Multiple metabolic changes mediate the response of Caenorhabditis elegans to the complex I inhibitor rotenone

Abstract

Rotenone, a mitochondrial complex I inhibitor, has been widely used to study the effects of mitochondrial dysfunction on dopaminergic neurons in the context of Parkinson's disease. Although the deleterious effects of rotenone are well documented, we found that young adult Caenorhabditis elegans showed resistance to 24 and 48 h rotenone exposures. To better understand the response to rotenone in C. elegans, we evaluated mitochondrial bioenergetic parameters after 24 and 48 h exposures to 1 μM or 5 μM rotenone. Results suggested upregulation of mitochondrial complexes II and V following rotenone exposure, without major changes in oxygen consumption or steady-state ATP levels after rotenone treatment at the tested concentrations. We found evidence that the glyoxylate pathway (an alternate pathway not present in higher metazoans) was induced by rotenone exposure; gene expression measurements showed increases in mRNA levels for two complex II subunits and for isocitrate lyase, the key glyoxylate pathway enzyme. Targeted metabolomics analyses showed alterations in the levels of organic acids, amino acids, and acylcarnitines, consistent with the metabolic restructuring of cellular bioenergetic pathways including activation of complex II, the glyoxylate pathway, glycolysis, and fatty acid oxidation. This expanded understanding of how C. elegans responds metabolically to complex I inhibition via multiple bioenergetic adaptations, including the glyoxylate pathway, will be useful in interrogating the effects of mitochondrial and bioenergetic stressors and toxicants.

Keywords: Caenorhabditis elegans (C. elegans); Complex I; Glyoxylate; Metabolomics; Mitochondrial metabolism; Rotenone.

Figure 1:. Contribution of specific ETC complexes and metabolic pathways to ATP levels in rotenone-exposed PE255 C. elegans.

ATP decreases after exposure to malonate (B), DCCD (E), and FCCP (F) suggest increased reliance on Complex II and ATP synthase, as well as increased sensitivity to uncoupling-mediated ATP depletion, in rotenone-exposed nematodes. For malonate, one-way ANOVA p=0.0419*, Dunnett’s test vs control for 1 μM p=0.0279*. For DCCD, one-way ANOVA p=0.0218*, Dunnett’s test vs control for 0.25 μM p=0.0300*, for 1 μM p=0.0219*. For FCCP, one-way ANOVA p=0.0423*, Dunnett’s test vs control for 1 μM p=0.0261*. Bars ± SEM of three to six independent experiments.

Figure 2:. Rotenone caused a statistically significant increase in proton leak, but not basal respiration, ATP-linked respiration or spare capacity.

For proton leak, one-way ANOVA p<.0001*, Tukey-Kramer HSD vs control: p=0.0107* for 0.25 μM, p<.0001* for 5 μM). Bars ± SEM of duplicate independent experiments.

Figure 3:. Rotenone exposure induced isocitrate lyase (icl-1) and both complex two subunits (mev-1 and sdha-1).

Two-tailed t-test p-value: icl-1 p=0.0041*, mev-1 0.0201*, sdha-1 0.0097*. Bars ± SEM of triplicate independent experiments.

Figure 4:. The glyoxylate mutant strain icl-1 did not exhibit additional sensitivity to rotenone exposure in growth assays.

Bars ± SEM of 2-3 independent experiments.

Figure 5.. Metabolomics analysis of amino acid levels in C. elegans after a 48h rotenone exposure.

For alanine levels after the 5 μM rotenone exposure: one-way ANOVA p=0.0001*, Tukey-Kramer HSD vs control p=0.0001* for 5 μM. Bars ± SEM of three independent experimental replicates, with 2-3 technical replicates per experiment.

Figure 6.. Metabolomics analysis of organic acid levels in C. elegans after a 48h rotenone exposure.

For malate levels after the 5 μM exposure: one-way ANOVA p=0.0165*, Tukey-Kramer HSD vs control p=0.0140*. For lactate levels after the 5 μM exposure: one-way ANOVA p=0.0008*, Tukey-Kramer HSD vs control p=0.0011*. For pyruvate levels after the 5 μM exposure: one-way ANOVA p<.0001*, Tukey-Kramer HSD vs control p<.0001*. Bars ± SEM of three independent experimental replicates, with 2-3 technical replicates per experiment.

Figure 7:. Metabolomics analysis of acylcarnitine levels in C. elegans after a 48h rotenone exposure.

A, short chain acylcarnititnes. B, medium chain acylcarnititnes. C, long chain acylcarnititnes. Bars ± SEM of three independent experimental replicates, with 2-3 technical replicates per experiment. P-values for statistically significant changes are listed in Supplemental Table 1.