Ketogenic Diet Reduces Age-Induced Chronic Neuroinflammation in Mice

Abstract

The ketone body beta-hydroxybutyrate (BHB) is an acidic energy metabolite that is synthesized during periods of fasting or exercise. Our previous study demonstrated that an every other week cyclic ketogenic diet (Cyclic KD), which induces blood BHB levels similar to those observed during fasting, reduces midlife mortality and improves memory in aging mice. In addition to its canonical role as an energy metabolite, BHB regulates gene expression and inflammatory activation through non-energetic signaling pathways. The precise mechanisms by which BHB or KD affects brain function during aging remain incompletely understood. Using bulk RNA-sequencing (RNA-Seq), we examined whole brain gene expression of 12-month-old C57BL/6JN male mice fed KD for either one week or 14 months. While one-week KD increases some inflammatory gene expression, the 14-month Cyclic KD largely reduces age-induced neuroinflammatory gene expression. Next, a gene expression analysis of human primary brain cells (microglia, astrocytes, and neurons) using RNA-Seq revealed that BHB alone induces a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced inflammatory gene expression induced by lipopolysaccharide (LPS) in microglia. BHB exhibits a comparable inhibitory effect on LPS-induced inflammation in mouse primary microglia, which we used as an in vitro model to test and exclude known mechanisms by which BHB regulates inflammation and gene expression as responsible for this modulation of LPS-induced inflammatory gene expression. An acidic milieu resulting from BHB may be required for or contribute to the effect. Overall, we observe that BHB has the potential to attenuate the microglial response to inflammatory stimuli, such as LPS. This may contribute to an observed reduction in chronic inflammation in the brain following long-term Cyclic KD treatment in aging mice.

Figure 1.. Fourteen-month Cyclic ketogenic diet (Cyclic KD) reduces age-induced neuroinflammation.

(A) Diet composition and experimental timeline. Brains were harvested at 12 months old (12-month-old mice maintained on control diet [CD] [CD (12 months), n = 5] or KD [KD (12 months), n = 7] for one week) and at 26 months old (12-month-old mice maintained on CD [CD (26 months), n = 8] or Cyclic KD [Cyclic KD (26 months), n = 8] for 14 months), collected at a dark cycle during a CD-fed week. (B) Gene set enrichment analysis (GSEA) (Molecular Signatures Database [MSigDB]) in the brain (Cyclic KD [26 months] vs. CD [26 months]). (C) Heatmap of gene expressions in the brain. (D) Rank–Rank Hypergeometric Overlap analysis comparing aging (CD [26 months] vs. CD [12 months]) with Cyclic KD (Cyclic KD [26 months] vs. CD [26 months]) in the brain. Abbreviation: NES, normalized enrich score.

Figure 2.. R-beta-hydroxybutyrate (R-BHB) reduces lipopolysaccharide (LPS)-induced inflammation in human primary microglia.

(A) Experimental timeline. (B,C) GSEA (MSigDB) analysis (R-BHB vs. Ctrl) (B) without or (C) with LPS treatment in human primary neurons, astrocytes, and microglia (n = 3 per group). Gray squares: not significant.

Figure 3.. BHB acids reduce LPS-induced inflammation in mouse primary microglia.

(A) Experimental timeline. (B) mRNA expression in mouse primary microglia (n = 3 per group). All data are presented as mean ± standard deviation (SD). One-way ANOVA with Dunnet’s correction for multiple comparisons. Compare the mean of each sample with the mean of “Ctrl.” (C) Protein expression and the quantification in mouse primary microglia (n = 2 per group). All data are representative of two independent experiments. Compare the mean of each sample with the mean of “Ctrl.” Abbreviations: R-BHB, R-BHB acid; S-BHB, S-BHB acid; Na-R-BHB, R-BHB sodium salt; Na-S-BHB, S-BHB sodium salt; Bu, butyric acid; and Na-Bu, butyrate sodium salt.

Figure 4.. BHB acids reduce LPS-induced inflammation potentially through minor pH fluctuations.

(A) mRNA expression in mouse primary microglia (n = 3 per group). All data are presented as mean ± SD. One-way ANOVA with Dunnet’s correction for multiple comparisons. Compare the mean of each sample with the mean of “Ctrl + LPS.” (B) IL-1β enzyme-linked immunosorbent assay analysis of IL-1β secretion after NLRP3 inflammasome activation in mouse primary microglia (n = 2 per group). All data are presented as mean ± SD. One-way ANOVA with Dunnet’s correction for multiple comparisons. Compare the mean of each sample with the mean of “Ctrl + nigericin.” All data are representative of two independent experiments.