Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage
Abstract
Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by
immunoresponsive gene 1 (
Irg1), acting on the citric acid cycle intermediate
cis-aconitate. In mitochondria, itaconate can be converted by succinate–coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of
Irg1 in murine bone marrow–derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of
in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)—but not scrambled siRNA—treatment directed against
Irg1. LPS dose-dependently inhibited oxygen consumption rates (61–91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5–2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3–19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a “CoA trap” in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that
Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.—Németh, B., Doczi, J., Csete, D., Kacso, G., Ravasz, D., Adams, D., Kiss, G., Nagy, A. M., Horvath, G., Tretter, L., Mócsai, A., Csépányi-Kömi, R., Iordanov, I., Adam-Vizi, V., Chinopoulos, C. Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced
Irg1 expression in cells of murine macrophage lineage.
Itaconic acid (2-methylidenebutanedioic acid, methylenesuccinic acid; CAS registry number: 97-65-4) is an unsaturated dicarboxylic acid. It is produced in industrial scale from
cis-aconitate by the extramitochondrial
cis-aconitate decarboxylase, an enzyme encoded by the
cadA gene in
Aspergillus terreus (
1), and is used as a monomer for the production of a plethora of products including resins, plastics, paints, and synthetic fibers (
1–
3); it is reviewed elsewhere (
4). Itaconic acid has been identified in a small number of metabolomic studies of mammalian tissue specimens, such as activated macrophages (
5),
Mycobacterium tuberculosis–infected lung tissue (
6), urine and serum samples (
7), and glioblastomas (
8). More recently, it has been shown that human and mouse macrophages produce itaconic acid from
cis-aconitate through an enzyme exhibiting
cis-aconitate decarboxylase activity, coded by the
immunoresponsive gene 1 (
Irg1, NM_001258406.1 for mice) (
9). The latter finding confirmed earlier reports suggesting the presence of itaconate in macrophage-like tumor cell lines and primary murine macrophages in the low millimolar range (
10). The expression profile of
Irg1 is reviewed in (
4).
Irg1-mediated itaconate production contributes to the antimicrobial activity of macrophages by inhibiting isocitrate lyase, a key enzyme of the glyoxylate shunt (
11,
12). The glyoxylate shunt is not present in animals, but is essential for the survival of bacteria growing on fatty acids or acetate (
13).
Although the applications of itaconic acid and its derivatives extend to dental, ophthalmic, and drug delivery fields (
2), and in complexation with benzylammonium it is used to prepare water-soluble coating for food packaging to reduce bacterial contamination, it does not enter the food chain to an appreciable degree. Still, it was shown to be extensively metabolized when administered
per os to cats, dogs, and murine animals (
14,
15). Adler and colleagues reported more than 50 yr ago that exogenously added itaconate to isolated mitochondria is oxidized as most members of the citric acid cycle (
15). The same group elucidated the pathway of itaconate metabolism toward pyruvate and acetyl coenzyme A (CoA) (see below) (
16); however, at the time, the identity of succinate-CoA ligase (referred to as “succinate-activating enzyme” or “P enzyme”) and its role in substrate-level phosphorylation (SLP) was not yet revealed (
17–
19).
Mindful of the critical role of matrix SLP in the protection against cytosolic/nuclear ATP depletion during impaired respiration (
20–
24), we investigated the possibility that itaconate exerts an effect on adenine (or guanine) nucleotide production in the mitochondrial matrix
via succinate-CoA ligase. Our results show that itaconate abolishes SLP. As such, the mitochondrial ATP output of macrophage cells that endogenously generate itaconate when immunologically challenged, is expected to become affected, especially during concomitant respiratory chain impairment.
