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J Biol Chem, Vol. 275, Issue 1, 223-228, January 7, 2000
From the Laboratoire de Bioénergétique Fondamentale et
Appliquée, Université Joseph Fourier, F-38041
Grenoble-Cedex 09, France and § the Institut de Biochimie et
de Génétique Cellulaires du CNRS, Université de
Bordeaux II, F-33077 Bordeaux-Cedex, France
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.
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 Ca2+ and is related
to the Ca2+-dependent mitochondrial
dehydrogenases that regulate the supply of substrates to the
respiratory chain (3). It has been reported that lipopolysaccharide
plus interferon- 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 biguanides inhibit respiration in intact cells,
dimethylbiguanide is 10 times less potent than phenethylbiguanide
(phenformin) and has no direct effect on isolated mitochondria
(12-15). 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 (4-6, 16), 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
(17), these results suggest the existence of a new cell signaling
pathway targeted to the respiratory chain complex I.
Hepatocytes were isolated according to the method of Berry and
Friend (18) as modified by Groen et al. (19). 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 mM
KCl, 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. 20. 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 mM
dimethylbiguanide. 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 or
in vivo subcutaneous injection, liver mitochondria were then
prepared according to Klingenberg and Slencza (21) 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 (22, 23) and evaporated under nitrogen stream. Final liver nonpolar soluble fraction was resuspended in Me2SO and kept
in the dark at 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 mM
dihydroxyacetone 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. 24. 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+ and
36Cl Lactate, pyruvate, 3-hydroxybutyrate, and acetoacetate were measured
enzymatically as described in Ref. 25 on sample of cell suspension
previously quenched in HClO4 (4% mass/volume final concentration) and neutralized with KOH (2 M)/MOPS (0.3 M). Intramitochondrial and cytosolic NADH/NAD+
ratios were calculated assuming thermodynamic equilibrium with 3-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios,
respectively (26).
ATP and ADP were measured by chemoluminescence on mitochondrial and
cytosolic spaces previously separated using the digitonin fractionation
method (27, 28).
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,
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).
Inhibition of Oxygen Uptake by Dimethylbiguanide Requires Intact
Cells--
The data shown in Fig. 1
illustrate the inhibitory effect of dimethylbiguanide on oxygen
consumption in isolated hepatocytes with respect to dimethylbiguanide
concentration and incubation time. As shown in panel A, the
inhibition induced by dimethylbiguanide was already observed after 5 min of cell incubation, whereas the maximal effect was reached after
20-30 min whatever the dimethylbiguanide concentration used,
i.e. 0.1, 1, 5, and 10 mM.
Conversely, incubation of permeabilized hepatocytes (panel
A, filled squares) or isolated liver mitochondria
(panel A, open squares) with 10 mM
dimethylbiguanide did not affect respiratory rate over 30 min.
Panel B shows a dose-dependent decrease in the
respiratory rate of intact cells for dimethylbiguanide concentrations
between 0.1 and 10 mM after 30 min of incubation. This
finding confirms that dimethylbiguanide has no direct effect on the
mitochondrial oxidative phosphorylation pathway (12) and indicates that
the cellular integrity is required for an effect of dimethylbiguanide
on respiration to be observed.
Studies in the pharmacokinetics of dimethylbiguanide have established
that it is totally excreted in an unchanged form (17, 30-32), and so
far, no dimethylbiguanide metabolite has been evidenced. However, to
investigate the possibility that the respiratory effect of
dimethylbiguanide might be due to some putative metabolite, we
incubated control mitochondria in the presence of water-soluble fraction or of nonpolar soluble fraction extracts prepared as described
under "Materials and Methods." Irrespective of the added fraction,
the respiratory rate of mitochondria energized with glutamate and
malate (see below) was not affected (data not shown). Although a
short-lived intermediate cannot be excluded, the lack of effect of both
polar and of nonpolar fractions indicate that the dimethylbiguanide
effect was not related to the accumulation of a stable cellular
factor(s) and may suggest the existence of a more integrated signaling pathway.
We next studied the consequences of the dimethylbiguanide-induced
inhibitory effect on the energy metabolism of intact hepatocytes. As
shown in Table I, dimethylbiguanide
decreased the mitochondrial membrane potential ( Persistence of the Dimethylbiguanide Effect after Drug Removal in
Both Permeabilized Hepatocytes and Isolated Mitochondria--
To
investigate the reversibility of the dimethylbiguanide-induced
respiratory effect, hepatocytes were incubated 30 min with 10 mM dimethylbiguanide, then washed (three times) and
re-incubated in the absence of dimethylbiguanide. Under such
conditions, the respiratory effect was seen to persist for over 30 min
after removal of the drug (data not shown). In addition, hepatocytes
permeabilized after 30 min of preincubation in the presence of 10 mM dimethylbiguanide, as well as liver mitochondria
isolated from either ex vivo perfused liver with
dimethylbiguanide, or in vivo dimethylbiguanide-treated rats, were all characterized by a clear respiratory inhibition when
incubated in the presence of glutamate and malate as respiratory substrates (states 4 and 3 and uncoupled state, see Fig.
2, panels A-C). Thus, in
addition to the finding that dimethylbiguanide-induced effect on
respiration requires cells to be intact another striking feature is its
persistence after cell permeabilization or mitochondria-isolation procedures.
Complex I as the Sole Target of the Dimethylbiguanide-induced
Cellular Respiratory Inhibition--
Contrary to observations in the
presence of glutamate and malate, respiration of either
dimethylbiguanide-exposed permeabilized hepatocytes or mitochondria
isolated from pretreated livers or animals was unchanged in the
presence of succinate (Fig. 2, panels D-F) or
TMPD/ascorbate (Fig. 2, all panels), indicating that
respiratory chain complexes II, III, and IV were not affected by
dimethylbiguanide pretreatment. Moreover, because the phosphorylating
respiratory rate (i.e. after addition of ADP) was not
affected when succinate was the substrate (Fig. 2, panels
D-F), it can be concluded that neither the ATP carrier nor the
F1F0-ATPase are affected by dimethylbiguanide pretreatment.
The finding that the dimethylbiguanide-induced respiratory inhibition
only occurred in the presence of complex I substrate strongly suggests
that complex I was the selective target of this process. To further
examine this question we functionally isolated complex I (NADH quinone
oxidoreductase, EC 1.6.99.3) from the remaining part of the respiratory
chain using decylubiquinone (oxidized quinone) as complex I electron
acceptor, in the presence of KCN. As shown Fig.
3, panel A, electron transfer
through complex I was inhibited in liver mitochondria isolated from
liver previously perfused with dimethylbiguanide as compared with that
of mitochondria isolated from a liver perfused in absence of
dimethylbiguanide. On the other hand when decylubiquinol (reduced
quinone) was used as complex III electron donor, no difference was
observed between the dimethylbiguanide-treated and the nontreated group
(Fig. 3, panel B), confirming previous results obtained with
succinate. Similar results were obtained when using mitochondria
isolated from in vivo dimethylbiguanide-treated rats (data
not shown). It can thus be concluded that the treatment by
dimethylbiguanide in vivo or in vitro (in
intact isolated hepatocytes) affects the mitochondrial respiratory
chain specifically at the complex I site.
Dimethylbiguanide-induced Respiratory Inhibition Is
Temperature-dependent--
In our attempt to clarify the
mechanism by which dimethylbiguanide affects complex I in intact cells,
we studied the effects of the incubating temperature performing
prolonged incubations of up to 3 h at 15, 25, or 37 °C in the
presence or not of dimethylbiguanide or of respiratory chain inhibitors
(rotenone and myxothiazol). Cell incubations were performed at the
adequate temperature and at the indicate time a sample of the
suspension was removed from the vial and placed in an oxygraph vessel
thermostated at 37 °C to quickly adjust the temperature of the
suspension to 37 °C before recording oxygen consumption rate (see
"Materials and Methods"). As shown in Fig.
4, incubations of up to 3 h at
15 °C (panel A) exhibited no effect of dimethylbiguanide,
whereas incubations at 25 and 37 °C showed a decreased respiration
by 20 and 50%, respectively. It is striking that the maximal
dimethylbiguanide-induced inhibition was reached after 30 min of
incubation and remained stable thereafter indicating that amplitude but
not kinetics of the response is temperature-dependent.
Regardless of the incubation temperature, rotenone and myxothiazol were
seen to be identically effective at either 15, 25, or 37 °C leading
to conclude that the effect of rotenone and myxothiazol were almost not
temperature-dependent (data not shown).
Dimethylbiguanide-induced Effect Does Not Involve Standard Cell
Signaling Pathways--
In the light of recent findings showing nitric
oxide to induce a persistent inhibition of the respiratory chain (4-6,
16), we also carried out a series of experiments using NO synthase inhibitor (L-NAME) or NO precursor
(L-arginine). As shown in Table II, neither inhibition nor stimulation of
the NO pathway affected the dimethylbiguanide-induced effect.
Reactive oxygen intermediates (mainly superoxides and hydroxyl
radicals) have been shown to inhibit mitochondrial respiration (33-36). To evaluate the role of oxygen radicals on the
dimethylbiguanide-induced effect, oxygen radical scavengers were added
to the incubation medium 20 min before addition of the drug. Neither
ascorbate (a superoxide scavenger), mannitol (a hydroxyl radical
scavenger), nor catalase (hydrogen peroxide scavenger) succeeded in
preventing the effect of dimethylbiguanide on cellular respiration
(Table II).
Although the cellular mechanism of action of dimethylbiguanide remains
largely unknown, results from several studies pointed to a connection
between dimethylbiguanide and cellular insulin signaling (30, 31,
37-40). Activation of the insulin receptor tyrosine kinase and
tyrosine phosphorylation of intracellular substrates are important
steps in insulin signaling (41, 42). However, inhibition of the
phosphatidylinositol 3-kinase pathway by specific inhibitors such as
wortmannin (43, 44) or LY294002 (45, 46) and of the MAP kinase pathway
by PD98059 (47) did not affect the dimethylbiguanide-induced inhibition
of respiration (Table II), leading us to exclude these two pathways in
the signaling effects of dimethylbiguanide.
Because it has been shown that dimethylbiguanide interferes with
cellular Ca2+ homeostasis (48, 49), we also incubated
hepatocytes with different free Ca2+ concentrations (0, 2.60, and 4 mM) or in the presence of EGTA (1 mM) in Ca2+-free medium. As shown in Table II,
none of the higher Ca2+ concentration conditions affected
the dimethylbiguanide-induced effect observed at the physiological
concentration (1.3 mM). Similarly, results also showed
preincubation in the presence of BAPTA (an intracellular calcium
chelator) did not affect the dimethylbiguanide effect. Inhibition of
phospholipase C by U73211 had no effect on the dimethylbiguanide
inhibitory effect. Hence, neither chelation of intra- or extracellular
Ca2+ nor inhibition of phospholipase C signaling pathway
affected the dimethylbiguanide-induced inhibition of respiration (Table II).
Finally, we have tested the possibility that ceramides could be
involved in this effect, although the inhibitory effect reported on
mitochondrial respiration is located on complex III (50). As shown in
Table II the ceramide synthesis inhibitors fumosine B1,
L-cycloserine, and 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 (6, 51). 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
(52, 53).
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 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.
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.
*
This work was supported by the Grant EP000983-01 from the
Fondation pour la Recherche Médicale, France (to M.-Y. El-Mir) and by the Ministère de l'Enseignement, de la Recherche et de la
Technologie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Laboratoire de
Bioénergétique Fondamentale et Appliquée,
Université Joseph Fourier, B. P. 53X, F-38041 Grenoble-Cedex 09, France. Fax: 33-4-76-51-42-18; E-mail:
xavier.leverve@ujf-grenoble.fr.
The abbreviations used are:
NO, nitric oxide;
CCCP, carbonyl cyanide p-trichloromethoxyphenylhydrazone;
TPMP+, triphenylmethylphosphonium;
TMPD, N,N,N',N'-tetramethyl-1,4-phenylenediamine
dihydrochloride;
L-NAME, N
Dimethylbiguanide Inhibits Cell Respiration via an Indirect
Effect Targeted on the Respiratory Chain Complex I*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C until used.
, respectively.
-DL-alanine, and all other reagents from Sigma-Aldrich.
Dimethylbiguanide was a gift from Merck-Lipha Co. Decylubiquinol was
prepared as described in Ref. 29 by chemical reduction of
decylubiquinone with sodium borohydride.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of dimethylbiguanide on oxygen uptake
in intact isolated hepatocytes, permeabilized hepatocytes, and isolated
liver mitochondria. Panel A, time course. Hepatocytes
(approximately 10 mg of dry cells/ml) were incubated in closed vials at
37 °C in 2.5 ml of Krebs-bicarbonate buffer (120 mM
NaCl, 4.8 mM KCl, 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) supplemented with 20 mM lactate, 2 mM pyruvate, 4 mM
octanoate, and different concentrations of dimethylbiguanide:
filled circles, 10 mM; filled
triangles, 5 mM; open circles, 1 mM; and open triangles, 0.1 mM.
Permeabilized hepatocytes (open squares) or isolated
mitochondria (2 mg of protein/ml, filled squares) were
incubated in closed vials at 37 °C in a medium containing 250 mM sucrose, 20 mM Tris-HCl, 1 mM
EGTA, 5 mM Pi, 5 mM glutamate, 1 mM malate, and 10 mM dimethylbiguanide.
Dimethylbiguanide was added after 3-min incubation, and where
indicated, oxygen consumption rate was measured. Results are expressed
as percentage of oxygen uptake obtained in the absence of
dimethylbiguanide for similar incubation times. Panel A
illustrates one typical experiment, similar results were obtained in
three others. Panel B, dose dependence. Hepatocytes were
incubated as described in the legend for panel A. Oxygen
consumption rate was measured after 30 min of incubation. Results are
mean ± S.E. (n = 15, 5 rats). Error
bars lie within the symbols.
) measured
in situ by approximately 30 mV and dramatically reduced
ATP/ADP ratio in both cytosolic and mitochondrial compartments.
Furthermore, the lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios, which are in thermodynamic equilibrium with cytosolic and
mitochondrial NADH concentrations, respectively (26), were significantly increased by dimethylbiguanide. This data indicates that
the respiratory inhibition is not due to a decrease in the supply of
energy substrates but rather suggests that the inhibition is related to
respiratory chain function. Identical results were obtained when
respiration was inhibited in similar proportion with standard
respiratory inhibitors such as rotenone or myxothiazol (see Table I).
It is noteworthy that the lactate/pyruvate ratio was significantly
higher with rotenone than with either dimethylbiguanide or myxothiazol
exposures.
Comparative effects of dimethylbiguanide, myxothiazol, or rotenone on
cellular energy metabolism
), cytosolic (cyto) and mitochondrial (mito) ATP/ADP ratios,
lactate/pyruvate ratio (Lac/Pyr), and 3-hydroxybutyrate/acetoacetate
ratio (3-OH/AcAc) were measured. Results are expressed as mean ± S.E. (n = 15, five rats). Statistical
comparisons were made using analysis of variance (Fisher PLSD test) by
using Statview software®.
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Fig. 2.
Consequence of dimethylbiguanide pretreatment
on oxygen uptake of permeabilized hepatocytes or isolated
mitochondria. The incubation medium contained 250 mM
sucrose, 1 mM EGTA, 5 mM Pi, 20 mM Tris-HCl, pH 7.4, 37 °C. Experiments were started by
the addition of permeabilized hepatocytes obtained by digitonin
treatment of intact liver cells (see "Materials and Methods")
previously incubated 30 min with or without 10 mM
dimethylbiguanide (panels A and D), of
mitochondria isolated from ex vivo perfused liver for 30 min
with a medium containing or not 10 mM dimethylbiguanide
(panels B and E), or of mitochondria isolated
from in vivo dimethylbiguanide-treated rat (panels
C and F). In this latter case and for the typical
experiment shown in panels C and F, plasma
dimethylbiguanide concentration at the sacrifice was 3.6 mM. Where indicated 5 mM glutamate and 1 mM malate (panels A-C) or 5 mM
succinate plus 1.25 µM rotenone (panels D-F),
1 mM ADP, 0.36 µM CCCP, and 1 mM
TMPD plus 5 mM ascorbate were added. One typical experiment
is presented (black line, control; gray line,
dimethylbiguanide); similar results were obtained in four other
different preparations.
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Fig. 3.
Consequence of dimethylbiguanide pretreatment
on complex I and complexes III-IV activities. The incubation
mediums contained 500 µM NADH, 1 mM EGTA, 5 mM KCN, 20 mM Tris-HCl, pH 7.4, 25 °C
(panel A) or 250 mM sucrose, 1.25 µM rotenone, 1 mM EGTA, 5 mM
Pi, 20 mM Tris-HCl, pH 7.4, 25 °C
(panel B). Experiments were started after addition of
mitochondria isolated from liver perfused 30 min with a medium
containing or not 10 mM dimethylbiguanide (not shown).
Where indicated, either 100 µM decylubiquinone
(panel A) or 300 µM decylubiquinol
(panel B) were added. NADH oxidation (panel A) or
oxygen consumption rate (panel B) were followed as described
under "Materials and Methods." Results represent one typical
experiment, similar results were obtained in three other different
preparations. Moreover similar results were also obtained when
mitochondria were isolated from in vivo
dimethylbiguanide-treated rat (data not shown).
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Fig. 4.
Effect of temperature on
dimethylbiguanide-induced respiratory inhibition. Hepatocytes were
incubated at 15 °C (panel A), 25 °C (panel
B), or 37 °C (panel C) in the medium described in
the legend to Fig. 1 supplemented with 20 mM
dihydroxyacetone and 4 mM octanoate in the absence
(open symbols) or in the presence of 10 mM
dimethylbiguanide (filled symbols). After 0, 30, 60, 120, and 180 min of incubation, a sample of the suspension was quickly
removed from the vial and placed in an oxygraph vessel thermostated at
37 °C permitting to adjust the temperature of the suspension
precisely to 37 °C before oxygen consumption rate was recorded.
Results are expressed as mean ± S.E. (n = 12, three rats). Inset shows the percentage of inhibition
respectively at 15, 25, and 37 °C. Statistical comparisons were made
using analysis of variance; dimethylbiguanide significantly decreased
oxygen consumption rate at 25 and 37 °C (p < 0.001)
but not at 15 °C.
Lack of effect of cell-signaling inhibitors on
dimethylbiguanide-induced inhibition of cellular respiration
-DL-alanine, 2.5 mM) were added 20 min
before 10 mM dimethylbiguanide addition. Oxygen consumption
rate was measured after 30-min incubation. Results are mean ± S.E. (n 
9 for at least three rats). Whatever the
experimental conditions, dimethylbiguanide always significantly
inhibited the respiratory rate by 40-50% (p < 0.001), and no significant difference was found concerning the extent
of the inhibitory effect among the various conditions.
-DL-alanine had no effect
on the dimethylbiguanide-induced inhibition of respiration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-DL-alanine) does not
support this hypothesis.
ACKNOWLEDGEMENTS
FOOTNOTES
On leave from the Departamento de Fisiologia y Farmacologia,
Facultad de Farmacia, Universidad de Salamanca-E-37007, Spain.
ABBREVIATIONS
-nitro-L-arginine methyl ester;
BAPTA-AM, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
MOPS, 4-morpholinepropanesulfonic acid.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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