Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure

Abstract

[Figure: see text].

Keywords: bile acids; dysbiosis; fasting; metabolomics; microbiota.

Figure. 1. EODF attenuates elevated BP in SHRSP.

(a) Diagram depicting feeding schedule for control and EODF groups. (b)Daily and (c) cumulative food intake. (d) Body weights were measured daily over the course of the study. (e) Weekly SBP of WKY and SHRSP fed ad libitum or EODF. Data presented as mean ± SEM. Three-way ANOVA with Tukey’s multiple comparisons test. Main effects of strain, feeding group, age, age x strain, age x diet, age x strain x feeding group p≤0.05 for all (b-d). n=6-8, **p≤0.01, ***p≤0.0005, ****p≤0.0001, ^#p≤0.05 vs SHRSP Control, ^##p≤0.005 vs SHRSP Control

Figure. 2. EODF prevents elevated SBP in SHRSPs through manipulation of the gut microbiota.

Weekly (a) body weight and (b) SBP of germ-free rats transplanted with microbiota from WKY and SHRSP controls or EODF. Data presented as mean ± SEM. Three-way ANOVA with Tukey’s multiple comparisons test. Main effects of strain, feeding group, strain x feeding group p≤0.0001 for (b). n=6-8, ****p≤0.0001

Figure. 3. The dysbiotic gut microbiota of SHRSP is altered by EODF.

(a) Principal Coordinate Analysis plots of Bray-Curtis dissimilarity of four groups (left), WKY (middle), and SHRSP (right). (b) Relative abundance of taxa at phyla level for individual samples. (c) LEfSe plots to identify species that characterize control groups (top) and effects of EODF on SHRSP (bottom). n=6-8

Figure. 4. Multi-omics integration of WGS and untargeted metabolomics.

(a) Pearson correlation between component one of each dataset (bottom-left) and Sparse partial least squares discriminant analysis (sPLS-DA) of two omics (top-right). (b) Sparse partial least squares discriminant analysis (sPLS-DA) of plasma (left) and cecal (right) metabolites in four groups. (c) Plasma (left) and cecal (right) metabolite sub pathways identified by random forest classification as important for four group separation (Gini index as feature importance). (d) Spearman correlation between the top 50 most important metabolites (left:plasma, right:cecal) and species. Bar graphs indicate metabolite importance, and color blocks indicate super pathways. Comparison of total counts per millions of (e) 7α-dehydroxylase and (f) bile salt hydrolase (bsh). (g) Species stratified counts per million (cpm) of bsh gene. Two-way ANOVA with robust estimator followed by Mann-Whitney U test with Benjamin-Hochberg post hoc correction. Main effects of treatment p<0.0001. and interaction p= 0.0267 for (e). Main effects of genotype p=0.0267 for (f). Main effect of genotype for B.uniformis p=0.0083. Main effect of interaction for L.reuteri p=0.0345. Main effect of genotype p=0.0134 and interaction p=0.0334 for L.johnsonii. n=6-8. * p<0.05 vs WKY Con; ** p<0.01 vs WKY Con; ## p<0.01 vs WKY EODF; $ p<0.05 vs SHRSP Con; $$ p<0.01 vs SHRSP Con; @ p<0.05 vs WKY Con (B.uniformis); # p<0.05 vs SHRSP Con (B.uniformis); %% p<0.01 vs WKY EODF (B.uniformis)

Figure. 5. Reduced bile acids in SHRSP are improved by EODF.

(a) SHRSP control plasma bile acid measurements relative to WKY controls. (b) SHRSP EODF plasma bile acids during fed and fasted states compared to SHRSP control levels. Mann-Whitney test with FDR correction (a), one-way ANOVA with Tukey’s multiple comparisons test or Kruskal-Wallis with Dunn’s multiple comparisons test (b). *p≤0.05 vs WKY Con, **p≤0.01 vs WKY Con ***p≤0.005 vs WKY Con, ****p≤0.001. n=6-8

Figure. 6. Cholic acid supplementation attenuates elevated blood pressure in SHRSP.

(a) SHRSP CA plasma bile acid measurements relative to SHRSP controls. (b) Weekly SBP measurements in WKY or SHRSP on control or 0.5% CA supplemented diet. Data presented as mean ± SEM. Mann-Whitney test with FDR correction (a), Three-way ANOVA with Tukey’s multiple comparisons test (b). Main effects of strain (p≤0.0001), diet (p=0.0014), age (p≤0.0001), age x strain (p≤0.0001), age x diet (p≤0.0001), strain x diet (p=0.0024),diet x strain x feeding group (p=0.0084). *p≤0.05, ***p≤0.005, ****p≤0.0001, ^%p=0.0003, ^#p≤0.05 vs SHRSP Control. n=6-8

Figure. 7. TGR5 agonism attenuates elevated blood pressure and improves vascular function in SHRSP.

(a) SBP measurements in SHRSP injected daily with vehicle or the TGR5 agonist OA. (b) Acetylcholine (Ach_induced vasodilation of mesenteric arteries isolated from vehicle- or OA-treated SHRSPs. (c) Calculated EC₅₀ values for acetylcholine induced vasodilation of mesenteric arteries isolated from vehicle or OA treated SHRSPs. Data presented as mean ± SEM. (a) Two-way ANOVA main effects of age (p<0.0001), treatment (p=0.0317), and age x treatment (p=0.0023). (b) Two-way ANOVA main effects of Ach dose p<0.0001, treatment p=0.0262, dose x treatment p=0.0125. (c) unpaired t-test. *p≤0.05 vs SHRSP vehicle, **p≤0.01 vs SHRSP vehicle, n=5-6