D. E. Mills

The role of n−3 essential fatty acids in brain and behavioral development: A cross-fostering study in the mouse

A cross-fostering design was used to examine the effects on brain and behavioral development in mice of pre-and/or postnatal dietary supplementation with n−3 fatty acids. Pregnant mice were fed either of two liquid diets, control (con) or experimental (exp). Each diet provided 3% of the calories in the form of n−6 fatty acids; the experimental diet was supplemented with an additional 1.5% from long chain n−3 fatty acids derived from fish oil. There were four treatment groups, with all pups fostered at birth. These groups were (prenatal diet/ postnatal diet): Group 1, exp/exp; Group 2, exp/con; Group 3, con/exp; Group 4, con/con; a fifth control group (unfostered) was fed lab chow (LC) throughout the study. Animals from the exp/exp and con/con groups were weaned onto lab chow for later behavioral assessment. Prenatal n−3 supplementation resulted in a small acceleration of behavioral development. The adult animals did not differ in visual discrimination learning nor did they differ in visual acuity. During development the fatty acid composition of the brain membrane phospholipids reflected closely that of the pre- and postnatal dietary conditions. Levels of 22∶5n−3 and 22∶6n−3 increased in the n−3 supplemented groups, accompanied by a decrease in levels of 22∶4n−6 and 22∶5n−6; the net effect of these changes was to increase the total levels of C22 fatty acids. While these results support considerable plasticity of the fatty acid composition of the developing brain with respect to the immediate dietary availability of n−3 compounds, they do not support long term effects on learning capacity of n−3 supplementation during the developmental period.

The Effects of dietary n−3/n−6 ratio on brain development in the mouse: a dose response study with long-chain n−3 fatty acids

This study examines the effects of the ratio of n−3/n−6 fatty acids (FA) on brain development in mice when longchain n−3 FA are supplied in the diet. From conception until 12 days after birth, B6D2F1 mice were fed liquid diets, each providing 10% of energy from olive oil, and a further 10% from different combinations of free FA concentrates derived from safflower oil (18∶2n−6), and fish oil (20∶5n−3 and 22∶6n−3). The range of dietary n−3/n−6 ratios was 0,025, 0.5, 1.0, 2.0, and 4.0, with an n−6 content of greater than 1.5% of energy in all diets, and similar levels of total polyunsaturated fatty acids (PUFA). In an additional group of ratio 0.5, 18∶2n−6 was partially replaced by its δ6 desaturation product, 18∶3n−6. Biochemical analyses were conducted on 12-day-old pup brains, as well as on samples of maternal milk. No obvious effects on overall pup growth and development were observed, apart from a smaller litter size at ratio 1. Co-variance analysis indicated that increasing the n−3/n−6 ratio was associated with slightly smaller brains, relative to body weight. We found that 18∶2n−6 and 20∶5n−3 were the predominant n−6 and n−3 FA in the milk; in the brain these were 20∶4n−6 and 22∶6n−3, respectively. Increasing dietary n−3/n−6 ratios generally resulted in an increase in n−3 FA, with a corresponding decrease in n−6 FA. The n−3/n−6 ratio of the milk lipids showed a strong linear relationship with the diet, but in the brain the rate of increase tended to decrease beyond 0.5 (phosphatidylcholine, PC) and 0.25 (phosphatidylethanolamine, PE), such that there was a significant quadratic contribution to the relationship. The partial replacement of dietary 18∶2n−6 with 18∶3n−6 raised levels of 20∶4n−6 in milk, brain PC, and brain PE. These results indicate that the n−3/n−6 ratio of the phospholipids in the developing mouse brain responds maximally to maternal dietary long-chain n−3/n−6 ratios of between 0.25 and 0.5.

Psychosocial Stress, Catecholamines, and Essential Fatty Acid Metabolism in Rats

Abstract To examine the effects of psychosocial stress and the “stress hormone,” epinephrine, on essential fatty acid metabolism in rats, two studies were conducted. In the first, the effects of four weeks of (i) social isolation and (ii) group housing (control) on liver microsomal Δ6 and Δ5 n-6 desaturase activity were studied in group-reared male normotensive (Wistar Kyoto) and spontaneously hypertensive (SHR) rats (n = 5/group). The second study examined the effects of acute ip epinephrine (0.0, 1.0, 2.0, and 4.0 mg/kg) 6 hr prior to and following an ig dose (4 g/kg) of safflower oil (rich in 18:2n-6, LA) on plasma and liver LA, 20:4n-6 (AA), and LA/AA ratios in adult essential fatty acid deficient Sprague-Dawley rats (n = 6/group). In the first experiment, isolation stress significantly inhibited the activity of Δ6 (P < 0.05) and Δ5 (P < 0.01) desaturase in the normotensive rats and of Δ5 desaturase in the SHR (P < 0.05). In the second study, epinephrine increased plasma and liver LA at doses 1.0 and 2.0 mg/kg in most of the fractions examined, and suppressed AA levels. The response of the LA/AA ratio to epinephrine varied between tissues and among lipid fractions, but increased this ratio at the moderate doses (2.0-4.0 mg/kg) of epinephrine in most cases. These data suggest that psychosocial stressors are capable of inhibiting the rate limiting steps of essential fatty acid metabolism and that this response is more pronounced in the SHR than in the Wistar Kyoto. They also suggest that epinephrine is capable of altering the in vivo metabolism of essential fatty acids in the rat.

Effect of maternal dietary fats with variable n-3/n-6 ratios on tissue fatty acid composition in suckling mice

This report examines the distribution of n−3 and n−6 fatty acids in heart, kidney and liver phosphatidylcholine and phosphatidylethanolamine of suckling mice from dams fed a fat-supplemented diet with variable n−3/n−6 ratios. After conception and throughout the pregnancy and lactation period, dams were fed a fat-free liquid diet supplemented with 20% by energy of oil mixtures (fish oil concentrate, rich in 20∶5n−3 and 22∶6n−3, and safflower oil concentrate, rich in 18∶2n−6). The diets contained similar amounts of combined n−3 and n−6 fatty acids but variable ratios of n−3 to n−6 fatty acids (0,025, 0.5, 1, 2, and 4). In 12-day-old suckling mice, as the n−3nn−6 ratio in the maternal diet increased (up to approx. 0.5), the tissue levels of 20∶5n−3, 22∶5n−3 and 22∶6n−3 increased, whereas those of 18∶2n−6 and 20∶4n−6 decreased. The responses were similar in both phospholipid subclasses, but varied between different tissues. Generally, the n−3/n−6 ratios were significantly greater in pup tissues than in milk fat, indicating preferential incorporation of n−3 over n−6 fatty acids into phospholipids during growth. However, the incorporation of n−3 fatty acids in pups was significantly suppressed whereas that of n−6 fatty acids was increased when 18∶2n−6 was replaced by its δ6-desaturation product, 18∶3n−6 (concentrated from evening primrose oil), as the source on n−6 fatty acid. This result suggests that δ6 desaturase activity in neonate tissues is low, and consequently, the metabolism of 18∶2n−6 to longer chain n−6 fatty acids is reduced. The preformed long-chain n−3 fatty acids, which bypass δ6-desaturation, were thus, preferentially incorporated into tissue phospholipids.

Interactive effects of prenatal ethanol and N−3 fatty acid supplementation on brain development in mice

This study assesses the combined effects on brain and behavioral development of ethanol administration and supplementation of the maternal diet with long chain n−3 polyunsaturated fatty acids. From day 7 to 17 of gestation, pregnant mice were fed equivalent daily amounts of isocaloric liquid diets; 20% of the energy was provided by either ethanol or maltose-dextrin, and a further 20% by either safflower oil (rich in linoleic acid, 18∶2n−6), or a combination of safflower oil with a fish oil concentrate (rich in eicosapentaenoic acid, 20∶5n−3, and docosahexaenoic acid, 22∶6n−3). On day 18 the liquid diets were replaced by lab chow; a fifth group was maintained on lab chow throughout the experiment. Measures on the pups included brain weight and the fatty acid composition of the brain phospholipids on days 22 and 32 post-conception (birth=day 19), as well as behavioral development. Maternal weight gain during gestation was decreased by ethanol relative to maltose-dextrin, and increased by fish relative to safflower oil. On day 32, the brain weight of ethanoltreated animals fed fish oil was greater than their safflower oil controls, whereas the reverse was true in the two maltose-dextrin groups; a similar trend was apparent on day 22. The brain phospholipid content of the longer chain fatty acids (20∶4n−6, 22∶4n−6, 22∶5n−6, 20∶5n−3, 22∶5n−3, 22∶6n−3) on day 22 reflected that of the prenatal diet, with the proportion of n−3 compounds being higher and that of n−6 floer in the fish oil than safflower oil groups. Prenatal dietary effects were absent by day 32, with the exception of lower 22∶5n−6 in fish oil groups. Dietary supplementation with n−3 fatty acids increased the ratio of 20∶3n−6 to 20∶4n−6, which is consistent with a blockade of the activity of Δ-5 desaturase. On day 22 the incorporation of dietary long chain n−3 fatty acids into the brain phosphatidylcholine fraction was enhanced in the ethanol-treated animals; by day 32 the animals treated prenatally with ethanol also showed increased levels of long chain n−6 compounds. Behavioral development was retarded by ethanol, but there was no effect of the dietary oils. These results support the hypothesis that effects of ethanol on the developing brain may be modified by the availability of an exogenous supply of long chain fatty acids.

Attenuation of Psychosocial Stress-Induced Hypertension by Gamma-Linolenic Acid (GLA) Administration in Rats

Abstract This study investigated a model of psychosocial stress-induced hypertension in the rat, and examined effects of the prostaglandin E precursor, gamma-linolenic acid (GLA) on the development of hypertension during psychosocial stress. In the first study, male rats were housed four/cage for an acclimation period of 21 days, followed by a 14-day control period. An experimental group (N = 12) was then placed in isolation cages for 14 days, then regrouped for a 7-day recovery period. Controls (N = 12) remained group-housed. Eight animals per group were sacrificed after the experimental period, and four per group after recovery for organ weight analysis. Mean systolic blood pressure (BP) was similar between groups during the control period (126 ± 2 and 125 ± 2 mm Hg), but increased during isolation, reaching 140 ± 2 mm Hg (P < 0.001) by Day 14. During recovery BP returned to control levels. No changes in heart rate, heart weight/body weight or adrenal weight were seen. The second study utilized a protocol similar to that of the experimental group of the first study, minus the recovery period. On Day 1 of the control period 28-day osmotic pumps were implanted ip, releasing olive oil or GLA in olive oil. Four groups of rats (N = 8/group) received either (i) olive oil (controls), (ii) 0.018 mg GLA/hr, (iii) 0.040 mg GLA/hr, or (iv) 0.040 mg GLA/hr with no stress. Organ weights were obtained following stress in groups 1-3. Controls developed a sustained elevation in BP within 24 hr of isolation. Animals receiving 0.018 mg GLA/hr developed elevated BP upon isolation, but the BP was less than that of controls on Days 1 (P < 0.05) and 14 (P < 0.001) of isolation. Animals receiving 0.040 mg GLA/hr demonstrated a greatly attenuated rise in BP vs controls (P < 0.001) on all isolation days. GLA in unstressed rats had no effect on BP. Heart rate, heart weight/body weight, and adrenal weight were unchanged in all groups. These data suggest that (i) isolation is a useful tool for investigating reversible psychosocial stress-induced hypertension, and (ii) GLA, while not affecting BP in unstressed animals, produces a dose-dependent attenuation of the BP response to chronic stress.

Effects of Eicosapentaenoic Acid (20:5ω3) on Stress Reactivity in Rats:

Abstract This study examined the effects of eicosapentaenoic acid (EPA) on cardiovascular responses to isolation stress in male rats. Group-reared rats, on a fat-free diet, were given olive oil (OL), or EPA in OL (1.47 x 10−7 mol/hr) via 8 week osmetic pumps, or a dummy pump (DUM), 2 weeks prior to a 4 week isolation period. Blood pressure (BP) , heart rate, and body weight were monitored weekly and pressor responses to i.a. norepinephrine and angiotensin were assessed at the end of the study. BP increased during stress in all animals vs. pre-stress conditions, but was attenuated by EPA (p<0.001). Heart rate also increased during stress in all groups, but was greater in the EPA group (p<0.001). In contrast, body weight gain during stress was similar in DUM and EPA groups, but depressed by OL (p<0.001). Vascular response to norepinephrine was enhanced by EPA vs. DUM and OL, whereas the response to angiotensin was similar in EPA and DUM groups, but reduced by OL. These data suggest that EPA may attenuate cardiovascular responses to psychological Stress.

Effect of essential fatty acid depletion on tissue phospholipid fatty acids in spontaneously hypertensive and normotensive rats

Weanling male spontaneously hypertensive (SHR) and normotensive (WKY) rats were maintained on a fat-free semisynthetic diet and killed at various intervals. The effects of fat-depletion on the appearance of essential fatty acid (EFA) deficiency symptoms, the progressive changes of major fatty acids in plasma, liver, heart, and kidney phospholipids (PL), and in skin total lipids were compared between these two strains. After five weeks on the diet, the slower growth and the appearance of EFA deficiency symptoms became evident in SHR. In general, fat-depletion reduced the levels of n−6 fatty acids, whereas it increased those of 20∶3n−9. However, the fat-depletion induced reduction of 18∶2n−6 in heart PL and 20∶4n−6 in kidney, while the elevation of 20∶3n−9 in plasma, heart, and kidney PL were greater in WKY than in SHR. As a result, the elevation of biochemical EFA deficiency index—20∶3n−9/20∶4n−6 ratio—was greater in WKY than in SHR. In comparison with WKY, the concentrations of liver triacylglycerols and the weights of adipose tissues in SHR were reduced to a greater extent, indicating an active catabolism of triacylglycerols in SHR. This study suggests that the earlier appearance of morphological symptoms of EFA deficiency in SHR was not associated with the reducing n−6 EFA levels or with an elevation of triene/tetraene ratio, but possibly to a reduced supply of n−6 EFA for skin prostaglandin synthesis.

Effects of bromocriptine on sweat gland function during heat acclimatization

This study examined the involvement of the hormones aldosterone and prolactin in sweat gland function during heat acclimatization. Two groups of male subjects (n = 8) were tested - one receiving a placebo (control), the other receiving bromocriptine. Both groups performed cycle ergometer exercise at 50% of maximal oxygen uptake over 10 consecutive days in an environmental chamber maintained at 39 degrees C and 30% relative humidity. Duration of exercise was 90 min on days 2-4 and 6-9, and 45 min on test days 1, 5 and 10. Electrolyte concentrations were determined by total body washdown. Prolactin increased (p less than 0.001) during exercise on day 1 in the control group but not on days 5 and 10. In contrast, prolactin was suppressed by bromocriptine and did not rise in response to exercise or heat exposure. Plasma aldosterone increased during exercise in both groups, showing no differences between groups. The sodium concentration in sweat decreased significantly (p less than 0.05) in the control group from day 1 to 10 but was unchanged in the treatment group. These data suggest that acclimatization-related changes in sweat gland function may be attenuated by increases in central dopaminergic activity and implicate prolactin in control of sweat gland function.

Stress modulates cholesterol-induced changes in plasma and liver fatty acid composition in rats fed N-6 fatty acid-rich oils

Abstract The effects of dietary cholesterol (CH) and isolation stress on fatty acid compositions of plasma and liver cholesteryl ester and phospholipids were compared in growing rats fed an 18:2n-6 or an 18:3n-6 enriched semisynthetic diet for 2 weeks. Stress, CH-feeding, and dietary fats had no significant effects on plasma CH level, but CH-feeding alone elevated the liver CH concentrations. CH-feeding also modulated the liver polyunsaturated fatty acid compositions, i.e., increasing 18:2n-6 levels, and reducing 20:4n-6 levels, indicating an inhibition of the enzymes, δ-6 and δ-5-desaturases. The extent of these changes was less in rats fed 18:3n-6 than in those fed 18:2n-6. Stress, which alone had no significant effects on plasma and liver fatty acid compositions, attenuated the CH-induced changes of fatty acid levels.