Ketone body mediated histone β-hydroxybutyrylation is reno-protective

Abstract

Starvation, intermittent fasting and exercise, all of which are recommended lifestyle modifiers share a common metabolic signature, ketogenesis to generate the ketone bodies, predominantly β-hydroxybutyrate. β-hydroxybutyrate exerts beneficial effects across various contexts, preventing or mitigating disease. We hypothesized that these dynamic health benefits of β-hydroxybutyrate might stem from its ability to regulate genome architecture through chromatin remodeling via histone β-hydroxybutyrylation, thereby influencing the transcriptome. Focusing on the kidney, which is an end organ protected by β-hydroxybutyrate, we examined histone β-hydroxybutyrylation-mediated chromatin remodeling. Notably, regions of the genome associated with lipid catabolism were predominantly in an open chromatin configuration, leading to active transcription and translation. Significant β-hydroxybutyrylation was observed in the kidneys and the most highly upregulated gene actively transcribed and translated was 3-hydroxy-3-methyglutaryl CoA Synthase 2 (Hmgcs2), a gene responsible for the biosynthesis of β-hydroxybutyrate in mitochondria. In contrast, regions with more compact chromatin structures were enriched with genes related to immune function such as protein tyrosine phosphatase receptor type C (Ptprc) and lymphocyte cytosolic protein 1 (Lcp1), which exhibited reduced transcription and translation. These results reveal that renal epigenetic histone β-hydroxybutyrylation is a novel mechanism by which transcriptional regulation of both energy metabolism and immune function occur concomitantly to protect kidneys and lower hypertension.

Keywords: Blood pressure; chromatin remodeling; energy metabolism; epigenetics; histone; ketone body.

Figure 1:. Histone β-hydroxybutyrylation is elevated with 1,3-butanediol treatment.

(A-B) Serum levels of β-hydroxybutyrate in males and females. (C-D) Immunoblotting for β-hydroxybutyrylation. (E-F) Quantification of the blots. BHB: β-hydroxybutyrate, H3K9BHB: Histone 3 lysine 9 β-hydroxybutyrylation, H3: Histone 3. All data are mean±SEM, *p<0.05, **p<0.01.

Figure 2:. Chromatin accessibility, gene expression, and protein levels are altered with 1,3-butanediol treatment.

(A) Volcano plot for BHB vs. control groups. Each data point is a tested locus. The vertical lines correspond to the logFC cutoff. (B) Annotation summary of loci tested in BHB vs control groups. X-axis: genomic annotations; Y-axis proportion of loci in each category within each type of annotation. All Tested Loci (red bars), serve as a basis by which to compare the significant loci (blue and green bars). (C) RNAseq data: Volcano plot representing the significantly differential transcripts. The X-axis: log2 normalized fold change in BHB group compared to control; The Y-axis: −log10 of adjusted p value. (D) Differentially expressed transcripts in numbers. The black bar indicates upregulated transcripts, and the gray bar indicates downregulated transcripts in the BHB group compared to the control. (E) Pathways associated with the upregulated genes of BHB group. (F) Proteomics data: Volcano plot depicting significantly altered protein levels. X-axis: log2 normalized fold change in the BHB group compared to control. Y-axis: −log10 of adjusted p value. (G) Differentially expressed proteins in numbers. The black bar indicates upregulated proteins, and the grey bar indicates downregulated proteins in BHB groups compared to the control. (H) Pathways associated with upregulated proteins in the BHB group. (I) Venn diagram showing common upregulated genes and corresponding proteins leading to increased metabolism. Different shades of blue circles depict ATACseq, RNAseq, and Proteomics data and numbers within the circles are numbers of genes/proteins significantly upregulated. The gray box provides the name for common upregulated genes and proteins; Underneath are the associated upregulated pathways. (J) Chromatin accessibility for genes of interest. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group.

Figure 2:. Chromatin accessibility, gene expression, and protein levels are altered with 1,3-butanediol treatment.

(A) Volcano plot for BHB vs. control groups. Each data point is a tested locus. The vertical lines correspond to the logFC cutoff. (B) Annotation summary of loci tested in BHB vs control groups. X-axis: genomic annotations; Y-axis proportion of loci in each category within each type of annotation. All Tested Loci (red bars), serve as a basis by which to compare the significant loci (blue and green bars). (C) RNAseq data: Volcano plot representing the significantly differential transcripts. The X-axis: log2 normalized fold change in BHB group compared to control; The Y-axis: −log10 of adjusted p value. (D) Differentially expressed transcripts in numbers. The black bar indicates upregulated transcripts, and the gray bar indicates downregulated transcripts in the BHB group compared to the control. (E) Pathways associated with the upregulated genes of BHB group. (F) Proteomics data: Volcano plot depicting significantly altered protein levels. X-axis: log2 normalized fold change in the BHB group compared to control. Y-axis: −log10 of adjusted p value. (G) Differentially expressed proteins in numbers. The black bar indicates upregulated proteins, and the grey bar indicates downregulated proteins in BHB groups compared to the control. (H) Pathways associated with upregulated proteins in the BHB group. (I) Venn diagram showing common upregulated genes and corresponding proteins leading to increased metabolism. Different shades of blue circles depict ATACseq, RNAseq, and Proteomics data and numbers within the circles are numbers of genes/proteins significantly upregulated. The gray box provides the name for common upregulated genes and proteins; Underneath are the associated upregulated pathways. (J) Chromatin accessibility for genes of interest. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group.

Figure 3:. 1,3-butanediol treatment reduces respiratory exchange ratio (RER) and inhibits mammalian target of rapamycin complex 1 (mTORC1).

(A-C) Metabolic CLAMS data showed reduced VO₂, VCO₂, and RER level in the BHB group compared to controls. Black line: control group and green line: BHB group. (D-E) Reduced phospho-S6 ribosomal protein levels were found in male rats treated with BHB compared to control. See also Supplementary Figure S4 for data from female rats. BHB: β-hydroxybutyrate. All data are mean±SEM; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 4:. Validation of common upregulated genes through CHIP-qPCR, Real time PCR, and proteomics.

(A) CHIP-qPCR data demonstrating enrichment of Hmgcs2, Acaa1b, Cyp2d4, and Cyp2e1 in the BHB group compared to control (n=3 replicates/group). Black bar: Control, Green bar: BHB. (B) Real time PCR showed higher expression of Hmgcs2, Acaa1b, Cyp2d4, and Cyp2e1 in BHB group compared to control (housekeeping gene is L36a). (C) Normalized relative abundance of Hmgcs2, Acaa1b, Cyp2d4, Cyp2e1 proteins. See also Supplementary Figure S2 for female data. Black open circle- control, green closed circle- BHB. All data are mean±SEM; *p< 0.05, **p< 0.01, ***p< 0.001, ****p <0.0001 and ^#p=0.0567.

Figure 5:. Kidney proximal tubule epithelial cell mitochondrial circulatory index was found to be higher in BHB group compared to control.

(A) Representative transmission electron microscopy images of renal mitochondria. Orange arrows point to mitochondria. (B-D) Bar graph shows quantification (B) Average mitochondrial area (C) Area/Perimeter, and (D) Circularity index. BHB: β-hydroxybutyrate. n=30 images per group. All data are mean±SEM, ***p< 0.001, and ****p<0.0001.

Figure 6:. Chromatin accessibility, gene expression, protein levels, and CD3⁺ T cells proliferation were altered with 1,3-butanediol treatment.

(A) Venn diagrams showing common downregulated genes and proteins leading to downregulation of immune function pathways. Different shades of blue circles are used to indicate ATACseq, RNAseq, and Proteomics data. The gray box lists the names of common downregulated genes and proteins, beneath which are shown the associated downregulated pathways. (B) Chromatin accessibility at the promoter region of Ptprc and (C) Chromatin accessibility at the Lcp1 promoter. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group. (D-E) Real time PCR data showing reduced expression with Ptprc and Lcp1 in the 1,3-butanediol supplementation. (F-G) Normalized relative abundances of Ptprc (Cd45) and Lcp1 detected in the quantitative proteomics study. Black open circle–control, green closed circle- BHB. (H-I) Complete blood count (H) Peripheral white blood cells and (I) Lymphocytes in the control and BHB treated male S rats. (J) Representative histogram for percent CFSE-positive CD3+ T cells. (K) Quantification for percent CFSE-positive CD3⁺ T cells in control and BHB groups after 5 days. Data represented as Mean ± SEM and N was plotted for control and BHB group. WBC: white blood cells, Lym: Lymphocytes, CFSE: carboxyfluorescein diacetate succinimidyl ester, CD3: Cluster of differentiation 3, P= Parent population, D1= Daughter 1, and D2= Daughter 2 population All data are mean±SEM, *p<0.05, **p< 0.01, ***p< 0.001 and ****p<0.0001.

Figure 6:. Chromatin accessibility, gene expression, protein levels, and CD3⁺ T cells proliferation were altered with 1,3-butanediol treatment.

(A) Venn diagrams showing common downregulated genes and proteins leading to downregulation of immune function pathways. Different shades of blue circles are used to indicate ATACseq, RNAseq, and Proteomics data. The gray box lists the names of common downregulated genes and proteins, beneath which are shown the associated downregulated pathways. (B) Chromatin accessibility at the promoter region of Ptprc and (C) Chromatin accessibility at the Lcp1 promoter. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group. (D-E) Real time PCR data showing reduced expression with Ptprc and Lcp1 in the 1,3-butanediol supplementation. (F-G) Normalized relative abundances of Ptprc (Cd45) and Lcp1 detected in the quantitative proteomics study. Black open circle–control, green closed circle- BHB. (H-I) Complete blood count (H) Peripheral white blood cells and (I) Lymphocytes in the control and BHB treated male S rats. (J) Representative histogram for percent CFSE-positive CD3+ T cells. (K) Quantification for percent CFSE-positive CD3⁺ T cells in control and BHB groups after 5 days. Data represented as Mean ± SEM and N was plotted for control and BHB group. WBC: white blood cells, Lym: Lymphocytes, CFSE: carboxyfluorescein diacetate succinimidyl ester, CD3: Cluster of differentiation 3, P= Parent population, D1= Daughter 1, and D2= Daughter 2 population All data are mean±SEM, *p<0.05, **p< 0.01, ***p< 0.001 and ****p<0.0001.

Figure 6:. Chromatin accessibility, gene expression, protein levels, and CD3⁺ T cells proliferation were altered with 1,3-butanediol treatment.

(A) Venn diagrams showing common downregulated genes and proteins leading to downregulation of immune function pathways. Different shades of blue circles are used to indicate ATACseq, RNAseq, and Proteomics data. The gray box lists the names of common downregulated genes and proteins, beneath which are shown the associated downregulated pathways. (B) Chromatin accessibility at the promoter region of Ptprc and (C) Chromatin accessibility at the Lcp1 promoter. The y-axis represents chromatin cut sites and thus open chromatin, and x-axis represents the chromatin location of genes of interest. Black peaks represent the control group, and green peaks represent BHB group. (D-E) Real time PCR data showing reduced expression with Ptprc and Lcp1 in the 1,3-butanediol supplementation. (F-G) Normalized relative abundances of Ptprc (Cd45) and Lcp1 detected in the quantitative proteomics study. Black open circle–control, green closed circle- BHB. (H-I) Complete blood count (H) Peripheral white blood cells and (I) Lymphocytes in the control and BHB treated male S rats. (J) Representative histogram for percent CFSE-positive CD3+ T cells. (K) Quantification for percent CFSE-positive CD3⁺ T cells in control and BHB groups after 5 days. Data represented as Mean ± SEM and N was plotted for control and BHB group. WBC: white blood cells, Lym: Lymphocytes, CFSE: carboxyfluorescein diacetate succinimidyl ester, CD3: Cluster of differentiation 3, P= Parent population, D1= Daughter 1, and D2= Daughter 2 population All data are mean±SEM, *p<0.05, **p< 0.01, ***p< 0.001 and ****p<0.0001.