Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor

Abstract

Concentrations of acetyl-coenzyme A and nicotinamide adenine dinucleotide (NAD(+)) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

Figures

Fig. 1

Inhibition of HDACs by βOHB in vitro and in vivo. (A) Structures of β-hydroxybutyrate and butyrate. (B) Effect of βOHB, TSA, or butyrate on acetylation of histone H3 and tubulin. HEK293 cells were treated with the indicated concentrations of drugs for 8 hours. Histones were acid-extracted, and their acetylation was assessed by protein immunoblotting with anti-AcH3K9, anti-AcH3K14, or anti-acetyllysine (AcLys). Proteins from whole-cell extracts were analyzed by immunoblotting with antibodies to α-tubulin or Ac-α-Tubulin. (C) Quantification of acetylation levels from blots in (B), shown relative to untreated cells (βOHB 0 mM). (D) Inhibition of immunopurified HDACs by βOHB in vitro. Flag-tagged HDACs were expressed in HEK293 cells, immunoprecipitated, and incubated in vitro with a 3H-labeled acetylated histone H4 peptide and the indicated concentrations of βOHB. HDAC activity is relative to the activity of each enzyme without βOHB. The IC50 values of βOHB are shown.

Fig. 2

Serum βOHB and global histone acetylation in kidney of mice deprived of food. (A) Serum concentration of βOHB was determined in age-matched groups of three C57Bl6 mice fed or fasted (16 weeks old, fasted for 24 hours), implanted with a pump delivering either PBS or βOHB (16 weeks old, 24 hours of pump treatment), fed ad libitum (AL) or on calorie restriction to 60% of AL (CR) (8 months old, 6 months on CR). Mean ± SE, *P < 0.05 by t test between paired conditions. (B) Histones were purified from the kidneys of the same mice as in (A). Acetylation was assessed with anti-AcH3K9, anti-AcH3K14, or anti-Ac-α-tubulin (fig. S8). Acetylation is normalized to total histone H3 and shown relative to the control condition in each pair (e.g., fed versus fasted). Mean ± SE, *P < 0.05 by t test between paired conditions. (C) Serum βOHB concentrations [from (A)] plotted against acetylation of histone H3K9 and H3K14 [from (B)]; correlation coefficient R2 = 0.772 for H3K9 and 0.863 for H3K14.

Fig. 3

Increased expression of oxidative stress resistance genes in cells exposed to βOHB. (A) Expression of Foxo3a under various conditions (see Fig. 2 for details) measured by QPCR. Foxo3 expression is normalized to abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mean ± SE, *P < 0.05 by t test between paired conditions. (B) Expression of Mt2, measured as in (A). (C) Promoters of Mt2 and Foxo3a are enriched for acetylated his-tone H3K9 after βOHB treatment. HEK293 cells were treated with 10 mM βOHB or PBS for 24 hours. Chromatin was immunoprecipitated with anti-H3 or anti-AcH3K9, and the purified DNA was analyzed with primer pairs specific for the Foxo3a or Mt2 promoters. Results are the ratios of AcH3K9 to total histone H3. Mean ± SE, *P < 0.05 by t test between βOHB and PBS conditions. (D) HDAC depletion increases Foxo3a and Mt2 mRNAs abundance. HEK293 cells were transfected with shRNAs specific for each class I or class II HDAC, and mRNA abundance was measured by QPCR 72 hours after transfection. Mean ± SE, *P < 0.05 by t test versus control shRNA. (E) HDAC1, but not HDAC6, is enriched at the promoters of Mt2 and Foxo3a. ChIP analysis of the Foxo3a and Mt2 promoters (two primer pairs per promoter) and Gapdh (one primer pair) from HEK293 cells with control immunoglobulin G (IgG), anti-HDAC1, or anti-HDAC6. Relative promoter binding of each HDAC is normalized to input Gapdh. Mean ± SE, *P < 0.05 by t test versus IgG control.

Fig. 4

Protective effect of βOHB treatment against oxidative stress. (A) Amounts of catalase, MnSOD, or FOXO3A measured by protein immunoblotting in kidney tissue from 16-week-old mice implanted with an osmotic pump delivering PBS or βOHB (as in Fig. 2; n = 3); mean ± SE, *P < 0.05 by t test between PBS and βOHB conditions. (B) Protein carbonylation in kidney samples from mice implanted with an osmotic pump delivering PBS or βOHB (as in Fig. 2; n = 3) and treated with paraquat (50 mg/kg) or vehicle for 2 hours. Car-bonylation was measured by immunoblotting with anti-DNP. All samples were run on a single gel; after imaging, lanes were rearranged for presentation. (C) Quantification of protein carbonylation in (B). Mean ± SE, *P < 0.05 by t test between PBS and βOHB conditions. (D) Sections of kidney obtained from the same mice as in (B) were stained with anti-4-HNE and quantified (see fig. S16 for primary picture). Mean ± SE, *P < 0.05 by t test between PBS and βOHB conditions. (E). Lipid peroxides were quantified in mice kidneys (LPO assay kit, Cayman, Ann Arbor, MI). Mean ± SE, *P < 0.05 by t test between PBS and βOHB conditions.