Atsushi Ikemoto

Dietary n-3 fatty acid deficiency decreases nerve growth factor content in rat hippocampus

Dietary deprivation of alpha-linolenic acid (n-3) through two generations has been shown to lower performance in an operant-type brightness-discrimination learning test in rats. Here, we examined a possible correlation between nerve growth factor (NGF) content and n-3 fatty acid status in the brain. Female rats were fed a semipurified diet supplemented with safflower oil (n-3 fatty acid-deficient) and their offsprings were fed a diet supplemented with either 3% safflower oil (Saf group) or a mixture of 2.4% safflower oil plus 0.6% ethyl eicosapentaenoate (Saf+EPA group) after weaning. The brain docosahexaenoic acid (22:6n-3, DHA) content in the Saf group was less than half of that in the Per group fed a diet supplemented with 3% perilla oil (n-3 fatty acid-sufficient) throughout the duration of the experiment. The DHA level of the Saf+EPA group was restored to the level of the Per group. However, the NGF contents in the hippocampus of the Saf and Saf+EPA groups were half that of the Per group. In the piriform cortex, the NGF content tended to be higher in the Saf and Saf+EPA groups than in the Per group. These results indicate that dietary n-3 fatty acid deficiency and restoration affect NGF levels differently among different brain regions.

Membrane Fatty Acid Modifications of PC12 Cells by Arachidonate or Docosahexaenoate Affect Neurite Outgrowth But Not Norepinephrine Release

The relationships between membrane fatty acid modification and neurite outgrowth and norepinephrine release were evaluated in PC12 cells. [3H]Norepinephrine release evoked by carbachol was unaffected by the modifications. Basal spontaneous release was elevated with increases in the degree of unsaturation using cells supplemented with n-3 fatty acids; a reverse correlation was observed for [3H]norepinephrine uptake. Supplementation of PC12 cells with either n-6 fatty acids or 18:1 also increased the basal release and decreased the uptake. Docosahexaenoic acid promoted and arachidonic acid suppressed neurite outgrowth induced by nerve growth factor. Choline acetyltransferase activity was slightly influenced by these fatty acids. Thus, modifications of PC12 cells with arachidonic acid and docosahexaenoic acid had a relatively small effect on the degree of differentiation but had pronounced but opposite effects on neurite elongation. Ethanolamine glycerophospholipid synthesis was elevated during differentiation induced by nerve growth factor and it was suppressed by added arachidonic acid but not by docosahexaenoic acid. Our results raise the possibility that the decreased phospholipid synthesis caused by arachidonate may lead to the suppression of neurite elongation.

Dietary, but not Topical, Alpha-linolenic Acid Suppresses UVB-induced Skin Injury in Hairless Mice when Compared with Linoleic Acid¶

Abstract Peroxidizability of fatty acids in the air is roughly proportional to the number of double bonds, but in vivo peroxidation proceeds in a more complex manner. Here, we compared the effects of dietary and topically applied oils enriched with linoleic acid (LA, 18:2n-6) or alpha-linolenic acid (ALA, 18:3n-3) on UV-induced skin injury in a strain of hairless mice. The UVB-induced erythema score was significantly lower in mice with topically applied creams containing LA and ALA than in mice with the basal cream; no significant increase in the score was detected in the ALA group compared with the LA group. However, dietary ALA inhibited the increase in erythema score after UVB irradiation compared with LA. The peroxidizability index of the skin total lipids was significantly higher, but UVB-induced prostaglandin E2 (PGE2) production was significantly lower in the group fed an ALA-rich diet compared with the group fed an LA-rich diet. The levels of thiobarbituric acid–reactive substances, estimated in the presence of butylated hydroxytoluene in the assay mixture, were not affected by UVB treatment or by the dietary fatty acids, but the severity of the skin lesion was associated with PGE2 levels. These results indicate that the type of fatty acids, n-6 or n-3, is critical for the suppression of UVB-induced skin lesion when the skin fatty acids are modified by dietary manipulation. Anti-inflammatory activity of dietary flaxseed oil with relatively high ALA and low LA contents was demonstrated in UVB-irradiated hairless mice.

Effect of n-3 fatty acid deficiency on fatty acid composition and metabolism of aminophospholipids in rat brain synaptosomes.

Docosahexaenoic acid (DHA, 22∶6n−3) is one of the major polyunsaturated fatty acids esterified predominantly in aminophospholipids such as ethanolamine glycerophospholipid (EtnGpl) and serine glycerophospholipid (SerGpl) in the brain. Synaptosomes prepared from rats fed an n−3 fatty acid-deficient safflower oil (Saf) diet had significantly decreased 22∶6n−3 content with a compensatory increased 22∶5n−6 content when compared with rats fed an n−3 fatty acid-sufficient perilla oil (Per) diet. When the Saf group was shifted to a diet supplemented with safflower oil plus 22∶6n−3 (Saf+DHA) after weaning, 22∶6n−3 content was found to be restored to the level of the Per group. The uptake of [3H]ethanolamine and its conversion to [3H]EtnGpl did not differ significantly among the three dietary groups, whereas the formation of [3H]lysoEtnGpl from [3H]ethanolamine was significantly lower in the Saf group than in the other groups. The uptake of [3H]serine, its incorporation into [3H]SerGpl, and the conversion into [3H]EtnGpl by decarboxylation of [3H]SerGpl did not differ among the three dietary groups. The observed decrease in lysoEtnGpl formation associated with a reduction of 22∶6n−3 content in rat brain synaptosomes by n−3 fatty acid deprivation may provide a clue to reveal biochemical bases for the dietary fatty acids-behavior link.

Effect of dietary oils enriched with n-3 fatty acids on survival of mice

Female mice were fed a conventional diet, shifted at 119 days of age to a diet supplemented with 10 wt % lard (Lar), high-linoleic (n-6) safflower oil (Saf), rapeseed oil (low-erucic, Rap), high-alpha-linolenic (n-3) perilla oil (Per) or a mixture (1:9) of ethyl docosahexaenoate (n-3) and soybean oil (DHA/Soy). Weight gain was less in the Per group than in the other groups at 497 days of age. In the Rap group, proteinuria was more severe than in the Saf, Per and DHA/Soy group, and hepatic triacylglycerol accumulation was greater than in the other groups. The mean survival time of the DHA/Soy group (753 days) was significantly longer than in the Lar group (672 days) and Saf group (689 days); the differences among other groups (e.g., 701 days in the Per group and 712 days in the Rap group) were not statistically significant. Although DHA is more susceptible to auto-oxidation than other major fatty acids in the air, an oil containing DHA was found to increase the survival of mice. Rapeseed oil that decreases the survival time of SHRSP rats was found to be safe in the mouse strain used in this study when survival was an end point.

Dietary alpha-linolenic acid suppresses the formation of lysophosphatidic acid, a lipid mediator, in rat platelets compared with linoleic acid.

Rats fed a high linoleic acid (LA, 18:2n-6) diet or a high alpha-linolenic acid (ALA, 18:3n-3) diet for 4 months after weaning. Platelets from the high-LA group contained more arachidonic acid (AA, 20:4n-6) and less eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared with those from the high-ALA group. Incorporation of [32P]orthophosphate into platelet phospholipids was increased by thrombin-treatment, and was greater by ca. 30% in the high-LA group than in the high-ALA group both in the presence and absence of thrombin. The formation of [32P]lysophosphatidic acid (LPA), a lipid messenger, in [32P]orthophosphate-labeled platelets was increased 6.6-fold in the high-LA group and 4.1-fold in the high-ALA-group by thrombin-treatment. The formation of [32P] LPA in activated platelets was reduced by 35% in the high-ALA group.

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n‐3 polyunsaturated fatty acid‐deficient (safflower oil group; S group) or ‐rich (perilla oil group; P group) diet before and after brightness‐discrimination learning tasks. The amount of concanavalin A‐binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex‐type oligosaccharides became dominant after the learning task and especially a tetra‐antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S‐group than the P group. When polysialylated glycans were analyzed on serotonin‐conjugated HPLC column, the glycans in the S‐group microsomes before the learning task contained larger amount of higher affinity‐polysialylated glycans to serotonin column than those in the P‐group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P‐group. Removal of mannoside from microsomes by α‐mannosidase‐treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1‐anilinonaphthalene‐8‐sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho‐glycans were changed by dietary n‐3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin. J. Neurosci. Res. 63:185–195, 2001.

Brightness—Discrimination Learning Behavior and Retinal Function Affected by Long-Term α-Linolenic Acid Deficiency in Rat

Major fatty acids in food are classified into three series depending on their metabolism in mammals (Fig. 1). Saturated fatty acids with 16- and 18-carbon chain lengths are synthesized de novo and are easily desaturated to monounsaturated fatty acids in mammals. Linoleic acid (18:2n−6, LA) and α-linolenic acid (18:3n−3, ALA) are synthesized in plants but not mammals. When ingested, LA is desaturated and elongated to form arachidonic acid (20:4n−6, ARA) and other n−6 fatty acids. ALA is metabolized similarly to form other n−3 fatty acids such as eicosapentaenoic acid (20:5n−3, EPA) and docosahexaenoic acid (22:6n−3, DHA). No interconversion occurs among the three series, although retroconversion occurs in mammals (e.g., from DHA to ALA) (Sprecher, 1995; Moor, 1995). Various types of food contain different proportions of these three series of fatty acids, and tissue fatty acid composition, particularly those of n−6 and n−3 fatty acids, varies depending on the choice of food. In the central nervous system, the major polyunsaturated fatty acids are ARA and DHA. In the desaturation, elongation, and chain-shortening steps, n−3 fatty acids are the preferred substrates leading to DHA. When the supply of n−3 fatty acids is limited, ARA is further metabolized to form docosapentaenoic acid (DPA, 22:5n−6). When the supply of both n−3 and n−6 fatty acids is limited, oleic acid (18:1n−9) is desaturated and elongated to form eicosatrienoic acid (20:3n−9, Mead acid), which is ineffective in suppressing the symptoms of LA deficiency (e.g., growth retardation and reproductive failure). ALA is essential for the maintenance of brain and retinal function.

Differential utilization of the ethanolamine moiety of phosphatidylethanolamine derived from serine and ethanolamine during NGF-induced neuritogenesis of PC12 cells

Neurite elongation involves the expansion of the plasma membrane and phospholipid synthesis. We investigated membrane phosphatidylethanolamine (PE) biosynthesis in PC12 cells during neurite outgrowth induced by nerve growth factor (NGF). When PE was prelabeled with [3H]ethanolamine and the radioactivity was chased by incubation with 1 mM unlabeled ethanolamine, the radioactivity of [3H]PE steadily declined and [3H]ethanolamine was released into the medium in NGF-treated cells during neurite outgrowth; in the absence of unlabeled ethanolamine, the radioactivity of [3H]PE remained relatively constant for at least 24 hr. In undifferentiated cells but not in NGF-treated cells, [3H]phosphoethanolamine accumulated in significant amounts during pulse labeling, and was converted partly to PE but largely released into the medium irrespective of incubation with unlabeled ethanolamine. The decline in the radioactivity of [3H]PE and release of [3H]ethanolamine following incubation with unlabeled ethanolamine were also observed in undifferentiated cells. Thus, the ethanolamine moiety of PE derived from ethanolamine is actively recycled in both differentiated and undifferentiated cells. When PE was derived from [3H]serine through phosphatidylserine (PS) decarboxylation, the decrease in radioactivity of [3H]PE and release of [3H]ethanolamine into the medium following incubation with unlabeled ethanolamine were observed only in NGF-treated cells, but not in undifferentiated cells, indicating that the ethanolamine moiety of PE derived from PS is actively recycled only in the cells undergoing NGF-induced neuritogenesis. Thus, in PC12 cells, the ethanolamine moiety of PE derived from PS is regulated differently from that of PE derived from ethanolamine.

Partial purification and characterization of phosphatidic acid-specific phospholipase A1 in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A(1) type (PA-PLA(1)) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA(1) was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca(2+) and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA(1) in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA(2) inhibitor, a Ca(2+)-independent phospholipase A(2) inhibitor, and a DG lipase inhibitor.