Figure 2.

Itaconate impacted substrate utilization for central carbon metabolism and promoted glutamine metabolism in cultured brain cells. A) Relative intracellular metabolite abundances in primary cortical neurons exposed to exogenous itaconate for 48 h. B) Primary cortical neurons did not metabolize ¹³C itaconate into pyruvate or TCA cycle intermediates in a detectable amount. Cells were exposed to 2 mM [U–¹³C]itaconate for 48 h and fractional enrichment from ¹³C itaconate is depicted in dark gray. C) Schematic depicting substrate utilization for TCA cycle metabolism using [U–¹³C]glucose (red), [U–¹³C]β-hydroxybutyrate (blue), [U–¹³C]leucine (green), and [U–¹³C]glutamine (brown). Open circles depict ¹²C, and closed circles represent ¹³C. D) Itaconate decreased ¹³C incorporation into citrate from ¹³C substrates [U–¹³C]leucine (green), [U–¹³C]β-hydroxybutyrate (blue, bHb), and [U–¹³C]glucose (red) in primary cortical neurons, while incorporation from [U–¹³C]glutamine (brown) increased. E) Itaconate increased glutamine metabolism in primary cortical neurons cultured in medium containing [U–¹³C]glutamine. Graph depicts M4 or M5 labeling on metabolite from ¹³C glutamine. F) Intracellular metabolite abundances in primary astrocytes exposed to 2 mM exogenous itaconate relative to control condition without itaconate. G) Itaconate increased glutamine metabolism in primary astrocytes cultured in medium containing [U–¹³C]glutamine. Graph depicts M4 or M5 labeling on metabolite from ¹³C glutamine. Cells were cultured for 48 h in ¹³C medium supplemented with 0 mM or 2 mM itaconate. Data are represented as box (25th to 75th percentile with median line) and whiskers (min. to max. values) with six (A) or three (F) biological replicates, or means ± s.e.m. with three biological replicates (B, D, E, and G). All of the experiments were repeated three independent times with the exception of D. Leucine and bHb trace were performed once. Student's t-test with *P < 0.05, **P < 0.01, and ***P < 0.001.