Dimethyl Itaconate Is Not Metabolized into Itaconate Intracellularly

Abstract

Edited by Luke O'NeillItaconic acid is an important metabolite produced by macrophages after stimulation with LPS. the cell culture medium leads to elevated intracellular levels of unlabeled succinate, with no evidence of intracellular uptake. The goal of this study is to encourage the development of effective pro-drug strategies to increase the intracellular levels of itaconate, which will enable more conclusive analysis of its action on macrophages and other cell and tissue types.Itaconic acid is a dicarboxylic acid polar metabolite originally characterized in Aspergillus terreus, but is also produced in mammalian cells (1). After LPS stimulation, itaconic acid is secreted by macrophages, where it can inhibit bacterial cell growth (1). In macrophages, itaconate synthesis is catalyzed by the immune-responsive gene 1 (IRG1) protein, which mediates the decarboxylation of cis-aconitate to itaconate (2). Metabolomic and fluxomic analysis of LPS-stimulated macrophages demonstrated reduced Isocitrate dehydrogenase-1 (IDH1) expression and increased IRG1 expression, resulting in a diversion of citrate from the TCA cycle 2 toward itaconate production (3). This metabolic remodeling results in glutamate serving as the anaplerotic substrate to maintain or elevate succinate levels (3). Moreover, elevated succinate acts as an inflammatory signal that induces secretion of IL-1␤ and stabilization of HIF-1␣ (4). Based on the similarity between itaconate (methylene succinic acid) and succinate, recent investigations have focused on the link between itaconate synthesis and succinate accumulation. Two complementary studies reported that itaconate inhibits succinate dehydrogenase and drives succinate accumulation (5, 6). Studying the role of itaconate requires either lowering its intracellular concentration by using IRG1 knock-out mouse models (5, 6) or increasing its intracellular concentration by using either itaconate (5) or a "cell-permeable" analog, dimethyl itaconate (DMI) (6). However, there is no direct evidence that itaconate or DMI can cross cell membranes and increase intracellular itaconate. Without direct evidence of intracellular delivery, it remains unclear whether itaconate-mediated metabolic and inflammatory effects are induced by increasing intracellular itaconate or by an extracellular mechanism.Here we synthesized isotopically labeled [ 13 C]itaconate and dimethyl [13 C]itaconate ([ 13 C]DMI) to directly profile itaconate metabolism and uptake (Fig. 1). This analysis suggests that exogenous itaconate is not taken up into cells and [13 C]DMI is not metabolized into [13 C]itaconate in bone marrow-derived macrophages. We also report that [13 C]itaconate in the cell culture medium leads to elevated intracellular levels of unlabeled succinate, yet there is no evidence of intracellular uptake. Overall, this study highlights current limitations in intracellular itaconate delivery, and emphasizes the development of effective pro-drug strategies to conclusively define its action on macrophages and other cell and tissue t...

Itaconate: an emerging determinant of inflammation in activated macrophages

“…It is known that cytoplasmic esterases catalyze OI to produce itaconate. However, whether exogenous DI can be directly metabolized into itaconate in cells is still controversial . This highlights current limitations in the intracellular delivery of itaconate and emphasizes the development of effective prodrug strategies to precisely define its anti‐inflammatory function.…”

“…LPS‐tolerized wild‐type BMDMs that can produce substantial amounts of itaconate compared with IRG1‐deficient cells display a significant decrease in IL‐6 levels, suggesting that endogenous itaconate acts in a manner consistent with exogenous DI . However, a recent study showed that DI cannot be directly converted to itaconate, instead it promotes itaconate biosynthesis in LPS‐stimulated BMDMs . It is believed that DI is unlikely to mimic endogenous itaconate.…”

“…7 However, a recent study showed that DI cannot be directly converted to itaconate, instead it promotes itaconate biosynthesis in LPS-stimulated BMDMs. 39 It is believed that DI is unlikely to mimic endogenous itaconate. Thus, additional research needs to determine whether DI is a true itaconate donor.…”

Regulation of leukocyte function by citric acid cycle intermediates

“…Furthermore, the exact mechanism of action of exogenously administered itaconate analogs, DMI and OI, is not clearly understood. It is postulated that these molecules might bind to the SUCNR1 receptor or other unknown cell surface receptor, leading to the observed effects …”

“…Furthermore, a study by El Azzouny et al. showed that cell‐permeable DMI is not metabolized to itaconate by mouse bone marrow‐derived macrophages and treatment with 13 C labelled DMI elevated intracellular succinate only, with no evidence of intracellular itaconate uptake . Therefore, suitable molecules aimed at modulating intracellular itaconate levels remain to be developed.…”