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1.  Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder 
Biochimica et biophysica acta  2013;1831(4):880-886.
Background
Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action.
Methods
Rat Acsl4-flag protein was expressed in E. coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics.
Results
Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4 mM compared to a published Ki of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect.
Conclusions
PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.
doi:10.1016/j.bbalip.2013.01.008
PMCID: PMC3593989  PMID: 23354024
bipolar disorder; valproate; arachidonic acid; acyl-CoA synthetase 4; mood stabilizer; Acsl4; brain; MTMCD; N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide; PIA; propylisopropylacetic; PID; propylisopropylacetamide; rat; butyrate; inhibition; uncompetitive; enzyme; anticonvulsant
2.  Aging is associated with altered inflammatory, arachidonic acid cascade and synaptic markers, influenced by epigenetic modifications, in the human frontal cortex 
Journal of neurochemistry  2013;125(1):63-73.
Aging is a risk factor for Alzheimer’s disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, since DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro- and anti-apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle-aged (41 ± 1 (SEM) years) and ten aged subjects (70 ± 3 years). The aged compared with middle-aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti-apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of BDNF, CREB, and synaptophysin and hypomethylation of BAX. These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated.
doi:10.1111/jnc.12153
PMCID: PMC3606672  PMID: 23336521
Aging; arachidonic acid; BDNF; CREB; DNA methylation; Synaptophysin
3.  Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability 
Journal of neurochemistry  2012;124(3):376-387.
Chronic administration of mood stabilizers to rats downregulates the brain arachidonic acid (AA) cascade. This downregulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E2 concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1-14C]AA was infused intravenously for 5 min, arterial plasma was collected and microwaved brain was analyzed. CLZ increased incorporation coefficients ki∗ and rates Jin,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly downregulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by downregulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.
doi:10.1111/jnc.12078
PMCID: PMC3540173  PMID: 23121637
clozapine; antipsychotic; arachidonic acid; phospholipid; incorporation; bipolar disorder; plasma; brain
4.  Neuroinflammation and synaptic loss 
Neurochemical research  2012;37(5):903-910.
Neuroinflammation plays a critical role in the progression of many neurodegenerative diseases and neuropsychiatric illnesses. It is evident that microglia in particular are central to mediating the effects of neuroinflammation. Activated microglia release a number of cytokines and chemokines, which in turn activate many signal transduction pathways. For instance, interleukin-1 beta and tumor necrosis factor alpha regulate transcription of a number of genes within the brain including proinflammatory products of the arachidonic acid (AA) cascade. Co-activation of pro-inflammatory markers and associated cytotoxic products during neuroinflammation process are detrimental to neurons by altering the synaptic proteins. In this review, we discuss both neuroinflammation as well as excitotoxicity insults reduce synaptic markers such as synaptophysin and drebrin in rat brain. Further we discuss here, neurodegenerative and neuropsychiatric illness are associated with increased neuroinflammatory and excitotoxicity markers as well as upregulated brain arachidonic acid markers and the loss of synaptic markers. The decrease in synaptic markers might contribute to reported cognitive defects in neurodegenerative and neuropsychiatric illnesses.
doi:10.1007/s11064-012-0708-2
PMCID: PMC3478877  PMID: 22311128
5.  TRANSLATIONAL STUDIES ON REGULATION OF BRAIN DOCOSAHEXAENOIC ACID (DHA) METABOLISM IN VIVO 
One goal in the field of brain polyunsaturated fatty acid (PUFA) metabolism is to translate the many studies that have been conducted in vitro and in animal models to the clinical setting. Doing so should elucidate the role of PUFAs in the human brain, and effects of diet, drugs, disease and genetics. This review briefly discusses new in vivo radiotracer kinetic and neuroimaging techniques that allow us to do this, with a focus on docosahexaenoic acid (DHA). We illustrate how brain PUFA metabolism is influenced by graded reductions in n-3 PUFA content in unanesthetized rats. We also show how kinetic tracer techniques in rodents have helped to identify mechanisms of action of mood stabilizers used in bipolar disorder, how DHA participates in neurotransmission,regulated by calcium-independent iPLA2β. In humans, regional rates of brain DHA metabolism can be measured with positron emission tomography and following intravenous injection of [1-11C]DHA.
doi:10.1016/j.plefa.2012.05.003
PMCID: PMC3467358  PMID: 22766388
docosahexaenoic acid; iPLA2; cPLA2; brain; biomarker; imaging; positron emission tomography; arachidonic; human; PET; diet; PUFA; phospholipase A2
6.  Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans 
Summary
Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in human diets, a major component of human tissues, and the direct precursor to the bioactive oxidized LA metabolites (OXLAMs), 9- and 13 hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE). These four OXLAMs have been mechanistically linked to pathological conditions ranging from cardiovascular disease to chronic pain. Plasma OXLAMs, which are elevated in Alzheimer’s dementia and non-alcoholic steatohepatitis, have been proposed as biomarkers useful for indicating the presence and severity of both conditions. Because mammals lack the enzymatic machinery needed for de novo LA synthesis, the abundance of LA and OXLAMs in mammalian tissues may be modifiable via diet. To examine this issue in humans, we measured circulating LA and OXLAMs before and after a 12-week LA lowering dietary intervention in chronic headache patients. Lowering dietary LA significantly reduced the abundance of plasma OXLAMs, and reduced the LA content of multiple circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. These results show that lowering dietary LA can reduce the synthesis and/or accumulation of oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the clinical implications of diet-induced OXLAM reductions are warranted.
doi:10.1016/j.plefa.2012.08.004
PMCID: PMC3467319  PMID: 22959954
Linoleic acid; HODE; hydroxy-octadecadienoic acid; oxoODE; oxo-octadecadienoic acid; oxidation; OXLAM; PUFA; polyunsaturated fatty acid
7.  Altered lipid concentrations of liver, heart and plasma but not brain in HIV-1 transgenic rats 
doi:10.1016/j.plefa.2012.07.006
PMCID: PMC3467364  PMID: 22939288
HIV-1 transgenic rat; fatty acid; brain; liver; heart; plasma; arachidonic; docosahexaenoic; polyunsaturated fatty acids (PUFA); concentration; composition; metabolism
8.  CAN THE RAT LIVER MAINTAIN NORMAL BRAIN DHA METABOLISM IN THE ABSENCE OF DIETARY DHA?2,3 
Background
Docosahexaenoic acid (DHA) is required for normal brain function. The concentration of DHA in the brain depends on both diet and liver metabolism.
Objective
To determine rat brain DHA concentration and consumption in relation to dietary n-3 (omega-3) polyunsaturated fatty acid (PUFA) content and liver secretion of DHA derived from circulating α-linolenic acid (α-LNA).
Design
Following weaning, male rats were fed for 15 weeks either: (1) a diet with a high DHA and α-LNA content, (2) an n-3 PUFA “adequate” diet containing 4.6% α-LNA but no DHA, or (3) an n-3 PUFA “deficient” diet containing 0.2% α-LNA and no DHA. Brain DHA consumption rates were measured following intravenous infusion in unanesthetized rats of [1-14C]DHA, whereas liver and brain DHA synthesis rates were measured by infusing [1-14C]α-LNA.
Results
Brain DHA concentrations equaled 17.6 μm/g, 11.4 μm/g and 7.14 μm/g in rats on diets 1, 2 and 3, respectively. With each diet, the rate of brain DHA synthesis from α-LNA was much less than the brain DHA consumption rate, whereas the liver synthesis-secretion rate was 5-10 fold higher. Higher elongase 2 and 5 and desaturase Δ5 and Δ6 activities in liver than in brain accounted for the higher liver DHA synthesis rates; these enzymes were transcriptionally upregulated in liver but not in brain of rats fed the deficient diet.
Conclusions
While DHA is essential to normal brain function, this need might be covered by dietary α-LNA when liver metabolic conversion machinery is intact and the diet has a high α-LNA content.
doi:10.1016/j.plefa.2009.05.021
PMCID: PMC2967256  PMID: 19540098
docosahexaenoic acid; liver; brain; rat; n-3; omega-3; PUFA; imaging; metabolism; diet; synthesis; α-linolenic acid
9.  Fifteen weeks of dietary n-3 polyunsaturated fatty acid deprivation increases turnover of n-6 docosapentaenoic acid in rat-brain phospholipids 
Biochimica et Biophysica Acta  2011;1821(9):1235-1243.
Docosapentaenoic acid (DPAn-6, 22:5n-6) is an n-6 polyunsaturated fatty acid (PUFA) whose brain concentration can be increased in rodents by dietary n-3 PUFA deficiency, which may contribute to their behavioral dysfunction. We used our in vivo intravenous infusion method to see if brain DPAn-6 turnover and metabolism also were altered with deprivation. We studied male rats that had been fed for 15 weeks post-weaning an n-3 PUFA adequate diet containing 4.6% alpha-linolenic acid (α-LNA, 18:3n-3) or a deficient diet (0.2% α-LNA), each lacking docosahexaenoic acid (22:6n-3) and arachidonic acid (AA, 20:4n-6). [1-14C]DPAn-6 was infused intravenously for 5 min in unanesthetized rats, after which the brain underwent high-energy microwaving, and then was analyzed. The n-3 PUFA deficient compared with adequate diet increased DPAn-6 and decreased DHA concentrations in plasma and brain, while minimally changing brain AA concentration. Incorporation rates of unesterified DPAn-6 from plasma into individual brain phospholipids were increased 5.2–7.7 fold, while turnover rates were increased 2.1–4.7 fold. The observations suggest that increased metabolism and brain concentrations of DPAn-6 and its metabolites, together with a reduced brain DHA concentration, contribute to behavioral and functional abnormalities reported with dietary n-3 PUFA deprivation in rodents.
doi:10.1016/j.bbalip.2011.11.002
PMCID: PMC3348251  PMID: 22142872
docosapentaenoic; docosahexaenoic; deficient; turnover; kinetics; brain; diet; n-6; PUFA; arachidonic; rat; phospholipase A2
10.  Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice 
Biochimica et Biophysica Acta  2012;1821(9):1278-1286.
Calcium-independent phospholipase A2 group VIA (iPLA2β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA2β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA2β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA2β+/+) and knockout (iPLA2β−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA2, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA2β+/+ mice, iPLA2β−/− mice showed decreased rates of incorporation of unesterified DHA in plasma into brain phospholipids, reduced concentration of several fatty acid residues (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover rates in brain phospholipids did not differ between genotypes. In iPLA2β−/− mice, brain levels of iPLA2β mRNA, protein, and activity were decreased, as was the iPLA2γ (Group VIB PLA2) mRNA level. Brain levels of secretory sPLA2-V mRNA, protein, and activity and cytosolic cPLA2-IVA mRNA were increased in iPLA2β−/− mice. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, genetic iPLA2β deficiency in mice is associated with profound reorganization of lipid-metabolizing enzyme expression and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to the neurologic abnormalities in humans with iPLA2β mutations.
doi:10.1016/j.bbalip.2012.02.003
PMCID: PMC3393806  PMID: 22349267
iPLA2; knockout; mice; docosahexaenoic acid; brain; turnover; incorporation; lipid; PLA2G6; phospholipid metabolism
11.  Gabapentin’s minimal action on markers of rat brain arachidonic acid metabolism agrees with its inefficacy against bipolar disorder 
In rats, FDA-approved mood stabilizers used for treating bipolar disorder (BD) selectively downregulate brain markers of the arachidonic acid (AA) cascade, which are upregulated in postmortem BD brain. Phase III clinical trials show that gabapentin (GBP) is ineffective in treating BD. We hypothesized that GBP would not alter the rat brain AA cascade. Chronic GBP (10 mg/kg body weight, injected i.p. for 30 days) compared to saline vehicle did not significantly alter brain expression or activity of AA-selective cytosolic phospholipase A2 (cPLA2) IVA or secretory (s) PLA2 IIA, activity of cyclooxygenase-2, or prostaglandin or thromboxane concentrations. Plasma AA concentration was unaffected. These results, taken with evidence of an upregulated AA cascade in the BD brain and that approved mood stabilizers downregulate rat brain AA cascade, support the hypothesis that effective anti-BD drugs act by targeting the AA cascade, and suggest that the rat model might be used for drug screening
doi:10.1016/j.plefa.2012.06.003
PMCID: PMC3431015  PMID: 22841517
gabapentin; arachidonic acid; bipolar disorder; mood stabilizer; drug screening
12.  The utility of 11C-arachidonate PET to study in vivo dopaminergic neurotransmission in humans 
We developed a novel method to study dopaminergic neurotransmission using positron emission tomography (PET) with [1-11C]arachidonic acid ([1-11C]AA). Previous preclinical studies have shown the utility of [1-11C]AA as a marker of signal transduction coupled to cytosolic phospholipase A2 (cPLA2). Using [1-11C]AA and [15O]water PET, we measured regional incorporation coefficients K* for AA and regional cerebral blood flow (rCBF), respectively, in healthy male volunteers given the D1/D2 agonist (10 or 20 μg/kg subcutaneous) apomorphine. We confirmed a robust central dopaminergic response to apomorphine by observing significant increases in the serum concentration of growth hormone. We observed significant increases, as well as decreases in K* and increases in rCBF in response to apomorphine. These changes remained significant after covarying for handedness and apomorphine dosage. The magnitude of increases in K* was lower than those in our previous animal experiments, likely reflecting the smaller dose of apomorphine used in the current human study. Changes in K* may reflect neuronal signaling downstream of activated D2-like receptors coupled to cPLA2. Changes in rCBF are consistent with previous studies showing net functional effects of D1/D2 activation. [1-11C]AA PET may be useful for studying disturbances of dopaminergic neurotransmission in conditions such as Parkinson's disease and schizophrenia.
doi:10.1038/jcbfm.2011.171
PMCID: PMC3318145  PMID: 22167235
apomorphine; cPLA2; D1/D2 receptors; [1-11C]arachidonate PET; regional cerebral blood flow
13.  DIETARY N-6 POLYUNSATURATED FATTY ACID DEPRIVATION INCREASES DOCOSAHEXAENOIC ACID METABOLISM IN RAT BRAIN 
Journal of Neurochemistry  2012;120(6):985-997.
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 A2 (cPLA2-IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective Ca2+-independent iPLA2-VIA expression. We hypothesized that these changes are accompanied by upregulated 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. [1-14C]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 acidn-3 (DPAn-3, 22:5n-3) and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid. 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 promote neuroprotection. (199 words)
doi:10.1111/j.1471-4159.2011.07597.x
PMCID: PMC3296886  PMID: 22117540
linoleic acid; arachidonic PUFA; diet; turnover; metabolism; docosahexaenoic; kinetics; brain; alpha-linolenic; rat
14.  IMAGING BRAIN SIGNAL TRANSDUCTION AND METABOLISM VIA ARACHIDONIC AND DOCOSAHEXAENOIC ACID IN ANIMALS AND HUMANS 
Brain Research Bulletin  2011;87(2-3):154-171.
The polyunsaturated fatty acids (PUFAs), arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), important second messengers in brain, are released from membrane phospholipid following receptor-mediated activation of specific phospholipase A2 (PLA2) enzymes. We developed an in vivo method in rodents using quantitative autoradiography to image PUFA incorporation into brain from plasma, and showed that their incorporation rates equal their rates of metabolic consumption by brain. Thus, quantitative imaging of unesterified plasma AA or DHA incorporation into brain can be used as a biomarker of brain PUFA metabolism and neurotransmission. We have employed our method to image and quantify effects of mood stabilizers on brain AA/DHA incorporation during neurotransmission by muscarinic M1,3,5, serotonergic 5-HT2A/2C, dopaminergic D2-like (D2, D3, D4) or glutamatergic N-methyl-D-aspartic acid (NMDA) receptors, and effects of inhibition of acetylcholinesterase, of selective serotonin and dopamine reuptake transporter inhibitors, of neuroinflammation (HIV-1 and lipopolysaccharide) and excitotoxicity, and in genetically modified rodents. The method has been extended for the use with positron emission tomography (PET), and can be employed to determine how human brain AA/DHA signaling and consumption are influenced by diet, aging, disease and genetics.
doi:10.1016/j.brainresbull.2011.12.001
PMCID: PMC3274571  PMID: 22178644
arachidonic acid; bipolar disorder; brain imaging; docosahexaenoic acid; mood stabilizers; neuroinflammation
15.  Chronic Valproate Treatment Blocks D2-like Receptor-Mediated Brain Signaling via Arachidonic Acid in Rats 
Neuropharmacology  2011;61(8):1256-1264.
Background and Objective
Hyperdopaminergic signaling and an upregulated brain arachidonic acid (AA) cascade may contribute to bipolar disorder (BD). Lithium and carbamazepine, FDA-approved for the treatment of BD, attenuate brain dopaminergic D2-like (D2, D3, and D4) receptor signaling involving AA when given chronically to awake rats. We hypothesized that valproate (VPA), with mood-stabilizing properties, would also reduce the D2-like-mediated signaling via AA.
Methods
An acute dose of quinpirole (1 mg/kg) or saline was administered to unanesthetized rats that had been treated for 30 days with a therapeutically relevant dose of VPA (200 mg/kg/day) or vehicle. Regional brain AA incorporation coefficients, k*, and incorporation rates, Jin, markers of AA signaling and metabolism, were measured by quantitative autoradiography after intravenous [1-14C]AA infusion. Whole brain concentrations of prostaglandin (PG)E2 and thromboxane (TX)B2 also were measured.
Results
Quinpirole compared to saline significantly increased k* in 40 of 83 brain regions, and increased brain concentrations of PGE2 in chronic vehicle-treated rats. VPA treatment by itself reduced concentrations of plasma unesterified AA and whole brain PGE2 and TXB2, and blocked the quinpirole-induced increments in k* and PGE2.
Conclusion
These results further support our hypothesis that similar to lithium and carbamazepine, VPA downregulates brain dopaminergic D2-like receptor-signaling involving AA.
doi:10.1016/j.neuropharm.2011.07.025
PMCID: PMC3190603  PMID: 21839100
arachidonic acid; phospholipase A2; valproate; D2-like receptor; quinpirole; bipolar disorder
16.  Regulation of Rat Brain Polyunsaturated Fatty Acid (PUFA) Metabolism during Graded Dietary n-3 PUFA Deprivation 
Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation may elucidate dietary regulation of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (α-LNA). Compared with control diet (4.6% α-LNA), plasma DHA fell significantly at 1.7% dietary α-LNA while brain DHA remained unchanged down to 0.8% α-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA2 and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA2, sPLA2 and COX-2 activities were increased at or below 0.8% dietary α-LNA, possibly in response to elevated brain DPAn-6. In summary, homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary DHA despite reduced plasma DHA, when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA2 and COX-1 activities may reduce brain DHA loss.
doi:10.1016/j.plefa.2011.08.002
PMCID: PMC3208751  PMID: 21880477
n-3 PUFA deprivation; rat brain; phospholipase A2; docosapentaenoic acid
17.  The Synthesis and In Vivo Pharmacokinetics of Fluorinated Arachidonic Acid: Implications for Imaging Neuroinflammation 
Arachidonic acid (AA) is found in high concentrations in brain phospholipids and is released as a second messenger during neurotransmission and much more so during neuroinflammation and excitotoxicity. Upregulated brain AA metabolism associated with neuroinflammation has been imaged in rodents using [1-14C]AA and with PET in Alzheimer disease patients using [1-11C]AA. Radiotracer brain AA uptake is independent of cerebral blood flow, making it an ideal tracer despite altered brain functional activity. However, the 20.4-min radioactive half-life of 11C-AA and challenges of routinely synthesizing 11C fatty acids limit their translational utility as PET biomarkers.
Methods
As a first step to develop a clinically useful 18F-fluoroarachidonic acid (18F-FAA) with a long radioactive half-life of 109.8 min, we report here a high-yield stereoselective synthetic method of non-radioactive 20-19F-FAA. We tested its in vivo pharmacokinetics by infusing purified nonradioactive 19F-FAA intravenously for 5 min at 2 doses in unanesthetized mice and measured its plasma and brain distribution using gas chromatography–mass spectrometry.
Results
Incorporation coefficients of injected 19F-FAA into brain phospholipids (ratio of brain 19F-FAA concentration to plasma input function) were 3- to 29-fold higher for choline glycerophospholipid and phosphatidylinositol than for ethanolamine glycerophospholipid and phosphatidylserine at each of the 2 tested doses. The selectivities and values of incorporation coefficients were comparable to those reported after [1-14C]AA (the natural arachidonate) infusion in mice.
Conclusion
These results suggest that it would be worthwhile to translate our stereoselective synthetic method for 19F-FAA to synthesize positron-emitting 18F-FAA for human brain AA metabolism in neuroinflammatory disorders such as Alzheimer disease.
doi:10.2967/jnumed.112.105734
PMCID: PMC3509950  PMID: 22851635
positron emitting tomography (PET); fluorinated arachidonic acid; fluoroarachidonic acid; brain; phospholipids; arachidonic acid signaling; imaging; neuroinflammation; excitotoxicity; biomarker; Alzheimer’s disease; cancer
18.  Upregulated expression of brain enzymatic markers of arachidonic and docosahexaenoic acid metabolism in a rat model of the metabolic syndrome 
BMC Neuroscience  2012;13:131.
Background
In animal models, the metabolic syndrome elicits a cerebral response characterized by altered phospholipid and unesterified fatty acid concentrations and increases in pro-apoptotic inflammatory mediators that may cause synaptic loss and cognitive impairment. We hypothesized that these changes are associated with phospholipase (PLA2) enzymes that regulate arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-6) acid metabolism, major polyunsaturated fatty acids in brain. Male Wistar rats were fed a control or high-sucrose diet for 8 weeks. Brains were assayed for markers of AA metabolism (calcium-dependent cytosolic cPLA2 IVA and cyclooxygenases), DHA metabolism (calcium-independent iPLA2 VIA and lipoxygenases), brain-derived neurotrophic factor (BDNF), and synaptic integrity (drebrin and synaptophysin). Lipid concentrations were measured in brains subjected to high-energy microwave fixation.
Results
The high-sucrose compared with control diet induced insulin resistance, and increased phosphorylated-cPLA2 protein, cPLA2 and iPLA2 activity and 12-lipoxygenase mRNA, but decreased BDNF mRNA and protein, and drebrin mRNA. The concentration of several n-6 fatty acids in ethanolamine glycerophospholipids and lysophosphatidylcholine was increased, as was unesterified AA concentration. Eicosanoid concentrations (prostaglandin E2, thromboxane B2 and leukotriene B4) did not change.
Conclusion
These findings show upregulated brain AA and DHA metabolism and reduced BDNF and drebrin, but no changes in eicosanoids, in an animal model of the metabolic syndrome. These changes might contribute to altered synaptic plasticity and cognitive impairment in rats and humans with the metabolic syndrome.
doi:10.1186/1471-2202-13-131
PMCID: PMC3531256  PMID: 23110484
Arachidonic acid; Docosahexaenoic acid; BDNF; Brain; Polyunsaturated fatty acids (PUFA); Metabolic syndrome; Drebrin; Sucrose; Insulin resistance
19.  Effects of chronic clozapine administration on markers of arachidonic acid cascade and synaptic integrity in rat brain 
Psychopharmacology  2012;222(4):663-674.
The mode of action of clozapine, an atypical antipsychotic approved for treating schizophrenia and bipolar disorder (BD) mania, remains unclear. We tested for overlap with the actions of the mood stabilizers, lithium, carbamazepine and valproate, which downregulate arachidonic acid (AA) cascade markers in rat brain and upregulate BDNF. AA cascade markers are upregulated in the postmortem BD brain in association with neuroinflammation and synaptic loss, while BDNF is decreased. Rats were injected intraperitoneally with a therapeutically relevant dose of clozapine (10 mg/kg/day) or with saline for 30 days, and AA cascade and synaptic markers and BDNF were measured in the brain. Compared with saline-injected rats, chronic clozapine increased brain activity, mRNA and protein levels of docosahexaenoic acid (DHA)-selective calcium-independent phospholipase A2 type VIA (iPLA2), mRNA and protein levels of BDNF and of the postsynaptic marker, drebrin, while decreasing cyclooxygenase (COX) activity and concentration of prostaglandin E2 (PGE2), a proinflammatory AA metabolite. Activity and expression of AA-selective calcium-dependent cytosolic cPLA2 type IVA and of secretory sPLA2 Type II were unchanged. These results show overlap with effects of mood stabilizers with regard to downregulation of COX activity and PGE2 and to increased BDNF, and suggest a common action against the reported neuropathology of BD. Additionally, the increased iPLA2 expression following clozapine suggests increased production of anti-inflammatory DHA metabolites, consistent with reports that dietary n-3 polyunsaturated fatty acid supplementation is beneficial in BD.
doi:10.1007/s00213-012-2671-7
PMCID: PMC3478065  PMID: 22414961
atypical; antipsychotic; arachidonic acid; BDNF; bipolar disorder; drebrin; cyclooxygenase; rat; clozapine; brain; docosahexaenoic; schizophrenia; mood stabilizer; iPLA2; PGE2
20.  Chronic lithium feeding reduces upregulated brain arachidonic acid metabolism in HIV-1 transgenic rat 
Background
HIV-1 transgenic (Tg) rats, a model for human HIV-1 associated neurocognitive disorder (HAND), show upregulated markers of brain arachidonic acid (AA) metabolism with neuroinflammation after 7 months of age. Since lithium decreases AA metabolism in a rat lipopolysaccharide model of neuroinflammation, and may be useful in HAND, we hypothesized that lithium would dampen upregulated brain AA metabolism in HIV-1 Tg rats.
Methods
Regional brain AA incorporation coefficients k* and rates Jin, markers of AA signaling and metabolism, were measured in 81 brain regions using quantitative autoradiography, after intravenous [1-14C] AA infusion in unanesthetized 10-month-old HIV-1 Tg and age-matched wildtype rats that had been fed a control or LiCl diet for 6 weeks.
Results
k* and Jin for AA were significantly higher in HIV-1 Tg than wildtype rats fed the control diet. Lithium feeding reduced plasma unesterified AA concentration in both groups and Jin in wildtype rats, and blocked increments in k* (19 of 54 regions) and Jin (77 of 81 regions) in HIV-1 Tg rats.
Conclusion
These in vivo neuroimaging data indicate that lithium treatment dampened upregulated brain AA metabolism in HIV-1 Tg rats. Lithium may improve cognitive dysfunction and be neuroprotective in HIV-1 patients with HAND through a comparable effect.
doi:10.1007/s11481-012-9381-0
PMCID: PMC3478068  PMID: 22760927
HIV-1; lithium; arachidonic acid; brain imaging; phospholipase A2; metabolism
21.  Bilateral common carotid artery ligation transiently changes brain lipid metabolism in rats 
Neurochemical research  2012;37(7):1490-1498.
Brain lipid metabolism was studied in rats following permanent bilateral common carotid artery ligation (BCCL), a model for chronic cerebral hypoperfusion. Unesterified (free) fatty acids (uFA) and acyl-CoA concentrations were measured 6 h, 24 h, and 7 days after BCCL or sham surgery, in high energy-microwaved brain. In BCCL compared to sham rats, cPLA2 immunoreactivity in piriform cortex, and concentrations of total uFA and arachidonoyl-CoA, an intermediate for arachidonic acid reincorporation into phospholipids, were increased only at 6 h. At 24 h, immunoreactivity for secretory phospholipase A2 (sPLA2), which may regulate blood flow, was increased near cortical and hippocampal blood vessels. BCCL did not affect difference brain IB4+ microglia, glial fibrillary acidic protein (GFAP)+ astrocytes, cyclooxygenase-2 (COX-2) immunoreactivity at any time, but increased cytosolic cPLA2 immunoreactivity in one region at 6 h. Thus, BCCL affected brain lipid metabolism transiently, likely because of compensatory sPLA2-mediated vasodilation, without producing evidence of neuroinflammation.
doi:10.1007/s11064-012-0740-2
PMCID: PMC3478069  PMID: 22422289
arachidonic acid; ischemia; carotid; ligation; acyl-CoA; brain; cPLA2; sPLA2; rat; brain; neuroinflammation
22.  Knocking out the dopamine reuptake transporter (DAT) does not change the baseline brain arachidonic acid signal in the mouse 
Background
Dopamine transporter (DAT) homozygous knockout (DAT−/−) mice have a 10-fold higher extracellular DA concentration in the caudate-putamen and nucleus accumbens than do wildtype (DAT+/+) mice, but show reduced presynaptic DA synthesis and fewer postsynaptic D2 receptors. One aspect of neurotransmission involves DA binding to postsynaptic D2-like receptors coupled to cytosolic phospholipase A2 (cPLA2), releasing second messenger arachidonic acid (AA) from synaptic membrane phospholipid. We hypothesized that tonic overactivation of D2-like receptors in DAT−/− mice due to elevated DA would not increase brain AA signaling, because of compensatory downregulation of postsynaptic signaling mechanisms.
Methods
[1-14C]AA was infused intravenously for 3 min in unanesthetized DAT+/+, heterozygous (DAT+/−) and DAT−/− mice. AA incorporation coefficients k* and rates Jin, markers of AA metabolism and signaling, were imaged in 83 brain regions using quantitative autoradiography brain cPLA2-IV activity also was measured.
Results
Neither k* nor Jin for AA in any brain region, or in brain cPLA2-IV activity, differed significantly between DAT−/−, DAT+/− and DAT+/+ mice.
Conclusions
These results differ from reported increases in k* and Jin for AA, and brain cPLA2 expression, in serotonin reuptake transporter (5-HTT) knockout mice, and suggest that postsynaptic dopaminergic neurotransmission mechanisms involving AA are downregulated despite elevated DA in DAT−/− mice.
doi:10.3109/00207454.2012.665972
PMCID: PMC3464054  PMID: 22376027
arachidonic acid; phospholipase A2; dopamine; dopamine transporter; knockout; mouse
23.  Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E2 concentration in rat brain 
Journal of neurochemistry  2011;119(2):364-376.
Olanzapine (OLZ) is used to treat bipolar disorder, but its therapeutic mechanism of action is not clear. Arachidonic acid (AA, 20:4n-6) plays a critical role in brain signaling and an upregulated AA metabolic cascade was reported in postmortem brains from bipolar disorder patients. Here we tested whether, similar to the action of the mood stabilizers lithium, carbamazepine and valproate, chronic OLZ treatment would reduce AA turnover in rat brain. We administered OLZ (6 mg/kg/day) or vehicle i.p. to male rats once daily for 21 days. A washout group received 21 days of OLZ followed by vehicle on day 22. Two hours after the last injection, [1-14C]AA (170 µCi/kg) was infused intravenously for 5 min, and timed arterial blood samples were taken. After the rat was euthanized at 5 min, its brain was microwaved, removed and analyzed. Chronic OLZ decreased plasma unesterified AA concentration, AA incorporation rates and AA turnover in brain phospholipids. These effects were absent after washout. Consistent with reduced AA turnover, OLZ decreased brain cyclooxygenase activity and the brain concentration of the proinflammatory AA-derived metabolite, prostaglandin E2. In view of upregulated brain AA metabolic markers in bipolar disorder, the abilities of OLZ and the mood stabilizers to commonly decrease prostaglandin E2 and AA turnover in rat brain phospholipids, albeit by different mechanisms, may be related to their efficacy against the disease.
doi:10.1111/j.1471-4159.2011.07410.x
PMCID: PMC3188676  PMID: 21812779
bipolar disorder; antipsychotic; washout; phospholipid; cyclooxygenase; prostaglandin E2
24.  Lamotrigine blocks NMDA receptor-initiated arachidonic acid signalling in rat brain: Implications for its efficacy in bipolar disorder 
An upregulated brain arachidonic acid (AA) cascade and a hyperglutamatergic state characterize bipolar disorder (BD). Lamotrigine (LTG), a mood stabilizer approved for treating BD, is reported to interfere with glutamatergic neurotransmission involving N-methyl-D-aspartate receptors (NMDARs). NMDARs allow extracellular calcium into the cell, thereby stimulating calcium-dependent cytosolic phospholipase A2 (cPLA2) to release arachidonic acid (AA) from membrane phospholipid. We hypothesized that LTG, like other approved mood stabilizers, would reduce NMDAR-mediated AA signaling in rat brain. An acute subconvulsant dose of NMDA (25 mg/kg) or saline was administered intraperitoneally to unanesthetized rats that had been treated p.o. daily for 42 days with vehicle or a therapeutically relevant dose of LTG (10 mg/kg/.d). Regional brain AA incorporation coefficients k* and rates Jin, AA signals, were measured using quantitative autoradiography after intravenous [1-14C]AA infusion, as were other AA cascade markers. In chronic vehicle-treated rats, acute NMDA compared to saline increased k* and Jin in widespread regions of the brain, as well as prostaglandin (PG)E2 and thromboxane B2 concentrations. Chronic LTG treatment compared to vehicle reduced brain cyclooxygenase (COX) activity, PGE2 concentration, and DNA binding activity of the COX-2 transcription factor, NF-κB. Pretreatment with chronic LTG blocked the acute NMDA effects on AA cascade markers. In summary, chronic LTG like other mood stabilizers blocks NMDA-mediated signaling involving the AA metabolic cascade. Since markers of the AA cascade and of NMDAR signaling are up-regulated in the postmortem BD brain, mood stabilizers generally may be effective in BD by dampening NMDAR signalling and the AA cascade.
doi:10.1017/S1461145711001003
PMCID: PMC3204186  PMID: 21733229
arachidonic acid; cyclooxygenase; mood stabilizer; NMDA receptor; phospholipase A2
25.  Dose-dependent changes in neuroinflammatory and arachidonic acid cascade markers with synaptic marker loss in rat lipopolysaccharide infusion model of neuroinflammation 
BMC Neuroscience  2012;13:50.
Background
Neuroinflammation, caused by six days of intracerebroventricular infusion of bacterial lipopolysaccharide (LPS), stimulates rat brain arachidonic acid (AA) metabolism. The molecular changes associated with increased AA metabolism are not clear. We examined effects of a six-day infusion of a low-dose (0.5 ng/h) and a high-dose (250 ng/h) of LPS on neuroinflammatory, AA cascade, and pre- and post-synaptic markers in rat brain. We used artificial cerebrospinal fluid-infused brains as controls.
Results
Infusion of low- or high-dose LPS increased brain protein levels of TNFα, and iNOS, without significantly changing GFAP. High-dose LPS infusion upregulated brain protein and mRNA levels of AA cascade markers (cytosolic cPLA2-IVA, secretory sPLA2-V, cyclooxygenase-2 and 5-lipoxygenase), and of transcription factor NF-κB p50 DNA binding activity. Both LPS doses increased cPLA2 and p38 mitogen-activated protein kinase levels, while reducing protein levels of the pre-synaptic marker, synaptophysin. Post-synaptic markers drebrin and PSD95 protein levels were decreased with high- but not low-dose LPS.
Conclusions
Chronic LPS infusion has differential effects, depending on dose, on inflammatory, AA and synaptic markers in rat brain. Neuroinflammation associated with upregulated brain AA metabolism can lead to synaptic dysfunction.
doi:10.1186/1471-2202-13-50
PMCID: PMC3464147  PMID: 22621398
Arachidonic acid; Cytokine; Synapse; Drebrin; Lipopolysaccharide; Synaptophysin; Phospholipase A2; Neuroinflammation; NF-κB

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