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BACKGROUND: Factors other than dietary fatty acids could be involved in the variability observed in blood docosahexaenoate (22:6n-3) and arachidonate (20:4n-6) status in formula-fed infants. OBJECTIVE: We considered the 22:6n-3 and 20:4n-6 status at birth to be one of these factors and studied its influence on postnatal changes in term infants fed 4 different diets. DESIGN: The blood phospholipid composition was determined at birth and on day 42 of feeding in 83 term infants fed breast milk, nonsupplemented formula, or 2 different 22:6n-3-supplemented formulas. Relations between 22:6n-3 and 20:4n-6 status at birth and their relative postnatal changes, calculated by the difference between status at the end of the feeding period (6 wk of age) and at birth, were assessed. RESULTS: Postnatal changes in the plasma and erythrocyte phospholipids 22:6n-3 and 20:4n-6 were negatively related to their respective concentrations at birth (P < 0.01) and the slopes of the regression lines were not significantly affected by the type of milk ingested. Adjusted mean values for phospholipid 22:6n-3 in nonsupplemented-formula-fed infants and for 20:4n-6 in formula-fed infants decreased significantly more than they did in the other infant groups (P < 0.02). The status at birth and the type of milk ingested explained 33-64% and 7-47%, respectively, of the variability in postnatal changes. CONCLUSIONS: The status of 22:6n-3 and 20:4n-6 at birth in term infants is one of the major determinants of postnatal changes in these fatty acids. This finding indicates that research is required to characterize environmental, genetic, or both factors, which, in addition to maternal diet, could influence fatty acid status at birth. Copyright (c) 1999 American Society for Clinical Nutrition [ABSTRACT FROM AUTHOR]

Decreased brain content of DHA, the most abundant long-chain n-3 polyunsaturated fatty acid (n-3 LCPUFA) in the brain, is accompanied by severe neurosensorial impairments linked to impaired neurotransmission and impaired brain glucose utilization. In the present study, we hypothesized that increasing n-3 LCPUFA intake at an early age may help to prevent or correct the glucose hypometabolism observed during aging and age-related cognitive decline. The effects of 12 months' supplementation with n-3 LCPUFA on brain glucose utilization assessed by positron emission tomography was tested in young adult mouse lemurs (Microcebus murinus). Cognitive function was tested in parallel in the same animals. Lemurs supplemented with n-3 LCPUFA had higher brain glucose uptake and cerebral metabolic rate of glucose compared with controls in all brain regions. The n-3 LCPUFA-supplemented animals also had higher exploratory activity in an open-field task and lower evidence of anxiety in the Barnes maze. Our results demonstrate for the first time in a nonhuman primate that n-3 LCPUFA supplementation increases brain glucose uptake and metabolism and concomitantly reduces anxiety., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Specific mechanisms for maintaining docosahexaenoic acid (DHA) concentration in brain cells but also transporting DHA from the blood across the blood-brain barrier (BBB) are not agreed upon. Our main objective was therefore to evaluate the level of gene expression of fatty acid transport and fatty acid binding proteins in the cerebral cortex and at the BBB level during the perinatal period of active brain DHA accretion, at weaning, and until the adult age. We measured by real time RT-PCR the mRNA expression of different isoforms of fatty acid transport proteins (FATPs), long-chain acyl-CoA synthetases (ACSLs), fatty acid binding proteins (FABPs) and the fatty acid transporter (FAT)/CD36 in cerebral cortex and isolated microvessels at embryonic day 18 (E18) and postnatal days 14, 21 and 60 (P14, P21 and P60, respectively) in rats receiving different n-3 PUFA dietary supplies (control, totally deficient or DHA-supplemented). In control rats, all the genes were expressed at the BBB level (P14 to P60), the mRNA levels of FABP5 and ACSL3 having the highest values. Age-dependent differences included a systematic decrease in the mRNA expressions between P14-P21 and P60 (2 to 3-fold), with FABP7 mRNA abundance being the most affected (10-fold). In the cerebral cortex, mRNA levels varied differently since FATP4, ACSL3 and ACSL6 and the three FABPs genes were highly expressed. There were no significant differences in the expression of the 10 genes studied in n-3 deficient or DHA-supplemented rats despite significant differences in their brain DHA content, suggesting that brain DHA uptake from the blood does not necessarily require specific transporters within cerebral endothelial cells and could, under these experimental conditions, be a simple passive diffusion process., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Optimal levels of unsaturated fatty acids have positive impacts on the use of prolonged bouts of hypothermia in mammalian hibernators, which generally have to face low winter ambient temperatures. Unsaturated fatty acids can maintain the fluidity of fat and membrane phospholipids at low body temperatures. However, less attention has been paid to their role in the regulation of shallow hypothermia, and in tropical species, which may be challenged more by seasonal energetic and/or water shortages than by low temperatures. The present study assessed the relationship between the fatty acids content of white adipose and liver tissues and the expression of shallow hypothermia in a tropical heterothermic primate, the gray mouse lemur (Microcebus murinus). The adipose tissue is the main tissue for fat storage and the liver is involved in lipid metabolism, so both tissues were expected to influence hypothermia dependence on fatty acids. As mouse lemurs largely avoid deep hypothermia (i.e. torpor) use under standard captive conditions, the expression of hypothermia was triggered by food-restricting experimental animals. Hypothermia depth increased with time, with a stronger increase for individuals that exhibited higher contents of unsaturated fatty acids suggesting that they were more flexible in their use of hypothermia. However these same animals delayed the use of long hypothermia bouts relative to individuals with a higher level of saturated fatty acids. This study evidences for the first time that body fatty acids unsaturation levels influence the regulation of body temperature not only in cold-exposed hibernators but also in tropical, facultative heterotherms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as transcription factors that regulate gene expression in numerous biological processes. Although the PPARβ/δ subtype is highly expressed in the brain, its physiological roles in neuronal function remain to be elucidated. In this study, we examined the presence of PPARβ/δ in the master circadian clock of the Syrian hamster and investigated its putative functional role in this structure. In mammals, the central circadian clock, located in the suprachiasmatic nucleus (SCN), is entrained by the light-dark (LD) cycle via photic6 signals conveyed by a direct pathway whose terminals release glutamate. Using immunocytochemical and qRT-PCR analysis, we demonstrated that the rhythmic expression of PPAR β/δ within the SCN of hamsters raised under an LD cycle was detectable only at the transcriptional level when the hamsters were maintained under constant darkness (DD). The increase in the number of immunoreactive PPARβ/δ cells observed under DD after light stimulation during the early subjective night (CT14), but not during the subjective day (CT06), demonstrated that the expression of PPARβ/δ can be up-regulated according to the photosensitive phase of the circadian clock. All of the PPARβ/δ-positive cells in the SCN also expressed the glutamate receptor NMDAR1. Moreover, we demonstrated that at the photosensitive point (CT14), the administration of L-16504, a specific agonist of PPARβ/δ, amplified the phase delay of the locomotor response induced by a light pulse. Taken together, these data suggest that PPARβ/δ activation modulates glutamate release that mediates entrainment of the circadian clock by light.

The particular interest in supplementing human foods with n-3 fatty acids has arisen from the findings that this series of polyunsaturated fatty acids (PUFA) have an impact on neuronal functions. Indeed vertebrates, including humans, preferentially use docosahexaenoic acid (DHA, 22:6n-3) over other long-chain n-3 PUFA for the genesis of their neuronal and retinal membranes. The grey mouse lemur is a nocturnal prosimian primate originating from Madagascar. The increased use of this omnivorous primate in nutritional studies (chronic caloric restriction, n-3 fatty acids supplementation), justifies the interest of determining their fatty acids body composition. In the present study, we report the fatty acid composition in lipid classes from the main target tissues (brain, retina, liver and adipose tissue) of six adult mouse lemurs raised under laboratory nutritional conditions. Among the main findings, n-6-docosapentaenoic acid (n-6-DPA; 22:5n-6) is very low in the brain cortex and retina, whereas there is a very high accumulation of docosahexaenoic acid (DHA, 22:6n-3) in the neural tissues compared to liver and plasma. In particular, DHA accounts for about one half of the total fatty acids in the retina ethanolamine glycerophospholipids. This high concentration clearly indicates that DHA is efficiently transferred from blood lipids to the outer segment of the mouse lemur retina. We conclude that the mouse lemur n-3 PUFA metabolism efficiently drives DHA to neural tissues, through the blood-brain barrier and the blood-retina barrier.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: The conversion rate of α-linolenic acid (ALA) into docosahexaenoic acid (DHA) is determined by dietary and non-dietary factors. Higher capacity of DHA synthesis has been evidenced in females, indicating that sex factors influence the conversion pathway. To evaluate the extent to which sexual dimorphism of DHA synthesis is subordinated to nutritional handling, we measured the ω3 ∆4-desaturation index in male and female rats receiving adequate or inadequate amounts of ALA. The ω3 ∆4-desaturation index was drawn from the DHA to docosapentaenoic acid (ω3DPA) ratio in liver phospholipids., Methods: Male and female rats born to ω3-deficient dams were fed a supplemented diet supplying low, inadequate, intermediate, or adequate ALA (5, 20, 100, or 300 mg ALA/100 g diet, respectively). Control rats from both gender received the adequate diet from fetal life., Results: Compared with control, low ALA feeding induced the ω3 ∆4-desaturation index to increase by 38 and 70% in the phosphatidylethanolamine fraction of males and females, respectively, and by 67% in phosphatidylcholine in females only. Supplementations with increased doses of ALA progressively smoothed this gender effect. Moreover, the analysis of our data from a previous study shows that ovariectomy decreased, whereas estradiol treatment increased the ω3 index to values comparable with those of diet-matched males and intact females, respectively., Conclusion: Females are more prone than males to increase their index of ω3 ∆4-desaturation, especially in response to low supplies in ALA. Estradiol supports the ω3 index, suggesting that this hormone plays a role in the effect of gender on DHA synthesis.

The protection of the developing organism from oxidative damage is ensured by antioxidant defense systems to cope with reactive oxygen species (ROS), which in turn can be influenced by dietary polyunsaturated fatty acids (PUFAs). PUFAs in membrane phospholipids are substrates for ROS-induced peroxidation reactions. We investigated the effects of dietary supplementation with omega-3 PUFAs on lipid peroxidation and antioxidant enzyme activities in rat cerebrum, liver and uterus. Pups born from dams fed a diet low in omega-3 PUFAs were fed at weaning a diet supplying low α-linolenic acid (ALA), adequate ALA or enriched with eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Malondialdehyde (MDA), a biomarker of lipid peroxidation, and the activities of superoxide dismutase 1 (SOD1), SOD2, catalase (CAT) and glutathione peroxidase (GPX) were determined in the three target organs. Compared to low ALA feeding, supplementation with adequate ALA or with EPA+DHA did not affect the cerebrum MDA content but increased MDA content in liver. Uterine MDA was increased by the EPA+DHA diet. Supplementation with adequate ALA or EPA+DHA increased SOD2 activity in the liver and uterus, while only the DHA diet increased SOD2 activity in the cerebrum. SOD1, CAT and GPX activities were not altered by ALA or EPA+DHA supplementation. Our data suggest that increased SOD2 activity in organs of the growing female rats is a critical determinant in the tolerance to oxidative stress induced by feeding a diet supplemented with omega-3 PUFAs. This is may be a specific cellular antioxidant response to ROS production within the mitochondria., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements., Methods: over 7wk, young rats consumed an EFA diet containing either 0en% linoleate (0LA) and 0en% α-linolenate (0LNA) or a diet containing 0.5en% LNA plus one of seven levels of added LA (0.12-4.0en%; n=6/group)., Results: Rats consuming the 0LA-0LNA diet had the lowest final body weight, 34-68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8-20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA-0LNA group., Conclusion: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5en% instead of 2en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The accretion of docosahexaenoic acid (DHA) in membranes of the central nervous system is required for the optimum development of retina and brain functions. DHA status is determined by the dietary intake of n-3 polyunsaturated fatty acids (PUFA), both the metabolic precursor α-linolenic acid (α-LNA) and DHA. Clinical studies have shown that feeding term or premature infants with formula low in total n-3 PUFA may alter the maturation of visual acuity. Moreover, feeding infants over the first 6 mon of life with formula containing adequate α-LNA, but no DHA, did not sustain the same cerebral accretion of DHA as that of breast-fed infants. Whether lower DHA accretion in brain of formula-fed term infants impairs neurophysiological performances is not clearly established. Contradictory data have been published, possibly owing to confounding factors such as maternal intakes and/or genetic variations in PUFA metabolism. Nevertheless, a large corpus of data is in favor of the recommendation of regular dietary intakes of DHA (during at least the first 6 mon of life) and suggest that DHA should be added in formulas at the level generally found in human milk (0.2-0.3 wt% of total fatty acids). The maternal intake of n-3 PUFA during pregnancy and lactation is also crucial, since the n-3 PUFA are provided during perinatal development through placental transfer and maternal milk, which determines the DHA status of the newborn and consequently impacts on post-natal development of brain and visual functions. Whether more clinical studies are needed to control and improve the impact of DHA maternal intakes on the progeny's neurodevelopment, several commissions recommended by precaution that DHA average intake for pregnant and lactating women should be of 200-300 mg/day., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

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.

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.

The goal of this study was to seek the relations between baseline n-3 PUFA status and the later occurrence of depressive episodes in a French cohort of middle-aged men and women, the SU.VI.MAX study. A nested case-control study was designed within the cohort: cases with at least two depressive episodes during the 8-year follow-up were paired to non-depressed controls, antidepressant prescriptions being taken as markers of depressive episodes. The fatty acid profiles of baseline serum phospholipids have been determined. Results were analyzed using logistic regression and principal component analysis, taking into account depression history and demographic and lifestyle confounders. There was no consistent association of depression risk with any serum fatty acid, and in particular there was no association of depression risk with the long-chain n-3 PUFA eicosapentaenoic, docosapentaenoic and docosahexaenoic acids. This study does not support the hypothesis of a predictive value of n-3 PUFA status for depression in population settings.

Polyunsaturated fatty acids (PUFA) are crucial for proper functioning of cell membranes, particularly in brain. Biologically important PUFA include docosahexaenoic acid (n-3 series) and arachidonic acid (n-6 series) which can be formed from their respective dietary essential precursors, alpha-linolenic acid (ALA) and linoleic acid (LA). Steroid hormones are thought to modulate PUFA synthesis in humans but whether they regulate PUFA status in brain and/or in neural membranes is unknown. In human neuroblastoma SH-SY5Y cells, we compared the effect of estradiol, testosterone, and progesterone on PUFA synthesis. Cells were incubated with ALA and/or LA 7 microM in combination with estradiol, testosterone, or progesterone at 10 nM without serum. The fatty acid composition was determined by gas chromatography and the mRNA expression of genes involved in PUFA metabolism by real-time RT-PCR. Estradiol affected both the n-3 and the n-6 PUFA conversion, the n-3 PUFA pathway being more sensitive to the estradiol treatment. In ALA-supplemented cells, estradiol increased while testosterone decreased the long-chain n-3 PUFA content (+17% and -15%, respectively) and the mRNA expression of the Delta5-desaturase (+11% and -9%), these two events being strongly correlated. Progesterone did not affect the PUFA composition. The positive effect of estradiol was blocked by the estrogen receptor antagonist ICI-182,780. We conclude that steroids have differential effects on PUFA synthesis and that their mode of action could involve the modulation of the Delta5-desaturase mRNA expression in neuroblastoma cells. These results help our understanding of the regulation of brain PUFA metabolism by steroid hormones.

Several studies suggest that (n-3) PUFA may play a role in the regulation of cognitive functions, locomotor and exploratory activity, and affective disorders. Additionally, (n-3) PUFA affect pineal function, which is implicated in the sleep-wake rhythm. However, no studies to our knowledge have explored the role of PUFA on the circadian system. We investigated the effect of an (n-3) PUFA-deficient diet on locomotor and pineal melatonin rhythms in Syrian hamsters used as model species in circadian rhythm research. To assess the possible relationship between voluntary wheel running activity and dopaminergic neurotransmission, we also measured endogenous monoamine concentrations in the striatum. Two-month-old male hamsters, fed either an (n-3) PUFA-deficient or an (n-3) PUFA-adequate diet, were housed individually in cages equipped with run wheels. At 3 mo, cerebral structures were extracted for biochemical and cellular analysis. In (n-3) PUFA-deficient hamsters, the induced changes in the pineal PUFA membrane phospholipid composition were associated with a reduction in the nocturnal peak level of melatonin that was 52% lower than in control hamsters (P < 0.001). The (n-3) PUFA-deficient hamsters also had higher diurnal (P < 0.01) and nocturnal (P = 0.001) locomotor activity than the control hamsters, in parallel with activation of striatal dopaminergic function (P < 0.05). The (n-3) PUFA-deficient hamsters exhibited several symptoms: chronic locomotor hyperactivity, disturbance in melatonin rhythm, and striatal hyperdopaminergia. We suggest that an (n-3) PUFA-deficient diet lessens the melatonin rhythm, weakens endogenous functioning of the circadian clock, and plays a role in nocturnal sleep disturbances as described in attention deficit/hyperactivity disorder.

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.

Whether neurosteroids regulate the synthesis of long chain polyunsaturated fatty acids in brain cells is unknown. We examined the influence of 17-beta-estradiol (E2) on the capacity of SH-SY5Y cells supplemented with alpha-linolenic acid (ALA), to produce eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Cells were incubated for 24 or 72 h with ALA added alone or in combination with E2 (ALA + E2). Fatty acids were analyzed by gas chromatography of ethanolamine glycerophospholipids (EtnGpl) and phosphatidylcholine (PtdCho). Incubation for 24 h with ALA alone increased EPA and DPA in EtnGpl, by 330 and 430% compared to controls (P < 0.001) and DHA by only 10% (P < 0.05). Although DHA increased by 30% (P < 0.001) in ALA + E2-treated cells, the difference between the ALA and ALA + E2 treatments were not significant after 24 h (Anova-1, Fisher's test). After 72 h, EPA, DPA and DHA further increased in EtnGpl and PtdCho of cells supplemented with ALA or ALA + E2. Incubation for 72 h with ALA + E2 specifically increased EPA (+34% in EtnGpl, P < 0.001) and DPA (+15%, P < 0.001) compared to ALA alone. Thus, SH-SY5Y cells produced membrane EPA, DPA and DHA from supplemental ALA. The formation of DHA was limited, even in the presence of E2. E2 significantly favored EPA and DPA production in cells grown for 72 h. Enhanced synthesis of ALA-elongation products in neuroblastoma cells treated with E2 supports the hypothesis that neurosteroids could modulate the metabolism of PUFA.

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood-brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (-23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 microM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [(3)H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood-brain barrier, possibly via changes in GLUT1 protein expression and activity.

The importance of a high fat intake in the increasing prevalence of childhood and adult obesity remains controversial. Moreover, qualitative changes (i.e. the fatty acid composition of fats) have been largely disregarded. Herein is reviewed the role of polyunsaturated fatty acids (PUFAs) of the n-6 series in promoting adipogenesis in vitro and favouring adipose tissue development in rodents during the gestation/suckling period. Epidemiological data from infant studies as well as the assessment of the fatty acid composition of mature breast milk and infant formulas over the last decades in the Western industrialized world are revisited and appear consistent with animal data. Changes over decades in the intake of n-6 and n-3 PUFAs, with a striking increase in the linoleic acid/alpha-linolenic ratio, are observed. In adults, using a consumption model based upon production data, similar changes in the PUFA content of ingested lipids have been found for France, and are associated with an increase of fat consumption over the last 40 years. These profound quantitative and qualitative alterations can be traced in the food chain and shown to be due to changes in human dietary habits as well as in the feeding pattern of breeding stock. If prevention of obesity is a key issue for future generations, agricultural and food industry policies should be thoroughly reevaluated.

The altered neuron activity of rats deficient in (n-3) PUFAs may be due in part to a decrease in brain glucose utilization and glucose transport. We measured the glucose transporter protein GLUT1 isoforms at the blood-brain barrier (55-kDa) and in astrocytes (45-kDa) by Western immunoblotting and their mRNA by real time RT-PCR analysis in the cerebral cortex of adult male rats fed diets lacking (n-3) fatty acids (1st generation). The neuron glucose transporter GLUT3 was also assayed. The fatty acids in the phosphatidylcholine (PC), ethanolamine phosphoglycerolipid (EPG), and phosphatidylserine (PS) fractions of isolated microvessels and homogenates of the cerebral cortex were determined. The levels of (n-6) PUFAs [mainly arachidonic acid, 20:4(n-6)] in the phospholipid fractions of microvessels were higher and the levels of (n-3) PUFAs [mainly docosahexaenoic acid, 22:6(n-3)] were lower than in cerebral cortex homogenates. The microvessels and cortex of rats fed the (n-3) PUFA-deficient diet had 50% of the control 22:6(n-3) contents; 22:6(n-3) was replaced by 22:5(n-6). The 55-kDa GLUT1 immunoreactivity in (n-3) PUFA-deficient microvessels was decreased (down 25%, P < 0.01), as was the 45 kDa-GLUT1 in the homogenate (down 30%, P < 0.01). But the amount of immunoreactivity of GLUT3 did not change. The amount of GLUT1 mRNA was not affected by the (n-3) PUFA-deficient diet. These results suggest that the decreased glucose utilization in the cerebral cortex of (n-3) PUFA-deficient rats is due to reduced amounts of the 2 isoforms of GLUT1, indicating post-transcriptional regulation of GLUT1 synthesis.

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 down-regulate the incorporation of newly formed DHA within human neuroblastoma cells. SH-SY5Y cells were incubated with gradual doses of alpha-linolenic acid (alpha-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 alpha-LNA, EPA, or DPA peaked at concentrations of precursors below 15-30 microM 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 beta-oxidation, L-bifunctional protein and peroxisomal acyl-CoA oxidase, were expressed at lower levels than fatty acyl-CoA ligase 3 (FACL3) and delta6-desaturase (delta6-D). The delta6-D mRNA slightly increased between 16 and 48 h of culture, and this effect was abolished in the presence of 70 microM 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.

Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) are the major polyunsaturated fatty acids in the membranes of brain and retinal cells. Animals specifically deficient in dietary n-3 fatty acids have low DHA content in their membranes, reduced visual acuity and impaired learning ability. Studies on bottle-fed human infants have shown that adding DHA and AA to milk replacer-formulas can bring their concentrations in the infant blood lipids to values as high as those produced by breast-feeding and significantly improves mental development and maturation of visual function. In older subjects, diverse neuropsychiatric and neurodegenerative diseases have been associated to decreased blood levels of n-3 PUFA. Low intakes of fish or of n-3 PUFA in populations have been associated with increased risks of depression and Alzheimer disease, and n-3 PUFA, especially eicosapentaenoic acid (EPA, 20:5n-3), have shown efficacy as adjunctive treatment - and in some cases as the only treatment--in several psychiatric disorders. The mechanisms by which polyunsaturated fatty acids have an impact on neuronal functions will be reviewed: the modulation of membrane biophysical properties, regulation of neurotransmitter release, synthesis of biologically active oxygenated derivatives, and nuclear receptor-mediated transcription of genes responsive to fatty acids or to their derivatives.

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 alpha-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., (Copyright 2003 Wiley-Liss, Inc.)

The mRNA expression levels of acyl-CoA oxidase (AOX), a key enzyme in very-long-chain fatty acid peroxisomal oxidation, and of peroxisome proliferator-activated receptor-delta (PPAR-delta), a nuclear receptor possibly involved in the gene regulation of brain lipid metabolism, were determined in human Y79 retinoblastoma cells by using real-time quantitative polymerase chain reaction. Cells were dosed with alpha-linolenic acid (18:3n-3), the essential metabolic precursor of the n-3 polyunsaturated fatty acid series that normally gives rise through terminal peroxisomal oxidation to the synthesis of membrane docosahexaenoic acid (22:6n-3, or DHA). The AOX and PPAR-delta relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 microM 18:3n-3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 microM 18:3n-3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine-phosphoglycerides from cells dosed with 7 microM 18:3n-3, whereas it decreased by 38% upon dosing with 70 microM 18:3n-3. The DHA's upstream precursors (20:5n-3 and 22:5n-3) both accumulated in cells dosed with 7 or 70 microM 18:3n-3. The 18:3n-3-induced changes in membrane phospholipid fatty acid composition support the hypothesis that the terminal peroxisomal step of n-3 conversion is rate limiting in the Y79 line. The concurrent 7 microM 18:3n-3-induced increase of mRNAs encoding for AOX and for PPAR-delta suggests that 18:3n-3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR-delta-mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70-microM 18:3n-3-dosed cells corroborated the relationship between AOX expression and DHA synthesis and suggested that simultaneous down-regulating events occurred at high concentrations of 18:3n-3., (Copyright 2003 Wiley-Liss, Inc.)

Background: Functional maturation of nervous tissues depends on membrane accretion of docosahexaenoic acid (DHA). Animal studies have shown that incorporation of dietary DHA into membrane phospholipids is dose dependent. The molecular effects of DHA are commonly studied in cultured cells, but questions remain about the physiologic connection between animal and cell models., Objective: We developed a linear model for comparing the responses of rat nervous tissues to dietary DHA with the responses of human cell lines to DHA in medium., Design: Rats were rendered chronically deficient in n-3 fatty acids by being reared on a peanut oil diet. DHA status was replenished in the F2 generation by using increasing supplements of a microalgal oil. Human retinoblastoma and neuroblastoma cells were dosed with unesterified DHA. DHA accumulation into phospholipids was defined by the plateau of the dose-response curve (DHA(max)) and by the supplement required to produce one-half the DHA(max) (DHA(50))., Results: The DHA(max) values for 4 brain regions and 2 neuroblastoma lines were similar, and the value for the retinoblastoma line was similar to the retinal value. Expressing the DHA input as micro mol/10 g diet and as micro mol/L medium resulted in similar values for the ratio of DHA(max) to DHA(50) in the 4 brain regions and the 3 cell lines. The DHA(max)-DHA(50) ratios in the ethanolamine phosphoglyceride and phosphatidylcholine fractions in retinal phospholipids were 6 and 10 times, respectively, those in the brain and cultured cells., Conclusions: The dose-dependent responses of cells and the brain to DHA supplements can be compared by using DHA(max)-DHA(50) ratios. We propose a counting frame that allows the comparison of the dose responses of the brain and cells to exogenous DHA.

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.

Alterations in lipid composition occur in the retinal pigment epithelium and photoreceptor cells of the Royal College of Surgeons (RCS) dystrophic rat, a model for inherited retinal degeneration. With respect to lipid composition of nonretinal tissues, the developmental timing of lipid alterations and the incidence of dystrophy are unknown. We determined the fatty acid composition in choline phosphoglycerides (ChoGpl) and ethanolamine phosphoglycerides (EtnGpl) in the brain, liver, and retina from dystrophic RCS rats and from their nondystrophic congenics (controls) at the ages of 3 and 6 wk. At 3 wk, the fatty acid compositions were specific to individual phospholipid classes without any difference between dystrophic and nondystrophic tissues. In plasma phospholipids, there was an age-related increase in the relative contents of monounsaturated and n-3 polyunsaturated fatty acids, with only minor differences between dystrophic and nondystrophic rats. At 6 wk, the fatty acid compositions in ChoGpl and EtnGpl from dystrophic brain and retina were significantly different from those of nondystrophics. The effect of strain on developmental changes in brain fatty acid composition was significant for 18:0 and 22:6n-3 in EtnGpl and for 16:0, 18:0, 18:1n-9, and 20:4n-6 in ChoGpl. The brain ChoGpl fatty acid composition in nondystrophic rats was similar at 6 wk to that of normal rats, and there were almost no postweaning changes in the dystrophics. In retinal phospholipids, the effect of dystrophy was to increase the 20:4n-6 content in EtnGpl and to decrease 22:6n-3 in ChoGpl. The 18:2n-6 and 22:6n-3 contents in dystrophic liver ChoGpl were also significantly affected, while no difference was observed in the EtnGpl fraction. The dystrophy affected the phospholipid fatty acid developmental changes in a tissue- and class-specific manner. Fatty acid metabolism could be selectively altered in neural and nonneural tissues of developing dystrophic RCS rats.

Animal and human studies have indicated that developing mammals fed only alpha-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 18:3n-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 18:3n-3 and 22:6n-3 lead us to speculate that these two n-3 polyunsaturated fatty acids were beta-oxidized in the same amount.

The metabolic conversion of n-3 fatty acids was studied in the human Y79 retinoblastoma cell line. Cultured cells were exposed to increasing concentrations of either 18:3n-3, 22:5n-3, or 22:6n-3, and their phospholipid fatty acid composition was analyzed after 72 hr. Cells internalized the supplemental fatty acids and proceeded to their metabolic conversion. Supplemental 22:6n-3 was directly esterified into cell phospholipids, at levels typical for normal neural retinas (41% by weight of phosphatidylethanolamine fatty acids, and 24% of phosphatidylcholine fatty acids). In contrast, 18:3n-3 was mainly converted to 20:5n-3 and 22:5n-3, both of which appeared in cell phospholipids after exposure to low external concentrations of 18:3n-3 (10 microg/ml). Y79 cells can proceed to the metabolic conversion of 18:3n-3 through elongation and Delta6- and Delta5-desaturation. When cells were exposed to high external concentrations of 18:3n-3 (30 microg/ml), the supplemental fatty acid was directly incorporated, and its relative content increased in both phospholipid classes to the detriment of all other n-3 fatty acids. Cells cultured in the presence of 22:5n-3 did not incorporate 22:6n-3 into their phospholipids but did incorporate 20:5n-3 and 22:5n-3. The data suggest that Y79 cells can proceed to the microsomal steps of n-3 metabolism, involving elongation, desaturation, and chain shortening of 22C fatty acids. Although Y79 cells avidly used supplemental 22:6n-3 for phospholipid incorporation at levels typical for normal photoreceptor cells, they failed to match such levels through metabolic conversion of n-3 parent fatty acids. The terminal step of the very long-chain polyunsaturated fatty acid synthesis, consisting in Delta6-desaturation followed by peroxisomal chain shortening of 24C-fatty acids, could be rate-limiting in Y79 cells., (Copyright 2000 Wiley-Liss, Inc.)

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.

We investigated the influence of four different culture media: 20% fetal bovine serum (FBS), 5% FBS, 5% FBS supplemented with 10 mg x L(-1) linoleic acid (18:2(n-6)) or alpha-linolenic acid (18:3(n-3)) on alpha-linolenic acid apical uptake in clone TC7 of human intestinal Caco-2 cell line. Neither cellular viability nor cell monolayer integrity and permeability were altered by the four culture conditions. Our results show that the different culture media led to changes in alpha-linolenic acid maximal rate of uptake (Vmax) but did not alter the apparent transport constant (Km). Reducing FBS concentration from 20% to 5% increased significantly the rate of alpha-linolenic acid uptake, which was further increased by supplementation of the medium with 18:2(n-6) or 18:3(n-3). Supplementation with essential fatty acids led to a marked enrichment of brush-border membrane phospholipids in polyunsaturated fatty acids of the corresponding series and decreased significantly the levels of monounsaturated fatty acids. Saturated fatty acids, unsaturation index, and cholesterol/fatty acid ratios were unchanged. No clear relation could be established between the changes in membrane lipid composition and the alterations of alpha-linolenic acid uptake. These results indicate a weak influence of membrane lipid composition in the modulation of the uptake. Therefore, the increase of uptake following long-term supplementation of TC7 cells with essential fatty acids could be attributed to an increase of the expression of membrane protein(s) involved in the apical uptake of long-chain fatty acids. This remains to be established.

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, 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 of 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.

The incorporation and conversion of polyunsaturated fatty acids (PUFA) of n-3 and n-6 families were examined in the T47D breast cancer cell line in parallel with their effects on cell proliferation. In low serum-containing medium, PUFA exerted differential growth effects, depending both on their affiliation and unsaturation degree. The study of PUFA processing suggested that T47D cells are deficient in delta 6 and delta 4-desaturation activities whereas they can process to delta 5-desaturation. Thus, the PUFA growth effect on T47D cells appeared to be associated with a lack of desaturation.

This study was designed to determine whether dietary lipids influence the development of intestinal cell glycosylation, in relationship to diet-induced changes in phospholipid fatty acid composition. The ability of two different lectins, wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA), to combine specifically with particular carbohydrate residues was used to investigate the surface characteristics of epithelial cells of rats fed different dietary lipids from birth to 6 weeks of age. Diets contained 5% (weight) peanut oil (PO), rich in n-6 fatty acids; salmon oil (SO), rich in n-3 fatty acids; hydrogenated palm oil (HPO), deficient in both n-6 and n-3 fatty acids or a PO and rapeseed oil (RO) mixture (PRO), the control diet. Pieces of jejunal and ileal villi were excised from postweanling rats and prepared for lectin histochemical study. Concurrently, epithelial cells were removed from jejunal and ileal segments for determining their phospholipid fatty acid compositions. Polyunsaturated fatty acid (PUFA) deficiency was evidenced in the HPO group by the appearance of eicosatrienoic acid (20:3n-9) in both jejunal and ileal phospholipids, which paralleled the decrease in arachidonic acid content. Accretion of 18:1n-9 and 20:3n-9 in cell phospholipids of group HPO was not sufficient to match the unsaturation level in rats fed nonhydrogenated vegetable oils (PRO, PO) or fish oil (SO). The lectin histochemical study showed that WGA strongly labelled the brush border membrane microvilli whereas binding of MAA was specific to goblet cells and mucus. Regardless of the type of diet, WGA binding was weaker in the ileum than in the jejunum. In comparison to all other groups, WGA-labelling of villi was less intense in the jejunum and disappeared almost completely in the ileum of HPO-fed rats. Although SO- and PO-fed rats had, respectively, very low and high ratios of n-6 to n-3 in their intestinal phospholipids, binding of WGA in both groups was not markedly different from that in the control (PRO). MAA-labelling was very intense in jejunal and ileal villi of n-3-fed (SO) rats, whereas it was strongly attenuated in the n-3- and n-6 deficient (HPO) group. These results suggest that intestinal glycosyltransferase activities involved in cell differentiation were altered relative to the overall unsaturation index of dietary fatty acids. Alterations of epithelial glycosylation mainly resulted from a drop in total n-6 and n-3 fatty acids, although it may be speculated that there is a specific effect of n-3 fatty acids.

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterations might be related to the appearance of specific functions.

The lipid components of columnar cells harvested from rat small intestine were analyzed at each step of cell maturation. The effect of dietary lipids on the evolution of lipids in differentiating cells was studied using two diets representative either of a control or of an essential polyunsaturated fatty acid deficient lipid supply. Two groups of weanling rats were fed a semisynthetic diet composed of corn oil (control diet) or hydrogenated coconut oil (deficient diet) for 11 weeks. Intestinal cells were extracted according to their position along the villus column. Linoleic and arachidonic acids constitutive of cell phospholipids increased as cells migrated from the lower to the mid-part of the crypt-villus axis only with the control diet. This gradient disappeared after a 3-week feeding period with hydrogenated coconut oil diet so that columnar cells of essential fatty acid deficient rats exhibited the same overall fatty acid composition all along the crypt-villus axis. Essential fatty acid deficiency resulted in an increase of both the triene to tetraene and arachidonic acid to linoleic acid weight ratios regardless of the maturational step of cells. As compared to the control diet, the essential fatty acid deficient diet induced a decrease of both the cholesterol and free fatty acid to phospholipid molar ratios only in lower villus cells. We conclude that (a) progressive compositional modifications of lipids occur while ascending the crypt-villus axis and (b) essential fatty acid deficiency induces substantial alterations of the lipid evolution normally linked to cell differentiation.

Alterations in lipids linked to intestinal maturation and enterocyte differentiation were reviewed. The 3 main lipid components of cell membranes, ie cholesterol, phospholipids and glycolipids, were examined. Cell phospholipid content increases from the crypts to the mid-villus, which accounts for membrane development and organelle growth in differentiating cells. Changes in the proportion of phospholipid polar head groups occur in brush border membrane during postnatal maturation of the small intestine. The possibility that phospholipid fatty acid composition in differentiating cells might be altered by dietary lipids is discussed. Cholesterol biosynthesis mainly occurs in crypt and lower villus cells whereas its absorption from luminal content and esterification into lipoproteins occur in upper villus mature cells. Cholesterol cell content increases in mature cells in comparison to immature cells on the one hand, and in the distal by comparison with proximal parts of the intestine on the other. Increasing cholesterol content is generally correlated with decreasing membrane fluidity, which in turn could modulate functional properties of the mucosa. Glycosphingolipids are mainly found in the brush border membrane, which contains 20-30% glycolipids by weight of total lipids. These components tend to reinforce the membrane stability and significantly contribute to the surface properties of epithelial cells. The latter undergo noticeable changes during cell differentiation and postnatal maturation. Significant changes in both the glycosidic and lipophilic parts of glycosphingolipid molecules occur in differentiating cells and are of possible importance in the process of mucosal maturation. It is possible that the addition of a terminal sialic acid (sialyltransferase activity) instead of a terminal galactose (galactosyltransferase) to an endogenous acceptor (lactosylceramide) could constitute an important event in the differentiation process, and may account for the increasing content of hematosides along the intestinal villus of rat. Alterations in lipid counterpart mainly consist of hydroxylation of fatty acids in hematosides during postnatal maturation or in glucosylceramides during cell differentiation. Collectively these intestinal lipid changes may contribute in part to the development of mucosal barrier, selective permeability and functional properties of the mature intestinal mucosa.

Affinity chromatography of lipase on a colipase-coupled gel was studied in the present paper. The elution volume of the associable lipase increased when the loaded amount decreased. A KD value of 1.9 X 10(-6) M at pH 6.2 was thus deduced. A minimum value of 1.5 X 10(-6) M was obtained at pH 5.1-5.3. Mixed micelles associated with coupled colipase, but no modifications of lipase-colipase interactions took place when mixed micelles were added to the elution buffer. DMMA-modified coupled colipase failed to interact with lipase, owing to the specific orientation of the modified cofactor in the gel.

The technique of zonal high-performance affinity chromatography applied to the lipase-colipase system (lipase B as eluted acceptor and colipase as silica-bonded ligand) gave qualitatively the same results as conventional affinity chromatography. The elution volume of the acceptor increases with decreasing load introduced at constant volume into the column of ligand-bonded silica. This led to the use of a mathematical treatment for calculating the dissociation constant (KD) of the lipase-colipase complex. The influence of some physical and chemical chromatographic parameters was studied. Increasing temperature and flow-rate reduced the affinity of lipase for colipase, whereas it was only slightly modified by increasing the ionic strength. The KD value was minimal and equal to 0.1 X 10(-6) M at pH 4.7 and 0.38 X 10(-6) M at pH 6.5, after correction for the flow-rate. The latter value is similar to that obtained by more conventional techniques. The absence of some marked KD modifications by ionic strength and the value of delta S for the complex association obtained by temperature studies suggest the intervention of mixed hydrophobic-ionic interactions in the formation of the lipase-colipase complex. Their respective importances are discussed.

The surface properties of small intestine brush-border membranes (BBMs) were examined by frontal affinity chromatography using three types of unsolubilized ligands: phlorizin polymer, immobilized lectins and linolenic acid bound to agarose gel. BBM vesicles were purified from piglets fed a corn oil diet (control diet) or a hydrogenated coconut oil diet. The second diet was representative of a deficient supply of essential polyunsaturated fatty acid (EPUFA). It induced a marked decrease in 18:2n-6 content in membrane choline phosphoglycerides and ethanolamine phosphoglycerides, whereas 20:3n-9 appeared in each class of phospholipids. Control and EPUFA-deprived BBM vesicles bound to phlorizin polymer, linolenic acid-agarose and wheat germ agglutinin (WGA) gel. In contrast, concanavalin A gel and Lens culinaris A gel exhibited a low binding capacity towards the two types of vesicles. EPUFA deficiency induced a slight decrease in binding on phlorizin polymer and a marked increase in binding on WGA gel, whereas the two types of vesicles similarly bound to linolenic acid-agarose. Desorption of phlorizin polymer-bound membranes was performed using several detergents with special regard to sodium deoxycholate (NaDOC) micelles. Sucrase activity recovery showed that the efficiency of NaDOC desorption was diminished in the case of EPUFA-deprived vesicles. EPUFA-deprived membrane domains involved in the binding would be less sensitive to the detergent attack. This assumption agrees with the putative decrease in membrane fluidity induced by the deficient diet. The possibility that fatty acid compositional changes induced by dietary lipids are extensive enough to alter some chromatographic properties of BBM vesicles is discussed.

Procedures for purification of porcine colipase II (Gly6-Gly89) and for obtaining purified anti-colipase antibodies are described. The interactions between antibodies immobilized on an Ultrogel AcA 22 column and colipase were investigated and colipase radioimmunoassay carried out. The immobilized antibody-colipase binding was preserved in the presence of mixed micelles, lipase, or both when added to the elution mixture. Bound colipase maintained its capability of interacting with mixed micelles, but not with lipase in either the presence or the absence of mixed micelles. It could be inferred that the antigenic site(s) is independent of the interfacial recognition site and close to the site of lipase recognition. Results are reported suggesting that one or both colipase histidyl residue-containing sequences are involved as antigenic determinant(s). Immunoreactive colipase, bound to a macromolecular protein complex, was found in the plasma of pig. This finding could be explained by an endocrine 'leakage' of colipase from the exocrine pancreatic cell rather than by passage through the intestinal mucosa.