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.
Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1
α-subunit and a substrate-specific Sucla2 or Suclg2 β-subunit
yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme
leads to encephalomyopathy with or without methylmalonyl aciduria, in addition
to resulting in mitochondrial DNA depletion. We generated mice lacking either
one Sucla2 or Suclg2 allele.
Sucla2 heterozygote mice exhibited tissue- and
age-dependent decreases in Sucla2 expression associated with decreases in
ATP-forming activity, but rebound increases in cardiac Suclg2 expression and
GTP-forming activity. Bioenergetic parameters including substrate-level
phosphorylation (SLP) were not different between wild-type and
Sucla2 heterozygote mice unless a submaximal
pharmacological inhibition of SUCL was concomitantly present. mtDNA contents
were moderately decreased, but blood carnitine esters were significantly
elevated. Suclg2 heterozygote mice exhibited decreases in
Suclg2 expression but no rebound increases in Sucla2 expression or changes in
bioenergetic parameters. Surprisingly, deletion of one Suclg2
allele in Sucla2 heterozygote mice still led to a rebound but
protracted increase in Suclg2 expression, yielding double heterozygote mice with
no alterations in GTP-forming activity or SLP, but more pronounced changes in
mtDNA content and blood carnitine esters, and an increase in succinate
dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits
rebound increases in Suclg2 expression, which is sufficiently dominant to
overcome even a concomitant deletion of one Suclg2 allele, pleiotropically
affecting metabolic pathways associated with SUCL. These results as well as the
availability of the transgenic mouse colonies will be of value in understanding
SUCL deficiency.
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