A Walnut Diet in Combination with Enriched Environment Improves Cognitive Function and Affects Lipid Metabolites in Brain and Liver of Aged NMRI Mice

Abstract

This in vivo study aimed to test if a diet enriched with 6% walnuts alone or in combination with physical activity supports healthy ageing by changing the oxylipin profile in brain and liver, improving motor function, cognition, and cerebral mitochondrial function. Female NMRI mice were fed a 6% walnut diet starting at an age of 12 months for 24 weeks. One group was additionally maintained in an enriched environment, one group without intervention served as control. After three months, one additional control group of young mice (3 weeks old) was introduced. Motor and cognitive functions were measured using Open Field, Y-Maze, Rotarod and Passive Avoidance tests. Lipid metabolite profiles were determined using RP-LC-ESI(-)-MS/MS in brain and liver tissues of mice. Cerebral mitochondrial function was characterized by the determination of ATP levels, mitochondrial membrane potential and mitochondrial respiration. Expression of genes involved with mito- and neurogenesis, inflammation, and synaptic plasticity were determined using qRT-PCR. A 6% walnut-enriched diet alone improved spatial memory in a Y-Maze alternation test (p < 0.05) in mice. Additional physical enrichment enhanced the significance, although the overall benefit was virtually identical. Instead, physical enrichment improved motor performance in a Rotarod experiment (p* < 0.05) which was unaffected by walnuts alone. Bioactive oxylipins like hydroxy-polyunsaturated fatty acids (OH-PUFA) derived from linoleic acid (LA) were significantly increased in brain (p** < 0.01) and liver (p*** < 0.0001) compared to control mice, while OH-PUFA of α-linolenic acid (ALA) could only be detected in the brains of mice fed with walnuts. In the brain, walnuts combined with physical activity reduced arachidonic acid (ARA)-based oxylipin levels (p < 0.05). Effects of walnut lipids were not linked to mitochondrial function, as ATP production, mitochondrial membrane potential and mitochondrial respiration were unaffected. Furthermore, common markers for synaptic plasticity and neuronal growth, key genes in the regulation of cytoprotective response to oxidative stress and neuronal growth were unaffected. Taken together, walnuts change the oxylipin profile in liver and brain, which could have beneficial effects for healthy ageing, an effect that can be further enhanced with an active lifestyle. Further studies may focus on specific nutrient lipids that potentially provide preventive effects in the brain.

Keywords: Ageing; Behaviour; Brain; Cognition; Liver; Mitochondrial function; NMRI; Neurodegeneration; Oxylipins.

Fig. 1

Simplified PUFA synthesis and origin of oxylipins analysed within this study formed by cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 monooxygenase (CYP) (Gabbs ; Ostermann et al. ; Coras 2020). Bold arrows connecting PUFA indicate a multiple-step elongation process between each of them. Thin arrows indicate oxylipins formed by the exemplary indicated enzymes from the PUFA in single or multiple-step reactions. It should be noted that several of the oxylipins can also be formed by other pathways as well as autooxidation. n6-PUFA are shown on the left, n3-PUFA on the right. The oxylipins act as potent lipid mediators showing diverse biological activity ranging from pro-inflammatory (e.g. PG, LT) to analgesic, vasodilatory, anti-inflammatory action (Epoxy-PUFA). Oxylipins from n3-PUFA are commonly considered to be less potent inflammatory mediators compared to n6-PUFA oxylipins or even anti-inflammatory. Displayed are only a few selected oxylipins, not a complete set of all oxylipins known to be produced. Data for underlined oxylipins can be found in Table 5 or Fig. 5

Fig. 2

Y-Maze alternation test. a Number of entries into the three different arms of the Y-Maze. Duration of Y-Maze experiment was 5 min. b Number of full alternations. One full alternation is defined as a mouse entering all arms of the maze before re-entering the already visited one. c Alternation rate of Y-Maze test, calculated as the number of full alternations divided by the number of entries. d Rotarod test. Time in [s] of mice being able to stay on the rotating rod. Speed of the rod gradually increased until 60 s into the experiment; experiment was stopped if mice were able to stay on the rod for 120 s. e Passive Avoidance test. Mice were put in an illuminated chamber and time was measured until they retreated into a dark chamber. On day one, a weak stimulus was applied upon entering the dark chamber. On the second day, time until mice entered the dark chamber was recorded to investigate mice’s memory. Displayed are mean ± SEM of n = 12–15 in a–d and n = 10–12 in e. Full range of data is displayed using dots for each individual mice. Statistical significance was tested via one-way ANOVA and post hoc Dunnett’s test comparing all groups with oldCon (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001). oldCon = aged Control; youCon = Young control; Wal = Walnut group; WalEE = Walnut + Enriched Environment group. Parameters for the statistical tests can be found in the supplementary Table S1

Fig. 3

a Oxygen consumption of mitochondria isolated from the brain adjusted to protein. Activity of OXPHOS complexes were assessed via addition of several substrates, inhibitors or uncouplers. Which substance was added in which stage of the experiment is marked with “ + ”; n = 12. b Basal MMP levels were measured as R123 fluorescence after incubation of DBC samples. c MMP levels after 0.5 mM SNP-induced nitrosative stress. d Basal ATP concentration measured in µM per mg protein in the DBC samples. Measured signal stems from bioluminescence reaction of luciferin and ATP. e ATP levels after SNP-induced nitrosative stress. DBCs have been incubated for 3 h prior to measurement. f Citrate synthase activity as an indicator of mitochondrial content. Amount of protein was measured via BCA method. Displayed are means ± SEM. Statistical significance was tested via one-way ANOVA with Dunnett’s post hoc test using oldCon as reference for statistical comparisons. Each group in C-F consisted of 12–15 female NMRI mice. Parameters for the statistical tests can be found in the supplementary Table S2

Fig. 4

Heatmap of hydroxy-PUFA, epoxy-PUFA and dihydroxy-PUFA oxylipins derived from LA, ALA, ARA, EPA and DHA in brain (a) and liver (b). Note Data are displayed relative to results for old control group (oldCon, marked in white). Green colouring and colour strength indicates an increase compared to the aged control group, while brown colouring indicates a decrease. < LLOQ indicate oxylipin concentrations below the lower limit of quantification. Full disclosure of results including significance levels can be found in the supportive information/appendix

Fig. 5

Concentration of ARA-derived prostaglandins in brain a and liver b; n = 15; Displayed are means ± SEM; the LLOQ is indicated if an analyte was < LLOQ in > 50% of the samples. One-way ANOVA with Dunnett’s multiple comparison post-test, comparing all groups with oldCon, was used to calculate significance; Significance is displayed as: *p < 0.05, **p < 0.01, ***p < 0.001. Statistical parameters can be found in supplementary Table S7