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.