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

Open AccessPublished:January 07, 2000DOI:https://doi.org/10.1074/jbc.275.1.223
      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 dimethylbiguanide-pretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complex I. The dimethylbiguanide effect was temperature-dependent, 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 Ca2+. 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.
      NO
      nitric oxide
      CCCP
      carbonyl cyanide p-trichloromethoxyphenylhydrazone
      TPMP+
      triphenylmethylphosphonium
      TMPD
      N,N,N′,N′-tetramethyl-1,4-phenylenediamine dihydrochloride
      l-NAME
      N ω-nitro-l-arginine methyl ester
      BAPTA-AM
      1,2-bis(aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
      MOPS
      4-morpholinepropanesulfonic acid
      Mitochondria are intracellular organelles devoted mainly to energy metabolism (ATP production) that also play a pivotal role in the onset of cell death (
      • Kroemer G.
      • Petit P.
      • Zamzami N.
      • Vayssiere J.L.
      • Mignotte B.
      ,
      • Green D.R.
      • Reed J.C.
      ). 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 Ca2+ and is related to the Ca2+-dependent mitochondrial dehydrogenases that regulate the supply of substrates to the respiratory chain (
      • McCormack J.G.
      • Halestrap A.P.
      • Denton R.M.
      ). It has been reported that lipopolysaccharideplus interferon-γ can persistently inhibit respiratory chain complex IV in intact astrocytes (
      • Bolanos J.P.
      • Peuchen S.
      • Heales S.J.
      • Land J.M.
      • Clark J.B.
      ) and that activation of glutamate receptors induces a persistent inhibition of complexes II, III, and IV in intact neurons (
      • Almeida A.
      • Heales S.J.R.
      • Bolanos J.P.
      • Medina J.M.
      ). 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 (
      • Clementi E.
      • Brown G.C.
      • Feelisch M.
      • Moncada S.
      ).
      Dimethylbiguanide (metformin) is an oral antihyperglycemic drug widely used in the treatment of type-II diabetes (
      • Bailey C.J.
      • Turner R.C.
      ,
      • Bailey C.J.
      ,
      Anonymous
      ,
      • Lee A.J.
      ), the action mechanism of which remains largely unknown (see Refs.
      • Argaud D.
      • Roth H.
      • Wiernsperger N.
      • Leverve X.M.
      and
      • Schafer G.
      for review). Dimethylbiguanide inhibits hepatic gluconeogenesis, possibly through a decrease in the cytosolic ATP/ADP ratio (
      • Schafer G.
      • Bojanowski D.
      ). Although it has been long known that biguanides inhibit respiration in intact cells, dimethylbiguanide is 10 times less potent than phenethylbiguanide (phenformin) and has no direct effect on isolated mitochondria (
      • Schafer G.
      ,
      • Schafer G.
      • Bojanowski D.
      ,
      • Schafer G.
      ,
      • Jalling O.
      • Olsen C.
      ). Therefore the mechanism by which high concentrations of dimethylbiguanide inhibit oxidative phosphorylation remained unclear.
      The present results indicate that dimethylbiguanide decreases oxygen consumption and mitochondrial membrane potential in intact hepatocytes, whereas it has no effect on isolated mitochondria or on permeabilized hepatocytes. Contrary to the previously identified long term mitochondrial regulators (
      • Bolanos J.P.
      • Peuchen S.
      • Heales S.J.
      • Land J.M.
      • Clark J.B.
      ,
      • Almeida A.
      • Heales S.J.R.
      • Bolanos J.P.
      • Medina J.M.
      ,
      • Clementi E.
      • Brown G.C.
      • Feelisch M.
      • Moncada S.
      ,
      • Bolanos J.P.
      • Almeida A.
      • Stewart V.
      • S. P.
      • Land J.M.
      • Clark J.B.
      • Heales S.J.R.
      ), the mitochondrial inhibitory effect of dimethylbiguanide is purely located on the respiratory chain complex I and does not affect the oxidative phosphorylation machinery downstream complex I. This effect is not affected by a variety of cell signaling inhibitors but is completely prevented when cells are incubated at 15 °C. Because dimethylbiguanide is not metabolized (
      • Pentikainen P.J.
      • Neuvonen P.J.
      • Penttila A.
      ), these results suggest the existence of a new cell signaling pathway targeted to the respiratory chain complex I.

      MATERIALS AND METHODS

      Hepatocytes were isolated according to the method of Berry and Friend (
      • Berry M.N.
      • Friend D.S.
      ) as modified by Groen et al. (
      • Groen A.K.
      • Sips H.J.
      • Vervoorn R.C.
      • Tager J.M.
      ). Hepatocytes (final concentration 10 mg dry cells/ml) were incubated in closed vials at 37 °C in a shaking water bath (60 strokes/min) in 2.5 ml of Krebs-bicarbonate buffer (120 mm NaCl, 4.8 mmKCl, 1.2 mm KH2PO4, 1.2 mm MgSO4, 24 mm NaHCO3, 1.3 mm CaCl2, pH 7.4) saturated with a mixture of O2/CO2 (19:1 by volume). For some experiments incubations were performed simultaneously at 37, 25, and 15 °C.
      Isolated hepatocytes were permeabilized using digitonin (6 μg/mg dry cells, 2 min, at room temperature) as described in Ref.
      • Fontaine E.M.
      • Keriel C.
      • Lantuejoul S.
      • Rigoulet M.
      • Leverve X.M.
      • Saks V.A.
      . Cell membrane permeabilization was always evidenced by the lack of trypan blue exclusion.
      Two different approaches were used to study the effect of dimethylbiguanide on liver mitochondria. In one case, mitochondria were prepared from rat liver previously perfused for 30 min with Krebs-Bicarbonate medium containing or not 10 mmdimethylbiguanide. In the second case pharmacological (sublethal) dose of dimethylbiguanide (60 mg/100 g of body weight) dissolved in saline solution buffer (NaCl, 0.9%) or saline solution alone were injected subcutaneously 30 min before sacrifice. When using the latter method, plasma concentration of dimethylbiguanide at the sacrifice was determined by high performance liquid chromatography (3.6 ± 0.3 mm, n = 4). After liver perfusion orin vivo subcutaneous injection, liver mitochondria were then prepared according to Klingenberg and Slencza (
      • Klingenberg M.
      • Slenczka W.
      ) and resuspended in a medium containing 250 mm sucrose, 1 mm EGTA, 20 mm Tris-HCl, pH 7.2.
      Water-soluble fraction and nonpolar soluble fraction of livers were obtained as follows: rat livers were first isolated and perfused during 30 min at 37 °C with Krebs-bicarbonate buffer saturated with a mixture of O2/CO2 (19:1 by volume) supplemented or not with 10 mm dimethylbiguanide. Livers were then homogenized in a small volume of Krebs-bicarbonate buffer and centrifuged at 2000 g for 10 min then supernatant and pellet were separated. The supernatant was the soluble fraction, and the pellet lipids were extracted with chloroform/methanol (2:1) as described elsewhere (
      • Shoukry M.I.
      ,
      • Kiselev G.V.
      • Pavlinova L.I.
      ) and evaporated under nitrogen stream. Final liver nonpolar soluble fraction was resuspended in Me2SO and kept in the dark at −80 °C until used.
      Oxygen consumption rate was measured polarographically in a stirred oxygraph vessel thermostated at 37 °C and equipped with a Clark oxygen electrode. For the study of the temperature effect, we performed prolonged incubations of up to 3 h at 15, 25, or 37 °C in the medium described above supplemented with 20 mmdihydroxyacetone and 4 mm octanoate in the presence or not of 10 mm dimethylbiguanide. After 0, 30, 60, 120, and 180 min of incubation, 1 ml of the suspension was removed from the vial and placed in the oxygraph vessel containing 1 ml of the same medium saturated with O2/CO2 gas mixture and thermostated at 37 °C. This procedure permitted to adjust precisely the temperature of the suspension to 37 °C in less than 2 min before recording oxygen consumption rate.
      Measurement of mitochondrial membrane potential in intact cells was performed as described in Ref.
      • Espie P.
      • Guerin B.
      • Rigoulet M.
      . Briefly, after determination of mitochondrial and cellular volumes using 3H2O plus [14C]mannitol and 3H2O plus [14C]carboxymethyl-inulin respectively, mitochondrial and plasmic membrane potentials were determined by measuring accumulation of [3H]TPMP+ and36Cl, respectively.
      Lactate, pyruvate, 3-hydroxybutyrate, and acetoacetate were measured enzymatically as described in Ref.
      • Bergmeyer H.
      on sample of cell suspension previously quenched in HClO4 (4% mass/volume final concentration) and neutralized with KOH (2 m)/MOPS (0.3m). Intramitochondrial and cytosolic NADH/NAD+ratios were calculated assuming thermodynamic equilibrium with 3-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, respectively (
      • Williamson D.H.
      • Lund P.
      • Krebs H.A.
      ).
      ATP and ADP were measured by chemoluminescence on mitochondrial and cytosolic spaces previously separated using the digitonin fractionation method (
      • Pison C.M.
      • Chauvin C.
      • Fontaine E.
      • Catelloni F.
      • Keriel C.
      • Paramelle B.
      • Leverve X.M.
      ,
      • Leverve X.M.
      • Fontaine E.
      • Putod Paramelle F.
      • Rigoulet M.
      ).
      For complex I assay, mitochondria (0.5 mg/ml) were incubated in a 1 mm EGTA, 20 mm Tris-HCl, pH 7.2, solution (to break inner membrane by hypoosmotic shock) in the presence of 500 μm NADH and 5 mm KCN. Complex I activity was assessed by the oxidation rate of NADH (measuring absorbance at 340 nm in a Uvikon-Kontron 941-plus spectrophotometer equipped with thermostatic control and magnetic stirring) after addition of 100 μm decylubiquinone as electron acceptor.
      Complex III plus IV activity was assessed by measuring oxygen consumption with decylubiquinol (300 μm) as the respiratory substrate in presence of rotenone (1.25 μm).
      Pyruvate, digitonin, and ADP were purchased from Roche Molecular Biochemicals; phospholipase C inhibitor U73122 from Calbiochem; octanoate from Janssen; collagenase type IV, lactate, myxothiazol, CCCP, TMPD, rotenone, wortmannin, LY294002, PD98059,l-NAME, l-arginine, decylubiquinone, BAPTA-AM, EGTA, fumosine B1, l-cycloserine, β-dl-alanine, and all other reagents from Sigma-Aldrich. Dimethylbiguanide was a gift from Merck-Lipha Co. Decylubiquinol was prepared as described in Ref.
      • Rieske J.S.
      by chemical reduction of decylubiquinone with sodium borohydride.
      Results are expressed either as typical experiment or as indicated as mean ± S.E. of the number of incubation from at least three rats. Statistical analyses were made using analysis of variance followed by Fisher's protected least significant difference post hoc test (Stat View®, Abacus concepts, Inc., Berkley, CA, 1992).

      DISCUSSION

      In this study we have shown that dimethylbiguanide specifically inhibits respiratory chain complex I through an indirect mechanism that (i) does not operate through traditional cell signaling pathways but (ii) requires cells to be intact to be initiated and (iii) persists after removal of the drug or after isolation of the mitochondria. The amplitude, but not the kinetics, of this effect is temperature-dependent. Although the chain of cellular reactions triggering such a mitochondrial effect has not been identified and its physiological role remains unknown, we conclude that hepatocytes have a signaling pathway targeted to the respiratory chain complex I.

      Dimethylbiguanide Indirectly Affects Respiratory Chain Complex I

      Electron transfer through complex I can be modulated by numerous substances including poisons such as rotenone and physiological molecules such as NO (
      • Clementi E.
      • Brown G.C.
      • Feelisch M.
      • Moncada S.
      ,
      • Degli Esposti M.
      ). Although the molecular mechanisms by which such compounds inhibit complex I are not totally resolved, a direct interaction between the inhibitors and the enzymatic complex is essential. The novelty of the results presented in this work is based on the finding that the dimethylbiguanide-induced complex I inhibition is not a consequence of a direct interaction with the respiratory chain, because dimethylbiguanide, which is known for being not metabolized, has no effect on isolated mitochondria.
      Among the cell signaling pathways capable of mitochondrial regulation, we have clearly shown that neither the NO pathway nor Ca2+homeostasis are involved in the dimethylbiguanide-induced respiratory inhibition. The absence of a link between the dimethylbiguanide effect and NO pathway is not surprising considering that the NO effect seems to be related to a decrease in glutathione level, whereas dimethylbiguanide is known to increase the liver glutathione content (
      • Ewis S.A.
      • Abdel-Rahman M.S.
      ,
      • Ewis S.A.
      • Abdel-Rahman M.S.
      ).
      The finding that oxygen radical scavengers do not neutralize the dimethylbiguanide-induced effect suggests that oxygen radicals are not involved in this process. However, because complex I can be both a source and a target of oxygen radicals, this hypothesis cannot be definitively ruled out.
      Dimethylbiguanide signaling effect could operate via ceramide formation, because it has been reported recently to inhibit respiration. But ceramide affects complex III conversely to the highly specific effect toward complex I reported here. Moreover the lack of effect of ceramide synthesis inhibitors (Fumosine B1,l-cycloserine, and β-dl-alanine) does not support this hypothesis.

      Mechanism of Mitochondrial Regulation by Dimethylbiguanide

      The finding that hydrophylic or lipophylic extracts of dimethylbiguanide-treated livers do not affect mitochondria strongly suggests that dimethylbiguanide does not simply induce an accumulation of stable natural compounds or putative metabolites. This conclusion associated with the compelling evidence that dimethylbiguanide inhibits respiration in intact cells lead us to propose that dimethylbiguanide acts via a complex signaling pathway, the first step of which may be an interaction between the drug and a membrane receptor. This hypothesis is supported by the observed logarithmic dose-dependent effect of dimethylbiguanide on cellular respiration (Fig. 1, panel B) such as seen for hormone-receptor interaction. Although, the temperature-dependent nature of this inhibition is difficult to explain, it could suggest an effect related to the physicochemical state of the plasmic membrane. Indeed, the temperature dependence of an enzymatic reaction is generally expected to influence the kinetics but not the final amplitude of the reaction. Considering the rapid onset (5 min) and the short time to maximal effect (20–30 min), the data are more consistent with a phosphorylation-dephosphorylation or a protein degradation mechanism.

      ACKNOWLEDGEMENTS

      We express our gratitude to Drs. Nicolas Wiernsperger, Gilles Mithieux, and Juan P. Bolanos for their helpful discussions and also thank Hélène Perrault for revision of English text of the manuscript.

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