Dimethylbiguanide Inhibits Cell Respiration via an Indirect Effect Targeted on the Respiratory Chain Complex I

Abstract

We report here a new mitochondrial regulation occurring only in intact cells. We have investigated the effects of dimethylbiguanide on isolated rat hepatocytes, permeabilized hepatocytes, and isolated liver mitochondria. Addition of dimethylbiguanide decreased oxygen consumption and mitochondrial membrane potential only in intact cells but not in permeabilized hepatocytes or isolated mitochondria. Permeabilized hepatocytes after dimethylbiguanide exposure and mitochondria isolated from dimethylbiguanide pretreated livers or animals were characterized by a significant inhibition of oxygen consumption with complex I substrates (glutamate and malate) but not with complex II (succinate) or complex IV (N,N,N,N-tetramethyl-1,4-phenylenediamine dihydrochloride (TMPD)/ascorbate) substrates. Studies using functionally isolated complex I obtained from mitochondria isolated from dimethylbiguanidepretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complexI.Thedimethylbiguanideeffectwastemperaturedependent, oxygen consumption decreasing by 50, 20, and 0% at 37, 25, and 15°C, respectively. This effect was not affected by insulin-signaling pathway inhibitors, nitric oxide precursor or inhibitors, oxygen radical scavengers, ceramide synthesis inhibitors, or chelation of intra-or extracellular Ca 2؉ . Because it is established that dimethylbiguanide is not metabolized, these results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.Mitochondria are intracellular organelles devoted mainly to energy metabolism (ATP production) that also play a pivotal role in the onset of cell death (1, 2). The regulation of such functions is essential and has been well characterized in isolated mitochondria, whereas much less is known in intact cells. Short term regulation of intact cell respiration has been established with Ca 2ϩ and is related to the Ca 2ϩ -dependent mitochondrial dehydrogenases that regulate the supply of substrates to the respiratory chain (3). It has been reported that lipopolysaccharide plus interferon-␥ can persistently inhibit respiratory chain complex IV in intact astrocytes (4) and that activation of glutamate receptors induces a persistent inhibition of complexes II, III, and IV in intact neurons (5). Both inhibitions can be prevented by nitric-oxide synthase inhibitors. Furthermore, it has been shown that prolonged direct exposure to nitric oxide (NO) 1 in intact J774 cells leads to a persistent inhibition of respiratory chain complex I, whereas inhibition of complex IV was reversible (6).Dimethylbiguanide (metformin) is an oral antihyperglycemic drug widely used in the treatment of type-II diabetes (7-10), the action mechanism of which remains largely unknown (see Refs. 11 and 12 for review). Dimethylbiguanide inhibits hepatic gluconeogenesis, possibly through a decrease in the cytosolic ATP/ADP ratio (13). Although it has been long known that big...

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