Dietary inulin alters the gut microbiome, enhances systemic metabolism and reduces neuroinflammation in an APOE4 mouse model

Abstract

The apolipoprotein ε4 allele (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD). APOE4 carriers develop systemic metabolic dysfunction decades before showing AD symptoms. Accumulating evidence shows that the metabolic dysfunction accelerates AD development, including exacerbated amyloid-beta (Aβ) retention, neuroinflammation and cognitive decline. Therefore, preserving metabolic function early on may be critical to reducing the risk for AD. Here, we show that inulin increases beneficial microbiota and decreases harmful microbiota in the feces of young, asymptomatic APOE4 transgenic (E4FAD) mice and enhances metabolism in the cecum, periphery and brain, as demonstrated by increases in the levels of SCFAs, tryptophan-derived metabolites, bile acids, glycolytic metabolites and scyllo-inositol. We show that inulin also reduces inflammatory gene expression in the hippocampus. This knowledge can be utilized to design early precision nutrition intervention strategies that use a prebiotic diet to enhance systemic metabolism and may be useful for reducing AD risk in asymptomatic APOE4 carriers.

Fig 1. Effect of inulin on food intake, body weight, cognition and Aβ retention in E4FAD mice.

Inulin intake increased daily energy uptake (kcal/d) from the diet in the E4FAD mice (A) but did not change bodyweight (B). (C) The mice showed no performance differences on the RAWM test. Wrong entries were recorded as errors. There were no significant differences in performance among E3FAD-Control, E4FAD-Control and E4FAD-Inulin mice in any of the 6 blocks. (D) Representative images of Aβ immunohistochemical staining from the three groups of mice (Top) and quantitation of the Aβ load (Bottom). There were no significant differences in Aβ load among the three groups of mice. Data are presented as the mean ± SEM. n.s. = not significant; *p < 0.05.

Fig 2. Analysis of inulin-induced changes in gut microbiome diversity.

All analyses were performed on annotated sequence data at the genus level, rarefied to 10,000 sequences per sample. (A) E4FAD-Inulin mice had significantly lower α-diversity (shown here as the Shannon index) than E4FAD-Control mice (p = 0.019). (B) E4FAD-Control and E4FAD-Inulin mice exhibited a significant difference in β-diversity (p = 0.001). (C) E3FAD-Control and E4FAD-Control mice exhibited a significant difference in β-diversity (p = 0.013). (D) Mice with and without FAD mutations (FAD (+) vs. FAD(-)) exhibited no significant difference in β-diversity (p = 0.546). (E) Male and female mice did not exhibit a significant difference in β-diversity (p = 0.07).

Fig 3. Analysis of inulin-induced changes in metabolism and weight in the cecum.

E4FAD-Inulin fed mice had significantly enhanced production of the short-chain fatty acids (A) acetate; (B) butyrate; and (C) propionate compared to the controls. E3FAD-Control and E4FAD-Control mice did not exhibit significant differences in these measures. (D) Representative images showing the significantly increased cecum size of the inulin-fed E4FAD mice compared to the E4FAD control mice. (E) Quantitation of data in (D) comparing the cecum weights among the three groups. Data are presented as the mean ± SEM. n.s. = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4. Analysis of inulin-induced changes in microbial metabolism changes in the periphery.

E4FAD-Inulin mice had a significant increase in (A) acetate and trend-level increases in (B) butyrate and (C) propionate. E4FAD-Inulin mice had increased tryptophan metabolism, as indicated by the levels of (D) indolepropionate (IPA) and (E) indoleacrylate, and increased bile acid metabolism, as indicated by the levels of (F) cholate and (G) deoxycholate. n.s. = not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 5. Analysis of inulin-induced changes in glycolytic metabolism in the periphery.

(A) An illustration of the glycolytic pathway through the TCA cycle and PPP. E4FAD mice fed prebiotic inulin showed changes in the levels of TCA cycle metabolites, including (B) succinate, (C) fumarate, and (D) malate, compared to E4FAD mice fed the control diet. E4FAD mice fed the prebiotic inulin showed a significant increase in the levels PPP-associated metabolites including (E) ribose, (F) ribulose, (G) ribonate, and (H) arabonate compared to E4FAD mice fed the control diet. n.s. = not significant; *p < 0.05; **p < 0.01.

Fig 6. Analysis of inulin-induced changes in microbial metabolism in the brain.

Scyllo-inositol was dramatically increased in the hippocampus of E4FAD mice fed the prebiotic inulin compared to the E4FAD mice fed the control diet. (A) Shows the voxel that was used for MRS. Representative spectra are shown for (B) the E3FAD-Control, E4FAD-Control, and E4FAD-Inulin groups. The arrow indicates scyllo-inositol, which was dramatically increased in mice fed the prebiotic inulin. Scyllo-inositol (C) was significantly increased in E4FAD inulin mice compared to control fed E3FAD and E4FAD mice. Myo-inositol (D) was significantly decreased in E4FAD mice fed inulin. Data are the mean ± SEM, n.s. = not significant; ****p < 0.0001.

Fig 7. Analysis of inulin-induced changes in inflammatory gene expression in the hippocampus.

(A) In APOE4 mice with and without the FAD mutations, data are plotted as the mean expression ratios with their respective FDR-adjusted p-value. This revealed 52 genes that were significantly differentially enriched (magenta) with log₂ (fold change) greater than one due to the FAD mutations. (B) The 52 genes that were enriched due to the FAD mutations were further examined in the context of diet manipulation. Overall, there was a trend for most of the 52 genes to be downregulated due to the prebiotic inulin in E4FAD mice (e.g., gray bars). Furthermore, an approximately 2-fold decrease in expression was observed in 4 genes (aquamarine). Among these, (C) CCL4, and (D) Fcgr4 were significantly decreased. Although there was a strong visual trend for both (E) CXCL10 and (F) Itgax to be decreased due to the prebiotic inulin, these decreases were not statistically significant. Data are mean ± SEM, n.s. = not significant; *p < 0.05.