic acid (AA) taken together as the sole source of dietary fatty acids can prevent biochemical essential fatty acid deficiency (EFAD). This study evaluates the effect of varying dietary ratios of DHA:AA in the prevention and reversal of biochemical EFAD in a murine model.
Methods
Using a murine model of EFAD, we provided mice with 2.1% of daily caloric intake in varying DHA:AA ratios (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) for 19 days in association with a liquid high-carbohydrate fat-free diet to evaluate the effect on fatty acid profiles. In a second experiment, we evaluated the provision of varying DHA:AA ratios (20:1, 200:1, 100:0) on the reversal of biochemical EFAD.
Results
Mice provided with DHA and AA had no evidence of biochemical EFAD, regardless of the ratio (1:1, 5:1, 10:1, 20:1, 200:1, 100:0) administered. Biochemical EFAD was reversed with DHA:AA ratios of 20:1, 200:1, and 100:0 following 3 and 5 weeks of dietary provision, although the 20:1 ratio was most effective in the reversal and stabilization of the triene:tetraene ratio.
Conclusion
Provision of DHA and AA, at 2.1% of daily caloric intake in varying ratios can prevent biochemical evidence of EFAD and hepatic steatosis over the short-term, with a ratio of 20:1 DHA:AA most effectively reversing EFAD.
doi:10.1016/j.metabol.2012.10.003
PMCID: PMC3608741  PMID: 23151438
omega-3; polyunsaturated fatty acid; triene:tetraene ratio; dietary lipid; mouse
14.  Omega 3 fatty acid inhibition of inflammatory cytokine-mediated Connexin43 regulation in the heart 
Background: The proinflammatory cytokine Interleukin-1β (IL-1β), which increases in the heart post myocardial infarction (MI), has been shown to cause loss of Connexin43 (Cx43) function, an event known to underlie formation of the arrhythmogenic substrate. Omega 3 Fatty acids exhibit antiarrhythmic properties and impact IL-1β signaling. We hypothesize that Omega-3 fatty acids prevent arrhythmias in part, by inhibiting IL-1β signaling thus maintaining functional Cx43 channels. Methods: Rat neonatal myocytes or Madin-Darby Canine Kidney Epithelial (MDCK) cells grown in media in the absence (Ctr) or presence of 30 μM docosahexaenoic acid (DHA, an Omega-3 Fatty acid) were treated with 0.1 μM activated IL-1β. We determined Cx43 channel function using a dye spread assay. Western blot and immunostaining were used to examine Cx43 levels/localization and downstream effectors of IL-1β. In addition we used a murine model of MI for 24 h to determine the impact of an Omega-3 fatty acid enriched diet on Cx43 levels/localization post MI. Results: IL-1β significantly inhibited Cx43 function in Ctr cells (200.9 ± 17.7 μm [Ctr] vs. 112.8 ± 14.9 μm [0.1 uM IL-1β], p<0.05). However, DHA-treated cells remained highly coupled in the presence of IL-1β [167.9 ± 21.9 μm [DHA] vs. 164.4 ± 22.3 μm [DHA + 0.1 uM IL-1β], p<0.05, n = 4]. Additionally, western blot showed that IL-1β treatment caused a 38.5% downregulation of Cx43 [1.00 au [Ctr] vs. 0.615 au (0.1 μM IL-1β) which was completely abolished in DHA-treated cells (0.935 au [DHA] vs. 1.02 au [DHA + 0.1 μM IL-1β), p < 0.05, n = 3]. Examination of the downstream modulator of IL-1β, NFκβ showed that while hypoxia caused translocation of NFκβ to the nucleus, this was inhibited by DHA. Additionally we found that a diet enriched in Omega-3 Fatty acids inhibited lateralization of Cx43 in the post-MI murine heart as well as limited activation of fibroblasts which would lead to decreased fibrosis overall. Conclusions: Omega 3 Fatty acid treatment inhibited IL-1β-stimulated loss of Cx43 protein, and more importantly, inhibited loss of Cx43 function by inhibiting translocation of NFκβ. In the intact heart a diet enriched in Omega 3 Fatty Acids limited loss of Cx43 at the intercalated disk in the heart following MI. These data suggest that one of cardio-protective mechanisms by which Omega 3 Fatty acids work includes prevention of the pro-arrhythmic loss of Cx43 post MI and the attenuation of cardiac fibrosis after injury.
doi:10.3389/fphys.2012.00272
PMCID: PMC3429046  PMID: 22934026
arrhythmia; fibrosis; gap junction; inflammation; interleukin; myocardial infarction
15.  Docosahexaenoic acid prevents dendritic cell maturation and in vitro and in vivo expression of the IL-12 cytokine family 
Background
Acute and chronic inflammation play essential roles in inflammatory/autoimmune conditions. Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in animal models of colitis, sepsis, and stroke. Since dendritic cells (DC) represent the essential cellular link between innate and adaptive immunity and have a prominent role in tolerance for self-antigens, we sought to investigate the impact of DHA on DC maturation and proinflammatory cytokine production.
Methods
Murine bone marrow-derived DC were treated with DHA and stimulated with various toll-like receptor (TLR) ligands. Flow cytometry was used to determine the levels of surface maturation markers and endocytic activity. Cytokine expression and secretion were measured by real-time RT-PCR and ELISA assays. PPARγ and NFκB activity in nuclear extracts were determined by binding to specific oligonucleotide sequences using ELISA-based assays. In vivo effects of DHA were assessed in splenic DC from LPS-inoculated mice maintained on a DHA-enriched diet.
Results
DHA maintained the immature phenotype in bone marrow-derived DC by preventing the upregulation of MHCII and costimulatory molecules (CD40, CD80 and CD86) and maintaining high levels of endocytic activity. DHA inhibited the production of pro-inflammatory cytokines, including the IL-12 cytokine family (IL-12p70, IL-23, and IL-27), from DC stimulated with TLR2, 3, 4, and 9 ligands. DHA inhibition of IL-12 expression was mediated through activation of PPARγ and inhibition of NFκBp65 nuclear translocation. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet.
Conclusions
Exposure of bone marrow-derived DC to DHA resulted in the maintenance of an immature phenotype and drastic reduction in proinflammatory cytokine release. DHA inhibited the expression and secretion of the IL-12 cytokine family members (IL-12p70, IL-23 and IL-27), which play essential roles in the differentiation of the proinflammatory Th1/Th17 effector cells. The effect of DHA on IL-12 expression was mediated through activation of PPARγ and inhibition of NFκB. Inhibition of IL-12 and IL-23 expression was also evident in splenic DC from mice fed a DHA-enriched diet, suggesting that dietary DHA acts as an anti-inflammatory agent in vivo.
doi:10.1186/1476-511X-9-12
PMCID: PMC2827414  PMID: 20122166
16.  Docosahexaenoic Acid Neurolipidomics 
Mediator lipidomics is a field of study concerned with the characterization, structural elucidation and bioactivity of lipid derivatives generated by enzymatic activity. Omega-3 fatty acids have beneficial effects for vision, brain function, cardiovascular function, and immune-inflammatory responses. Docosahexaenoic acid [DHA; 22:6(n-3)], the most abundant essential omega-3 fatty acid in the human body, is selectively enriched and avidly retained in the central nervous system as an acyl chain of phospholipids. Brain-ischemia reperfusion and seizures trigger rapid release of DHA and of arachidonic acid (AA) as free, unesterified fatty acids. AA in turn generates eicosanoids, and DHA forms docosanoids. The stereoselective docosanoid neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,15E,19Z hexaenoic acid) is formed early in brain-ischemia reperfusion. Supplementation of NPD1 (intracerebroventricularly; i.c.v.) or of DHA (i.c.v. or systemically) results in decreased infarct size, polymorphonuclear neutrophil infiltration, ischemia-induced nuclear factor kappa B (NFκB) activation, and cyclooxygenase-2 (COX-2) induction. DHA involvement in cell function includes enhancing Akt translocation and activation, and binding to a peroxisome proliferator-activated receptor-gamma (PPAR-γ) family of ligand-activated nuclear receptors. Here we present an overview of recent DHA-mediator lipidomic studies in experimental brain ischemia-reperfusion and other conditions.
doi:10.1016/j.prostaglandins.2009.09.005
PMCID: PMC2905848  PMID: 19804838
17.  Docosahexaenoic acid in maternal and neonatal plasma phospholipids and milk lipids of Taiwanese women in Kinmen: fatty acid composition of maternal blood, neonatal blood and breast milk 
Background
Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid (LCPUFA) that is critically important for the structure, development and function of the retina and central nervous system (CNS), ultimately contributing to improved cognition. It is known that the DHA content of breast milk is positively correlated with maternal DHA intake. Since there is a lack of information about the DHA status of pregnant and lactating women in rural Taiwan. The aims of the present study were to: 1) assess the DHA status of mothers and babies in urban setting, and 2) determine the content of DHA in the milk of nursing mothers.
Methods
All pregnant women who attended the Obstetrics and Gynecology Outpatient Clinic of Kinmen Hospital on Kinmen Island in Taiwan between May 1 and May 30, 2011 were invited by research nurses to enroll in the study. The maternal blood sample was obtained on the day of their delivery. Cord blood was collected by the obstetrician following delivery. Participants were asked to visit the doctor forty-two days after the delivery, at which time a nurse collected breast milk on the day mothers were visiting the doctor for post-natal well-baby check-up.
Results
The DHA percentages of maternal and neonatal plasma phospholipids were 5.16% and 6.36%, respectively, which are higher than values reported for most populations elsewhere in the world. The DHA percentage for the breast milk of Kinmen mothers was also high (0.98%) relation to international norms. The DHA proportions in maternal and neonatal plasma phospholipids were positively correlated (r = 0.46, p = 0.01).
Conclusions
We show that the DHA status of mothers and newborns on Kinmen Island is satisfactory, thereby providing an evidence-based argument for promoting breastfeeding in Taiwan.
doi:10.1186/1476-511X-12-27
PMCID: PMC3608933  PMID: 23496882
Breast milk; Lactation; Neonates; Fish intake; Kinmen; Docosahexaenoic acid; Pregnancy; Fatty acids
18.  Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Modification of Caveolae/Lipid Rafts by Docosahexaenoic Acid 
PURPOSE.
Docosahexaenoic acid (DHA22:6,n3) is the principal n3 polyunsaturated fatty acid (PUFA) in the retina. The authors previously demonstrated that DHA22:6,n3 inhibited cytokine-induced adhesion molecule expression in primary human retinal vascular endothelial (hRVE) cells, the target tissue affected by diabetic retinopathy. Despite the importance of vascular inflammation in diabetic retinopathy, the mechanisms underlying anti-inflammatory effects of DHA22:6,n3 in vascular endothelial cells are not understood. In this study the authors address the hypothesis that DHA22:6,n3 acts through modifying lipid composition of caveolae/lipid rafts, thereby changing the outcome of important signaling events in these specialized plasma membrane microdomains.
METHODS.
hRVE cells were cultured in the presence or absence of DHA22:6,n3. Isolated caveolae/lipid raft–enriched detergent-resistant membrane domains were prepared using sucrose gradient ultracentrifugation. Fatty acid composition and cholesterol content of caveolae/lipid rafts before and after treatment were measured by HPLC. The expression of Src family kinases was assayed by Western blotting and immunohistochemistry.
RESULTS.
Disruption of the caveolae/lipid raft structure with a cholesterol-depleting agent, methyl-cyclodextrin (MCD), diminished cytokine-induced signaling in hRVE cells. Growth of hRVE cells in media enriched in DHA22:6,n3 resulted in significant incorporation of DHA22:6,n3 into the major phospholipids of caveolae/lipid rafts, causing an increase in the unsaturation index in the membrane microdomain. DHA22:6,n3 enrichment in the caveolae/raft was accompanied by a 70% depletion of cholesterol from caveolae/lipid rafts and displacement of the SFK, Fyn, and c-Yes from caveolae/lipid rafts. Adding water-soluble cholesterol to DHA22:6,n3-treated cells replenished cholesterol in caveolae/lipid rafts and reversed the effect of DHA22:6,n3 on cytokine-induced signaling.
CONCLUSIONS.
Incorporation of DHA22:6,n3 into fatty acyl chains of phospholipids in caveolae/lipid rafts, followed by cholesterol depletion and displacement of important signaling molecules, provides a potential mechanism for anti-inflammatory effect of DHA22:6,n3 in hRVE cells.
doi:10.1167/iovs.06-0619
PMCID: PMC1975816  PMID: 17197511
19.  Dietary saturated fat and docosahexaenoic acid differentially effect cardiac mitochondrial phospholipid fatty acyl composition and Ca2+ uptake, without altering permeability transition or left ventricular function 
Physiological Reports  2013;1(1):e00009.
High saturated fat diets improve cardiac function and survival in rodent models of heart failure, which may be mediated by changes in mitochondrial function. Dietary supplementation with the n3-polyunsaturated fatty acid docosahexaenoic acid (DHA, 22:6n3) is also beneficial in heart failure and can affect mitochondrial function. Saturated fatty acids and DHA likely have opposing effects on mitochondrial phospholipid fatty acyl side chain composition and mitochondrial membrane function, though a direct comparison has not been previously reported. We fed healthy adult rats a standard low-fat diet (11% of energy intake from fat), a low-fat diet supplemented with DHA (2.3% of energy intake) or a high-fat diet comprised of long chain saturated fatty acids (45% fat) for 6 weeks. There were no differences among the three diets in cardiac mass or function, mitochondrial respiration, or Ca2+-induced mitochondrial permeability transition. On the other hand, there were dramatic differences in mitochondrial phospholipid fatty acyl side chains. Dietary supplementation with DHA increased DHA from 7% to ∼25% of total phospholipid fatty acids in mitochondrial membranes, and caused a proportional depletion of arachidonic acid (20:4n6). The saturated fat diet increased saturated fat and DHA in mitochondria and decreased linoleate (18:2n6), which corresponded to a decrease in Ca2+ uptake by isolated mitochondria compared to the other diet groups. In conclusion, despite dramatic changes in mitochondrial phospholipid fatty acyl side chain composition by both the DHA and high saturated fat diets, there were no effects on mitochondrial respiration, permeability transition, or cardiac function.
doi:10.1002/phy2.9
PMCID: PMC3831937  PMID: 24303101
Cardiovascular; mitochondria; n3-polyunsaturated fatty acids; nutrition; phospholipid; saturated fatty acids
20.  Docosahexaenoic Acid and the Aging Brain123 
The Journal of nutrition  2008;138(12):2510-2514.
The dietary essential PUFA docosahexaenoic acid [DHA; 22:6(n-3)] is a critical contributor to cell structure and function in the nervous system, and deficits in DHA abundance are associated with cognitive decline during aging and in neurodegenerative disease. Recent studies underscore the importance of DHA-derived neuroprotectin D1 (NPD1) in the homeostatic regulation of brain cell survival and repair involving neurotrophic, antiapoptotic and antiinflammatory signaling. Emerging evidence suggests that NPD1 synthesis is activated by growth factors and neurotrophins. Evolving research indicates that NPD1 has important determinant and regulatory interactions with the molecular-genetic mechanisms affecting b-amyloid precursor protein (bAPP) and amyloid beta (Ab) peptide neurobiology. Deficits in DHA or its peroxidation appear to contribute to inflammatory signaling, apoptosis, and neuronal dysfunction in Alzheimer disease (AD), a common and progressive age-related neurological disorder unique to structures and processes of the human brain. This article briefly reviews our current understanding of the interactions of DHA and NPD1 on bAPP processing and Ab peptide signaling and how this contributes to oxidative and pathogenic processes characteristic of aging and AD pathology.
doi:10.3945/jn.108.096016
PMCID: PMC2666388  PMID: 19022980
21.  Pulmonary delivery of docosahexaenoic acid mitigates bleomycin-induced pulmonary fibrosis 
Background
Pulmonary fibrosis is an untreatable, fatal disease characterized by excess deposition of extracellular matrix and inflammation. Although the etiology of pulmonary fibrosis is unknown, recent studies have implicated dysregulated immune responses and wound healing. Since n-3 polyunsaturated fatty acids (n-3 PUFAs) may beneficially modulate immune responses in a variety of inflammatory disorders, we investigated the therapeutic role of docosahexaenoic acid (DHA), a single n-3 PUFA, in lung fibrosis.
Methods
Intratracheal DHA or PBS was administered to mouse lungs 4 days prior to intratracheal bleomycin treatment. Body weight and survival were monitored for 21 days. Bronchoalveolar fluid (BALF) and lung inflammatory cells, cytokines, eicosanoids, histology and lung function were determined on serial days (0, 3, 7, 14, 21) after bleomycin injury.
Results
Intratracheal administration of DHA mitigated bleomycin-induced lung injury. Mice pretreated with DHA had significantly less weight loss and mortality after bleomycin injury. The lungs from DHA-pretreated mice had markedly less fibrosis. DHA pretreatment also protected the mice from the functional changes associated with bleomycin injury. Bleomycin-induced cellular inflammation in BALF and lung tissue was blunted by DHA pretreatment. These advantageous effects of DHA pretreatment were associated with decreased IL-6, LTB4, PGE2 and increased IL-10.
Conclusions
Our findings demonstrate that intratracheal administration of DHA, a single PUFA, protected mice from the development of bleomycin-induced pulmonary inflammation and fibrosis. These results suggest that further investigations regarding the role of n-3 polyunsaturated fatty acids in fibrotic lung injury and repair are needed.
doi:10.1186/1471-2466-14-64
PMCID: PMC3998951  PMID: 24742272
22.  Cellular and molecular events mediated by docosahexaenoic acid-derived neuroprotectin D1 signaling in photoreceptor cell survival and brain protection 
Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid) contributes to understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Aβ peptide 42 (Aβ42), calcium ionophore A23187, interleukin (IL)-1 β, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the anti-apoptotic proteins of the Bcl-2 family and decreases the expression of pro-apoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1 β-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-β (Aβ) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer’s disease (AD) CA1 hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A2 (cPLA2), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Aβ42-triggered activation of pro-inflammatory genes and upregulated the anti-apoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that NPD1 promotes brain and retina cell survival via the induction of anti-apoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity and other forms of cell injury, which in turn fosters homeostasis during development in aging, as well as during the initiation and progression of neurodegenerative diseases.
doi:10.1016/j.plefa.2009.05.024
PMCID: PMC2756692  PMID: 19520558
n-3 (omega-3) fatty acid; n-6 (omega-6) fatty acid; retinal pigment epithelial cell; Aβ42; Bcl-2 proteins; eicosanoids; docosanoids; inflammation; photoreceptor renewal; liver; neurotrophins; aging; Alzheimer’s disease; macular degeneration
23.  Docosahexaenoic Acid (DHA): An Ancient Nutrient for the Modern Human Brain  
Nutrients  2011;3(5):529-554.
Modern humans have evolved with a staple source of preformed docosahexaenoic acid (DHA) in the diet. An important turning point in human evolution was the discovery of high-quality, easily digested nutrients from coastal seafood and inland freshwater sources. Multi-generational exploitation of seafood by shore-based dwellers coincided with the rapid expansion of grey matter in the cerebral cortex, which characterizes the modern human brain. The DHA molecule has unique structural properties that appear to provide optimal conditions for a wide range of cell membrane functions. This has particular implications for grey matter, which is membrane-rich tissue. An important metabolic role for DHA has recently been identified as the precursor for resolvins and protectins. The rudimentary source of DHA is marine algae; therefore it is found concentrated in fish and marine oils. Unlike the photosynthetic cells in algae and higher plants, mammalian cells lack the specific enzymes required for the de novo synthesis of alpha-linolenic acid (ALA), the precursor for all omega-3 fatty acid syntheses. Endogenous synthesis of DHA from ALA in humans is much lower and more limited than previously assumed. The excessive consumption of omega-6 fatty acids in the modern Western diet further displaces DHA from membrane phospholipids. An emerging body of research is exploring a unique role for DHA in neurodevelopment and the prevention of neuropsychiatric and neurodegenerative disorders. DHA is increasingly being added back into the food supply as fish oil or algal oil supplementation.
doi:10.3390/nu3050529
PMCID: PMC3257695  PMID: 22254110
docosahexaenoic acid; DHA; omega-3 fatty acids; n-3 fatty acids; brain evolution; erythrocyte phospholipids; algal oil; fish oil; nutritional supplementation
24.  DOCOSAHEXAENOIC ACID PREVENTS DENDRITIC CELL MATURATION, INHIBITS ANTIGEN-SPECIFIC Th1/Th17 DIFFERENTIATION AND SUPPRESSES EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS 
Brain, behavior, and immunity  2010;25(5):872-882.
Docosahexaenoic acid (DHA), the most abundant essential n-3 polyunsaturated fatty acid in the CNS, emerged recently together with eicosapentaenoic acid (EPA) and DHA/EPA metabolic derivatives as a major player in the resolution of inflammation. Protective antiinflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. Here we report for the first time a beneficial effect of dietary n-3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4+ T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6 and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent, and with decreases in Tbet, GATA-3 and RORγt, master transcription factors for Th1, Th2, and Th17. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. Similar to the in vitro results, the beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4+ T cells in both spleen and CNS.
doi:10.1016/j.bbi.2010.09.012
PMCID: PMC3031664  PMID: 20854895
Docosahexaenoic acid; Experimental autoimmune encephalomyelitis; IL-12; IL-23; Th1; Th17; Foxp3; Tbet; RORγt
25.  Docosanoids are multifunctional regulators of neural cell integrity and fate: significance in aging and disease 
The identification of neuroprotectin D1 (NPD1), a biosynthetic product of docosahexaenoic acid (DHA), in brain and retina as well as the characterization of its bioactivity, is generating a renewed interest in the functional role and pathophysiological significance of omega-3 fatty acids in the central nervous system.
Neurotrophins, particularly pigment epithelium-derived factor (PEDF), induce NPD1 synthesis and its polarized apical secretion, implying paracrine and autocrine bioactivity of this lipid mediator. Also, DHA and PEDF synergistically activate NPD1 synthesis and antiapoptotic protein expression and decreased proapoptotic Bcl-2 protein expression and caspase 3 activation during oxidative stress.
In experimental stroke, endogenous NPD1 synthesis was found to be upregulated, and the infusion of the lipid mediator into the brain under these conditions revealed neuroprotective bioactivity of NPD1.
The hippocampal CA1 region from Alzheimer’s disease (AD) patients (rapidly sampled) shows a major reduction in NPD1.
The interplay of DHA-derived neuroprotective signaling aims to counteract proinflammatory, cell-damaging events triggered by multiple, converging cytokine and amyloid peptide factors, as in the case of AD. Generation of NPD1 from DHA thereby appears to redirect cellular fate toward successful preservation of retinal pigment epithelial (RPE)-photoreceptor cell integrity and brain cell aging. The Bcl-2 pro- and antiapoptotic proteins, neurotrophins, and NPD1, lie along a cell fate-regulatory pathway whose component members are highly interactive, and have potential to function cooperatively in cell survival. Agents that stimulate NPD1 biosynthesis, NPD1 analogs, or dietary regimens may be useful as new preventive/therapeutic strategies for neurodegenerative diseases.
doi:10.1016/j.plefa.2007.10.022
PMCID: PMC2696125  PMID: 18060755

Results 1-25 (959072)