Monique Lavialle

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.

Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice.

Sickness behaviour is an adaptive behavioural response to the activation of the innate immune system. It is mediated by brain cytokine production and action, especially interleukin‐6 (IL‐6). Polyunsaturated fatty acids (PUFA) are essential fatty acids that are highly incorporated in brain cell membranes and display immunomodulating properties. We hypothesized that a decrease in n‐3 (also known as omega3) PUFA brain level by dietary means impacts on lipopolysaccharide (LPS)‐induced IL‐6 production and sickness behaviour. Our results show that mice exposed throughout life to a diet containing n‐3 PUFA (n‐3/n‐6 diet) display a decrease in social interaction that does not occur in mice submitted to a diet devoid of n‐3 PUFA (n‐6 diet). LPS induced high IL‐6 plasma levels as well as expression of IL‐6 mRNA in the hippocampus and cFos mRNA in the brainstem of mice fed either diet, indicating intact immune‐to‐brain communication. However, STAT3 and STAT1 activation, a hallmark of the IL‐6 signalling pathway, was lower in the hippocampus of LPS‐treated n‐6 mice than n‐3/n‐6 mice. In addition, LPS did not reduce social interaction in IL‐6‐knockout (IL‐6‐KO) mice and failed to induce STAT3 activation in the brain of IL‐6‐KO mice. Altogether, these findings point to alteration in brain STAT3 as a key mechanism for the lack of effect of LPS on social interaction in mice fed with the n‐6 PUFA diet. The relative deficiency of Western diets in n‐3 PUFA could impact on behavioural aspects of the host response to infection.

Long term adequate n-3 polyunsaturated fatty acid diet protects from depressive-like behavior but not from working memory disruption and brain cytokine expression in aged mice.

Converging epidemiological studies suggest that dietary essential n-3 polyunsaturated fatty acid (PUFA) are likely to be involved in the pathogenesis of mood and cognitive disorders linked to aging. The question arises as to whether the decreased prevalence of these symptoms in the elderly with high n-3 PUFA consumption is also associated with improved central inflammation, i.e. cytokine activation, in the brain. To answer this, we measured memory performance and emotional behavior as well as cytokine synthesis and PUFA level in the spleen and the cortex of adult and aged mice submitted to a diet with an adequate supply of n-3 PUFA in form of α-linolenic acid (α-LNA) or a n-3 deficient diet. Our results show that docosahexaenoic acid (DHA), the main n-3 PUFA in the brain, was higher in the spleen and cortex of n-3 adequate mice relative to n-3 deficient mice and this difference was maintained throughout life. Interestingly, high level of brain DHA was associated with a decrease in depressive-like symptoms throughout aging. On the opposite, spatial memory was maintained in adult but not in aged n-3 adequate mice relative to n-3 deficient mice. Furthermore, increased interleukin-6 (IL-6) and decreased IL-10 expression were found in the cortex of aged mice independently of the diets. All together, our results suggest that n-3 PUFA dietary supply in the form of α-LNA is sufficient to protect from deficits in emotional behavior but not from memory disruption and brain proinflammatory cytokine expression linked to age.

Docosahexaenoic acid (22:6n-3) enrichment of membrane phospholipids increases gap junction coupling capacity in cultured astrocytes.

Although it is agreed that n‐3 polyunsaturated fatty acids (PUFAs) are important for brain function, it has yet to be demonstrated how they are involved in precise cellular mechanisms. We investigated the role of enhanced n‐3 PUFA in astrocyte membranes on the gap junction capacity of these cells. Astrocytes isolated from newborn rat cortices were grown in medium supplemented with docosahexaenoic acid (DHA), the main n‐3 PUFA in cell membranes, or arachidonic acid (AA), the main n‐6 PUFA, plus an antioxidant (α‐tocopherol or N‐acetyl‐cystein) to prevent peroxidation. The resulting three populations of astrocytes differed markedly in their n‐3 : n‐6 PUFA ratios in phosphatidylethanolamine and phosphatidylcholine, the main phospholipids in membranes. DHA‐supplemented cells had a physiological high n‐3 : n‐6 ratio (1.58), unsupplemented cells had a low n‐3 : n‐6 ratio (0.66) and AA‐supplemented cells had a very low n‐3 : n‐6 ratio (0.36), with excess n‐6 PUFA. DHA‐supplemented astrocytes had a greater gap junction capacity than unsupplemented cells or AA‐supplemented cells. The enhanced gap junction coupling of DHA‐enriched cells was associated with a more functional distribution of connexin 43 at cell interfaces (shown by immunocytochemistry) and more of the main phosphorylated isoform of connexin 43. These findings suggest that the high n‐3 : n‐6 PUFA ratio that occurs naturally in astrocyte membranes is needed for optimal gap junction coupling in these cells.

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: evidence for a signalling role of docosahexaenoic acid.

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport. We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced D-[(3)H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with alpha-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[(3)H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on D-[(3)H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions. These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.

Omega-3 fatty acids deficiency aggravates glutamatergic synapse and astroglial aging in the rat hippocampal CA1.

Epidemiological data suggest that a poor ω3 status favoured by the low ω3/ω6 polyunsaturated fatty acids ratio in western diets contributes to cognitive decline in the elderly, but mechanistic evidence is lacking. We therefore explored the impact of ω3 deficiency on the evolution of glutamatergic transmission in the CA1 of the hippocampus during aging by comparing 4 groups of rats aged 6–22 months fed ω3‐deficient or ω3/ω6‐balanced diets from conception to sacrifice: Young ω3 Balanced (YB) or Deficient (YD), Old ω3 Balanced (OB) or Deficient (OD) rats. ω3 Deficiency induced a 65% decrease in the amount of docosahexaenoic acid (DHA, the main ω3 in cell membranes) in brain phospholipids, but had no impact on glutamatergic transmission and astroglial function in young rats. Aging induced a 10% decrease in brain DHA, a 35% reduction of synaptic efficacy (fEPSP/PFV) due to decreased presynaptic glutamate release and a 30% decrease in the astroglial glutamate uptake associated with a marked astrogliosis (+100% GFAP). The ω3 deficiency further decreased these hallmarks of aging (OD vs. OB rats: −35% fEPSP/PFV P < 0.05, −15% astroglial glutamate uptake P < 0.001, +30% GFAP P < 0.01). This cannot be attributed to aggravation of the brain DHA deficit because the brains of OD rats had more DHA than those of YD rats. Thus, ω3 deficiency worsens the age‐induced degradation of glutamatergic transmission and its associated astroglial regulation in the hippocampus.

Synergistic effects of stress and omega-3 fatty acid deprivation on emotional response and brain lipid composition in adult rats

The aim was to determine the consequences of multi-generational n-3 polyunsaturated fatty acids (PUFA) deficiency on emotional response in rats subjected to maternal separation (MS) as chronic early life stress. Pups fed a control or an n-3 PUFA deficient diet were daily separated for 2 weeks before weaning. In adult rats, reward response was assessed by sucrose consumption and reactivity to novelty using openfield test. Both n-3 PUFA deficiency and MS increased reward response and impulsivity. Moreover, nutritional deficiency and stress acted in synergy to elevate sucrose intake by 80%, compared to control conditions. n-3 PUFA deprivation induced a depletion of docosahexanoeic acid of brain membranes by 70% compensated by increase in 22:5 n-6 and arachidonic acid (AA) levels. The diet-induced AA increase was, however, significantly higher in MS rats. This suggests that n-3 PUFA deficit could be an environmental risk increasing vulnerability to depressive-like response induced by chronic stress.

Influence of Omega-3 Fatty acid status on the way rats adapt to chronic restraint stress.

Omega-3 fatty acids are important for several neuronal and cognitive functions. Altered omega-3 fatty acid status has been implicated in reduced resistance to stress and mood disorders. We therefore evaluated the effects of repeated restraint stress (6 h/day for 21 days) on adult rats fed omega-3 deficient, control or omega-3 enriched diets from conception. We measured body weight, plasma corticosterone and hippocampus glucocorticoid receptors and correlated these data with emotional and depression-like behaviour assessed by their open-field (OF) activity, anxiety in the elevated-plus maze (EPM), the sucrose preference test and the startle response. We also determined their plasma and brain membrane lipid profiles by gas chromatography. Repeated restraint stress caused rats fed a control diet to lose weight. Their plasma corticosterone increased and they showed moderate behavioural changes, with increases only in grooming (OF test) and entries into the open arms (EPM). Rats fed the omega-3 enriched diet had a lower stress-induced weight loss and plasma corticosterone peak, and reduced grooming. Rats chronically lacking omega-3 fatty acid exhibited an increased startle response, a stress-induced decrease in locomotor activity and exaggerated grooming. The brain omega-3 fatty acids increased as the dietary omega-3 fatty acids increased; diets containing preformed long-chain omega-3 fatty acid were better than diets containing the precursor alpha-linolenic acid. However, the restraint stress reduced the amounts of omega-3 incorporated. These data showed that the response to chronic restraint stress was modulated by the omega-3 fatty acid supply, a dietary deficiency was deleterious while enrichment protecting against stress.

Long chain-polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures.

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010.

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.