Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.

The products of these overflow pathways provide a valuable resource during conditions of nutrient limitation. Ketone bodies become fuel sources during fasting conditions, whereas lactate and alanine are readily metabolized in local microenvironments (). These unconventional fuel sources satisfy metabolic demands during nutrient scarcity. Interestingly, acetate is also a nutrient that has been found to be a major carbon source for central carbon metabolism in nutrient limited conditions. Acetate metabolism provides a parallel pathway for acetyl-coenzyme A (CoA) production separate from conversion of citrate to acetyl-CoA by ATP citrate lyase (ACLY) and thus acetate allows for protein acetylation and lipogenesis independent of citrate conversion to acetyl-CoA. This pathway is essential in nutrient-deprived tumor microenvironments and other diverse contexts, but the origin of acetate is unclear (). It has been postulated that acetate may be synthesized de novo in cells (), but the pathways and quantitative reaction mechanisms through which this may occur are unknown. Given that alternative carbon sources often arise from overflow metabolism, we suspected that such a mechanism may allow for acetate generation. This hypothesis led us to conduct a re-evaluation of mammalian central carbon metabolism.

In conditions of hyperactive cellular metabolism, excessive cellular nutrient uptake results in incomplete metabolism and excretion of intermediates. The carbon source for catabolic and anabolic processes is often incompletely catabolized and excreted into the extracellular space (). During the Warburg effect, a phenotype characterized by increased glucose uptake, the glycolysis rate (near 100 mM/hr) is 10 to 100 times faster than the rate at which complete oxidation of glucose occurs in the mitochondria (). As a result, the excess carbon from glycolysis is secreted as lactate. Other examples are seen in the cells in diabetic tissues, where excess fatty acid oxidation leads to incomplete lipid catabolism and excretion of acyl-carnitines and ketone bodies (). Numerous other examples, such as in the case of excess anabolic substrates released in nucleotide synthesis or the excretion of formic acid during excess one carbon metabolism (), are also apparent. In each case, overflow metabolism results from imbalanced metabolic supply and demand that results in limits to enzyme activity.

Results

13C 6 ]-glucose, harvested metabolites over time, subjected the extracts to chemical derivatization with 2-hydrazinoquinoline (HQ) to capture acetate and other organic acids, and analyzed the products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) (13C 3 ]-pyruvate, [13C 2 ]-acetate, and [13C 3 ]-lactate unexpectedly revealed that the rates of generation of pyruvate and lactate, the end products of glycolysis, were commensurate with the rate of acetate generation (13C 2 ]-acetate was comparable to the concentration of [13C 3 ]-pyruvate and on the order of the amount of [13C 3 ]-lactate generated from [13C 6 ]-glucose in these glycolytic cells. De novo acetate production from glycolysis was also observed in several other cell lines of diverse origins ( Faubert et al., 2017 Faubert B.

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et al. Lactate metabolism in human lung tumors. Sousa et al., 2016 Sousa C.M.

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et al. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. 13C source from [13C 6 ]-glucose to [13C 5 ]-glutamine, which labels [13C 3 ]-pyruvate to a far lesser extent, diminished the formation of [13C 2 ]-acetate ( Figure 1 Acetate Is Synthesized from Glucose Independent of Acetyl-CoA-Related Reactions Show full caption (A) Scheme of experimental setup for measuring glucose-derived acetate, pyruvate, and lactate. (B) Medium concentration of [13C 3 ]-pyruvate, [13C 2 ]-acetate, and [13C 3 ]-lactate secreted from HCT116 cells cultured in RPMI 1640 medium containing [13C 6 ]-glucose. Medium was switched to the [13C 6 ]-glucose medium at time 0. (C) The amount of [13C 2 ]-pyruvate, [13C 3 ]-lactate, and [13C 2 ]-acetate released from various cultured cells at 40 min after switching to the [13C 6 ]-glucose medium. (D) The relative levels of acetate production in HCT116 cells cultured in glucose-free medium containing different tracers: [13C 6 ]-glucose (11.1 mM), [2C 3 ]-lactate (5 mM), or [2C 3 ]-pyruvate (5 mM). (E) Intracellular concentrations of metabolites in HCT116 cells after incubation in [13C 6 ]-glucose medium for 5 hr. (F) Schematic depicting the workflow for HCT116 cells with [13C 6 ]-glucose and/or treatment with the histone deacetylation inhibitor SAHA. (G) Western blots of histone acetylation sites from HCT116 cells in the absence or the presence of SAHA (5 μM) for 1 hr. (H) Ponceau S staining (left panel) and quantitative proteomic analysis (right panel) of histone extracts from HCT116 cells cultured in the presence of 13C-glucose for 0, 6, 12 and 18 hr. An asterisk indicates 13C-labeled acetyl groups on histones. (I) Release of acetate from HCT116 cells with a 6-hr pretreatment with [13C 6 ]-glucose in the presence or the absence of histone deacetylation inhibitor (SAHA, 5 μM) for 1 hr (left panel). The release rate (right panel) is obtained by calculating the slope of the acetate release curve. Ac, acetate; Ac-histone, acetylated histone. The acetylated histone sites include lysine 14 (H3K14ac); lysine 18 (H3K18ac); lysine 18 and lysine 23 (H3K18acK23ac); lysine 23 (H3K23ac); lysine 27 (H3K27ac); lysine 9 (H3K9ac); lysine 9 and lysine 14 (H3K9acK14ac); lysine 14 containing mono-methylation at lysine 9 (H3K9me1K14ac); lysine 14 containing di-methylation at lysine 9 (H3K9me2K14ac); lysine 14 containing tri-methylation at lysine 9 (H3K9me3K14ac) of histone H3; and acetylation on lysine 16 (H4Kac), lysine 5 (H4K5ac), lysine 8 (H4K8ac), and lysine 12 (H4K12ac). Values are expressed as mean ± SD of n = 3 independent measurements. NS: p > 0.05 from a Student’s t test. See also Figure S1 Figure S1 Evaluation of the Effects of Glutamine and Pyruvate Carrier Inhibition on Acetate Production and the Levels of TCA Metabolites, Related to Figure 1 Show full caption (A) [13C 6 ]-glucose enrichment pattern of metabolites produced by HCT116 cells for 70 mins (acetate) or 40 mins (pyruvate and Ac-CoA). (B) Same as in (A) but for [13C 5 ]-glutamine. (C) Extracted ion chromatogram of acetate or the [2H 3 ]-acetate HQ derivative from samples prepared by incubating acetyl-carnitine, [2H 3 ]-acetyl-CoA or [2H 3 ]-acetate (100 μM) with HQ at 37°C for 70 mins. (D) 13C enrichment patterns of metabolites derived from [13C 6 ]-glucose. Red solid circle denotes 13C. Ac: Acetate; Ac-carnitine: Acetyl-carnitine; Pyr: Pyruvate. (E–H) The relative levels of 13C enriched Ac-CoA (E), Ac-carnitine (F), citrate (G), and acetate (H) in HCT116 cells cultured in RPMI 1640 medium containing [13C 6 ]-glucose with or without UK5099 treatment for 40 min. Values are expressed as mean ± SD of n = 3 independent measurements. NS: p > 0.05, ∗∗∗p < 0.001 in Student’s t test. To monitor glycolysis in the setting of acetate metabolism, we incubated exponentially growing human colorectal HCT116 cells with uniformly labeled []-glucose, harvested metabolites over time, subjected the extracts to chemical derivatization with 2-hydrazinoquinoline (HQ) to capture acetate and other organic acids, and analyzed the products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) ( Figure 1 A; STAR Methods ). Monitoring the kinetics of []-pyruvate, []-acetate, and []-lactate unexpectedly revealed that the rates of generation of pyruvate and lactate, the end products of glycolysis, were commensurate with the rate of acetate generation ( Figure 1 B). Strikingly, the concentration of []-acetate was comparable to the concentration of []-pyruvate and on the order of the amount of []-lactate generated from []-glucose in these glycolytic cells. De novo acetate production from glycolysis was also observed in several other cell lines of diverse origins ( Figure 1 C). Indeed, there are other sources of pyruvate, such as alanine or lactate (). Thus, the presence of exogenous pyruvate or lactate also increased acetate production ( Figure 1 D). Meanwhile, switching theC source from []-glucose to []-glutamine, which labels []-pyruvate to a far lesser extent, diminished the formation of []-acetate ( Figures S1 A and S1B). Together these findings suggest that acetate can be synthesized endogenously in quantitative amounts and with kinetics comparable to pyruvate and lactate production from glycolysis.

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Ballard F.J. Production and utilization of acetate in mammals. 13C 6 ]-glucose containing media for 6 hr, followed by incubation with the histone deacetylase inhibitor SAHA for an additional 1 hr (13C 6 ]-glucose at 6 hr that reached steady state (13C 6 ]-glucose medium was then replaced with 12C glucose medium, and the release of [13C 2 ]-acetate would represent the contribution from acetyl groups to acetate production. Histone deacetylase inhibition didn’t alter the [13C 2 ]-acetate release rate (13C 2 ]-acetate production rate from this assay was found to be nearly 5-fold less than the total amount (12C and 13C acetate) of acetate ( Acetate can be generated by the removal of acetyl groups from histones by histone deacetylases () and by hydrolysis of Ac-CoA (). We thus measured the concentrations of the metabolites involved in these processes and observed orders of magnitude lower concentrations of Ac-CoA than glucose-derived pyruvate, lactate, and acetate ( Figure 1 E), suggesting that de novo acetate production likely is uncoupled from these acetyl-CoA dependent reactions. Moreover, the acetate HQ derivative was not spontaneously formed by incubating Ac-CoA or acetyl (Ac)-carnitine with the HQ derivatization reagents indicating this potential artifact is sufficiently controlled ( Figure S1 C). To further evaluate the contribution of deacetylation reactions, including histone deacetylation, to acetate production, we cultured HCT116 cells in []-glucose containing media for 6 hr, followed by incubation with the histone deacetylase inhibitor SAHA for an additional 1 hr ( Figure 1 F). Treatment with SAHA suppressed histone deacetylation ( Figure 1 G), indicated by the increase of total histone 3 acetylation (H3_ac) as well as acetylation of H3 on lysines 9 (H3K9ac) and 27 (H3K27ac) of histone H3. Furthermore, a quantitative proteomics analysis across a diverse set of acetylation sites revealed a consistent 60%–80% incorporation of []-glucose at 6 hr that reached steady state ( Figure 1 H). []-glucose medium was then replaced withC glucose medium, and the release of []-acetate would represent the contribution from acetyl groups to acetate production. Histone deacetylase inhibition didn’t alter the []-acetate release rate ( Figure 1 I), and the measured []-acetate production rate from this assay was found to be nearly 5-fold less than the total amount (C andC acetate) of acetate ( Figure 1 I), indicating that hydrolysis of the acetyl group from histones is not a major source of acetate in these experiments. Together, these experiments allow us to conclude that, in these conditions, more than 80% of the acetate measured is rapidly generated de novo through glycolysis.

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et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. 13C-labeled Ac-CoA, Ac-carnitine (reversibly converted from Ac-CoA), and citrate (the first intermediate in the TCA cycle) were decreased by UK5099 treatment (13C 2 ]-acetate from [13C 6 ]-glucose was observed ( It is generally thought that Ac-CoA formation requires glucose to first enter the mitochondria, be exported as citrate and then metabolized to cytosolic Ac-CoA ( Figure S1 D). Thus, we tested whether the pyruvate carrier inhibitor (UK5099) () that perturbs entry of pyruvate into the mitochondria would affect acetate generation. As expected, theC-labeled Ac-CoA, Ac-carnitine (reversibly converted from Ac-CoA), and citrate (the first intermediate in the TCA cycle) were decreased by UK5099 treatment ( Figures S1 E–1G). Notably, no difference in the generation of []-acetate from []-glucose was observed ( Figure S1 H). Thus, these data indicate that acetate production is independent of Ac-CoA synthesis and breakdown.

Kim et al., 2016 Kim J.G.

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Yang S.H. Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells. 2 O 2 ) ( 2 O 2 via production of superoxides and then into acetate. H 2 O 2 thus obtains oxygen from molecular O 2 and therefore, culturing cells in the presence of 18O 2 and monitoring incorporation of 18O into acetate would enable quantitation of the endogenous contribution of reactive oxygen species (ROS) to acetate production. In this experimental design, other potential acetate production routes (deacetylation or aldehyde oxidation) would involve transferring the oxygen in a water molecule to acetate. 18O-labeled water is negligible in this setup, and thus these two possibilities could be resolved with this experiment ( 2 (18O 2 and 16O 2 ) and 80% N 2 (18O 1 ]-acetate from other isotopically labeled species. We used labeled kynurenine (produced through oxygenation) in SKOV3 cells with high indoleamine-2,3-dioxygenase (IDO) expression ( Litzenburger et al., 2014 Litzenburger U.M.

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et al. Constitutive IDO expression in human cancer is sustained by an autocrine signaling loop involving IL-6, STAT3 and the AHR. 18O 2 experienced by the cells which was found to be about 40% in this setup (18O 1 ]-acetate providing a direct observation in intact cells of the formation of [18O 2 ]-H 2 O 2 and oxidative decarboxylation of pyruvate by H 2 O 2 . Inhibiting the mitochondrial pyruvate carrier with UK5099 or addition of pyruvate to the culture medium substantially increased the contribution of H 2 O 2 to acetate production from pyruvate ( 2 O 2 ( , with tetrathiomolybdate (TTM), was done to decrease endogenous H 2 O 2 levels. Indeed, the presence of TTM decreased 18O incorporation into H 2 O 2 -coupled reactions, including methionine sulfoxide ( 2 O 2 contributes to acetate formation from pyruvate in cellular conditions. Figure 2 ROS Catalyzes the Oxidative Decarboxylation of Pyruvate to Generate Acetate in Mammalian Cells Show full caption (A) Schematic of the potential sources of elemental oxygen in acetate produced from different routes. 18O 2 tracing assay as described in the (B) Experimental setup oftracing assay as described in the STAR Methods (C) Incorporation of 18O into metabolites in tryptophan metabolism. (D) Schematic of 18O and 13C incorporation into acetate produced from H 2 O 2 -mediated pyruvate decarboxylation and representative chromatogram and tandem mass spectrum of [13C 2 , 18O 1 ]-acetate derivative. Blue-open circles and red-solid circles represent 18O and 13C, respectively. (E) Fraction of [13C 2 , 18O 1 ]-acetate out of the glucose-derived acetate pool. (F) Schematic of endogenous H 2 O 2 generated from superoxides and superoxide dismutase (SOD) and H 2 O 2 -mediated methionine oxidation and pyruvate decarboxylation. TTM, ammonium tetrathiomolybdate, a SOD inhibitor. (G and H) The effect of SOD inhibitor TTM on the relative levels of [18O 1 ]-MetO (G) and [13C 2 , 18O 1 ]-acetate (H) in SKOV3 cells cultured in [13C 6 ]-glucose and 18O 2 for 48 hr. (I) Secretion of [13C 2 ]-acetate from HCT116 cells in the presence or absence of TTM. (J) Release of [13C 2 , 18O 1 ]-acetate from HCT116 cells after addition of exogenous [18O 2 ]-H 2 O 2 . (K) Relative abundance of [13C 2 ]-acetate. The fractions in E used to represent the ROS contribution to acetate production were corrected for 18O natural abundance and 18O 2 enrichment. The data in (J) and (K) represent the acetate from the spent media collected at 10 min after addition of 0 or 300 μM [18O 2 ]-H 2 O 2 (J) or H 2 O 2 (K) to HCT116 cells that were pre-incubated in [13C 6 ]-glucose medium for 1 hr. Values are expressed as mean ± SD of n = 3 independent measurements. ∗∗p < 0.01; ∗∗∗p < 0.001 from a Student’s t test. See also Figure S2 One considerable possibility of a substrate for acetate production is pyruvate, a keto acid, that contains an electrophilic moiety. Keto acids have been reported to be chemical scavengers in both bacterial and mammalian cells (). A plausible reaction pathway for the generation of acetate could involve the nucleophilic attack of pyruvate by the reactive oxygen species generated from hydrogen peroxide (H) ( Figure 2 A, top), and the reaction would involve incorporation of the oxygen into Hvia production of superoxides and then into acetate. Hthus obtains oxygen from molecular Oand therefore, culturing cells in the presence ofand monitoring incorporation ofO into acetate would enable quantitation of the endogenous contribution of reactive oxygen species (ROS) to acetate production. In this experimental design, other potential acetate production routes (deacetylation or aldehyde oxidation) would involve transferring the oxygen in a water molecule to acetate.O-labeled water is negligible in this setup, and thus these two possibilities could be resolved with this experiment ( Figure 2 A, bottom). We cultured cells in the presence of 20% Oand) and 80% N Figure 2 B). We then employed LC-HRMS to enable spectral resolution of []-acetate from other isotopically labeled species. We used labeled kynurenine (produced through oxygenation) in SKOV3 cells with high indoleamine-2,3-dioxygenase (IDO) expression () to quantify the amount of heavyexperienced by the cells which was found to be about 40% in this setup ( Figure 2 C). We then identified ( Figure 2 D) and quantified ( Figure 2 E) []-acetate providing a direct observation in intact cells of the formation of []-Hand oxidative decarboxylation of pyruvate by H. Inhibiting the mitochondrial pyruvate carrier with UK5099 or addition of pyruvate to the culture medium substantially increased the contribution of Hto acetate production from pyruvate ( Figure 2 E). To further test this mechanism, inhibition of superoxide dismutase (SOD), which converts superoxides to H Figure 2 F)with tetrathiomolybdate (TTM), was done to decrease endogenous Hlevels. Indeed, the presence of TTM decreasedO incorporation into H-coupled reactions, including methionine sulfoxide ( Figure 2 G) and acetate ( Figures 2 H and 2I). These findings together demonstrate that endogenous Hcontributes to acetate formation from pyruvate in cellular conditions.

1H nuclear magnetic resonance spectroscopy (−1min−1) and could be accelerated with catalysts present in high concentrations in cells such as Cu2+, as Cu2+ stabilizes the intermediate of pyruvate decarboxylation ( Figure S2 2 O 2 Catalyzes Pyruvate Decarboxylation to Acetate, Related to Exogenous HCatalyzes Pyruvate Decarboxylation to Acetate, Related to Figure 2 Show full caption (A) Representative NMR spectra for pyruvate and acetate peaks from time 0 to 18.7 min. Time 0 min is when data acquisition began, and there is a delay from the actual reaction time due to temperature equilibration in the NMR tube. (B) Conversion rate of pyruvate (Pyr) to acetate under different conditions from 0 to 18.7 mins from the inception of the data acquisition. Conversion rate was calculated by dividing acetate peak area by the sum of acetate and pyruvate peak area. (C) Summary of reaction conditions, conversion rate (at 18.7 min), and reaction constant (mean ± SD). To further investigate the properties of this reaction, we performed in vitro assays by incubating pyruvate with hydrogen peroxide at 37°C and found the major product to be acetate as measured byH nuclear magnetic resonance spectroscopy ( Figure S2 A). Thus, in the presence of hydrogen peroxide, pyruvate is converted to acetate non-enzymatically with kinetics commensurate with values needed for the reaction to occur at appreciable amounts in cells ( Figure S2 B). The reaction followed second order kinetics (k = 0.19 ± 0.05 mMmin) and could be accelerated with catalysts present in high concentrations in cells such as Cu, as Custabilizes the intermediate of pyruvate decarboxylation ( Figures S2 B and S2C).

2 O 2 affects acetate production by adding [18O 2 ]-H 2 O 2 to cultured HCT116 cells (18O 2 ]-H 2 O 2 revealed a dose-dependent increase in acetate levels, and the acetate detected had 18O incorporation as measured by LC-HRMS (13C 6 ]-glucose and then treated with [18O 2 ]-H 2 O 2 showed a transient increase in the amount of [18O 1 ]-acetate peaking around 5 min post-induction of ROS with subsequent decay kinetics corresponding to the clearance of the ROS ( Winterbourn, 2008 Winterbourn C.C. Reconciling the chemistry and biology of reactive oxygen species. 13C 2 ]-acetate pool (18O 2 ]-H 2 O 2 . This analysis further revealed that acetate is synthesized from pyruvate in intact cells, and the activity of this reaction is mediated by ROS. As a further evaluation, we tested whether exogenous Haffects acetate production by adding []-Hto cultured HCT116 cells ( Figure 2 J). Titrating concentrations of []-Hrevealed a dose-dependent increase in acetate levels, and the acetate detected hadO incorporation as measured by LC-HRMS ( Figure 2 J). Additionally, HCT116 cells pre-incubated with []-glucose and then treated with []-Hshowed a transient increase in the amount of []-acetate peaking around 5 min post-induction of ROS with subsequent decay kinetics corresponding to the clearance of the ROS ( Figure 2 J) (). Importantly, it was observed that up to 30% of the total []-acetate pool ( Figures 2 J and 2K) could be derived from a transient increase in ROS from exogenous []-H. This analysis further revealed that acetate is synthesized from pyruvate in intact cells, and the activity of this reaction is mediated by ROS.