Silva 2019 MiPschool Coimbra
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Link: MitoEAGLE
Silva FSG, Komlodi T, Garcia-Souza LF, Bento G, Grilo L, Doerrier C, Oliveira PJ, Gnaiger E (2019)
Event: MiPschool Coimbra 2019
Etomoxir is an irreversible inhibitor of the mitochondrial transport of long-chain fatty acids via blockage of carnitine palmitoyltransferase-I (CPT-1) leading to inhibition of the mitochondrial fatty acid oxidation (FAO). Owing to its inhibitory effect, etomoxir is widely used (40-400 μM) in commercial kits [1,2], however, several studies shed light on the unspecific effect of etomoxir on the mitochondria [3,4].
To assess the specificity of etomoxir on FAO in comparison to its inhibitory effect on mitochondrial respiration involving other mitochondrial pathways, we tested different concentrations of etomoxir (1-200 µM) on permeabilized Huh7 human hepatocellular carcinoma cells and isolated liver mitochondria from mouse, using high-resolution respirometry. Different sources of fatty acids (palmitoylcarnitine, palmityl-CoA + carnitine) were used to test the specific effect of etomoxir towards FAO (F-pathway). We have also investigated the off-target effect of etomoxir on mitochondrial respiration using NADH- (N) and succinate- (S) linked substrates alone and in combination with F-pathway (FN), or FAO in the presence of anaplerotic reactions [F(N)] in different electron transfer (ET) pathway states. Substrate-uncoupler-inhibitor titration protocol was used to study oxidative phosphorylation (OXPHOS) and ET capacities. The effect of etomoxir on complex I activity was also spectrophotometrically determined.
Our results show that 200 µM etomoxir significantly inhibited F(N)-, FN- and N-pathways in the ET and OXPHOS states on Huh7 cells (Figure 1) and OXPHOS capacity in isolated liver mitochondria in the presence of palmitoylcarnitine (40 µM) and 2 mM malate, by more than 40%. After 30 min of incubation, 200 µM etomoxir also blocked S-linked ET capacity by ~40%. Low concentrations of etomoxir (2.5 µM - 40 µM) did not influence F-pathway in the OXPHOS state using palmitoylcarnitine and 0.1 mM of malate, while etomoxir (~20-40 µM) displayed a ~20% inhibitory effect on FAO using palmitoyl-CoA (40 µM) + carnitine (0.5 mM) + 0.1 mM of malate in the OXPHOS state in Huh7 cells. Surprisingly, low concentration of etomoxir (~20-40 µM) exerted a ~17% and 13% inhibitory effect on N- and S-linked respiration respectively in the OXPHOS state on Huh cells. Similarly, an immediate impairment of N- and S-linked pathways after addition of 5 and 10 µM of etomoxir on isolated liver mitochondria was also observed in the OXPHOS state, without minor inhibitory effect on FAO. These results suggest that FAO cannot be selectively blocked by etomoxir without inhibiting other mitochondrial ET-pathway states on permeabilized Huh7 cells and isolated liver mitochondria. In agreement, etomoxir (40 and 200 µM) also inhibited the complex I activity of isolated liver mitochondria by ~40 and 50%, respectively (Figure 2).
In conclusion, these results indicate an inhibitory effect of etomoxir downstream of the Q-junction, with an inhibition on complex I. This observation raises precaution in its application as specific inhibitor of FAO in respirometry and suggests that a profound characterization of etomoxir toxicity is required.
• Bioblast editor: Plangger M
Labels: MiParea: Respiration, Pharmacology;toxicology
Enzyme: Complex I
HRR: Oxygraph-2k
Affiliations and support
Silva Filomena SG1, Komlodi Timea2, Garcia-Souza Luiz F 2,3, Bento Guida1, Grilo Luís1, Doerrier Carolina2, Oliveira Paulo J1, Gnaiger Erich2,3 1CNC - Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Biocant Park, Cantanhede, Portugal; 2Oroboros Instruments, Innsbruck, Austria; 3Department Visceral, Thoracic Surgery, Medical University of Innsbruck, Austria [email protected]
