Pleiotropic Effects of Biguanides on Mitochondrial Reactive Oxygen Species Production

Abstract

Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10^-2-10^-1 M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain.

Figure 1

Characterization of brown adipose tissue (BAT) mitochondria. (a) SDS-PAGE and Western blot analysis of mitochondrial respiratory chain complexes in BAT and liver homogenates by polyclonal antibodies against mGPDH and VDAC and monoclonal antibodies against representative subunits of NDH (subunit NDUFA9), SDH (subunit SDHA), complex III (subunit UCQRC2), and actin. Two protein concentrations were loaded as indicated, representative image of three biological replicates. (b) Enzyme activities of mitochondrial dehydrogenases in mitochondria isolated from the BAT and liver. Activities were determined as rates of CoQ₁ reduction using respective substrates. Results are means ± SEM from three independent measurements. (c) Representative quality control curve of O₂ consumption and (d) representative trace of Δψ_m measurement in isolated BAT mitochondria. The following compounds were added: 10 mM glycerophosphate (GP), 0.3% BSA, 1 mM GDP, 1 mM ADP, 1 μM oligomycin (oligo), and 1 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

Figure 2

Respiratory chain dehydrogenase sensitivity to metformin titration. Specific enzyme activities (a, b, c) or oxygen consumption (d, e, f) of BAT mitochondria was titrated with 0–200 mM metformin using 100 μM NADH (a) or 10 mM pyruvate plus 2 mM malate (d), 10 mM succinate (b, e), and 25 mM (c) or 10 mM glycerophosphate (f). Individual points represent means ± SEM of at least three independent measurements.

Figure 3

Respiratory chain dehydrogenases sensitivity to phenformin titration. Specific enzyme activities (a, b, c) or oxygen consumption (d, e, f) of BAT mitochondria was titrated with 0–60 mM phenformin using 100 μM NADH (a) or 10 mM pyruvate plus 2 mM malate (d), 10 mM succinate (b, e), and 25 mM (c) or 10 mM glycerophosphate (f). Individual points represent means ± SEM of at least three independent measurements.

Figure 4

Comparison of iGP-1 effect on respiratory chain dehydrogenases mGPDH and SDH. Specific enzyme activities (a, b) or oxygen consumption (c, d) of BAT mitochondria was titrated with 0–100 μM iGP-1 using 25 mM (a) or 10 mM glycerophosphate (c) and 10 mM succinate (b, d). Individual points represent means ± SEM of at least three independent measurements.

Figure 5

Effect of biguanides on reactive oxygen species (ROS) production compared to typical inhibitors. The rate of H₂O₂ generation was estimated by Amplex red assay. H₂O₂ production with (a) 10 mM pyruvate and 2 mM malate (P + M) with or without 1 μM rotenone (ROT), (b) 10 mM succinate (saturating SUC) with or without 0.5 μM atpenin A5 (AA5) or 1 μM rotenone, (c) 0.4 mM succinate (physiological SUC) with or without 0.5 μM atpenin A5, and (d) 10 mM glycerophosphate (GP) with or without 100 μM iGP-1 (iGP) were indicated; BAT mitochondria were coupled with 1 mM guanosine diphosphate (GDP). (e) Titration of H₂O₂ production by biguanides metformin (MF, 0–200 mM) or (f) phenformin (PF, 0–60 mM) using the same substrate concentrations as in respective experiments in (a–d). Each point is the mean ± SEM of at least three independent measurements.

Figure 6

Proposed scheme of biguanide action. While at micromolar concentrations, biguanides confer antineoplastic action; at millimolar ones, they inhibit mitochondrial respiratory chain complexes and are toxic for all the cells.