Inhibition of complex I regulates the mitochondrial permeability transition through a
phosphate-sensitive inhibitory site masked by cyclophilin D
Bo Lia,1, Christiane Chauvinb,1, Damien De Paulisa, Frédéric De Oliveirab, Abdallah Ghariba,
Guillaume Vialb, Sandrine Lablancheb,c, Xavier Leverveb,2, Paolo Bernardid,
Michel Ovizea,e, Eric Fontaineb,c,⁎
aInserm, U1060 (CARMEN) at the University Claude Bernard Lyon 1, Lyon, F-69373, France
bInserm, U1055 (LBFA) at the Joseph Fourier University, Grenoble, F-38041, France
cGrenoble University Hospital, Grenoble, F-38043, France
dConsiglio Nazionale delle Ricerche, Institute of Neuroscience at the Department of Biomedical Sciences, University of Padova, I-35121 Padova, Italy
eLouis Pradel Hospital, Lyon Civil Hospitals, Bron F-69677, France
a b s t r a c ta r t i c l e i n f o
Received 13 March 2012
Received in revised form 15 May 2012
Accepted 24 May 2012
Available online 31 May 2012
Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for
preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here,
that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in
which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sen-
sitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone,
the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration;
and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably
(i)genetic ablation of cyclophilin DoritsdisplacementwithcyclosporinArestoredPTPinhibitionbyrotenonein
tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was
present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et
al. (2008) J. Biol. Chem. 283, 26307–26311]. These results indicate that inhibition of complex I by rotenone or
metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism;
D, a finding that has major implications for pore modulation in vivo.
© 2012 Elsevier B.V. All rights reserved.
The mitochondrial permeability transition (PT) is a sudden increase
in the permeability of the inner mitochondrial membrane, which
becomes non-selectively permeable to molecules smaller than 1500 Da
channel regulated by other mitochondrial proteins, which is referred to
as the permeability transition pore (PTP). Despite numerous efforts, the
exact molecular nature of the PTP remains elusive. The matrix protein
cyclophilin D (CypD) is the best defined regulatory component of PT
[2–5]. Although the molecular target of CypD on the PTP has not yet
been found, occurrence of the PT is easier when CypD binds to the PTP
. The classic PT-inhibitor cyclosporin A (CsA) appears to indirectly in-
hibit PTP opening by detaching CypD from the pore [6,7]. It has recently
been shown that CypD ablation, or detachment of CypD from the PTP by
CsA, does not prevent PT unless phosphate is present, revealing that
CypD masks an inhibitory site for phosphate .
Prolonged PT leads to cell death via the release of mitochondrial
pro-apoptotic mediators into thecytosol , while inhibitionof PT pre-
vents oxidative stress-induced cell death [2,4,5], including ischemia-
whyPTPregulation has extensivelybeenstudied overthe past decades.
However, these studies have mainly been performed in rat liver mito-
chondria , with the implicit assumption that PTP regulation is identi-
cal in every tissue.
by rotenone, piericidine or metformin [11–13] inhibited PT in U937, KB,
HMEC and INS-1 cells [11,13–15], whereas this type of regulation was
not observed in rat liver mitochondria . We therefore started an
Biochimica et Biophysica Acta 1817 (2012) 1628–1634
Abbreviations: ANT, adenine nucleotide translocator; COX, cytochrome oxidase;
CRC, Ca2+retention capacity; CsA, cyclosporin A; CypD, cyclophilin D; PT, permeability
transition; PTP, permeability transition pore
⁎ Correspondingauthorat: INSERM
Fondamentale et Appliquée, Université Joseph Fourier, BP 53, F-38041 Grenoble Cedex,
France. Tel.: +33 476 635601; fax: +33 476 514218.
E-mail address: email@example.com (E. Fontaine).
1These authors contributed equally to this work.
2Deceased November 8, 2010.
0005-2728/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
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PT by rotenone. Here we report that PT inhibition by rotenone and met-
formin is mediated by the same Pi-dependent site unmasked by CsA.
2. Material and methods
2.1. Animals and cell lines
Ppif−/−(CypDnull) micewerea generous giftfrom thelaboratoryof
the late Stanley Korsmeyer . Liver and heart mitochondria were iso-
lated from rat and mouse tissue as described previously [16,17].
Human fibroblasts were prepared from normal skin by trypsin treat-
ment. Human lymphocytes were extracted from whole blood collected
in the Grenoble medical centre. OV1 and CLTT cells were generous gifts
grown in a RPMI medium (Gibco) supplemented with 10% fetal calf
serum (FCS) and antibiotics. Isolated insulinoma cell lines INS-1, a gen-
erous gift of Dr. F. De Fraipont (CHU-Grenoble), were maintained in
RPMI 1640 medium supplemented with 10 mM HEPES, 10% heat-
inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml
streptomycin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol.
Mouse fibroblasts NIH/3T3 (CRL-1658™), human promyeloblasts HL-
60(CCL-240™),human epithelial KB (CCL-17™), human cervix carci-
noma Hela (CCL-2™), human erythroleukemia K-562 (CCL-243™),
human fibrosarcoma HT-1080 (CCL-121™), human hepatocellular car-
cinoma Hep G2 (HB-8065™), rat hepatoma MH1C1 (CCL-144™), and
rat glioma C6 (CCL-107™) were obtained from ATCC and grown in the
medium suggested by the supplier.
Freshly isolated rat hepatocytes were grown in a DMEM medium
(Gibco) containing 4.5 g/l glucose supplemented with 25% M199
(Sigma), 10% FCS, 0.2 mg/ml BSA, 10 μg/ml insulin, 2 mM glutamine,
and antibiotics (100 U/ml penicillin and streptomycin). After 24 h,
the medium was supplemented with 1 μM hydrocortisone, 10 ng/ml
EGF, and 1 mg/ml BSA. Rat hepatocytes (1 million cells) were
Fig. 1. Rotenone inhibits PTP opening in most of the tested tissues. CRC was measured in the presence of vehicle (control), 1 μM CsA, 1 μM rotenone or 1 μM CsA plus 1 μM rotenone in a
mediumsupplemented with 5 mMsuccinate-Tris. Results are expressed asmean±S.E.ofatleastthree independent experiments.*,pb0.05vs. control, #, pb0.05vs. CsA.Student's t test.
Fig. 2. CypD is more expressed in tissues in which rotenone does not inhibit PTP opening.
Representative immunoblot for the quantification of CypD, complex I, cytochrome
oxidase, and ANT-1 in the indicated cells. For an easier comparison between the different
cell lines, the ratios were normalized (divided by the corresponding ratio observed in KB
cells). Results are expressed as mean±S.E. of five independent experiments. *, pb0.05 vs.
CypD/COX. Student's t test.
B. Li et al. / Biochimica et Biophysica Acta 1817 (2012) 1628–1634
transfected in a serum and antibiotics-free DMEM transfection
medium by cationic liposomes, using Lipofectamine 2000 (Invitrogen).
CypD-targeting siRNA oligonucleotides CAAGAGCCCUGAGUUAUGU
siRNAs were obtained from Eurogentec. Oligonucleotides (300 pmol)
according to the manufacturer's protocol. After 20 min of incubation,
complexes wereadded to a 100 mmculturedishcontaining5 mltrans-
fection medium. Hepatocytes were then added at a density of 1 million
per dish and incubated at 37 °C for 4 h. After this period, the culture
medium was supplemented with FCS and antibiotics for 20 h.
2.3. Calcium retention capacity measurement
Unless otherwise specified, liver mitochondria and permeabilized
cells were incubated at a pH of 7.4, in a medium containing 250 mM
sucrose, 1 mM Pi-Tris, and 10 mM Tris-MOPS, while heart mitochon-
dria were incubated in a medium containing 150 mM sucrose, 50 mM
KCl, 2 mM KH2PO4, and 20 mM Tris/HCl. When the concentration of
phosphate was varied, vanadate was added to maintain the total
phosphate plus vanadate concentration constant . The incubation
medium was supplemented with 5 mM succinate-Tris and 0.25 μM
Calcium Green-5N. The final volume was 1 ml at 25 °C. Experiments
began by the addition of either 2×106cells followed by 40 μM
digitonin, or of 0.5 mg of isolated mitochondria, followed by the
addition of vehicle (control), 1 μM CsA, 1 μM rotenone or 1 μM CsA
plus 1 μM rotenone. After 2 min of incubation, the Ca2+retention
capacity (CRC) was measured by adding sequential Ca2+pulses
until PTP opening . Measurements of extramitochondrial Ca2+
were performed fluorimetrically (excitation: 506 nm; emission:
530 nm), using a spectrofluorometer equipped with magnetic stirring
and thermostatic control .
2.4. Respiratory chain activity
Oxygen consumption was measured polarographically at 25 °C
using a Clark-type electrode in the medium used for CRC measurement
supplemented with 5 mM glutamate-Tris and 2.5 mM malate-Tris.
Fig. 3. Detachment of CypD or decrease in CypD content allows rotenone to inhibit PTP opening. A: The measurement of bound (pellet) and unbound (supernatant) CypD and the
CRC was performed on rat liver mitochondria incubated in a medium containing the indicated concentration of sucrose in the presence or absence of 1 μM CsA. The pH was as in-
dicated. *, pb0.05 vs. the proper control (i.e., without rotenone). Student's t test. B: The quantification of CypD, complex I, and cytochrome oxidase was performed on rat hepato-
cytes transfected with CypD siRNA oligonucleotides. For an easier comparison, the ratios were divided by the proper ratio observed in control conditions (i.e., lipofectamine without
oligonucleotides). The CRC measurements were performed on digitonin-permeabilized rat hepatocytes. *, pb0.05 vs. control, left panel. Mann–Whitney. *, pb0.05 vs. control, right
panel. Student's t test. C: The quantification of CypD and actin, and the CRC measurements were performed on heart mouse mitochondria isolated from wild type or Ppif−/−animals.
In all the panels, results are expressed as mean±S.E. of at least five independent experiments. *, pb0.05 vs. control, #, pb0.05 vs. CsA. Student's t test.
B. Li et al. / Biochimica et Biophysica Acta 1817 (2012) 1628–1634
2.5. Protein quantification
Total proteins from cells or mitochondria lysates were separated
using 10% SDS-PAGE, transferred onto nitrocellulose membranes,
and probed with antibodies against human CypD [rabbit polyclonal
antibody (Affinity Bioreagent PA1028)], human complex I 39 kDa
subunit (mouse monoclonal Molecular Probes A21344), human cyto-
chromeoxidase subunit 4 (cox4) (mouse monoclonal MolecularProbes
A21348), ANT-1 (mouse monoclonal Mitosciences MSA02), and actin
(mouse monoclonal Sigma A3853). Detection was performed by
enhanced chemiluminescence (GE-Healthcare), and densitometry was
performed using NIH image software. For the quantification of bound
and unbound CypD, mitochondria were broken (sonicated for 30 s
twice) and centrifuged for 30 min at 8000 g. Supernatants and pellets
(resuspended in the same volume) were run on 10% SDS-PAGE gels,
blotted and probed with a polyclonal antibody against CypD.
2.6. Quantification of cell death by flow cytometry
Apoptosis analyses were performed with a double-stain system
using Annexin V (Interchim) combined with FluoProbes 488 and
Propidium Iodide (PI) (Sigma Aldrich). INS-1 cells were detached by
trypsinization, washed by centrifugation, and incubated with 100 μl
of Annexin V buffer 1× (10 mM HEPES NaOH, pH 7.4, 150 mM NaCl,
5 mM KCl, 1 mM MgCl2and 1.8 mM CaCl2). Cells were then incubated
for 15 min at room temperature in the dark in the presence of 5 μl of
AnnexinV-FP488. Labeled cells were transferred in a 5 ml propylene
tube containing 900 μl PBS. 10 μl from a 1 mg/ml stock solution of PI
was added to the suspension and immediately analyzed. Data acqui-
sition (~5000 cells) was carried out using a FACSCAN flow cytometer
tuned at 488 nm, using the Cell Quest Pro software (Becton Dickinson
Biosciences). Data were plotted as a function of fluorescence intensity
on FL-1 (530 nm/30 nm band‐pass filter) (Annexin V) and FL-3 chan-
was regarded as normal healthy cells.
Matrix Ca2+is the single most important factor for inducing PT.
“PT-inhibitors” and “PT-inducers” are compounds that increase and
decrease the amount of Ca2+required to induce PT, respectively
. CRC (i.e., the amount of Ca2+required to induce PT) was mea-
sured by loading mitochondria or digitonin-permeabilized cells with
a train of Ca2+pulses until a fast Ca2+release occurred, which
marks the onset of the PT and is accompanied by swelling . As
shown in Fig. 1, the complex I inhibitor rotenone did not inhibit PT
(i.e., it did not increase the CRC) in rat liver and heart mitochondria,
and in mouse heart mitochondria. This is in line with the general lit-
erature on the PTP, which has mainly studied rat liver and rat heart
mitochondria . In all the other tissues and cell lines tested, howev-
er, rotenone was more potent than, or at least as potent as, CsA at PTP
inhibition (Fig. 1). This effect was particularly prominent in mouse
liver mitochondria, rat hepatoma MH1C1 cells and rat glioma C6
cells, suggesting that the lack of effect of rotenone is not a feature of
a particular animal species (rat) or a particular tissue (liver). It is
also important to note that CsA is not a universal PT-inhibitor, as it
did not prevent PT in CLTTi, NIH/3T3, and HL60 cells (Fig. 1). Overall,
this set of data reveals (i) that PTP regulation by rotenone and CsA is
different in different tissues and organs, indicating that at least some
regulatory features of the PTP defined in rat liver and heart mitochon-
dria may not necessarily apply to other tissues; and (ii) that in tissues
orcellswhereboth rotenoneandCsAareeffectiveinhibition isadditive.
Indeed, in preparations where an effect of CsA was detected, the CRC
was always higher in the presence of both CsA and rotenone than
with CsA alone (Fig. 1). This was particularly evident in mitochondria
from rat liver and heart, and from mouse heart, where rotenone alone
did not affect the CRC.
CypD is the molecular target of CsA [2–5], while the adenine nucle-
otide translocator (ANT) and complex I have been suggested to be part
of the PTP [14,23]. We next quantified CypD, ANT, complex I, and cyto-
chrome oxidase in several cell lines in which rotenone does or does not
regulate PTP opening. The CypD/cytochrome oxidase ratio was the
same in all the cells tested, while the CypD/ANT and CypD/complex I
ratios were dramatically higher in preparations where rotenone did
not inhibit PT in the absence of CsA, as compared to cells in which rote-
nonealonewaseffective (Fig.2).Thissetof data suggeststhattheeffect
of rotenone at PTP regulation might depend on specific interactions
between CypD and the pore.
It has been reported that CypD can be detached from the inner
membrane of inside-out submitochondrial particles by incubating
mitochondria in the presence of CsA , in hyperosmotic media ,
or at alkaline pH . We have observed that sonication of mitochondria
releases a fraction of CypD, and that this fraction is dramatically
increased by treatment with CsA, largely increased by exposure to
hypertonic sucrose, and somewhat increased by alkaline pH but not
thePT in ratlivermitochondria once CypD had beendetached byany of
the above treatments, the effect being larger after treatment with CsA,
intermediate with hypertonic sucrose, and modest but significant
with pH 9.0 (Fig. 3A). PT inhibition by rotenone was also observed in
permeabilized rat hepatocytes after treatment with specific siRNAs de-
creasing the expression of CypD (Fig. 3B). In mouse heart mitochondria
Fig. 4. Rotenone and CsA require phosphate to inhibit PTP opening. The CRC was mea-
suredinthepresenceof 5 mM succinate-Trisattheindicatedconcentrationsofphosphate
rotenone (panel A) or on rat liver mitochondria in the presence of vehicle (control), 1 μM
CsA, or 1 μM CsA plus 1 μM rotenone (panel B). Results are mean±S.E. of three indepen-
dent experiments. *, pb0.05 vs. without rotenone. Student's t test.
B. Li et al. / Biochimica et Biophysica Acta 1817 (2012) 1628–1634
rotenone inhibited the PT only in the Ppif−/−(CypD null) genotype or
after treatment of the wild type littermates with CsA, but not in naïve
wild type mitochondria (Fig. 3C).
CypDablation in mouseheart mitochondria (Fig. 4A) or detachment
of CypD by CsA in rat liver mitochondria (Fig. 4B) did not inhibit PT in
the absence of phosphate, confirming results obtained with mouse
liver mitochondria . Interestingly, rotenone did not inhibit PT unless
phosphate was present (Fig.4A,B), suggesting that rotenone also
unmasks an inhibitory site for phosphate.
Rotenone inhibited complex I and PT with a similar concentration
dependence (Fig. 5A), suggesting that rotenone inhibits PTP opening
ing could be obtained when complex I was inhibited only partially.
Indeed, metformin (which partly inhibits complex I activity [11–13])
also increased the CRC in digitonin-permeabilized cells (Fig. 5B). More-
over, it should be mentioned that metformin did not inhibit PTP open-
ing in tissues with high expression of CypD such as rat liver (data not
shown), whereas metformin potentiated the action of CsA (Fig. 5B)
but not that of rotenone (Fig. 5C), further suggesting that metformin
and rotenone have a common mechanism of action.
In order to check whether the synergic action of CsA and complex I
inhibition on PTP opening could also be seen in cytoprotection, we
exposed INS-1 β-cell to 30 mM glucose, a condition that induces
PTP opening and cell death . Hyperglycemia-induced cell death
was partially prevented by treatment with CsA or metformin alone,
and totally prevented by the combined treatment with both drugs
In this manuscript we have shown that inhibition of the PT by
rotenone is maximal in tissues that express low levels of CypD. Like
inhibition by CsA , inhibition by rotenone requires Pi, suggesting
that rotenone and CsA eventually act on the same inhibitory site,
and that the cell can modulate its response to rotenone by shielding
this inhibitory site with increased expression of CypD.
Themoleculartargetof CypDforPTPregulation (i.e.,thebindingsite
for phosphate) has not been identified yet; and neither transcriptional
regulation of the Ppif gene (encoding for CypD) nor turnover of CypD
have been studied, so the reason why the CypD content varies in differ-
enttissues isnot known.CypDhas a varietyof well-characterized mito-
chondrial targets  including: (i) the F1FOATP synthase; binding of
CypD is favored by Pi and leads to inhibition of the enzyme, and the
effect is counteracted by CsA in striking analogy with the effects on
the PTP ; (ii) the ANT; this finding was taken to imply that the
ANT is part of the PTP , but this seems unlikely in view of the fact
that the pore is inhibited by CsA in ANT-null mitochondria ;
(iii) Hsp90and itsrelatedmoleculeTRAP-1;asCypDboundtothecom-
plex is no longer available for PTP opening, CypD displacement from
the PTP, which has been successfully used for the selective killing of
tumorcellsthatoverexpressTRAP-1;(iv) theantiapoptotic protein
Bcl2; binding could be displaced by CsA resulting in increased tBid-
dependent release of cytochrome c from mitochondria under condi-
tions that did not cause opening of the PTP .
Fig. 5. Mild inhibition of complex I inhibits PTP opening and potentiates the effects of CsA. A: Isolated rat liver mitochondria were incubated in the presence of 1 μM CsA. The oxygen
consumption rate in the presence of 5 mM glutamate-Tris plus 2.5 mM malate-Tris (JO2) and the CRC in the presence of 5 mM succinate-Tris were measured in parallel experiments
at the indicated concentration of rotenone. Results are mean±S.E. of three independent experiments. B: The CRC was measured as in Fig. 1 on digitonin-permeabilized HeLa cells
and on digitonin-permeabilized INS-1 cells incubated overnight in the presence or absence of 100 μM metformin. Where indicated, the CRC was measured in the presence of 1 μM
CsA. Results are mean±S.E. of three independent experiments. *, pb0.05 vs. control, #, pb0.05 vs. CsA. Student's t test. C: The CRC was measured as in Fig. 1 at the indicated con-
centration of rotenone on digitonin-permeabilized HeLa cells incubated overnight in the presence (closed symbols) or absence (open symbols) of 100 μM metformin. Results are
mean±S.E. of three independent experiments. D: INS-1 cells incubated in RPMI 1640 medium supplemented or not with either 1 μM CsA for 1 h, 100 μM metformin for 24 h, or
100 μM metformin for 24 h and 1 μM CsA for 1 h were then incubated in complete RPMI 1640 medium supplemented or not (control) with glucose (30 mM, final concentration)
for 72 h. Cell viability was assessed by double labeling as described in Material and methods. Histograms represent the results of three different experiments. Results are mean±SE;
* pb0.05 vs. control, # pb0.05 vs. Glucose 30 mM. Student's t test.
B. Li et al. / Biochimica et Biophysica Acta 1817 (2012) 1628–1634
rotenoneand metformin remainsunknown; yet, and irrespectiveof the
site of action, our findings represent a mechanistic advance in under-
standingofhow thePTP is regulatedin differenttissues. Thehypothesis
that rotenone regulates PT via a site located outside complex I cannot
indisputably be ruled out at present, but this hypothesis seems very
unlikely. The effect of rotenone on PTP opening can indeed be traced
to complex I inhibition per se because a similar outcome was caused
by both piericidine  and the antidiabetic drug metformin
[11,13,15], which partially inhibits complex I [11–13]. Since complex I
inhibition affects the redox status of complex I [30–32], we propose a
model (see Fig. 6) in which according to its conformation, complex I
interacts with the PTP, in turn modulating the number of accessible Pi
sites affected by CypD. The early recognition that several ligands of
the ANT regulate PT led to proposal that ANT might form the PTP .
However, the observation that PT persists in ANT deficient cells
questioned this assumption . It was then suggested that ANT is a
regulatory component of the PTP, or that PT might involve several
different inner membrane proteins. Therefore, an alternative hypothe-
sis should be that complex I might be one of the proteins able to
form a nonspecific inner membrane channel depending on its redox
is subjected to regulation by both covalent and non-covalent modifica-
tions [30,33,34]. Our demonstration that complex I impinges on the
same site(s) unmasked by CsA (and otherwise shielded by CypD) re-
veals a novel level of PTP regulation by modulation of the expression
levels and/or turnover rate of CypD. As mentioned above, CypD binds
to numerous proteins, most probably with different affinities. If we
assume that the binding sites on the PTP are of high affinity, once
these are saturated a further increase in CypD will not regulate the
PTP, unless rotenone unmasks new sites for phosphate. In the scenario
proposed in Fig. 6, a decrease in CypD is expected to increase the CRC
only once the binding sites of high affinity become vacant, which
requires a large decrease in CypD expression in tissues with normally
high levels of CypD.
This hypothesis is supported by the findings that a significant de-
crease in CypD did not inhibit PTP opening in rat liver (i.e., a tissue
with high levels of CypD) in the absence of rotenone (Fig. 3B); and that
PTP opening was not inhibited in heart mitochondria of Ppif+/−mice
, which is a tissue with high levels of CypD (Fig. 2) where rotenone
does not inhibit PTP opening (Fig. 1). On the contrary, PTP opening
was inhibited in liver mitochondria of Ppif+/−mice , i.e. a tissue
with low levels of CypD (Fig. 2) where rotenone is an effective inhibitor
Metformin is the most widely prescribed drug to treat patients
affectedbytype 2 diabetesandisrecommended asa first-lineoralther-
apy in both American and European guidelines . This recommenda-
tion is based on clinical studies suggestinga reduction ofcardiovascular
mortality by metformin compared with any other oral antidiabetic
treatment [36,37]. This suggests that beside its antihyperglycemic
effect, metformin may have other beneficial effects. Both ischemia-
reperfusion injury and hyperglycemia-induced cell death have been
shown to involve PTP opening [5,10,11,15]. It appears therefore plausi-
ble that some of the beneficial effects of metformin are due to the inhi-
bition of PT.
to involve an activation of AMPK . However, this assertion has
been questioned by the demonstration that the inhibition of liver
neoglucogenesis, which is one of the main effects of metformin in
diabetes, persists in liver-specific AMPK-deficient mice . Moreover,
the addition of cell-permeant succinate, a substrate bypassing the inhi-
bition of complex I, has been shown to abolish the metformin-induced
AMPK activation, suggesting that AMPK activation is due to Complex I
The finding that the protective effect of rotenone and metformin is
additive with that of CsA represents a major advancement that may
lead to a better treatment of PTP-dependent diseases with a combina-
tion of metformin and cyclophilin inhibitors, a protocol that may
proveextremely relevantfor diabetes. Mutations of thepancreatic duo-
denal homeobox gene-1, Pdx1, cause heritable diabetes in humans and
apoptosis and necrosis [42–44].
Genetic ablation of the Ppif gene in Pdx1+/−mice restored β-cell
mass and prevented onset of diabetes, demonstrating that CypD is a
viable target for therapy. Our current demonstration that the approved
antidiabetic drug metformin hits the same final molecular target on the
PTP as does CsA, and that the inhibitory effect is synergistic, opens new
perspectives to the study of β-cell death and to effective pharmacolog-
ical treatments for cytoprotection.
Conflict of interest
This work was supported in part by Grants from INSERM, Agence
Nationale de la Recherche (PTP-ischemia) and the Ministère de
l'Enseignement de la Recherche et de la Technologie (MERT).
Fig. 6. Model for PTP regulation by Pi, in which the number and the accessibility of Pi
sites are modulated by interaction with complex I and CypD, respectively. A: conforma-
tion of active Complex I, I: conformation of inactive Complex I. More Pi binds to the
PTP, more Ca2+is required to induce PTP opening.
B. Li et al. / Biochimica et Biophysica Acta 1817 (2012) 1628–1634
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