Silva 2019 ESCI2019
|Silva FSG, Komlodi T, Garcia-Souza LF, Bento G, Doerrier C, Oliveira PJ, Gnaiger E (2019) Can fatty acid oxidation be specifically blocked by the CPT1 inhibitor etomoxir?|
Link: ESCI 2019
Event: ESCI 2019 Coimbra PT
Etomoxir is an irreversible inhibitor of the mitochondrial transport of long-chain fatty acids via blockage of carnitine palmitoyltransferase-I (CPT-1) . Therefore, etomoxir is widely used in commercial kits to inhibit mitochondrial fatty acid oxidation (FAO). However, several studies shed light on the unspecific effect of etomoxir on the mitochondria in respirometry protocols [2,3,4].
To assess the specificity of etomoxir on mitochondrial respiration, different concentrations of etomoxir were tested using high-resolution respirometry on permeabilized Huh7 human hepatocellular carcinoma cells and mitochondria isolated from mouse liver. Different sources of fatty acids (palmitoylcarnitine, palmityl-CoA + carnitine) were used to test the specific effect of etomoxir towards FAO (F-pathway), while NADH (N)- and succinate (S) linked substrates were applied to evaluate mitochondrial side-effects of etomoxir at different electron transfer (ET) pathway states. Substrate-uncoupler-inhibitor titration protocol was used to study oxidative phosphorylation (OXPHOS) and ET capacity.
Our results show that 40 µM etomoxir in the presence of palmitoylcarnitine (40 µM) and high concentration of malate displayed a trend of inhibition towards FN-linked pathway in the OXPHOS and ET states on Huh7 cells. While 200 µM etomoxir significantly inhibited FN-pathways in the ET and OXPHOS states in Huh7 cells and OXPHOS capacity in liver mitochondria. After 30 min of incubation, 200 µM etomoxir also blocked ET capacity with S-linked substrates. Finally, we investigated the specific effect of etomoxir at low concentrations (2.5 µM - 40 µM) on FAO applying different F-linked substrate combinations. Using palmitoylcarnitine and low malate concentration (0.1 mM malate which kinetically saturates the F-pathway but is sufficiently low to prevent stimulation of the anaplerotic pathways ), etomoxir did not influence FAO in the OXPHOS state on Huh7 cells, while on mitochondria respiring on palmitoyl-CoA (40 µM) + carnitine (0.5 mM) etomoxir (~20-40µM) displayed an inhibitory effect on FAO. Noteworthy, this etomoxir concentration which fully inhibited FAO, blocked already N- and S-linked respiration as well.
In conclusion, these results indicate an inhibitory effect of etomoxir on downstream of the Q-junction, which is reflected in the blockage of N- and S-linked respiration. However, FAO cannot be blocked fully by etomoxir without inhibiting other mitochondrial ET-pathway states on permeabilized Huh7 cells and isolated liver mitochondria. This observation raises precaution in its application as specific inhibitor of FAO in respirometry. Our study suggests a profound characterization of etomoxir toxicity.
Affiliations and Support
Silva FSG (1), Komlodi T (2), Garcia-Souza LF (2,3), Bento G (1), Doerrier C(2), Oliveira PJ(1), Gnaiger E(2,3)
- CNC - Center Neuroscience Cell Biology, Univ Coimbra, UC Biotech Building, Biocant Park, Cantanhede, Portugal
- Oroboros Instruments
- Dept Visceral, Thoracic Surgery, Medical Univ; Innsbruck, Austria
The mtFOIE GRAS project received funding from the European Union’s Horizon-2020 Research and Innovation program under the Marie Skłodowska-Curie Grant Agreement No.734719. Filomena Silva is recipient of a Post-Doctoral Fellowship from the Foundation for Science and Technology, SFRH/ BPD/122648/2016. Filomena Silva was participated in short-term scientific mission supported by MitoEAGLE (COST Action CA15203).
- Lopaschuk GD, Wall SR, Olley PM and Davies NJ (1988) Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcarnitine. Circ Res 63:1036-43.
- Raud B, Roy DG, Divakaruni AS et al (2018) Etomoxir actions on regulatory and memory T cells are independent of Cpt1a-mediated fatty acid oxidation. Cell Metab 28:504-15.
- Divakaruni AS, Hsieh WY, Minarrieta L et al (2018) Etomoxir inhibits macrophage polarization by disrupting CoA homeostasis. Cell Metab 28:490-503.e7.
- Yao CH, Liu GY, Wang R et al (2018) Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation. PLoS Biol 16:e2003782.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Meszaros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.
Labels: MiParea: Respiration, Pharmacology;toxicology
Organism: Human, Mouse Tissue;cell: Liver Preparation: Permeabilized cells, Isolated mitochondria
Regulation: Inhibitor Coupling state: OXPHOS, ET Pathway: F, N, S HRR: Oxygraph-2k