Etomoxir Inhibits Macrophage Polarization by Disrupting CoA Homeostasis

Abstract

Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.

Fatty acid metabolism in CD8+ T cell memory: Challenging current concepts

“…Moreover, careful examination of the effect of etomoxir on T cell metabolism has suggested that, at high concentrations, this compound has multiple off-target effects on mitochondrial metabolism and other cellular processes, which may vary depending on the cellular context and requirements for activation, influencing the differentiation of not only T cells, but also other immune subsets such as macrophages (143, 146). …”

“…With a half-maximal inhibitory concentration in the nanomolar range (IC 50 = 10–700 nM), etomoxir is a potent inhibitor of CPT1 (140–143). Nonetheless, there are important caveats to consider when using this compound.…”

“…To identify off-target effects of etomoxir in cell differentiation, a recent study by Divakaruni and colleagues used permeabilized cells to measure the effect of etomoxir on substrate specific oxygen consumption, and thus better characterize its influence on mitochondrial metabolism (143). In accordance with previous findings (140, 141, 147), the authors reported that etomoxir is a very efficient inhibitor of CPT1 activity in multiple tested cell lines, primary bone marrow-derived macrophages (BMDM) and T cells, even at concentrations below 1 μM (25, 143).…”

“…During OxPhos, inhibition of the ANT prevents ADP import and ATP export from the mitochondrial matrix, with the consequent inhibition of ATP synthase activity. This in turn slows the rate of ETC activity and oxygen consumption regardless of the oxidizable substrate (25, 143) (Figure 2). The secondary inhibitory effect on complex I slows oxidation of all substrates providing NADH to the ETC.…”

Plasma cells: You are what you eat

“…To highlight these issues, hematopoietic stem cells were thought to possess very little mitochondrial mass based on mitochondrial dye stains. 166 Furthermore, genetic ablation of CPT2, which carries long-chain fatty acids into the inner matrix of the mitochondria, was unable to recapitulate findings obtained on etomoxir treatment of cells, implying therefore an inconsistency between pharmacological and genetic data. 163 As another example of how drug manipulations should be treated with caution, we performed pharmacological experiments with plasma cell subsets and etomoxir, an inhibitor of fatty acid oxidation, to define the carbon sources that drive basal respiration.…”