Acetate Production from Glucose and Coupling to Mitochondrial Metabolism in Mammals

Abstract

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.

Keywords: dehydrogenase; flux analysis; glycolysis; lipogenesis; metabolomics; mitochondria; pyruvate; reactive oxygen species; stable isotope tracing; thiamine.

Figure 1.. Acetate is synthesized from glucose independent of acetyl-CoA related reactions

(A) Scheme of experimental setup for measuring glucose-derived acetate, pyruvate and lactate. (B) Medium concentration of [¹³C₃]-pyruvate, [¹³C₂]-acetate, and [¹³C₃]-lactate secreted from HCT116 cells cultured in RPMI 1640 medium containing [¹³C₆]-glucose. Time 0 is when medium was switched to [¹³C₆]-glucose medium. (C) The amount of [¹³C₂]-pyruvate, [¹³C₃]-lactate and [¹³C₂]-acetate released from various cultured cells at time 40 min after switching to [¹³C₆]-glucose medium. (D) The relative levels of acetate production in HCT116 cells cultured in glucose free medium containing different tracers: [¹³C₆]-glucose (11.1 mM), [²C₃]-lactate (5 mM) or [²C₃]-pyruvate (5 mM). (E) Intracellular concentrations of metabolites in HCT116 cells after incubation in [¹³C₆]-glucose medium for 5 hrs. (F) Schematic depicting the workflow for HCT116 cells with [¹³C₆]-glucose and or treatment with the histone deacetylation inhibitor, SAHA. (G) Western blots of histone acetylation sites from HCT116 cells in the absence or 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 hrs. * denotes ¹³C labeled acetyl groups on histones. (I) Release of acetate from HCT116 cells with 6 hours pretreatment of [¹³C₆]-glucose in the presence or 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. Abbreviations: 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 in Student’s t test. See also Figure S1.

Figure 2.. ROS catalyzes the oxidative decarboxylation of pyruvate to generate acetate in mammalian cells

(A) Schematic of the potential sources of elemental oxygen in acetate produced from different routes. (B) Experimental setup of ¹⁸O₂ tracing assay as described in STAR Methods. (C) Incorporation of ¹⁸O into metabolites in tryptophan metabolism. (D) Schematic of ¹⁸O and ¹³C incorporation into acetate produced from H₂O₂-mediated pyruvate decarboxylation, and representative chromatogram and tandem mass spectrum of [¹³C₂, ¹⁸O₁]-acetate derivative. Blue open circles and red solid circles denote O and C, respectively. (E) Fraction of [¹³C₂, ¹⁸O₁]-acetate out of the glucose-derived acetate pool. (F) Schematic of endogenous H₂O₂ generated from superoxides and superoxide dismutase (SOD) and H₂O₂-mediated methionine oxidation and pyruvate decarboxylation. TTM, Ammonium tetrathiomolybdate, a SOD inhibitor. The effect of SOD inhibitor TTM on the relative levels of [¹⁸O₁]-MetO (G), [¹³C₂, ¹⁸O₁]-acetate (H) in SKOV3 cells cultured in [¹³C₆]-glucose and ¹⁸O₂ for 48 hrs. (I) Secretion of [¹³C₂]-acetate from HCT116 cells in the presence or absence of TTM. (J) Release of [¹³C₂, ¹⁸O₁]-acetate from HCT116 cells after addition of exogenous [¹⁸O₂]-H₂O₂. (K) Relative abundance of [¹³C₂]-acetate. The fractions in E used to represent the ROS contribution to acetate production were corrected for ¹⁸O natural abundance and the ¹⁸O₂ enrichment and. The data in J and K represent the acetate from the spent media collected at 10 min after addition of 0 or 300 μM [¹⁸O₂]-H₂O₂ (J) or H₂O₂ (K) to HCT116 cells which were pre-incubated in [¹³C₆]-glucose medium for 1 hour. Values are expressed as mean ± SD of n=3 independent measurements. ** p<0.01, *** p<0.001 in Student’s t test. See also Figure S2.

Figure 3.. Conversion from pyruvate to acetate is catalyzed by mammalian PDH in a thiamine dependent manner

(A) Release of acetate and acetaldehyde from pyruvate is possibly catalyzed by keto acid dehydrogenases, especially pyruvate dehydrogenase (PDH). (B) Intracellular CoA and GSH concentrations in HCT116 cells. (C) CoA and GSH concentrations in mouse sarcoma tumors. (D) Conversion of pyruvate (200 μM) to acetate and acetaldehyde by PDH over 30 min in the absence or presence of cofactors. (E) Production of pyruvate-derived acetate, acetaldehyde, Ac-CoA and Ac-GSH after 30 min incubation with PDH. Consumption of pyruvate and production of acetaldehyde and acetate by alpha-ketoglutarate dehydrogenase supplemented with TPP in the absence (F) or the presence (G) of other cofactors and alpha-ketoglutarate (aKG, 200 μM). (H) Proposed model for acetate release from pyruvate catalyzed by keto acid dehydrogenase. Values are expressed as mean ± SD of n=3 (HCT116 cells) and n=5 (mouse) independent measurements. See also Figure S3.

Figure 4.. Acetate production from pyruvate occurs in both cultured cells and in vivo

(A) ¹³C enrichment of acetate (Ac), acetaldehyde (ACE) and Acetyl-GSH (Ac-GSH) in HCT116 cultured in the presence of [¹³C₆]-glucose. (B) Intracellular and medium concentrations of ¹³C labeled Ac and ACE in HCT116 cells after incubation in[¹³C₆]-glucose medium for 1 hr. (C) Relative levels of ¹³C enriched Ac, ACE and Ac-GSH in mouse sarcoma cells with wild type PDH (PDH WT) or PDH knockout (PDH KO) using CRISPR/Cas9. (D) The presence of PDH and aKGDH proteins in the subcellular fractions of HCT116 cells. Abbreviations: E1, the E1 component of PDH or aKGDH; CS, citrate synthetase; MPC1, mitochondrial pyruvate carrier 1. (E) Relative levels of ¹³C enriched Ac, ACE and Ac-GSH in HCT116 cells cultured in medium with or without thiamine. (F) Relative levels of [¹³C₆]-glucose and [²H₃]-pyruvate-derived acetate after the addition of [¹⁸O₂]-H₂O₂ (200 μM) for 10 min. (G) Contribution of ROS to acetate production (represented by the fraction of ¹⁸O labeled acetate) upon thiamine starvation. (H) Schematic of ¹³C glucose infusion in soft tissue sarcoma-bearing mice. (I) ¹³C enrichment fraction of glucose and pyruvate in the serum of sarcoma free mouse after the [¹³C₆]-glucose infusion. (J) Concentrations of [¹³C₃]-Pyr, [¹³C₃]-Lac, [¹³C₂]-ACE and [¹³C₂]-Ac in sarcoma tumors. (K) ¹³C enrichment of AC-GSH and [¹³C₂]-Ac-GSH in sarcoma. (l) Concentrations of [¹³C₃]-Pyr, [¹³C₃]-Lac, [¹³C₂]-ACE and [¹³C₂]-Ac in serum of tumor-bearing mice. J to L were obtained from sarcoma mouse samples collected 3 hrs after [¹³C₆]-glucose infusion. Values are expressed as mean ± SD of n=3 (HCT116 cells) and n=5 (mouse) independent measurements. See also Figure S4.

Figure 5.. De novo acetate production rescues proliferation defects in cells with ACLY deficiency

(A) Western blot of ATP citrate lyase (ACLY) in MEF (ACLY WT or KO) cells. (B) Growth curve of MEF (ACLY WT or KO) cells cultured in RPMI 1640 supplemented with 10% dialyzed FBS without acetate supplement. (C) Picture of co-culture system. (D) Medium acetate concentration in co-culture system in the absence or presence of HCT116 cells. Representative images and cell number of MEF ACLY KO (E) or WT cells (F) co-cultured with or without HCT116 for 3 days. (G) Western blot confirming the knockdown of ACLY and ACSS2 in MEF cells. (H) Representative photos and cell numbers of MEF ACSS2 WT or KD cells co-cultured with or without HCT116 for 30 hrs. (I) The incorporation of ¹³C into palmitate in MEF ACLY KO cells co-cultured with or without HCT116 cells in RPMI medium containing [¹³C₆]-glucose and 10% dialyzed FBS (bottom). (J), Model of restoration proliferation in MEF ACLY KO cells by acetate released from HCT116 cells in a co-culture system. Values are expressed as mean ± SD of n=3 independent measurements. NS: p>0.05, * p<0.05, ** p<0.01, *** p<0.001 in Student’s t test.

Figure 6.. Endogenous acetate contributes to acetyl-CoA metabolism

(A) Western blot confirming the knockdown of ACLY and ACSS2 in HCT116 cells. The effect of ACLY and ACSS2 knockdown (B) or ACSS2 inhibitor (20 μM) (C) on lipogenesis in HCT116 cells cultured in RPMI 1640 supplemented with 10% dialyzed FBS, without exogenous acetate. (D) Schematic of ¹⁸O incorporation to acetyl groups via ROS mediated pyruvate decarboxylation. (E) Extracted ion chromatogram (left) and mass spectra (right) of ac-carnitine mass isotopologues from HCT116 cells treated with [¹⁸O₂]-H₂O₂ (300 μM) for 30 mins. (F) The relative levels of ¹⁸O labeled acetyl-aspartate in from HCT116 cells treated with [¹⁸O₂]-H₂O₂ (300 μM) for 30 mins in the presence and absence of UK5099. (G-H) Carbon incorporation from deuterium labeled acetaldehyde into acetyl groups by HCT116 cells treated with control siRNA or ACLY siRNA cultured in low (1%) or regular (10%) D-FBS. Values are expressed as mean ± SD of n=3 independent measurements. ND: not detected. ** p<0.001 in Student’s t test. See also Figure S5.