Distinct Post-Exposure Metabolic States Following Pharmacological Perturbation of Mitochondrial Respiratory Complexes I-V in SH-SY5Y Cells: A Positional 13C-NMR Study

Abstract

Mitochondrial dysfunction is a recurring mechanism in neuronal vulnerability and in the toxicity of several neuroactive compounds, yet lesions at different respiratory-chain nodes are commonly treated as a single bioenergetic defect. Whether pharmacological perturbation at distinct nodes leaves neuronal cells in distinguishable metabolic states has not been examined across all five complexes in one system. Undifferentiated SH-SY5Y human neuroblastoma cells were exposed to five compounds selected as pharmacological perturbagens nominally directed at Complexes I-V: MPTP, 3-nitropropionic acid (3-NP), antimycin A, sodium azide and oligomycin, each titrated to approximately 80% viability (24 h, except 3-NP at 12 h). Inhibitors were then removed, and cells were labelled for 24 h with [U-C-13(6)]glucose. Positional C-13-NMR was combined with pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) enzyme-capacity assays in parallel extracts from the same biological replicates (n = 3 per group). The design is a single-factor, six-level layout in which five exposures share one vehicle control; labelling data were log2-transformed and analysed by one-way ANOVA with Dunnett many-to-one contrasts (error df = 12), with Hedges' g and 95% confidence intervals, and group structure was tested by permutational multivariate analysis of variance (PERMANOVA). PERMANOVA demonstrated a significant overall effect of exposure group on the multivariate labelling matrix (pseudo-F = 18.77, p = 0.0001, 9999 permutations), and principal component analysis of the seven complete-case metabolites showed visually distinct group structure (PC1 65.9%, PC2 19.3%; 85.3% cumulative). Oligomycin produced the most distinct positional phenotype, preserving the PDH-associated glutamate C4 readout (107.2% of control, p = 0.971) while nearly doubling the PC-associated glutamate C3 readout (197.7%, p = 0.004; Hedges' g = 17.8, 95% CI 5.3-30.2), concordant with PC capacity at approximately 138% and PDH capacity at approximately 60% of control. Azide produced the strongest reduction in glutamate C4 labelling (31.9%, p < 0.001; g = -8.3); antimycin A produced the broadest suppression of labelling; 3-NP produced accumulation of C-13-labelled succinate that was undetectable in the control. MPTP gave the weakest phenotype, with most of its individual comparisons not surviving the shared-control analysis. Exposure to compounds directed at different respiratory nodes leaves neuronal cells in non-equivalent post-exposure C-13-labelling states that positional C-13-NMR resolves. Because tracing followed washout, because the compounds are not fully selective and because the enzyme assays report capacity rather than flux, these are compound-defined post-exposure metabolic states and not measurements of complex-specific flux.