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Background: Health authorities are near universal in their recommendation to replace sugar-sweetened beverages (SSBs) with water. Non-nutritive sweetened beverages (NSBs) are not as widely recommended as a replacement strategy due to a lack of established benefits and concerns they may induce glucose intolerance through changes in the gut microbiome. The STOP Sugars NOW trial aims to assess the effect of the substitution of NSBs (the “intended substitution”) versus water (the “standard of care substitution”) for SSBs on glucose tolerance and microbiota diversity. Design and Methods: The STOP Sugars NOW trial (NCT03543644) is a pragmatic, “head-to-head”, open-label, crossover, randomized controlled trial conducted in an outpatient setting. Participants were overweight or obese adults with a high waist circumference who regularly consumed ≥1 SSBs daily. Each participant completed three 4-week treatment phases (usual SSBs, matched NSBs, or water) in random order, which were separated by ≥4-week washout. Blocked randomization was performed centrally by computer with allocation concealment. Outcome assessment was blinded; however, blinding of participants and trial personnel was not possible. The two primary outcomes are oral glucose tolerance (incremental area under the curve) and gut microbiota beta-diversity (weighted UniFrac distance). Secondary outcomes include related markers of adiposity and glucose and insulin regulation. Adherence was assessed by objective biomarkers of added sugars and non-nutritive sweeteners and self-report intake. A subset of participants was included in an Ectopic Fat sub-study in which the primary outcome is intrahepatocellular lipid (IHCL) by 1H-MRS. Analyses will be according to the intention to treat principle. Baseline results: Recruitment began on 1 June 2018, and the last participant completed the trial on 15 October 2020. We screened 1086 participants, of whom 80 were enrolled and randomized in the main trial and 32 of these were enrolled and randomized in the Ectopic Fat sub-study. The participants were predominantly middle-aged (mean age 41.8 ± SD 13.0 y) and had obesity (BMI of 33.7 ± 6.8 kg/m2) with a near equal ratio of female: male (51%:49%). The average baseline SSB intake was 1.9 servings/day. SSBs were replaced with matched NSB brands, sweetened with either a blend of aspartame and acesulfame-potassium (95%) or sucralose (5%). Conclusions: Baseline characteristics for both the main and Ectopic Fat sub-study meet our inclusion criteria and represent a group with overweight or obesity, with characteristics putting them at risk for type 2 diabetes. Findings will be published in peer-reviewed open-access medical journals and provide high-level evidence to inform clinical practice guidelines and public health policy for the use NSBs in sugars reduction strategies. Trial registration: ClinicalTrials.gov identifier, NCT03543644.

Fatty acid-binding protein 7 (FABP7), one of the fatty acid (FA) chaperones involved in the modulation of intracellular FA metabolism, is highly expressed in glioblastoma, and its expression is associated with decreased patients' prognosis. Previously, we demonstrated that FABP7 requires its binding partner to exert its function and that a mutation in the FA-binding site of FABP7 affects tumour biology. Here, we explored the role of FA ligand binding for FABP7 function in tumour proliferation and examined the mechanism of FABP7 and ligand interaction in tumour biology. We discovered that among several FA treatment, oleic acid (OA) boosted cell proliferation of FABP7-expressing cells. In turn, OA increased FABP7 nuclear localization, and the accumulation of FABP7-OA complex in the nucleus induced the formation of nuclear lipid droplet (nLD), as well as an increase in colocalization of nLD with promyelocytic leukaemia (PML) nuclear bodies. Furthermore, OA increased mRNA levels of proliferation-related genes in FABP7-expressing cells through histone acetylation. Interestingly, these OA-boosted functions were abrogated in FABP7-knockout cells and mutant FABP7-overexpressing cells. Thus, our findings suggest that FABP7-OA intracellular interaction may modulate nLD formation and the epigenetic status thereby enhancing transcription of proliferation-regulating genes, ultimately driving tumour cell proliferation.

Accumulation of excess lipids in non-adipose tissues, such as the hypothalamus, is termed lipotoxicity and causative of free fatty acid-mediated pathology in metabolic disease. This study aimed to elucidate the molecular mechanisms behind oleate (OA)- and palmitate (PA)-mediated changes in hypothalamic neurons. Using the well-characterized hypothalamic neuronal cell model, mHypoE-46, we assessed gene changes through qRT-PCR, cell death with quantitative imaging, PA metabolism using stable isotope labeling, and cellular mechanisms using pharmacological modulation of lipid metabolism and autophagic flux. Palmitate (PA) disrupts gene expression, including Npy, Grp78, and Il-6 mRNA in mHypoE-46 hypothalamic neurons. Blocking PA metabolism using triacsin-C prevented the increase of these genes, implying that these changes depend on PA intracellular metabolism. Co-incubation with oleate (OA) is also potently protective and prevents cell death induced by increasing concentrations of PA. However, OA does not decrease U

Background PUFAs play vital roles in the development, maintenance, and functioning of circuitries that regulate reward and social behaviors. Therefore, modulations in PUFA concentrations of these brain regions may disrupt reward and social circuitries contributing to mood disorders, developmental disabilities, and addictions. Though much is known about regional and phospholipid-pool-specific PUFA concentrations, less is known about the effects of dietary interventions that concurrently lowers n-6 PUFA and supplements n-3 PUFA, on brain PUFA concentrations. There is even less knowledge on the effects of sex on brain PUFA concentrations. Objective This study aimed to comprehensively examine the interaction effects of diet (D), sex (S), brain regions (BR), and phospholipid pools (PL) on brain PUFA concentrations. Methods Male and female C57BL/6J mice were fed 1 of 4 custom-designed diets varying in linoleic acid (LNA) (8 en% or 1 en%) and eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) (0.4 en% or 0 en%) concentrations from in utero to 15 weeks old. At 15 weeks old, the prefrontal cortex, dorsal striatum, and cerebellum were collected. Fatty acids of 5 major PL were quantified by GC-flame ionization detection. Repeated measures ANOVA was used to test for differences among the groups for D, S, BR, and PL. Results No significant 4-way interactions on PUFA concentrations. DHA, predominant n-3 PUFA, concentrations were dependent on significant D × BR × PL interactions. DHA concentration was not affected by sex. Arachidonic acid (ARA; predominant n-6 PUFA) concentrations were not dependent on 3-way interactions. However, significant 2-way D × PL, BR × PL, and D × Sinteractions affected ARA concentrations. Brain fatty acid concentrations were differentially affected by various combinations of D, S, BR, and PL interactions. Conclusion Though DHA concentrations are not affected by sex, ARA concentrations are affected by interactions of the 4 variables examined. This study provides comprehensive references in the investigation of complex interactions between factors that affect brain PUFA concentrations in mice.

Protein arginine methyltransferases (PRMTs) are a family of enzymes involved in gene regulation and protein/histone modifications. PRMT8 is primarily expressed in the central nervous system, specifically within the cellular membrane and synaptic vesicles. Recently, PRMT8 has been described to play key roles in neuronal signaling such as a regulator of dendritic arborization, synaptic function and maturation, and neuronal differentiation and plasticity. Here, we examined the role of PRMT8 in response to hypoxia-induced stress in brain metabolism. Our results from liquid chromatography mass spectrometry, mitochondrial oxygen consumption rate, and protein analyses indicate that PRMT8(-/-) knockout mice presented with altered membrane phospholipid composition, decreased mitochondrial stress capacity, and increased neuroinflammatory markers, such as tumor necrosis factor alpha and ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) after hypoxic stress. Furthermore, adenovirus-based overexpression of PRMT8 reversed the changes in membrane phospholipid composition, mitochondrial stress capacity, and neuroinflammatory markers. Together, our findings establish PRMT8 as an important regulatory component of membrane phospholipid composition, short-term memory function, mitochondrial function, and neuroinflammation in response to hypoxic stress.

Wnt signaling pathway suppresses vascular endothelium transcytosis in the BRB through activating MFSD2A., Breakdown of the blood-retinal barrier (BRB) causes retinal edema and vision loss. We investigated the role of Wnt signaling in maintaining the BRB by limiting transcytosis. Mice lacking either the Wnt co-receptor low-density lipoprotein receptor–related protein 5 (Lrp5−/−) or the Wnt ligand Norrin (Ndpy/−) exhibit increased retinal vascular leakage and enhanced endothelial transcytosis. Wnt signaling directly controls the transcription of an endothelium-specific transcytosis inhibitor, major facilitator superfamily domain–containing protein 2a (MFSD2A), in a β-catenin–dependent manner. MFSD2A overexpression reverses Wnt deficiency–induced transcytosis in endothelial cells and in retinas. Moreover, Wnt signaling mediates MFSD2A-dependent vascular endothelium transcytosis through a caveolin-1 (CAV-1)–positive caveolae pathway. In addition, levels of omega-3 fatty acids are also decreased in Wnt signaling–deficient retinas, reflecting the basic function of MFSD2A as a lipid transporter. Our findings uncovered the Wnt/β-catenin/MFSD2A/CAV-1 axis as a key pathway governing endothelium transcytosis and inner BRB integrity.

There is a gap in understanding the effect of the essential ω-3 and ω-6 long-chain polyunsaturated fatty acids (LCPUFA) on Phase I retinopathy of prematurity (ROP), which precipitates proliferative ROP. Postnatal hyperglycemia contributes to Phase I ROP by delaying retinal vascularization. In mouse neonates with hyperglycemia-associated Phase I retinopathy, dietary ω-3 (vs. ω-6 LCPUFA) supplementation promoted retinal vessel development. However, ω-6 (vs. ω-3 LCPUFA) was also developmentally essential, promoting neuronal growth and metabolism as suggested by a strong metabolic shift in almost all types of retinal neuronal and glial cells identified with single-cell transcriptomics. Loss of adiponectin (APN) in mice (mimicking the low APN levels in Phase I ROP) decreased LCPUFA levels (including ω-3 and ω-6) in retinas under normoglycemic and hyperglycemic conditions. ω-3 (vs. ω-6) LCPUFA activated the APN pathway by increasing the circulating APN levels and inducing expression of the retinal APN receptor. Our findings suggested that both ω-3 and ω-6 LCPUFA are crucial in protecting against retinal neurovascular dysfunction in a Phase I ROP model; adequate ω-6 LCPUFA levels must be maintained in addition to ω-3 supplementation to prevent retinopathy. Activation of the APN pathway may further enhance the ω-3 and ω-6 LCPUFA’s protection against ROP.

The rate at which dietary α-linolenic acid (ALA) is desaturated and elongated to its longer-chain n-3 polyunsaturated fatty acid (PUFA) in humans is not agreed upon. In this study, we applied a methodology developed using rodents to investigate the whole-body, presumably hepatic, synthesis-secretion rates of esterified n-3 PUFA from circulating unesterified ALA in 2 healthy overweight women after 10 weeks of low-linoleate diet exposure. During continuous iv infusion of d5-ALA, 17 arterial blood samples were collected from each subject at -10, 0, 10, 20, 40, 60, 80, 100, 120, 150, 180, and 210 min, and at 4, 5, 6, 7, and 8 h after beginning infusion. Plasma esterified d5-n-3 PUFA concentrations were plotted against the infusion time and fit to a sigmoidal curve using nonlinear regression. These curves were used to estimate kinetic parameters using a kinetic analysis developed using rodents. Calculated synthesis-secretion rates of esterified eicosapentaenoate, n-3 docosapentaenoate, docosahexaenoic acid, tetracosapentaenate, and tetracosahexaenoate from circulating unesterified ALA were 2.1 and 2.7; 1.7 and 5.3; 0.47 and 0.27; 0.30 and 0.30; and 0.32 and 0.27 mg/day for subjects S01 and S02, respectively. This study provides new estimates of whole-body synthesis-secretion rates of esterified longer-chain n-3 PUFA from circulating unesterified ALA in human subjects. This method now can be extended to study factors that regulate human whole-body PUFA synthesis-secretion in health and disease.

Deficiency in retinoid acid receptor-related orphan receptor alpha (RORα) of staggerer mice results in extensive granule and Purkinje cell loss in the cerebellum as well as in learned motor deficits, cognition impairments and perseverative tendencies that are commonly observed in autistic spectrum disorder (ASD). The effects of RORα on brain lipid metabolism associated with cerebellar atrophy remain unexplored. The aim of this study is to examine the effects of RORα deficiency on brain phospholipid fatty acid concentrations and compositions. Staggerer mice (Rora(sg/sg)) and wildtype littermates (Rora(+/+)) were fed n-3 polyunsaturated fatty acids (PUFA) containing diets ad libitum. At 2 months and 7 or more months old, brain total phospholipids fatty acids were quantified by gas chromatography-flame ionization detection. In the cerebellum, all fatty acid concentrations were reduced in 2 months old mice. Since total fatty acid concentrations were significantly different at 2-month-old, we examined changes in fatty acid composition. The composition of ARA was not significantly different between genotypes; though DHA composition remained significantly lowered. Despite cerebellar atrophy at >7-months-old, cerebellar fatty acid concentrations had recovered comparably to wildtype control. Therefore, RORα may be necessary for fatty acid accretions during neurodevelopment. Specifically, the effects of RORα on PUFA metabolisms are region-specific and age-dependent.

The light‐sensitive photoreceptors in the retina are extremely metabolically demanding and have the highest density of mitochondria of any cell in the body. Both physiological and pathological retinal vascular growth and regression are controlled by photoreceptor energy demands. It is critical to understand the energy demands of photoreceptors and fuel sources supplying them to understand neurovascular diseases. Retinas are very rich in lipids, which are continuously recycled as lipid‐rich photoreceptor outer segments are shed and reformed and dietary intake of lipids modulates retinal lipid composition. Lipids (as well as glucose) are fuel substrates for photoreceptor mitochondria. Dyslipidemia contributes to the development and progression of retinal dysfunction in many eye diseases. Here, we review photoreceptor energy demands with a focus on lipid metabolism in retinal neurovascular disorders.

International audience; The results of several meta-analyses suggest that eicosapentaenoic acid (EPA) supplementation is therapeutic in managing the symptoms of major depression. It was previously assumed that because EPA is extremely low in the brain it did not cross the blood-brain barrier and any therapeutic effects it exerted would be via the periphery. However, more recent studies have established that EPA does enter the brain, but is rapidly metabolised following entry. While EPA does not accumulate within the brain, it is present in microglia and homeostatic mechanisms may regulate its esterification to phospholipids that serve important roles in cell signaling. Furthermore, a variety of signaling molecules from EPA have been described in the periphery and they have the potential to exert effects within the brain. If EPA is confirmed to be therapeutic in major depression as a result of adequately powered randomized clinical trials, future research on brain EPA metabolism could lead to the discovery of novel targets for treating or preventing major depression.

Background Docosahexaenoic acid (DHA) is an ω-3 (n-3) polyunsaturated fatty acid (PUFA) thought to be important for brain function. Although the main dietary source of DHA is fish, DHA can also be synthesized from α-linolenic acid (ALA), which is derived from plants. Enzymes involved in DHA synthesis are also active toward ω-6 (n-6) PUFAs to synthesize docosapentaenoic acid n-6 (DPAn-6). It is unclear whether DHA synthesis from ALA is sufficient to maintain brain DHA. Objective The objective of this study was to determine how different amounts of dietary ALA would affect whole-body DHA and DPAn-6 synthesis rates. Methods Male Long-Evans rats were fed an ALA-deficient diet (ALA-D), an ALA-adequate (ALA-A) diet, or a high-ALA (ALA-H) diet for 8 wk from weaning. Dietary ALA concentrations were 0.07%, 3%, and 10% of the fatty acids, and ALA was the only dietary PUFA that differed between the diets. After 8 wk, steady-state stable isotope infusion of labeled ALA and linoleic acid (LA) was performed to determine the in vivo synthesis-secretion rates of DHA and DPAn-6. Results Rats fed the ALA-A diet had an ∼2-fold greater capacity to synthesize DHA than did rats fed the ALA-H and ALA-D diets, and a DHA synthesis rate that was similar to that of rats fed the ALA-H diet. However, rats fed the ALA-D diet had a 750% lower DHA synthesis rate than rats fed the ALA-A and ALA-H diets. Despite enrichment into arachidonic acid, we did not detect any labeled LA appearing as DPAn-6. Conclusions Increasing dietary ALA from 3% to 10% of fatty acids did not increase DHA synthesis rates, because of a decreased capacity to synthesize DHA in rats fed the ALA-H diet. Tissue concentrations of DPAn-6 may be explained at least in part by longer plasma half-lives.

Docosahexaenoic acid (DHA) is thought to be important for brain function. The main dietary source of DHA is fish, however, DHA can also be synthesized from precursor omega-3 polyunsaturated fatty acids (n-3 PUFA), the most abundantly consumed being α-linolenic acid (ALA). The enzymes required to synthesize DHA from ALA are also used to synthesize longer chain omega-6 (n-6) PUFA from linoleic acid (LNA). The large increase in LNA consumption that has occurred over the last century has led to concern that LNA and other n-6 PUFA outcompete n-3 PUFA for enzymes involved in DHA synthesis, and therefore, decrease overall DHA synthesis. To assess this, rats were fed diets containing LNA at 53 (high LNA diet), 11 (medium LNA diet) or 1.5% (low LNA diet) of the fatty acids with ALA being constant across all diets (approximately 4% of the fatty acids). Rats were maintained on these diets from weaning for 8 weeks, at which point they were subjected to a steady-state infusion of labeled ALA and LNA to measure DHA and arachidonic acid (ARA) synthesis rates. DHA and ARA synthesis rates were generally highest in rats fed the medium and high LNA diets, while the plasma half-life of DHA was longer in rats fed the low LNA diet. Therefore, increasing dietary LNA, in rats, did not impair DHA synthesis; however, low dietary LNA led to a decrease in DHA synthesis with tissue concentrations of DHA possibly being maintained by a longer DHA half-life.

To determine how the level of dietary n-6 PUFA affects the rate of loss of arachidonic acid (ARA) and DHA in brain phospholipids, male rats were fed either a deprived or adequate n-6 PUFA diet for 15 weeks postweaning, and then subjected to an intracerebroventricular infusion of (3)H-ARA or (3)H-DHA. Brains were collected at fixed times over 128 days to determine half-lives and the rates of loss from brain phospholipids (J out). Compared with the adequate n-6 PUFA rats, the deprived n-6-PUFA rats had a 15% lower concentration of ARA and an 18% higher concentration of DHA in their brain total phospholipids. Loss half-lives of ARA in brain total phospholipids and fractions (except phosphatidylserine) were longer in the deprived n-6 PUFA rats, whereas the J out was decreased. In the deprived versus adequate n-6 PUFA rats, the J out of DHA was higher. In conclusion, chronic n-6 PUFA deprivation decreases the rate of loss of ARA and increases the rate of loss of DHA in brain phospholipids. Thus, a low n-6 PUFA diet can be used to target brain ARA and DHA metabolism.

The brain has a unique polyunsaturated fatty acid composition, with high levels of arachidonic and docosahexaenoic acids (DHA) while levels of eicosapentaenoic acid (EPA) are several orders of magnitude lower. As evidence accumulated that fatty acid entry into the brain was not selective and, in fact, that DHA and EPA enter the brain at similar rates, new mechanisms were required to explain their large concentration differences in the brain. Here we summarize recent research demonstrating that EPA is rapidly and extensively β-oxidized upon entry into the brain. Although the ATP generated from the β-oxidation of EPA is low compared to the use of glucose, fatty acid β-oxidation may serve to regulate brain fatty acid levels in the absence of selective transportation. Furthermore, when β-oxidation of EPA is blocked, desaturation of EPA increases and Land׳s recycling decreases to maintain low EPA levels.

Recent studies suggest that at least two pools of plasma docosahexaenoic acid (DHA) can supply the brain: non-esterified DHA (NE-DHA) and lysophosphatidylcholine (lysoPtdCho)-DHA. In contrast to NE-DHA, brain uptake of lysoPtdCho-DHA appears to be mediated by a specific transporter, but whether both forms of DHA supply undergo the same metabolic fate, particularly with regards to enrichment of specific phospholipid (PL) subclasses, remains to be determined. This study aimed to evaluate brain uptake of NE-DHA and lysoPtdCho-DHA into brain PL classes. Fifteen-week-old rats were infused intravenously with radiolabelled NE-14C-DHA or lysoPtdCho-14C-DHA (n=4/group) over five mins to achieve a steady-state plasma level. PLs were extracted from the brain and separated by thin layer chromatography and radioactivity was quantified by liquid scintillation counting. The net rate of entry of lysoPtdCho-DHA into the brain was between 59% and 86% lower than the net rate of entry of NE-DHA, depending on the PL class. The proportion of total PL radioactivity in the lysoPtdCho-14C-DHA group compared to the NE-14C-DHA group was significantly higher in choline glycerophospholipids (ChoGpl) (48% vs 28%, respectively) but lower in ethanolamine glycerophospholipids (EtnGpl) (32% vs 46%, respectively). In both groups, radioactivity was disproportionally high in phosphatidylinositol and ChoGpl but low in phosphatidylserine and EtnGpl compared to the corresponding DHA pool size. This suggests that DHA undergoes extensive PL remodeling after entry into the brain.

OBJECTIVES: Our objective is to examine the effects of lowering dietary omega-6 polyunsaturated fatty acids (n-6 PUFA, linoleic acid; LA) and/or supplementation of omega-3 PUFA (n-3 PUFA, eicosapentaenoic acid and docosahexaenoic acid; EPA and DHA) on voluntary ethanol binge drinking and pathway networks involved in the interactions of PUFA and ethanol in mesolimbic circuit of the brain. METHODS: Behavioral cohort: Time-pregnant C57BL/6J dams were randomized to one of four custom dietary interventions. The diets varied in the combination of n-6 PUFA (8 energy% LA or 1 en% LA) and n-3 PUFA (0.5 en% EPA + DHA or 0 en% EPA + DHA). The dietary fatty acid compositions are crafted based on contemporary American and evolutionary model intake. Male offspring continued their respective maternal diet for 8 weeks before ethanol exposure. During the 6-week ethanol binge drinking, mice were exposed to cycles of 5-day 20%v/v ethanol for 4 hours in the dark and 2-day abstinence. RNA-Sequencing cohort: Same dietary interventions were applied. 15-week-old male offspring was subjected to a 9-day 2 g/kg and 1-day 5 g/kg ethanol gavage paradigm. Striatum were collected for next generation RNA sequencing after the last dose of gastric gavage. RESULTS: Mice fed 1 en% LA and 0 en% EPA + DHA (L6L3) lowered voluntary ethanol binge drinking by 29% as compared to mice fed 8 en% LA and 0 en% EPA + DHA (H6L3) (one-way ANOVA; week 3, p = 0.0072; week 4–6, P

Docosahexaenoic acid (DHA) is important for brain function, however, the exact amount required for the brain is not agreed upon. While it is believed that the synthesis rate of DHA from α-linolenic acid (ALA) is low, how this synthesis rate compares with the amount of DHA required to maintain brain DHA levels is unknown. The objective of this work was to assess whether DHA synthesis from ALA is sufficient for the brain. To test this, rats consumed a diet low in n-3 PUFAs, or a diet containing ALA or DHA for 15 weeks. Over the 15 weeks, whole body and brain DHA accretion was measured, while at the end of the study, whole body DHA synthesis rates, brain gene expression, and DHA uptake rates were measured. Despite large differences in body DHA accretion, there was no difference in brain DHA accretion between rats fed ALA and DHA. In rats fed ALA, DHA synthesis and accretion was 100-fold higher than brain DHA accretion of rats fed DHA. Also, ALA-fed rats synthesized approximately 3-fold more DHA than the DHA uptake rate into the brain. This work indicates that DHA synthesis from ALA may be sufficient to supply the brain.

Schönfeld and Reiser recently hypothesized that fatty acid β-oxidation is a source of oxidative stress in the brain. To test this hypothesis, we inhibited brain mitochondrial β-oxidation with methyl palmoxirate (MEP) and measured oxidative polyunsaturated fatty acid (PUFA) metabolites in the rat brain. Upon MEP treatment, levels of several nonenzymatic auto-oxidative PUFA metabolites were reduced with few effects on enzymatically derived metabolites. Our finding confirms the hypothesis that reduced fatty acid β-oxidation decreases oxidative stress in the brain and β-oxidation inhibitors may be a novel therapeutic approach for brain disorders associated with oxidative stress.

Dietary lipids modify brain fatty acid profile, but evidence of their direct effect on neuronal function is sparse. The enthorinal cortex (EC) neurons connecting to the hippocampus play a critical role in learning and memory. Here, we have exposed mice to diets based on canola:soybean oils (40 : 10, g/kg) or safflower : corn oils (25 : 25, g/kg) to investigate the relationship between the lipid profile of brain fatty acids and the intrinsic properties of EC neurons. Consumption of canola : soybean oil-enriched diet led to the increase of the monounsaturated fatty acid oleic acid and to a decrease of arachidonic acid in ethanolamine glycerophospholipids of the white matter. We also found an important rise in docosahexaenoic acid (DHA) within ethanolamine glycerophospholipids and phosphatidylserine of gray matter. The canola:soybean oil treatment led to a shorter duration of action potential (-21%), a reduction in the duration of postsynaptic response (-21%) and increased firing activity (+43%). Data from additional experiments with animals fed DHA alone or DHA with canola oil suggested that dietary monounsaturated fatty acid may have contributed to these effects on EC neuron physiology. Since neuronal function within the enthorhinal-hippocampal loop is critical to learning and memory processes, the present data may provide a functional basis for the beneficial cognitive effects of canola oil-based diets.

J. Neurochem. (2011) 116, 363–373. Abstract Eicosapentaenoic acid (EPA, 20:5n-3) is being explored as a therapy in neurological diseases and disorders. Although it is known that palmitate is the most abundant fatty acid in the brain while EPA is one of the lowest, the mechanism by which the brain maintains this balance is unclear. Therefore, to trace the metabolism of these fatty acids in the brain, 14C-palmitate or 14C-EPA was administered via intracerebroventricular infusion to rats. From 4 to 128 days post-infusion, brains were collected after head-focused, high-energy microwave irradiation for biochemical analysis. At day 4 post-infusion, 57% (82 ± 26 nCi) of the total phospholipid radioactivity in 14C-palmitate-infused brains was intact palmitate; whereas in 14C-EPA-infused brains, 9% (2 ± 0.9 nCi) of the radioactivity was intact EPA. The half-life of esterified 14C-palmitate and 14C-EPA was 32 ± 4 (2% loss per day) and 5 ± 0.2 days (14% loss per day), respectively. Radioactivity was also detected in other saturates, monounsaturates, and cholesterol, suggesting that the infused radiolabeled fatty acids were β-oxidized. In conclusion, the low concentration of EPA in brain phospholipids may be the result of extensive metabolism of EPA, in part by β-oxidation, upon entry into the brain and upon de-esterification from phospholipids.

In the brain, polyunsaturated fatty acids (PUFA), especially arachidonic acid and docosahexaenoic acid (DHA), are required for regulating membrane fluidity, neuronal survival and signal transduction. Since the brain cannot synthesize n-6 and n-3 PUFA de novo, they must be supplied from the blood. However, the methods of PUFA entry into the brain are not agreed upon. This study tested the necessity of CD36, a candidate transporter of unesterified fatty acids, for maintaining brain PUFA concentrations by comparing brain PUFA concentrations in CD36(-/-) mice to their wild-type littermates. Because CD36(-/-) mice have been reported to have impaired learning ability, the PUFA concentrations in different brain regions (cortex, hippocampus, cerebellum and the remainder of brain) were investigated. At 9 weeks of age, the brain was separated into the four regions and fatty acid concentrations in total and phospholipid classes of these brain regions were analyzed using thin layer and gas chromatography. There were no statistical differences in arachidonic acid or DHA concentrations in the different brain regions between wild-type and CD36(-/-) mice, in total or phospholipid fractions. Concentrations of monounsaturated fatty acids were decreased in several phospholipid fractions in CD36(-/-) mice. These findings suggest that CD36 is not necessary for maintaining brain PUFA concentrations and that other mechanisms must exist.

Docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are n-3 polyunsaturated fatty acids with a therapeutic potential for CNS diseases. Here, using an in situ brain perfusion technique in mice, we show that [(14)C]-DHA and [(14)C]-EPA readily cross the mouse blood-brain barrier (BBB) with brain transport coefficients (Clup) of 48+/-3microlg(-1)s(-1) and 52+/-4microlg(-1)s(-1), respectively. Mechanical capillary depletion of brain homogenates showed that less than 10% of [(14)C]-DHA or [(14)C]-EPA remained in endothelial cells of the brain vasculature, demonstrating that both molecules fully crossed the BBB. Addition of bovine serum albumin decreased the Clup of [(14)C]-DHA to 0.6+/-0.3microlg(-1)s(-1), indicating that binding to albumin reduced importantly, but not totally, the passage of DHA through the BBB. The Clup of [(14)C]-DHA or [(14)C]-EPA was not saturable at concentration up to 100microM, suggesting that these compounds crossed the BBB by simple diffusion. However, long-term high-DHA dietary consumption reduced the Clup of [(14)C]-DHA to 33+/-6microlg(-1)s(-1) (-20%, p

Purpose The low-density lipoprotein receptor (LDLr) mediates the uptake of LDL particles enriched with cholesterol, into several tissues. In contrast to other tissues, the brain is thought to obtain cholesterol solely by de novo synthesis, yet certain brain regions such as the brainstem are highly enriched with the LDLr. The goal of the present study was to assess the role of the LDLr in maintaining cholesterol concentrations in the brainstem of wildtype and LDLr knockout (LDLr−/−) mice. Cholesterol concentrations were also measured in the cortex, which served as a reference point, due to the lower expression of the LDLr, as compared to the brainstem. Methods LDLr−/− and wildtype mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem were isolated for cholesterol analysis. Cholesterol was extracted into chloroform/methanol, derivatized in trimethylsilyl chloride and measured by gas chromatography/mass spectrometry. Results Concentrations of cholesterol in the brainstem did not differ statistically between LDLr−/− (18.8 ± 1.6 mg/g wet weight brain) and wildtype (19.1 ± 2.0). Cortical cholesterol concentrations also did not differ statistically between LDLr−/− (11.0 ± 0.4 mg/g wet weight brain) and wildtype (11.1 ± 0.2) mice. Conclusion The LDLr is not necessary for maintaining cholesterol concentrations in the cortex or brainstem, suggesting that other mechanisms are sufficient to maintain brain cholesterol concentrations.

The brain cannot synthesize n-6 or n-3 PUFAs de novo and requires their transport from the blood. Two models of brain fatty acid uptake have been proposed. One requires the passive diffusion of unesterified fatty acids through endothelial cells of the blood-brain barrier, and the other requires the uptake of lipoproteins via a lipoprotein receptor on the luminal membrane of endothelial cells. This study tested whether the low density lipoprotein receptor (LDLr) is necessary for maintaining brain PUFA concentrations. Because the cortex has a low basal expression of LDLr and the anterior brain stem has a relatively high expression, we analyzed these regions separately. LDLr knockout (LDLr(-/-)) and wild-type mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem (pons and medulla) were isolated for phospholipid fatty acid analyses. There were no differences in phosphatidylserine, phosphatidylinositol, ethanolamine, or choline glycerophospholipid esterified PUFA or saturated or monounsaturated fatty acid concentrations in the cortex or brain stem between LDLr(-/-) and wild-type mice. These findings demonstrate that the LDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFAs into the brain must exist.

Despite being critical for normal brain function, the pools that supply docosahexaenoic acid (DHA) to the brain are not agreed upon. Using multiple kinetic models in free-living adult rats, we first demonstrate that DHA uptake from the plasma non-esterified fatty acid (NEFA) pool predicts brain uptake of DHA upon oral administration, which enters the plasma NEFA pool as well as multiple plasma esterified pools. The rate of DHA loss by the brain is similar to the uptake from the plasma NEFA pool. Furthermore, upon acute iv administration, although more radiolabeled lysophosphatidylcholine (LPC)-DHA enters the brain than NEFA-DHA, this is due to the longer plasma half-life and exposure to the brain. Direct comparison of the uptake rate of LPC-DHA and NEFA-DHA demonstrates that uptake of NEFA-DHA into the brain is 10-fold greater than LPC-DHA. In conclusion, plasma NEFA-DHA is the major plasma pool supplying the brain.

Tracer studies suggest that phospholipid DHA (PL-DHA) more effectively targets the brain than triglyceride DHA (TAG-DHA), although the mechanism and whether this translates into higher brain DHA concentrations are not clear. Rats were gavaged with [U- 3 H]PL-DHA and [U- 3 H]TAG-DHA and blood sampled over 6 h prior to collection of brain regions and other tissues. In another experiment, rats were supplemented for 4 weeks with TAG-DHA (fish oil), PL-DHA (roe PL) or a mixture of both for comparison to a low-omega-3 diet. Brain regions and other tissues were collected, and blood was sampled weekly. DHA accretion rates were estimated using the balance method. [U- 3 H]PL-DHA rats had higher radioactivity in cerebellum, hippocampus and remainder of brain, with no differences in other tissues despite higher serum lipid radioactivity in [U- 3 H]TAG-DHA rats. TAG-DHA, PL-DHA or a mixture were equally effective at increasing brain DHA. There were no differences between DHA-supplemented groups in brain region, whole-body, or tissue DHA accretion rates except heart and serum TAG where the PL-DHA/TAG-DHA blend was higher than TAG-DHA. Apparent DHA β-oxidation was not different between DHA-supplemented groups. This indicates that more labeled DHA enters the brain when consumed as PL; however, this may not translate into higher brain DHA concentrations.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n − 3 PUFA) that has been shown to raise seizure thresholds in the maximal pentylenetetrazole model following acute subcutaneous (s.c.) administration in rats. Following s.c. administration, however, the dose–response relationship for DHA has shown an inverted U-pattern. The purposes of the present experiment were as follows: (1) to determine the pattern of serum unesterified concentrations resulting from the intravenous (i.v.) infusions of various doses of DHA, (2) to determine the time course of these concentrations following the discontinuation of the infusions, and (3) to determine whether seizure protection in the maximal PTZ model would correlate with serum unesterified DHA levels. Animals received 5-minute i.v. infusions of saline or 25, 50, 100, or 200 mg/kg of DHA via a cannula inserted into one of the tail veins. Blood was collected during and after the infusions by means of a second cannula inserted into the other tail vein (Experiment 1). A separate group of animals received saline or 12.5-, 25-, 50-, 100-, or 200 mg/kg DHA i.v. via a cannula inserted into one of the tail veins and were then seizure-tested in the maximal PTZ model either during infusion or after the discontinuation of the infusions. Slow infusions of DHA increased serum unesterified DHA concentrations in a dose-dependent manner, with the 200-mg/kg dose increasing the concentration approximately 260-fold compared with saline-infused animals. Following discontinuation of the infusions, serum concentrations rapidly dropped toward baseline, with half-lives of approximately 40 and 11 s for the 25-mg/kg dose and 100-mg/kg dose, respectively. In the seizure-tested animals, DHA significantly increased latency to seizure onset in a dose-dependent manner. Following the discontinuation of infusion, seizure latency rapidly decreased toward baseline. Overall, our study suggests that i.v. infusion of unesterified DHA results in transient anticonvulsant effects which parallel unesterified DHA serum concentrations.

Background Phosphoinositides, lipid-signaling molecules, participate in diverse brain processes within a wide metabolic cascade. Hypothesis Gene transcriptional networks coordinately regulate the phosphoinositide cascade during human brain Development and Aging. Methods We used the public BrainCloud database for human dorsolateral prefrontal cortex to examine age-related expression levels of 49 phosphoinositide metabolic genes during Development (0 to 20+ years) and Aging (21+ years). Results We identified three groups of partially overlapping genes in each of the two intervals, with similar intergroup correlations despite marked phenotypic differences between Aging and Development. In each interval, ITPKB, PLCD1, PIK3R3, ISYNA1, IMPA2, INPPL1, PI4KB, and AKT1 are in Group 1, PIK3CB, PTEN, PIK3CA, and IMPA1 in Group 2, and SACM1L, PI3KR4, INPP5A, SYNJ1, and PLCB1 in Group 3. Ten of the genes change expression nonlinearly during Development, suggesting involvement in rapidly changing neuronal, glial and myelination events. Correlated transcription for some gene pairs likely is facilitated by colocalization on the same chromosome band. Conclusions Stable coordinated gene transcriptional networks regulate brain phosphoinositide metabolic pathways during human Development and Aging.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n-3 PUFA) which has been shown to raise seizure thresholds following acute administration in rats. The aims of the present experiment were the following: 1) to test whether subchronic DHA administration raises seizure threshold in the maximal pentylenetetrazol (PTZ) model 24h following the last injection and 2) to determine whether the increase in seizure threshold is correlated with an increase in serum and/or brain DHA. Animals received daily intraperitoneal (i.p.) injections of 50mg/kg of DHA, DHA ethyl ester (DHA EE), or volume-matched vehicle (albumin/saline) for 14days. On day 15, one subset of animals was seizure tested in the maximal PTZ model (Experiment 1). In a separate (non-seizure tested) subset of animals, blood was collected, and brains were excised following high-energy, head-focused microwave fixation. Lipid analysis was performed on serum and brain (Experiment 2). For data analysis, the DHA and DHA EE groups were combined since they did not differ significantly from each other. In the maximal PTZ model, DHA significantly increased seizure latency by approximately 3-fold as compared to vehicle-injected animals. This increase in seizure latency was associated with an increase in serum unesterified DHA. Total brain DHA and brain unesterified DHA concentrations, however, did not differ significantly in the treatment and control groups. An increase in serum unesterified DHA concentration reflecting increased flux of DHA to the brain appears to explain changes in seizure threshold, independent of changes in brain DHA concentrations.

Dietary lipids modify brain fatty acid profile, but evidence of their direct effect on neuronal function is sparse. The enthorinal cortex (EC) neurons connecting to the hippocampus play a critical role in learning and memory. Here, we have exposed mice to diets based on canola:soybean oils (40 : 10, g/kg) or safflower : corn oils (25 : 25, g/kg) to investigate the relationship between the lipid profile of brain fatty acids and the intrinsic properties of EC neurons. Consumption of canola : soybean oil-enriched diet led to the increase of the monounsaturated fatty acid oleic acid and to a decrease of arachidonic acid in ethanolamine glycerophospholipids of the white matter. We also found an important rise in docosahexaenoic acid (DHA) within ethanolamine glycerophospholipids and phosphatidylserine of gray matter. The canola:soybean oil treatment led to a shorter duration of action potential (-21%), a reduction in the duration of postsynaptic response (-21%) and increased firing activity (+43%). Data from additional experiments with animals fed DHA alone or DHA with canola oil suggested that dietary monounsaturated fatty acid may have contributed to these effects on EC neuron physiology. Since neuronal function within the enthorhinal-hippocampal loop is critical to learning and memory processes, the present data may provide a functional basis for the beneficial cognitive effects of canola oil-based diets.

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic and arachidonic acids, as well as cholesterol are important for neural development and maintaining brain function. However, in contrast to cholesterol, the brain is unable to synthesize the required amounts of these PUFA de novo and requires a constant supply from plasma. Suggested pools of uptake include plasma unesterified PUFA or the uptake of PUFA-containing lipoproteins via lipoprotein receptors into endothelial cells of the blood brain barrier. Our study tested whether the very low density lipoprotein receptor (VLDLr) is necessary for maintaining brain PUFA and cholesterol concentrations. Moreover, since VLDLr knockout (VLDLr(-/-)) mice have been reported to have behavioural deficits, this study asked the question whether altered brain PUFA and cholesterol concentrations might be related to these deficits. VLDLr(-/-) and wild-type mice had ad libitum access to chow. At 7 weeks of age the mice were sacrificed, and the cortex, cerebellum, hippocampus, and the remainder of the brain were isolated for total fatty acid and cholesterol analyses. There were no differences in total lipid PUFA or cholesterol concentrations in any of the four brain regions between VLDLr(-/-) and wild-type mice. These findings demonstrate that the VLDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFA into the brain must exist.

The brain is particularly enriched in glycerophospholipids with either arachidonic or docosahexaenoic acid esterified in the stereospecifically numbered-2 position. In this paper, we review how combining a kinetic approach to study the uptake and turnover of arachidonic and docosahexaenoic acids within brain phospholipids of unanesthetized rats, along with chronic administration of antimanic drugs (lithium, valproate and carbamazepine), have advanced our understanding of how polyunsaturated fatty acids (PUFA) enter the brain, and the mechanisms that regulate their turnover within brain phospholipids. The incorporation rates of arachidonic and docosahexaenoic acid from the plasma unesterified pool into brain phospholipids closely approximate independent measures of their consumption rates by the brain, suggesting this is quantitatively the major pool for uptake of these PUFA. Antimanic drugs (lithium and carbamazepine) that downregulate the activity of the calcium-dependent cytosolic phospholipase A(2) (cPLA(2)) transcription factor AP-2, and in turn the expression and activity of cPLA(2,) lead to a selective downregulation in brain arachidonic acid turnover. Furthermore, targeting arachidonoyl-CoA formation via ordered, non-competitive inhibition of an acyl-CoA synthetase with valproate also selectively decreases brain arachidonic acid turnover. Drugs that increase brain cPLA(2) activity (N-methyl-d-aspartic acid and fluoxetine) are correlated with increased turnover of arachidonic acid in brain phospholipids. Altered PUFA metabolism has been implicated in several neurological disorders, including bipolar disorder and Alzheimer's disease. Identifying the enzymes that regulated brain PUFA metabolism could lead to new therapeutic approaches for these disorders.

Analyses of brain phospholipid fatty acid profiles reveal a selective deficiency and enrichment in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. In order to account for this difference in brain fatty acid levels, we hypothesized that EPA is more rapidly beta-oxidized upon its entry into the brain. Wild-type C57BL/6 mice were perfused with either (14)C-EPA or (14)C-DHA via in situ cerebral perfusion for 40s, followed by a bicarbonate buffer to wash out the residual radiolabeled polyunsaturated fatty acid (PUFA) in the capillaries. (14)C-PUFA-perfused brains were extracted for chemical analyses of neutral lipid and phospholipid fatty acids. Based on the radioactivity in aqueous, total lipid, neutral lipid and phospholipid fractions, volume of distribution (V(D), microl/g) was calculated. The V(D) between (14)C-EPA- and (14)C-DHA-perfused samples was not statistically different for total lipid, neutral lipids or total phospholipids. However, the V(D) of (14)C-EPA in the aqueous fraction was 2.5 times higher than that of (14)C-DHA (p=0.025), suggesting a more extensive beta-oxidation than DHA. Furthermore, radiolabeled palmitoleic acid, a fatty acid that can be synthesized de novo, was detected in brain phospholipids from (14)C-EPA but not from (14)C-DHA-perfused mice suggesting that beta-oxidation products of EPA were recycled into endogenous fatty acid biosynthetic pathways. These findings suggest that low levels of EPA in brain phospholipids compared to DHA may be the result of its rapid beta-oxidation upon uptake by the brain.