Metformin induces lactate production in peripheral blood mononuclear cells and platelets through specific mitochondrial complex I inhibition.

AIM: Metformin is a widely used antidiabetic drug associated with the rare side effect of lactic acidosis which has been proposed to be linked to drug-induced mitochondrial dysfunction. Using respirometry, the aim of this study was to evaluate mitochondrial toxicity of metformin to human blood cells in relation to that of phenformin, a biguanide analogue withdrawn in most countries due to a high incidence of lactic acidosis. METHODS: Peripheral blood mononuclear cells and platelets were isolated from healthy volunteers, and integrated mitochondrial function was studied in permeabilized and intact cells using high-resolution respirometry. A wide concentration range of metformin (0.1-100 mm) and phenformin (25-500 µm) was investigated for dose- and time-dependent effects on respiratory capacities, lactate production and pH. RESULTS: Metformin induced respiratory inhibition at complex I in peripheral blood mononuclear cells and platelets (IC50 0.45 mm and 1.2 mm respectively). Phenformin was about 20-fold more potent in complex I inhibition of platelets than metformin. Metformin further demonstrated a dose- and time-dependent respiratory inhibition and augmented lactate release at a concentration of 1 mm and higher. CONCLUSION: Respirometry of human peripheral blood cells readily detected respiratory inhibition by metformin and phenformin specific to complex I, providing a suitable model for probing drug toxicity. Lactate production was increased at concentrations relevant for clinical metformin intoxication, indicating mitochondrial inhibition as a direct causative pathophysiological mechanism. Relative to clinical dosing, phenformin displayed a more potent respiratory inhibition than metformin, possibly explaining the higher incidence of lactic acidosis in phenformin-treated patients.

Binding mitochondria to cryogel monoliths allows detection of proteins specifically released following permeability transition.

Following proapoptotic signals such as calcium-induced mitochondrial permeability transition or translocation of proapoptotic proteins, mitochondria induce cell death through release of apoptogenic proteins. The mechanism of release and the identity of the released proteins are currently debated. Earlier attempts at identification of the apoptogenic proteins have been hampered by a high nonspecific background. Our aim was to develop a novel method where background release was eliminated, allowing proteins specifically released from mitochondria following proapoptotic stimulation to be identified. Liver mitochondria were immobilized and washed on cryogel monoliths prior to induction of protein release (calcium or Bid/Bax). Immobilized mitochondria exhibited normal morphology and swelling response and retained respiratory activity. The released proteins were collected, concentrated, separated on polyacrylamide gels which were cut into pieces, trypsin-digested, and analyzed using liquid chromatography-tandem mass spectrometry. Control samples contained no protein, and stimulation with calcium and Bid/Bax resulted in identification of 68 and 82 proteins, respectively. We conclude that, in combination with the robust proteomic approach, immobilization on cryogel monoliths is a fruitful approach for studying specific protein release from isolated mitochondria. We propose that this method is a powerful tool to further characterize the role of mitochondria in cell death induction.