Figure 2.

Macrophage Efflux of TCA Cycle Intermediates into Itaconate, GABA, and 2-Hydroxyglutarate

(A–D) (A) A reference isotopologue enrichment scheme depicting labeling of TCA cycle and TCA-cycle-derived intermediates by [U-¹³C₆]glucose (Glc), through the first TCA cycle turn, via citrate synthase (CS, possibility a [black dots]), pyruvate carboxylase (PC, possibility b [orange dots]), or both together (possibility c). Subsequent rows in this scheme illustrate isotopologues obtained from second and third TCA cycle turns only from the citrate synthase incorporation (i.e., derived from CS, possibility a). Gray dots represent ¹²C. Light orange and green arrow paths depict efflux of the TCA cycle primarily in LPS- and IL-4-polarized macrophages, respectively. 10 mM [U-¹³C₆]glucose labeling of (B) itaconate, (C) gamma-aminobutyric acid (GABA), and (D) 2-hydroxyglutaric acid in unpolarized (control), LPS-polarized (25 ng/mL), or IL-4-polarized (25 ng/mL) WT BMDMs after 24 hr of exposure (n > 5/group). Data expressed as the mean ± standard error of the mean (SEM). PDH, pyruvate dehydrogenase; CAD, cis-aconitate decarboxylase. Significant differences determined by multiple Student's t test with Holm-Sidak correction when compared with control. *p < 0.05, **p < 0.01, ***p < 0.001, *****p < 0.00001, as indicated.