Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression

Abstract

Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.

Figure 1.

During mammalian cell culture compounds which promote histone hyperacetylation accumulate in the tissue culture medium. (A) The spectrum of post-translational modifications seen on histone H4 in HCT116 cells by FTMS. (B) Histone acetylation (peaks 7, 10, 13 and 16) increases in exhausted medium (after 48 h in culture). (C) Histone H4 acetylation levels increase during culture. (D) Confirmation (by Western blotting) of the changes in histone H4 acetylation detected by FTMS using modification-specific antibodies. Increased acetylation is also evident on lysine-9 of histone H3. (E) A complete fresh medium change is able to restore acetylation levels within 1 h. (F) Exhausted medium inhibits the restoration of acetylation levels after a medium change.

Figure 2.

Exhausted tissue culture medium exhibits HDAC inhibitory activity due to the accumulation of l-lactate and increasing acidity. (A) In vitro HDAC assays to test the HDAC-inhibitory activities of fresh (open circles) and used tissue culture medium (black diamonds). All activities are percentages of the reaction buffer control. The media contributed 40% by volume to the total HDAC reaction mix. The pH values of the reaction mixtures are the same throughout (range pH 8.09–8.12). (B) NMR spectroscopy of complete tissue culture medium at various times in culture following a medium change. The control profile is of fresh medium spiked with an l-lactate standard. (C) Lactate measurements in fresh and used tissue culture medium. (D) l-Lactate, d-lactate and low-pH inhibit histone deacetylase activity in vitro. The graph shows HDAC activity in the presence of increasing concentrations of NaCl (control, filled circles), butyrate (open circles), TSA (black crosses), l-lactate (black diamonds), d-lactate (black triangles) and pyruvate (black squares). (E) In vitro HDAC activity is inhibited at low pH.

Figure 3.

l-Lactate and d-lactate induce histone hyperacetylation in vivo. Histone H4 FTMS profiles of HCT116 cells 3 h after exposure to mock (control, (A), 25 mM l-lactate (B) and d-lactate (C)). For peak identification refer to Figure 1A. Peaks 7 and 10 and 13 and are one and two and 3 ε-amino acetylated histone H4 molecules and are clearly increased in the lactate-treated cells relative to the corresponding molecules in the control. The profiles have been scaled to the zero acetylated (two methylated) peak (peak 4). (D) The fold increase in one, two and three acetylated histone H4 molecules compared with control. (E) Bar graphs showing the overall increase in total histone acetylation (***P < 0.001, unpaired test).

Figure 4.

Gene expression changes induced by different HDAC inhibitors are positively correlated. (A–D) Correlations of the expression changes induced by different pairs of treatments. The data are taken from a refined dataset of 3496 probes. (A) Log₂ butyrate/C₂ versus TSA/C₁; B, Log₂ l-lactate/C₂ versus TSA/C₁; C, Log₂ Butyrate/C₂ versus d-Lactate/C₁; (D) Log₂, l-lactate/C₂ versus Zeocin/C₁. Regression lines are superimposed on the graphs together with their corresponding R² values (also see Table 2). (E) Hierarchical cluster analysis of the expression changes induced by HDAC inhibitors and zeocin using the Pearson correlation R as the distance metric. (F) Probes ordered on the x-axis according to their expression ratios (log₂, y-axis) in l-lactate (red line). Fifty-point moving averages of the log₂ expression ratios of the same probes on exposure to the other HDAC inhibitor treatments (and controls) are over-laid. See key.

Figure 5.

HDAC inhibition preferentially up-regulates HDAC-associated genes. (A) The probability of significant gene deregulation in CD4 cells after a 2 h exposure to high-dose butyrate and TSA. (B and C) Analysis of probes with expression values between 1000 and 5000 in CD4 cells. HDAC-associated genes/probes are more likely to be significantly upregulated (B) and less likely to be significantly downregulated (C) when compared with the overall probability of being up or downregulated on the array [horizontal lines (Chi-squared test)]. *P < 0.05; **P < 0.01; ***P < 0.001.