Jean-Marc Alessandri

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids.

Long‐chain polyunsaturated (n‐3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n‐3), the present study addresses the question of whether the 22:6 n‐3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto‐parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n‐3 fatty acids resulted in a 30–50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20–40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n‐3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n‐3 fatty acid deficient rats, GLUT1‐immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n‐3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n‐3 deficiency could result from functional alteration in glucose transporters.

Association of fish and long-chain n-3 polyunsaturated fatty acid intakes with the occurrence of depressive episodes in middle-aged French men and women

This study aimed to seek whether habitual fish and seafood or n-3 long-chain PUFA intake could influence the occurrence of depressive episodes. In a subsample from the French SU.VI.MAX cohort, dietary habits have been assessed during the first 2 years of the follow-up (six 24-h records) and declarations of antidepressant prescription, taken as markers of depressive episodes, have been recorded during the 8-year follow-up. Subjects consuming fatty fish or with an intake of long-chain n-3 PUFA higher than 0.10% of energy intake had a significantly lesser risk of any depressive episode and of recurrent depressive episodes, but not of single depressive episode. These associations were stronger in men and in non-smokers. In contrast, smokers eating fatty fish had an increased risk of recurrent depression. These results suggest that a usual intake of fatty fish or long-chain n-3 PUFA may decrease the risk of recurrent depression in non-smokers.

Comparative bioavailability of dietary α-linolenic and docosahexaenoic acids in the growing rat

Animal and human studies have indicated that developing mammals fed only α-linolenic acid (18∶3n−3) have lower docosahexaenoic acid (22∶6n−3) content in brain and tissue phospholipids when compared with mammals fed 18∶3n−3 plus 22∶6n−3. The aim of this study was to test the hypothesis that low bioavailability of dietary 18∶−3 to be converted to 22∶6n−3 could partly explain this difference in fatty acid accretion. For that purpose, we determined the partitioning of dietary 18∶3n−3 and 22∶6n−3 between total n−3 fatty acid body accumulation, excretion, and disappearance (difference between the intake and the sum of total n−3 fatty acids accumulated and excreted). This was assessed using the quantitative method of whole-body fatty acid balance in growing rats fed the same amount of a 5% fat diet supplying either 18∶3n−3 or 22∶6n−3 at a level of 0.45% of dietary energy (i.e., 200 mg/100 g diet). We found that 58.9% of the total amount of 18∶3n−3 ingested disappeared, 0.4% was excreted in feces, 21.2% accumulated as 18∶3n−3 (50% in total fats and 46% in the carcass-skin compartment), and 17.2% accumulated as long-chain derivatives (14% as 22∶6n−3 and 3.2% as 20∶5n−3+22∶5n−3). Similar results were obtained from the docosahexaenoate balance (as % of the total amount ingested): disappearance, 64.5%; excretion, 0.5%; total accumulation, 35% with 30.1% as 22∶6n−3. Thus, rats fed docosahexaenoate accumulated a twofold higher amount of 22∶6n−3, which was mainly deposited in the carcass-skin compartment (68%). Similar proportions of disappearance of dietary 18∶−3 and 22∶6n−3 lead us to speculate that these two n−3 polyunsaturated fatty acids were β-oxidized in the same amount.

n-3 and n-6 fatty acid enrichment by dietary fish oil and phospholipid sources in brain cortical areas and nonneural tissues of formula-fed piglets.

Sufficient availability of both n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) is required for optimal structural and functional development in infancy. The question has been raised as to whether infant formulae would benefit from enrichment with 20 and 22 carbon fatty acids. To address this issue, we determined the effect of fish oil and phospholipid (LCPUFA) sources on the fatty acid composition of brain cortical areas and nonneural tissues of newborn piglets fed artificially for 2 wk. They were fed sow milk, a control formula, or the formula enriched with n-3 fatty acids from a low-20:5n-3 fish oil added at a high or a low concentration, or the formula enriched with n-3 and n-6 fatty acids from either egg yolk- or pig brain-phospholipids. Both the fish oil- and the phospholipid-enriched formula produced significantly higher plasma phospholipid 22:6n-3 concentrations than did the control formula. The 22:6n-3 levels in the brain, hepatic, and intestinal phospholipids were significantly correlated with plasma values, whereas cardiac 22:6n-3 content appeared to follow a saturable dose-response. Feeding sow milk resulted in a much higher 20:4n-6 content in nonneural tissues than did feeding formula. Supplementation with egg phospholipid increased the 20:4n-6 content in the heart, red blood cells, plasma, and intestine in comparison to the control formula, while pig brain phospholipids exerted this effect in the heart only. The addition of 4.5% fish oil in the formula was associated with a decline in 20:4n-6 in the cortex, cerebellum, heart, liver, and plasma phospholipids, whereas using this source at 1.5% limited the decline to the cerebellum, liver, and plasma. Whatever the dietary treatment, the phosphatidylethanolamine 20:4n-6 level was 10–20% higher in the brain temporal lobe than in the parietal, frontal, and occipital lobes in the temporal lobe by administering the formula enriched with egg or brain phospholipids.In conclusion, feeding egg phospholipids to neonatal pigs increased both the 22:6n-3 content in the brain and the 20:4n-6 content in the temporal lobe cortex. This source also increased the 22:6n-3 levels in nonneural tissues with only minor alterations of 20:4n-6. These data support the notion that infant formulae should be supplemented with both 22:6n-3 and 20:4n-6 rather than with 22:6n-3 alone.

Modifying the n−3 fatty acid content of the maternal diet to determine the requirements of the fetal and suckling rat

During perinatal development, docosahexaenoic acid (22:6n−3) accumulates extensively in membrane phospholipids of the nervous system. To evaluate the n−3 fatty acid requirements of fetal and suckling rats, we investigated the accumulation of 22:6n−3 in the brain and liver of pup rats from birth to day 14 postpartum when their dams received increasing amounts of dietary 18:3n−3 (from 5 to 800 mg/100 g diet) during the pregnancy-lactation period. The fatty acid composition of brain and liver phospholipids of pups, as well as that of dam’s milk, was determined. At birth, brain 22:6n−3 increased regularly to reach the highest level when the maternal diet contained 800 mg 18:3n−3/100 g. On days 7 and 14 postpartum, brain 22:6n−3 plateaued at a maternal dietary supply of 200 mg/100 g. Docosapentaenoic acid (22:5n−6) had the opposite temporal pattern. The unusually high concentration of eicosapentaenoic acid (20:5n−3) in liver and dam’s milk observed at the highest 18:3n−3 intake suggests an excessive dietary supply of this fatty acid. All these data suggest that the n−3 fatty acid requirements of the pregnant rat are around 400 mg 18:3n−3 and those of the lactating rat at 200 mg (i.e., 0.9 and 0.45% of dietary energy, respectively). The values for 18:3n−3 and 22:6n−3 milk content which allowed brain 22:6n−3 to reach a plateau value in suckling pups were 1% of total fatty acids and 0.9% (colostrum) to 0.2% (mature milk), respectively. These levels are similar to those recommended for infant formulas.

Omega-3 Fatty Acids from Fish Oil Lower Anxiety, Improve Cognitive Functions and Reduce Spontaneous Locomotor Activity in a Non-Human Primate

Omega-3 (ω3) polyunsaturated fatty acids (PUFA) are major components of brain cells membranes. ω3 PUFA-deficient rodents exhibit severe cognitive impairments (learning, memory) that have been linked to alteration of brain glucose utilization or to changes in neurotransmission processes. ω3 PUFA supplementation has been shown to lower anxiety and to improve several cognitive parameters in rodents, while very few data are available in primates. In humans, little is known about the association between anxiety and ω3 fatty acids supplementation and data are divergent about their impact on cognitive functions. Therefore, the development of nutritional studies in non-human primates is needed to disclose whether a long-term supplementation with long-chain ω3 PUFA has an impact on behavioural and cognitive parameters, differently or not from rodents. We address the hypothesis that ω3 PUFA supplementation could lower anxiety and improve cognitive performances of the Grey Mouse Lemur (Microcebus murinus), a nocturnal Malagasy prosimian primate. Adult male mouse lemurs were fed for 5 months on a control diet or on a diet supplemented with long-chain ω3 PUFA (n = 6 per group). Behavioural, cognitive and motor performances were measured using an open field test to evaluate anxiety, a circular platform test to evaluate reference spatial memory, a spontaneous locomotor activity monitoring and a sensory-motor test. ω3-supplemented animals exhibited lower anxiety level compared to control animals, what was accompanied by better performances in a reference spatial memory task (80% of successful trials vs 35% in controls, p<0.05), while the spontaneous locomotor activity was reduced by 31% in ω3-supplemented animals (p<0.001), a parameter that can be linked with lowered anxiety. The long-term dietary ω3 PUFA supplementation positively impacts on anxiety and cognitive performances in the adult mouse lemur. The supplementation of human food with ω3 fatty acids may represent a valuable dietary strategy to improve behavioural and cognitive functions.

Astrocytes in culture require docosahexaenoic acid to restore the n-3/n-6 polyunsaturated fatty acid balance in their membrane phospholipids.

Docosahexaenoic acid (DHA), the main n‐3 polyunsaturated fatty acid (PUFA) in membranes, is particularly abundant in brain cells. Decreased cerebral concentrations of DHA, resulting from dietary n‐3 deficiency, are associated with impaired cognitive function. Because the cellular causes of this impairment are still unknown, we need in vitro models that mimic the variations in n‐3/n‐6 PUFA seen in vivo. We have compared the PUFA profiles of hamster astrocytes cultured in medium supplemented with long‐chain PUFA [DHA and/or arachidonic acid (AA)] with those of brain tissue from hamsters fed an n‐6/n‐3 PUFA‐balanced diet or one lacking n‐3 PUFA. Astrocytes were obtained from the brain cortex of newborn hamsters and cultured in minimum essential medium + 5% fetal calf serum (FCS) supplemented with DHA and/or AA for 10 days. The astrocytes cultured in medium + FCS had low n‐3 PUFA contents, comparable to those of brain tissue from hamsters fed an n‐3‐deficient diet. We have shown that astrocytes grown in medium supplemented with DHA and/or AA, plus α‐tocopherol to prevent lipid peroxidation, incorporated large amounts of these long‐chain PUFA, so that the n‐6/n‐3 PUFA compositions of the phosphatidylethanolamine and phosphatidylcholine, the two main classes of membrane phospholipids, were greatly altered. Astrocytes cultured in medium plus DHA had a more physiological n‐3 status, grew better, and retained their astrocyte phenotype. Thus astrocytes in culture are likely to be physiologically relevant only when provided with adequate DHA. This reliable method of altering membrane phospholipid composition promises to be useful for studying the influence of n‐6/n‐3 imbalance on astrocyte function.

n-3 PUFA status affects expression of genes involved in neuroenergetics differently in the fronto-parietal cortex compared to the CA1 area of the hippocampus: effect of rest and neuronal activation in the rat.

n-3 Polyunsaturated fatty acids (PUFA) support whole brain energy metabolism but their impact on neuroenergetics in specific brain areas and during neuronal activation is still poorly understood. We tested the effect of feeding rats as control, n-3 PUFA-deficient diet, or docosahexaenoic acid (DHA)-supplemented diet on the expression of key genes in fronto-parietal cortex and hippocampal neuroenergetics before and after neuronal stimulation (activated) by an enriched environment. Compared to control rats, n-3 deficiency specifically repressed GLUT1 gene expression in the fronto-parietal cortex in basal state and also during neuronal activation which specifically stimulated GLUT1. In contrast, in the CA1 area, n-3 deficiency improved the glutamatergic synapse function in both neuronal states (glutamate transporters, Na(+)/K(+) ATPase). DHA supplementation induced overexpression of genes encoding enzymes of the oxidative phosphorylation system and the F1F0 ATP synthase in the CA1 area. We conclude that n-3 deficiency repressed GLUT1 gene expression in the cerebral cortex, while DHA supplementation improved the mitochondrial ATP generation in the CA1 area of the hippocampus.

Changes of the transcriptional and fatty acid profiles in response to n-3 fatty acids in SH-SY5Y neuroblastoma cells

Synthesis of docosahexaenoic acid (DHA) from its metabolic precursors contributes to membrane incorporation of this FA within the central nervous system. Although cultured neural cells are able to produce DHA, the membrane DHA contents resulting from metabolic conversion do not match the high values of those resulting from supplementation with preformed DHA. We have examined whether the DHA precursors downregulate the incorporation of newly formed DHA within human neuroblastoma cells. SH-SY5Y cells were incubated with gradual doses of α-linolenic acid (α-LNA), EPA, or docosapentaenoic acid (DPA), and the incorporation of DHA into ethanolamine glycerophospholipids was analyzed as a reflection of synthesizing activity. The incorporation of EPA, DPA, and preformed DHA followed a dose-response saturating curve, whereas that of DHA synthesized either from α-LNA, EPA, or DPA peaked at concentrations of precursors below 15–30 μM and sharply decreased with higher doses. The mRNA encoding for six FA metabolism genes were quantified using real-time PCR. Two enzymes of the peroxisomal β-oxidation, L-bifunctional protein and peroxisomal acyl-CoA oxidase, were expressed at lower levels than fatty acyl-CoA ligase 3 (FACL3) and Δ6-desaturase (Δ6-D). The Δ6-D mRNA slightly increased between 16 and 48 h of culture, and this effect was abolished in the presence of 70 μM EPA. In contrast, the EPA treatment resulted in a time-dependent increase of FACL3 mRNA. The terminal step of DHA synthesis seems to form a “metabolic bottleneck,” resulting in accretion of EPA and DPA when the precursor concentration exceeds a specific threshold value. We conclude that the critical precursor-concentration window of responsiveness may originate from the low basal expression level of peroxisomal enzymes.

n-3 long-chain fatty acids and regulation of glucose transport in two models of rat brain endothelial cells.

Several in vivo studies suggest that docosahexaenoic acid (22:6 n-3), the main n-3 long-chain polyunsaturated fatty acids (LC-PUFA) of brain membranes, could be an important regulator of brain energy metabolism by affecting glucose utilization and the density of the two isoforms of the glucose transporter-1 (GLUT1) (endothelial and astrocytic). This study was conducted to test the hypothesis that 22:6 n-3 in membranes may modulate glucose metabolism in brain endothelial cells. It compared the impact of 22:6 n-3 and the other two main LC-PUFA, arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3), on fatty acid composition of membrane phospholipids, glucose uptake and expression of 55-kDa GLUT1 isoform in two models of rat brain endothelial cells (RBEC), in primary culture and in the immortalized rat brain endothelial cell line RBE4. Without PUFA supplementation, both types of cerebral endothelial cells were depleted in 22:6 n-3, RBE4 being also particularly low in 20:4 n-6. After exposure to supplemental 20:4 n-6, 20:5 n-3 or 22:6 n-3 (15microM, i.e. a physiological dose), RBEC and RBE4 avidly incorporated these PUFA into their membrane phospholipids thereby resembling physiological conditions, i.e. the PUFA content of rat cerebral microvessels. However, RBE4 were unable to incorporate physiological level of 20:4 n-6. Basal glucose transport in RBEC (rate of [(3)H]-3-o-methylglucose uptake) was increased after 20:5 n-3 or 22:6 n-3 supplementation by 50% and 35%, respectively, whereas it was unchanged with 20:4 n-6. This increase of glucose transport was associated with an increased GLUT1 protein, while GLUT1 mRNA was not affected. The different PUFA did not impact on glucose uptake in RBE4. Due to alterations in n-6 PUFA metabolism and weak expression of GLUT1, RBE4 seems to be less adequate than RBEC to study PUFA metabolism and glucose transport in brain endothelial cells. Physiological doses of n-3 LC-PUFA have a direct and positive effect on glucose transport and GLUT1 density in RBEC that could partly explain decreased brain glucose utilization in n-3 PUFA-deprived rats.