PMC

Glycolytic metabolism in response to lipopolysaccharide (LPS) and β-hydroxybutyrate (BHB) in BV2 cells. (A) Schematic representation of ¹³C enrichment derived from ¹³C₆-glucose. In (B–H), cells were seeded and the next day treated with 5 mM ¹²C₄-BHB for 24 h before the media was replaced by fresh media containing 5 mM ¹³C₆-glucose and immediately treated with either 100 ng/mL of LPS, 5 mM of ¹²C₄-BHB or both for 6 h. (B) ¹³C-glucose uptake rate in cells treated with 100 ng/mL LPS and/or 5 mM BHB for 6 h estimated by ¹H-NMR. Two-way ANOVA (LPS n.s., BHB p = 0.027, LPS × BHB n.s.) followed by Tukey’s test (no statistical significance reached). (C) m₊₃ ¹³C enrichment in intracellular pyruvate. Data were analysed by two-way ANOVA (LPS p < 0.0001, BHB n.s., LPS × BHB p = 0.023) followed by Tukey’s test. (D) m₊₃ ¹³C enrichment in intracellular lactate. Data were analysed by two-way ANOVA (LPS p < 0.0001, BHB n.s., LPS × BHB n.s.) followed by Tukey’s test. (E) Cytosolic NADH:NAD+ ratio estimated using the intracellular levels of lactate and pyruvate. Data were analysed by two-way ANOVA (LPS p = 0.001, BHB p = 0.0021, LPS × BHB n.s.) followed by Tukey’s test. (F) ¹³C-lactate export rate calculated from ¹³C-lactate exported into media analysed by ¹H-NMR. Data were analysed by two-way ANOVA (LPS p = 0.003, BHB p = 0.001, LPS × BHB p = 0.018) followed by Tukey’s test. (G) Metabolite levels normalised to untreated cells and expressed as fold-change as per colour-coded scale. Each data point represents mean of n = 3 biological replicates. Statistical significance was assessed by two-way ANOVA (DHAP: LPS p < 0.0001, BHB n.s., LPS × BHB p = 0.023; pyruvate, alanine, serine, glycine, and methionine: LPS n.s., BHB p < 0.05) followed by Tukey’s test (untreated vs. +LPS, untreated vs. +BHB, +LPS -BHB vs. +BHB +LPS). A complementary statistical analysis using Fisher’s LSD test followed by false discovery rate (FDR) correction to account for multiple comparisons both across treatment class and metabolite class can be found in . (H) Bar chart representation of intracellular levels of DHAP. In (B–E,H), bars represent mean ± SD of n = 3 biological replicates. Statistical significance is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001 with exception of G, where statistical significance between untreated and single treatments (untreated vs. +LPS, untreated vs. +BHB) is denoted as ## p < 0.01, and statistical significance between single treatment with LPS and double treatment (−BHB +LPS vs. +BHB +LPS) is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.

(A) Fluorescence spectra of 10 µM BHb solution (365 nm excitation) after irradiation with 532 nm laser at an intensity of 60 mW/cm² for 0 min (black line), 10 min (red line), and 30 min (blue line), respectively. (B) Real-time detection of fluorescence of 1 mM H₂O₂-pretreated (10 min) BHb solution at 595/2 nm with 532 nm excitation (green line) and at 464/2 nm with 365 nm excitation (purple line). (C) Fluorescence spectra of BHb solution with 365 nm excitation: black line means the pure BHb solution, purple line indicates BHb solution pretreated with 1 mM H₂O₂ for 10 min. (D) Fluorescence spectra of BHb solution with 532 nm excitation: black line corresponds to a BHb solution without any treatment, green line represents the BHb solution pretreated with 1 mM H₂O₂ for 10 min.

a, Model by which HOPX is induced through the HDAC-inhibitory activity of BHB. b, HDAC activity in organoids treated with BHB, with or without the pharmacological HDAC inhibitor vorinostat. c–e, Representative flow cytometry plot (c), relative proportion of H3K9ac (d), and normalized MFI of H3K9ac (e) in intestinal organoids treated with BHB. f, HDAC activity in intestinal epithelial cells from mice treated with BHB esters. g–i, Representative flow cytometry plot (g), relative proportion of H3K9ac (h), and normalized MFI of H3K9ac (i) in intestinal epithelial cells from KD-fed mice and controls. j, k, Representative immunofluorescence staining (j) and quantification (k) of pan-acetylation levels in colons from KD-fed mice and controls. l, Size of wild-type organoids treated with BHB and/or vorinostat. m, n, Size quantification of wild-type (m) and APKS organoids (n) exposed to BHB or butyrate. o, p, PCA plot (o) and k-means clustering (p) of global gene expression in organoids treated with BHB or the HDAC inhibitor (HDACi) vorinostat. q, Transcript abundance of Hopx determined by RNA-sequencing of organoids treated with BHB or vorinostat. r, Normalized profiles of H3K9ac in the Hopx locus from ChIP-sequencing of intestinal epithelial cells from mice treated with BHB or fed a 90% fat diet. s, Size of Hopx-deficient organoids after treatment with BHB and/or vorinostat. t, Size quantification of organoids from Hopx-deficient mice exposed to BHB or butyrate. u, Quantification of HDAC activity in wild-type and Hopx-deficient organoids treated with BHB and/or vorinostat. v, w, Tumour numbers (v) and representative colonoscopy images (w) in AOM/DSS-treated mice on KD and treated with the HDAC inhibitor vorinostat. Error bars indicate means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact n and p-values are presented in .

Exogenous BHB treatment rescues the neurogenesis defect in Kmt2d^+/βGeo mice. (A) Several distinct doses (0, 2.5, 5, 10 mM/kg) of BHB were injected i.p., followed by urine collection 1.5 h later (approximate time of BHB peak levels) (). The 5 mM/kg dose shows a BHB level that resembles the urine BHB profile (approximated in this figure with dashed lines) from KD-treated mice (n = 22 total). (B) A single 5 mM/kg BHB injection (9 AM) of BHB per day (for 2 wk) yielded comparable peak levels as mice on a KD but less total daily exposure, as seen from a urine time course (12 h; n = 4–5 per group). Also shown is the saline vehicle group. (C) Kmt2d^+/βGeo mice injected with a once-daily dose of 5 mM/kg BHB (for 2 wk) show a significant increase in the EdU⁺ cell numbers in the GCL of the DG of the mice compared with Kmt2d^+/βGeo mice injected with saline vehicle. However, this rescue did not show the same magnitude of rescue compared with the EdU⁺ cell numbers from KD treatment (n = 5–12). (D) Mice implanted with a 2-wk BHB pump that provided 2.5 mg/mL BHB at a rate of 0.25 µL/h showed a significant (P < 0.05) increase in BHB measured from urine (n = 10–12 per group). However, although the pump provided a constant stream of BHB, the urine BHB did not reach comparable levels to what was seen in mice on a KD (∼1–2 mM BHB). (E) The combination of osmotic pumps and three injections of 5 mM/kg BHB at 9 AM, 1 AM, and 5 AM showed a more similar BHB profile to KD-treated mice when treated for 2 wk (n = 4–5 per group). (F) When treated with this higher daily dose of BHB for 2 wk, we saw a more pronounced rescue of EdU⁺ cell numbers in the DG GCL in Kmt2d^+/βGeo mice, which more closely mirrors the rescue seen after KD treatment. (G and H) We further confirmed the neurogenesis rescue at both low and high doses of BHB (treated for 2 wk) by looking at the fraction of DCX⁺ cells in the DG GCL, and found that this measure showed similar neurogenesis rescues compared with EdU analysis (n = 7–10 per group). *P < 0.05; **P < 0.01; ^†P < 0.005. ^††P < 0.001. N.S., not significant, P > 0.05.