FIG 3.

Isotope labeling distribution pattern of U¹³C glucose and glutamine and U¹⁵N glutamine during the M1-like polarization. (A) Diagram of ¹³C distribution from the catabolism of U¹³C glucose and U¹³C glutamine in glycolysis and/or the TCA cycle. Catabolism of U¹³C glucose generates M + 3 glycolytic intermediates and M + 2 (via pyruvate oxidation) and M + 3 (via pyruvate carboxylation) TCA cycle intermediates/derivatives. M + 5 glutamate, the direct metabolite of U¹³C glutamine, can enter the TCA cycle in the form of alpha ketoglutarate (α-KG) and/or via the GABA shunt. Increased ¹³C distribution from U¹³C glucose catabolism in glycolysis (B) with the generation of M + 3 lactate and (C to I) from the catabolism of U¹³C glutamine in the oxidative and reductive TCA cycle resulting in the generation of TCA cycle intermediates/derivatives. M1-like polarization was marked by diverting the ¹³C glucose carbon distribution from the TCA cycle M + 2, M + 1, and M + 3 intermediates to the formation of M + 3 lactate (A). In contrast, catabolism of U¹³C glutamine led to increased ¹³C distribution in the form of M + 4 TCA cycle intermediates/derivatives, including succinate (D), fumarate (E), malate (F), itaconate (H), and aspartate (I), as well as M + 4 and M + 5 citrate (G), indicating the simultaneous operation of both the oxidative and reductive TCA cycle. Increased ¹⁵N distribution from U¹⁵N glutamine to M + 1 aspartate (I) also indicates glutamine being a nitrogen source for the formation of nonessential amino acid aspartate. Solid red and blue circles represent ¹³C from U¹³C glucose and U¹³C glutamine, respectively. Thick arrows indicate increased ¹³C carbon flux into the formation of lactate and the TCA cycle intermediates or derivatives, respectively. Enrichment calculated from U¹³C glucose was marked as 13CG_M1, 13CG_M2, 13CG_M3, and 13CG_Σ. Σ was calculated with M1 × 1 + M2 × 2…+ Mn × n. Enrichment calculated from U¹³C glutamine was marked as 13CGln_M3, 13CGln_M4, 13CGln_M5, and 13CGln_Σ. Enrichment calculated from ¹⁵N glutamine was marked as 15NGln_M1. Data are shown as means ± standard deviation (S.D.) from 3 to 4 biological replicates. Statistical significance at *, P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001 was based on two-tailed student’s t test.