Silva 2019 MiPschool Coimbra

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Filomena Pereira da Silva Grilo da Silva
Off-target effects of etomoxir on mitochondrial complex I.

Link: MitoEAGLE

Silva FSG, Komlodi T, Garcia-Souza LF, Bento G, Grilo L, Doerrier C, Oliveira PJ, Gnaiger E (2019)

Event: MiPschool Coimbra 2019

COST Action MitoEAGLE

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 O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E


Labels: MiParea: Respiration, Pharmacology;toxicology 


Organism: Human, Mouse  Tissue;cell: Liver  Preparation: Permeabilized cells, Isolated mitochondria  Enzyme: Complex I  Regulation: Inhibitor  Coupling state: OXPHOS, ET  Pathway: F, N, Other combinations  HRR: Oxygraph-2k 


Affiliations and support

Silva FSG(1), Komlodi T(2), Garcia-Souza LF(2,3), Bento G(1), Grilo (1), Doerrier (2), Oliveira PJ(1), Gnaiger E(2,3)
  1. CNC - Center Neuroscience Cell Biology, Univ Coimbra, UC Biotech Building, Biocant Park, Cantanhede, Portugal
  2. Oroboros Instruments, Innsbruck, Austria
  3. Department Visceral, Thoracic Surgery, Medical Univ Innsbruck, Austria. - fsgsilv@gmail.com
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).

Figures

SilvaFilomena Figure1.jpg
Figure 1: Effect of etomoxir on different mitochondrial pathway control states on permeabilized Huh7 human hepatocellular carcinoma cells. Effect of etomoxir was tested on the following mitochondrial pathways using high-resolution respirometry: 1) fatty acid oxidation (F-pathway) with 2 mM malate to support anaplerotic pathways, F(N); 2) NADH- (N) linked pathway in the presence of pyruvate, glutamate and 2 mM malate; 3) FN-pathway represents F-pathway in presence of pyruvate, glutamate and malate. Black dots represent the flux control ratios (FCR) of the control (untreated cells). Green dots show the FCR after 40 µM etomoxir treatment, while the red triangles demonstrate FCR in the presence of 200 µM etomoxir. (A) FCR in OXPHOS-state. (B) FCR in ET-state. The results are expressed as median and interquartile range of 4 independent experiments.


SilvaFilomena Figure2.jpg
Figure 2: Effect of etomoxir on complex I activity on isolated liver mitochondria from mouse. Complex I activity at different concentration of etomoxir (2-200 µM) is expressed in % respect to the control group. The results are expressed as median and interquartile range of 4 independent experiments.










References

  1. Clara R, Langhans W, Mansouri A (2016) Oleic acid stimulates glucagon-like peptide-1 release from enteroendocrine cells by modulating cell respiration and glycolysis. Metabolism 65:8-17.
  2. Kang HM, Ahn SH, Choi P, Ko YA, Han SH, Chinga F, Park AS, Tao J, Sharma K, Pullman J, Bottinger EP, Goldberg IJ, Susztak K (2015) Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development. Nat Med 21:37-46.
  3. Divakaruni AS, Hsieh WY, Minarrieta L, Duong TN, Kim KKO, Desousa BR, Andreyev AY, Bowman CE, Caradonna K, Dranka BP, Ferrick DA, Liesa M, Stiles L, Rogers GW, Braas D, Ciaraldi TP, Wolfgang MJ, Sparwasser T, Berod L, Bensinger SJ, Murphy AN (2018) Etomoxir inhibits macrophage polarization by disrupting CoA homeostasis. Cell Metab 28:490-503.
  4. Yao CH, Liu GY, Wang R, Moon SH, Gross RW, Patti GJ (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.