Prominent action of butyrate over β-hydroxybutyrate as histone deacetylase inhibitor, transcriptional modulator and anti-inflammatory molecule

Abstract

Butyrate and R-β-hydroxybutyrate are two related short chain fatty acids naturally found in mammals. Butyrate, produced by enteric butyric bacteria, is present at millimolar concentrations in the gastrointestinal tract and at lower levels in blood; R-β-hydroxybutyrate, the main ketone body, produced by the liver during fasting can reach millimolar concentrations in the circulation. Both molecules have been shown to be histone deacetylase (HDAC) inhibitors, and their administration has been associated to an improved metabolic profile and better cellular oxidative status, with butyrate inducing PGC1α and fatty acid oxidation and R-β-hydroxybutyrate upregulating oxidative stress resistance factors FOXO3A and MT2 in mouse kidney. Because of the chemical and functional similarity between the two molecules, we compared here their impact on multiple cell types, evaluating i) histone acetylation and hydroxybutyrylation levels by immunoblotting, ii) transcriptional regulation of metabolic and inflammatory genes by quantitative PCR and iii) cytokine secretion profiles using proteome profiling array analysis. We confirm that butyrate is a strong HDAC inhibitor, a characteristic we could not identify in R-β-hydroxybutyrate in vivo nor in vitro. Butyrate had an extensive impact on gene transcription in rat myotubes, upregulating PGC1α, CPT1b, mitochondrial sirtuins (SIRT3-5), and the mitochondrial anti-oxidative genes SOD2 and catalase. In endothelial cells, butyrate suppressed gene expression and LPS-induced secretion of several pro-inflammatory genes, while R-β-hydroxybutyrate acted as a slightly pro-inflammatory molecule. Our observations indicate that butyrate induces transcriptional changes to a higher extent than R-β-hydroxybutyrate in rat myotubes and endothelial cells, in keep with its HDAC inhibitory activity. Also, in contrast with previous reports, R-β-hydroxybutyrate, while inducing histone β-hydroxybutyrylation, did not display a readily detectable HDAC inhibitor activity and exerted a slight pro-inflammatory action on endothelial cells.

Figure 1

Effects of NaBut and β-hydroxybutyrate molecules on histone acetylation in multiple cell types. (A) HEK293 cells were incubated for 18 hours with 5 mM NaBut or increasing concentrations of NaR-βOHB (10–40 mM range). (B) HEK293 cells were incubated for 18 hours with NaBut, the racemic mix of β-hydroxybutyrate sodium salt (Na R/S-βOHB), R-β-hydroxybutyric acid (R-βOHB) and S-β-hydroxybutyric acid (S-βOHB) at the indicated concentrations. Separation bars indicate non-contiguous lanes on the same image acquisition (the original blots are shown in the supplementary information file). (C) HMEC-1 were incubated for 8 or 18 hours with SAHA, NaBut or NaR-βOHB at the indicated concentrations. (D) L6 myotubes were incubated with 10 mM NaBut, 40 mM R-βOHB or 40 mM S-βOHB for the indicated times. (A–D) Acid-extracted histones were immunoblotted with antibodies anti H3K9/14Ac, anti-acetyl-H3K9Ac, anti-acetyl-H3 (H3KAc), anti-acetyl lysine, anti total H3 or stained with coomassie blue as indicated. (A,B) quantification of acetyl immunoblotting signals normalized to total histone H3 content. n = 3. Error bars represent means ± SEM from 3 independent experiments. (*p < 0.05, versus control group (−) using one-way ANOVA). Quantifications for panels A (H3K9/14Ac and Acetyl lysine blots), C, D are shown in Supplementary Fig. 1.

Figure 2

Effect of butyrates on in vitro HDAC activity. (A) HDAC activity of crude nuclear extract was measured fluorimetrically (λ_ex 355 nm, λ_em 460 nm) for 30 minutes in the presence of 10 mM NaR-βOHB, 5 mM NaBut or 5 mM 4-PBA. Fluorescence was measured every minute. One representative trace of two independent measurements is shown. (B) Mean fluorescence (with SD) of the two independent reactions at 30 min. 1 μM TSA was also included as positive control for inhibition.

Figure 3

Effects of NaBut or NaR/S-β-hydroxybutyrate supplementation throughout L6 cells differentiation. L6 myoblasts were differentiated for 7 days into myotubes in the presence of increasing concentrations of NaBut or NaR/S-βOHB. Acid-extracted histones were immunoblotted with antibodies anti-acetyl lysine, anti-H3K9/14Ac, anti H3K9Ac and anti H3K9Me2. Loading was assessed by anti total histone H3 immunoblotting and coomassie staining of extracted histones. Signal quantification, relative to total H3 immunoblot signals, from representative western blots is shown on the right.

Figure 4

Effects of butyrate-related molecules on histone acetylation in L6 myotubes. (A) L6 myotubes were incubated for 18 hours with NaBut, the indicated butyrate-related molecules or TSA. Acid-extracted histones were immunoblotted with antibodies anti H2A.ZAc, anti H3K9/14Ac, anti H3K9Ac, anti H3K9Me2 or stained with coomassie blue as indicated. Signal quantification from representative western blots, relative to the coomassie blue staining signal, is shown next to each immunoblot. (B) Chemical formulas of NaBut and butyrate related molecules.

Figure 5

β-hydroxybutyrate and HDAC inhibitors induce histone β-hydroxybutyrylation. HEK293 cells (A), L6 myotubes (B) and HMEC-1 (C) were incubated for 18 hours with NaR-βOHB or the HDAC inhibitors NaBut, 4-PBA or SAHA as indicated. Acid-extracted histones were immunoblotted with antibodies anti H3K9/14Ac, anti β-hydroxybutyryl-histone H3 Lysine 9 (H3K9bhb) and anti total H3. (B, right panels) L6 myotubes were incubated for 18 hours with 10 mM R-βOHB (R) or 10 mM S-βOHB (S) and immunoblotted with antibodies anti H3K9bhb and anti total H3. Signal quantifications from representative western blots, relative to total H3 immunoblot signals, are shown. Empty bars: anti H3K9/14Ac, black bars: anti H3K9bhb).

Figure 6

Effects of NaBut and NaR-βOHB (R-βOHB in graphs) on transcription of genes involved in insulin action. Quantification of the mRNA levels by RT qPCR for Glut4, HK2, IRS1, Gs, Gsk-3β and Hmgcs1 in L6 myotubes treated for 24 h with 5 mM NaBut (grey bars) or 20 mM NaR-βOHB (black bars). Gene expressions are normalized to the Hprt1 housekeeping gene. Error bars represent the mean ± SEM from 3 independent experiments. (*p < 0.05, versus Ctrl group using one-way ANOVA).

Figure 7

Effects of NaBut and NaR-βOHB (R-βOHB in graphs) on transcription of stress resistance and oxidative stress – protective genes. Quantification of the mRNA levels by RT qPCR for stress-resistance genes (EIF2 and FOXO3a) and oxidative stress protecting genes (catalase, SOD1, SOD2) in L6 myotubes treated for 24 h with 5 mM NaBut (grey bars) or 20 mM NaR-βOHB (black bars). Data are presented and analyzed as in Fig. 6.

Figure 8

NaBut, but not NaR-βOHB (R-βOHB in graphs) affect transcription of multiple genes related to mitochondrial function. (A) Quantification of mRNA levels by RT qPCR for PGC1α CPT1b and SIRT1 in L6 myotubes treated for 24 h with 5 mM NaBut (grey bars) or 20 mM NaR-βOHB (black bars). (B) Quantification of mRNA levels of mitochondrial Sirtuins 3,4 and 5 in L6 myotubes treated for 24 h with 5 mM NaBut (grey bars) or 20 mM NaR-βOHB (black bars). (A,B) Data are presented and analyzed as in Fig. 6. (C) Mitochondrial to nuclear genome ratio as measured by amplification of two mitochondrial gene loci (Cox2 and Cox1) and a nuclear locus (on the Ppia gene) in a control or 5 mM NaBut conditions. Data are presented and analyzed as in Fig. 6.

Figure 9

Opposite actions of NaBut and NaR-βOHB on inflammatory markers in HMEC-1. (A) Analysis of inflammatory gene expression in quiescent HMEC-1 cells treated with 5 mM NaBut or 20 mM NaR-βOHB for 6 h. Results are the average +/− SD of 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by a Bonferroni post-hoc test. *significantly lower than control, p < 0.05; **significantly higher than control, p < 0.05. (B) Analysis of inflammatory gene expression in LPS-activated HMEC-1 cells treated with 5 mM NaBut and 20 mM NaR-βOHB. Cells were incubated with butyrates for 6 hours, with addition of 100 ng/ml LPS in the last 4 hours of incubation. Results are the average +/−SD of 3 independent experiments. Statistical analysis was as in panel A. *significantly higher than control, p < 0.05; ^#significantly lower than the LPS treatment condition, p < 0.05; ^##significantly higher than the LPS treatment condition, p < 0.05.

Figure 10

Effect of NaBut and NaR-βOHB on the release of the cytokines and chemokines in LPS-treated HMEC-1. (A) Secreted cytokines and chemokines in the cell culture supernatants from LPS-treated HMEC-1 cells in the presence/absence of butyrates. Cells were incubated with NaBut and NaR-βOHB for 6 hours, with addition of 100 ng/ml LPS in the last 4 hours of incubation. (B) The intensity of the signals of secreted factors was quantified using a CCD digital imaging system Alliance Mini HD4 (UVItec Limited, Cambridge, United Kingdom).