Lipids Reprogram Metabolism to Become a Major Carbon Source for Histone Acetylation

Abstract

Cells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part by increasing glucose-derived histone acetylation. Here, we find that lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. Using ¹³C-carbon tracing combined with acetyl-proteomics, we show that up to 90% of acetylation on certain histone lysines can be derived from fatty acid carbon, even in the presence of excess glucose. By repressing both glucose and glutamine metabolism, fatty acid oxidation reprograms cellular metabolism, leading to increased lipid-derived acetyl-CoA. Gene expression profiling of octanoate-treated hepatocytes shows a pattern of upregulated lipid metabolic genes, demonstrating a specific transcriptional response to lipid. These studies expand the landscape of nutrient sensing and uncover how lipids and metabolism are integrated by epigenetic events that control gene expression.

Keywords: acetylation; epigenetics; fatty acid; gene expression; histone; lipids; metabolism; metabolomics; proteomics.

Figure 1. Octanoate causes histone hyperacetylation

(A) Western blot of histone acetylation in AML12 cells treated for 24 hours with octanoate or PBS added to complete media. Data is presented as fold change relative to control, error bars are standard deviations of triplicate samples. Data is representative of experiments repeated at least 5 times. (B) Western blot of acetylated histone H3K9 in cell lysates from AML12 cells. Cells were serum starved overnight and then treated for 24 hours with base DMEM media (deplete of glucose, glutamine, or pyruvate; containing 10% FBS) alone or in combination with glucose (25 mM), glutamine (4 mM) or octanoate (2 mM). Graph shows the fold change of mean signal intensity of duplicate experiments relative to vehicle control +/− standard error mean (SEM) with representative western blot shown. (C) Quantitative proteomic analysis of protein acetylation in AML12 cells treated for 24 hours with vehicle or 2mM octanoate added to complete media, performed in triplicate. The graph shows log₂ fold-change of acetylated peptides in octanoate vs. PBS treated samples with an adjusted p-value (P_adjusted) ≤ 0.05 (5% FDR). (D) Pie chart representing the top 50 most hyperacetylated peptides upon octanoate treatment with P_adjusted ≤ 0.05. (E) Illustration summarizing the top 10 most acetylated histone peptides upon octanoate treatment on that make up the core nucleosome are labeled in red. (F) Western blot of histone H3K9 acetylation in AML12 cells treated with various fatty acids (2 mM) for 6 hours; the graph displays the mean fold-change compared to control of the signal intensity from 3 independent experiments; +/− SEM with representative western blot shown. (G) Western blot of histone acetylation in multiple cell lines treated for 24 hours with 2 mM octanoate. See also Fig. S1 and Table S1.

Figure 2. Lipid carbon directly acetylates histones

Representative MS¹ spectra from AML12 cells treated with vehicle (A) or [U-¹³C]octanoate (C) added to complete media, highlighting the isotope distribution of histone H3 peptides acetylated on lysines 9 and 14 (z=2). The same is shown for precursor ions (z=3) detecting histone H4 acetylation on lysines 8, 12 and 16 in vehicle (B) and [U-¹³C]octanoate (D) treated samples. Mass shifts to the right indicate presence of [U-¹³C]octanoate-derived carbon at those specific histone lysines. Relative enrichments of acetylated sites for histone H3 (E) and histone H4 (F) corrected for natural abundance. Single ¹³C-acetyl indicates one lysine in a given peptide was found to be modified with a ¹³C₂-acetyl group; double or triple acetyl means that two or three lysines of a given peptide are acetylated with [U-¹³C]octanoate-derived carbon. Red lower-case lysines indicate acetylated lysines (regardless of ¹³C enrichment). See also Figure S2 and Table S2.

Figure 3. Octanoate reprograms cellular metabolism

(A) Organic acid levels measured by tandem mass spectrometry of AML12 cells treated in quadruplicate for 24 hours with 2 mM octanoate compared to vehicle added to complete media. Data is fold-change over control, +/− SEM. ¹⁴C-labeled pyruvate (B) or glutamine (C) oxidation in AML12 cells treated with or without octanoate in complete media for 24 hours prior to capture and measurement of ¹⁴CO₂. Mean normalized data shown from 3 independent experiments +/− SEM. *p<0.05, **p<0.01 (Students t-test) (D) Volcano plot of protein phosphorylation changes measured by TMT-based quantitative mass spectrometry of whole cell AML12 lysates treated for 24 hours with octanoate or vehicle added to complete media; fold-change on a log₂ scale vs. p-value on a negative log₁₀ scale. Phoshopeptides showing statically significant (P_adjusted <0.1) increases or decreases in abundance in response to octanoate treatment are colored red or blue, respectively. Phosphopeptides identifying Pdha1 serine 232 phosphorylation residues are highlighted (E) ¹³C-labeling of TCA cycle intermediates in AML12 cells treated with [U-¹³C]glucose or [U-¹³C]glutamine in the presence or absence of unlabeled 2 mM octanoate compared to [U-¹³C]octanoate labeling in the presence of unlabeled glucose and glutamine. Mass isotopomer distribution (MID) of citrate from [U-¹³C]glucose (F) or [U-¹³C]glutamine (G) in the presence or absence of unlabeled 2 mM octanoate. (H) Citrate MID from [U-¹³C]octanoate in the presence of unlabeled glucose and glutamine. (I) Acetate levels in media collected from AML12 cells with [U-¹³C] labeled substrates for 24 hours. (J) Acetyl-CoA labeling pattern from AML12 cells treated with [U-¹³C] labeled substrates; all data collected after 24 hours. All experiments were done in complete media.

Figure 4. Octanoate causes a specific lipid metabolism gene expression signature

Microarray gene expression analysis of AML12 cells treated with vehicle or with 1 mM, 2 mM or 5 mM octanoate added to complete media for 24 hours. (A) Heat map of all genes identified with upregulated genes in red and downregulated genes in blue for the given doses of octanoate. (B) Gene set enrichment analysis (GSEA) of microarray data comparing vehicle to 2 mM octanoate. Ranked by p-value on a −log₁₀ scale with upregulated pathways in red and downregulated in blue. (C) qPCR analysis of lipid metabolic genes and genes found to be upregulated with octanoate treatment in microarray dataset. Cells were treated with vehicle or 2 mM octanoate for 24 hours. (D) qPCR analysis of genes associated with glucose-induced histone acetylation in AML12 cells treated with 2 mM octanoate. (E) qPCR data of AML12 cells treated with vehicle, 2 mM octanoate or 20 nM TSA alone or in combination for 24 hours in complete media. qPCR gene expression data are representative of repeated experiments each performed in triplicate +/− SD. All experiments were done in complete media. See also Figure S3 and Table S3.