Ketogenic diet enhances neurovascular function with altered gut microbiome in young healthy mice

Abstract

Neurovascular integrity, including cerebral blood flow (CBF) and blood-brain barrier (BBB) function, plays a major role in determining cognitive capability. Recent studies suggest that neurovascular integrity could be regulated by the gut microbiome. The purpose of the study was to identify if ketogenic diet (KD) intervention would alter gut microbiome and enhance neurovascular functions, and thus reduce risk for neurodegeneration in young healthy mice (12-14 weeks old). Here we show that with 16 weeks of KD, mice had significant increases in CBF and P-glycoprotein transports on BBB to facilitate clearance of amyloid-beta, a hallmark of Alzheimer's disease (AD). These neurovascular enhancements were associated with reduced mechanistic target of rapamycin (mTOR) and increased endothelial nitric oxide synthase (eNOS) protein expressions. KD also increased the relative abundance of putatively beneficial gut microbiota (Akkermansia muciniphila and Lactobacillus), and reduced that of putatively pro-inflammatory taxa (Desulfovibrio and Turicibacter). We also observed that KD reduced blood glucose levels and body weight, and increased blood ketone levels, which might be associated with gut microbiome alteration. Our findings suggest that KD intervention started in the early stage may enhance brain vascular function, increase beneficial gut microbiota, improve metabolic profile, and reduce risk for AD.

Figure 1

Ketogenic diet enhances neurovascular functions. (a) Representative cerebral blood flow (CBF) maps superimposed on structural images; color code indicates level of CBF in a linear scale. KD mice exhibited significantly higher CBF in the (b) ventromedial hypothalamus. Data are presented as mean ± SEM, ***p < 0.001. (c) Western blot (WB) images for mTOR, P-gp, and eNOS from the cortical vasculature, β-Actin was used as loading control. (d) The corresponding values of the levels of protein expression. WB data from KD mice were normalized to β-Actin and compared to the control mice (100%), *p < 0.05, **p < 0.01, ***p < 0.001. (e) Representative confocal images showing increased luminal accumulation of NBD-CSA fluorescence in brain capillaries isolated from KD mice compared to control mice, indicating higher P-gp transport activity. Corresponding quantitative fluorescence data; images are shown in arbitrary fluorescence units (scale 0–255). Data are mean ± SEM for 10 capillaries from one preparation of 10 mice per group, ***p < 0.001. mTOR: mechanistic target of Rapamycin; P-gp: P-glycoprotein; eNOS; endothelial nitric oxide synthase.

Figure 2

Ketogenic diet alters gut microbial diversity and increases pro-vascular microbiota. (a) Microbial diversity (Shannon index) was significantly higher in fecal samples from control relative to KD mice (Mann-Whitney U < 0.02). (b) Genus-level metric multi-dimensional scaling (mMDS) plot of 16S rRNA gene amplicon microbiome data was generated with a Bray-Curtis resemblance matrix. Fecal microbial communities of control and KD mice were significantly different in terms of individual taxa (ANOSIM R = 0.473; p = 0.0002), as described in the text and Table 1. All samples were standardized and square root transformed. 2D stress = 0.16.

Figure 3

Ketogenic diet modulates blood ketone and glucose, and decreases body weight. (a) KD mice had significantly lower blood glucose and (b) significantly higher blood ketone levels than control mice. (c) A scatter plot of blood ketone and glucose showing an inverse linear relationship (Pearson’s r = −0.5761, p < 0.01) where each point represents a mouse (n = 19). (d) KD mice had a significant decrease in weight over the 16 weeks compared to control mice.