Acetate supplementation modulates brain histone acetylation and decreases interleukin-1β expression in a rat model of neuroinflammation

Abstract

Background: Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression.

Methods: In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1β expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively.

Results: We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9), histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1β protein and mRNA levels were increased by 1.3- and 10-fold, respectively, and acetate supplementation reduced this expression to control levels.

Conclusion: Based on these results, we conclude that dietary acetate supplementation attenuates neuroglial activation by effectively reducing pro-inflammatory cytokine expression by a mechanism that may involve a distinct site-specific pattern of histone acetylation and histone deacetylase expression in the brain.

Figure 1

Changes in the acetylation state of brain H3K9, and H3K14 in control rats (aCSF), rats subjected to neuroinflammation (LPS) and rats treated with either water (H₂O) or glyceryl triacetate (GTA). (A) Representative images of the western blots. (B) Averaged proportion of brain H3K9 and H3K14, normalized to total H3. The data represent the means ± SD where statistical significance (a = compared to aCSF + H₂O, and b = compared to LPS + H₂O) was set at P ≤ 0.05, as determined by one-way ANOVA followed by Tukey's post-hoc test.

Figure 2

Changes in the acetylation state of brain H4K5, H4K8, H4K12 and H4K16 in control rats (aCSF), rats subjected to neuroinflammation (LPS) and rats treated with either water (H₂O) or glyceryl triacetate (GTA). (A) Representative images of the western blots (B) Averaged proportion of brain H4K5, H4K8, H4K12 andH4K16, normalized to total H4. The data represent the means ± SD where statistical significance (a = compared to aCSF + H₂O, and b = compared to LPS + H₂O) was set at P ≤ 0.05, as determined by one-way ANOVA followed by Tukey's post-hoc test.

Figure 3

The effect of long-term acetate supplementation on brain histone deacetylase and histone acetyltransferase activities in control rats (aCSF), rats subjected to neuroinflammation (LPS) and rats treated with either water (H₂O) or glyceryl triacetate (GTA). (A) Represents changes in brain HAT, and (B) represents changes in HDAC activities. HAT enzyme activity is expressed as the means ± SD of absorbance reading at 450 nm after 4 hours of incubation at 37°C, normalized to the protein assayed (μg). HDAC enzyme activity is expressed as the means ± SD of absorbance at 405 nm after 75 minutes of incubation at 37°C, normalized to the protein assayed (μg). Statistical significance (*compared to aCSF + H₂O controls) was set at P ≤ 0.05, as determined by nonparametric one-way ANOVA followed by Dunn's post-hoc test.

Figure 4

The effect of long-term acetate supplementation on the expression of individual brain histone deacetylase in control rats (aCSF), rats subjected to neuroinflammation (LPS) and rats treated with either water (H₂O) or glyceryl triacetate (GTA). (A) Representative images of the western blots. (B) Quantifications of HDAC1, 2, 3, 4, 5 and 7. Data in B represent means ± SD of the optical densities normalized to α-tubulin. Statistical significance (* compared to aCSF + H₂O controls) was set at P ≤ 0.05, as determined by one-way ANOVA followed by Tukey's post-hoc test for HDAC2, 3, 4 and 5, and nonparametric one-way ANOVA followed by Dunn's post-hoc test for HDAC1 and 7.

Figure 5

The effect of long-term acetate supplementation on the expression of brain IL-1β in control rats (aCSF), rats subjected to neuroinflammation (LPS) and rats treated with either water (H₂O) or glyceryl triacetate (GTA) as determined by Western blot analysis and quantitative real-time PCR. (A) Western blot representative images of bands for IL-1β and α-tubulin and (B) shows the means ± SD of the normalized optical density of IL-1β protein. (C) Representative images of the bands for IL-1β and β-actin cDNA and (D) shows the means ± SD of the normalized amplified IL-1β cDNA. Statistical significance (a = compared to aCSF + H₂O, and b = compared to LPS + H₂O) was set at P ≤ 0.05 (n = 6, per group), as determined by a one-way ANOVA followed by Tukey's post-hoc test.