Itaconate Alters Succinate and Coenzyme A Metabolism via Inhibition of Mitochondrial Complex II and Methylmalonyl-CoA Mutase

Abstract

Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing and mass spectrometry approaches to explore how itaconate impacts various metabolic pathways in cultured cells. Itaconate is a competitive and reversible inhibitor of Complex II/succinate dehydrogenase (SDH) that alters tricarboxylic acid (TCA) cycle metabolism leading to succinate accumulation. Upon activation with coenzyme A (CoA), itaconyl-CoA inhibits adenosylcobalamin-mediated methylmalonyl-CoA (MUT) activity and, thus, indirectly impacts branched-chain amino acid (BCAA) metabolism and fatty acid diversity. Itaconate, therefore, alters the balance of CoA species in mitochondria through its impacts on TCA, amino acid, vitamin B₁₂, and CoA metabolism. Our results highlight the diverse metabolic pathways regulated by itaconate and provide a roadmap to link these metabolites to potential downstream biological functions.

Keywords: TCA cycle metabolism; acetyl-CoA; branched-chain amino acids (BCAA); isotopic tracing; itaconate; itaconyl-CoA; methylmalonate; odd-chain fatty acids (OCFAs); propionyl-CoA; succinate; succinate dehydrogenase; vitamin B12.

Figure 1

Itaconate promotes succinate accumulation in diverse cell types. (a) Itaconate levels in RAW264.7 cells exposed to LPS or 2 mM exogenous itaconate (ita) for 24 h compared to control condition (Ctr). (b,c) Itaconate (b) and succinate (c) levels in cells exposed to 2 mM itaconate for 48 h. (d) Pearson correlation coefficient (r) of itaconate and succinate levels in Huh7 cells exposed to increasing itaconate concentrations for 48 h. (e) Labeling on itaconate and succinate in cell lines cultured in the presence of 2 mM [U-¹³C₅]itaconate. (f) Succinate levels in Huh7 cells exposed to 2 mM itaconate or 62.5 µM dimethyl-itaconate (DMI) for 48 h. Data are depicted as box and whiskers (b,c,e,f) or mean (d) ± s.e.m. (a) obtained from 3 cellular replicates. Students t-test (c) or one-way ANOVA (a,f) with no adjustment for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001.

Figure 2

Itaconate is a competitive and reversible SDH inhibitor in diverse cell types. (a) Schematic depicting plasma membrane permeabilizer reagent (PMP) to access mitochondrial respiration in permeabilized cells. (b) Succinate-driven respiration in Huh7 cells in the presence (red) or absence (black) of itaconate (ita). (c) Succinate-driven respiration with increasing itaconate concentrations in Huh7 and A549 cells. Significance is depicted compared to 0 mM itaconate. (d) Succinate-driven respiration after serial addition of Ita/Suc/Ita (red) or Ita/Suc/Suc (grey) in HuH7 cells. (e) Succinate driven respiration after serial addition of itaconate or succinate in Huh7 and A549 cells. (f) Schematic depicting the competitive inhibition of itaconate on succinate dehydrogenase (SDH) activity. Data depict mean ± s.e.m. (b,d) or box and whiskers (c,e) obtained from 5 cellular replicates. One-way ANOVA (c) relative to 0 mM itaconate with no adjustment for multiple comparisons and # p < 0.0001.

Figure 3

Itaconate influences glutamine metabolism. (a) Schematic depicting carbon incorporation into tricarboxylic acid (TCA) cycle intermediates from [U-¹³C₅]glutamine (blue). Open circles depict ¹²C, closed circles ¹³C carbon. (b) Labeled metabolite level (depicted as abundance times mole percent enrichment (MPE)) from [U-¹³C₅]glutamine in Huh7 cells cultured in the presence of 2 mM itaconate for 4 h. (c) ¹³C incorporation (mole percent enrichment, MPE) into metabolites from [U-¹³C₅]glutamine in Huh7 cells cultured for 48 h. (d) ¹³C incorporation into succinate from [U-¹³C₅]glutamine in diverse cell types in the presence of 0 mM or 2 mM itaconate after 48 h. Data are depicted as mean ± s.e.m. obtained from 3 cellular replicates. Students t-test with no adjustment for multiple comparisons and * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001.

Figure 4

Itaconate influences methylmalonyl-coenzyme A (CoA) mutase (MUT) dependent BCAA metabolism. (a) Schematic depicting nitrogen exchange from [α-¹⁵N]glutamate on branched-chain amino acids (BCAA) and branched-chain keto acids (BCKA). (b) Labeling on amino acids from [α-¹⁵N]glutamine in Huh7 cells cultured for 48 h in the presence of 2 mM itaconate. (c) Itaconyl-CoA levels depicted as ion counts (m/z = 880.1379) in Huh7 cells. N.D. not detectable. (d) Methylmalonate levels in Huh7 or HepG2 cells exposed to 2 mM itaconate for 48 h compared to 0 mM itaconate (dotted line). (e) Schematic depicting methylmalonyl-CoA mutase (MUT) dependent BCAA catabolism. Open circles depict ¹²C, closed circles ¹³C carbons from [U-¹³C₆]isoleucine tracer. (f) M3 label on fumarate from [U-¹³C₆]isoleucine in Huh7 and HepG2 cells exposed to 2 mM itaconate for 48 h. Data are depicted as box and whiskers (c,d) or mean ± s.e.m. (b,f) obtained from 3 cellular replicates. Students t-test with no adjustment for multiple comparisons and * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001.

Figure 5

Itaconate alters fatty acid diversity and CoA metabolism. (a) Levels of fatty acids in Huh7 cells cultured for 48 h in the presence of 2 mM ita. (b) Isotopologue distribution on C17:0 from [U-¹³C₆]isoleucine in Huh7 cells after 48 h. (c) Newly synthesized C15:0 and C17:0 from [U-¹³C₆]isoleucine in Huh7 cells exposed to itaconate or dimethyl-itaconate (DMI) for 48 h. (d) Levels of CoA species in Huh7 cells exposed to 5 mM itaconate for 48 h compared to 0 mM itaconate (dotted line). MeMal-CoA; Methylmalonyl-CoA. (e) Propionyl-CoA to acetyl-CoA metabolite ratio in Huh7 cells after 48 h. (f) Propionyl-carnitine to acetyl-carnitine metabolite ratio in Huh7 cells after 48 h. Data are depicted as box and whiskers (d–f), mean ± s.e.m. (a,b) or 95% confidence intervals from ISA model (c) obtained from 3 cellular replicates. Students t-test (a,b,d–f) with no adjustment for multiple comparisons * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001. Significance was considered as non-overlapping confidence intervals for (c).

Figure 6

Metabolic compensation induced by itaconate metabolism. Schematic depicts the impact of itaconate metabolism on glucose, glutamine, branched-chain amino acid (BCAA), CoA species, and fatty acid metabolism in mammalian cells.