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.

• MeSH
• Biguanides pharmacology   MeSH
• Phenformin pharmacology   MeSH
• Glycerolphosphate Dehydrogenase metabolism   MeSH
• Adipose Tissue, Brown cytology   MeSH
• Hypoglycemic Agents pharmacology   MeSH
• Rats MeSH
• Succinic Acid metabolism   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Metformin pharmacology   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Oxidation-Reduction drug effects   MeSH
• Hydrogen Peroxide pharmacology   MeSH
• Rats, Wistar MeSH
• Reactive Oxygen Species metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biguanides MeSH
• Phenformin MeSH
• Glycerolphosphate Dehydrogenase MeSH
• Hypoglycemic Agents MeSH
• Succinic Acid MeSH
• Metformin MeSH
• Hydrogen Peroxide MeSH
• Reactive Oxygen Species MeSH

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials physiology   MeSH
• Rhodotorula cytology   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ(m)) and the enzyme affinity to oxygen (p₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.

• MeSH
• Liver metabolism   MeSH
• Rats MeSH
• Potassium Cyanide pharmacology   MeSH
• Pyruvic Acid metabolism   MeSH
• Oxygen metabolism   MeSH
• Membrane Potential, Mitochondrial physiology   MeSH
• Mitochondria metabolism   MeSH
• Rats, Wistar MeSH
• Protons * MeSH
• Electron Transport Complex IV antagonists & inhibitors   metabolism   MeSH
• Oxygen Consumption physiology   MeSH
• Electron Transport physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium Cyanide MeSH
• Pyruvic Acid MeSH
• Oxygen MeSH
• Protons * MeSH
• Electron Transport Complex IV MeSH