Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2006/10/06/M604123200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 49, pp. 37361–37371, December 8, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Mitochondrial Creatine Kinase Activity Prevents Reactive
Oxygen Species Generation
ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP
RE-CYCLING ACTIVITY *□
S
Received for publication, May 1, 2006, and in revised form, September 5, 2006 Published, JBC Papers in Press, October 6, 2006, DOI 10.1074/jbc.M604123200
Laudiene Evangelista Meyer‡1, Lilia Bender Machado‡1, Ana Paula S. A. Santiago‡§, Wagner Seixas da-Silva‡2,
Fernanda G. De Felice‡, Oliver Holub‡, Marcus F. Oliveira‡1,3, and Antonio Galina‡1,4
From the ‡Instituto de Bioquı́mica Médica, Programa de Biofı́sica e Bioquı́mica Celular and Programa de Biologia Molecular e
Biotecnologia and the §Instituto de Biofı́sica Carlos Chagas Filho, Programa de Biologia Celular e Parasitologia, Universidade
Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Rio de Janeiro 21941-590, Brazil
* This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Fundação Carlos Chagas Filho
de Amparo à Pesquisa do Estado do Rio de Janeiro, Fundação Universitária
José Bonifácio, and the Third World Academy of Sciences. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S2.
1
These authors contributed equally to the results of this work.
2
Present address: Thyroid Section, Division of Endocrinology, Diabetes, and
Hypertension, Dept. of Medicine, Brigham and Women’s Hospital and
Harvard Medical School, Boston, MA 02115.
3
Research fellow of CNPq. To whom correspondence may be addressed: Av.
Brigadeiro Trompowsky, s/n, CCS, Bloco D, sub-solo sala D-013 and D-005,
Laboratory of Bioenergetic and Mitochondrial Physiology Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil. E-mail: maroli@bioqmed.ufrj.br.
4
Research fellow of CNPq. To whom correspondence may be addressed:
E-mail: galina@bioqmed.ufrj.br.
DECEMBER 8, 2006 • VOLUME 281 • NUMBER 49
ventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.
Mitochondrial electron transport chain is the major and continuous source of cellular reactive oxygen species (ROS),5
which are involved in several conditions, such as apoptosis,
ischemia-reperfusion injury, neurodegenerative diseases, and
toxicity induced by hyperglycemia (1–5). Electron leakage at
the complexes I (6, 7) and III (6, 8 –10) are the main sites for the
monoelectronic reduction of oxygen, which results in superoxide (O2. ) radical production in the respiratory chain. The rate of
mitochondrial ROS production is highly dependent on the
mitochondrial membrane potential (⌬⌿m) (9, 11) and evidence
supporting these observations have long demonstrated (9) that
pharmacological uncoupling of oxidative phosphorylation
caused a drastic reduction in mitochondrial H2O2 formation.
Similarly, activation of oxidative phosphorylation by ADP can
also reduce the ⌬␺m and ROS formation through activation of
F1F0-ATP synthase complex by using the energy of the ⌬⌿m to
drive ATP synthesis (9, 11). On the other hand, when mitochondrial ADP levels drop, the respiratory rate is reduced,
increasing the ⌬⌿m levels, which ultimately leads to ROS generation. There is a clear link between the increased levels of
oxidative stress markers and several neuropathies such as
amyotrophic lateral sclerosis, Parkinson and Alzheimer disease
and hyperglycemia-derived neuropathy (3, 12–15). However, if
oxidative stress is a major cause or just a consequence of associated neuron cell loss remains elusive (3). Growing evidence
indicates that ROS are involved in the propagation of cellular
damage leading to neuropathy and thus, modulation of key
enzymes that control oxidative stress is central for the develop5
The abbreviations used are: ROS, reactive oxygen species; mt-CK, mitochondrial-associated creatine kinase; PCr, phosphocreatine; Cr, creatine; mt-HK,
mitochondrial-associated hexokinase; MM-CK, cytosolic rabbit muscle creatine kinase; VDAC, voltage-dependent anion channel; ANT, adenine
nucleotide transporter; ⌬⌿m, mitochondrial membrane potential; 2-DOG,
2-deoxyglucose; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Ap5A, P1,P5-di(adenosine 5⬘)-pentaphosphate; Glc-6-P, glucose
6-phosphate; JC-1, 5,5⬘,6,6⬘-tetrachloro-1,1,3,3⬘-tetraethylbenzimidazolylcarbocyanine iodide; CM-H2DCFDA, 5-(and-6)-chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein diacetate, acetyl ester; G6PDH, glucose-6-phosphate dehydrogenase; RBM, rat brain mitochondria; RLM, rat liver mitochondria; MTP, mitochondrial permeability transitition.
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As recently demonstrated by our group (da-Silva, W. S.,
Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R.,
De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004)
J. Biol. Chem. 279, 39846 –39855) mitochondrial hexokinase
activity (mt-HK) plays a preventive antioxidant role because of
steady-state ADP re-cycling through the inner mitochondrial
membrane in rat brain. In the present work we show that ADP
re-cycling accomplished by the mitochondrial creatine kinase
(mt-CK) regulates reactive oxygen species (ROS) generation,
particularly in high glucose concentrations. Activation of
mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like
respiration in isolated brain mitochondria and prevention of
H2O2 production obeyed the steady-state kinetics of the enzyme
to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr
ratio. Rat liver mitochondria, which lack mt-CK activity, only
reduced state 4-induced H2O2 generation when 1 order of magnitude more exogenous CK activity was added to the medium.
Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose
phosphorylation via mt-HK, induced H2O2 production in a Crsensitive manner. Simulation of hyperglycemia in embryonic rat
brain cortical neurons increased both ⌬⌿m and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate
that mitochondrial kinase activity performed a key role as a pre-
Mitochondrial Kinases Prevent ROS Formation
37362 JOURNAL OF BIOLOGICAL CHEMISTRY
of mt-CK inhibits the mitochondrial permeability transition
(MPT), a process that is involved in apoptosis (42). The postulated protective mechanism of mt-CK activity against MPT
pore opening lies on functional coupling between the mt-CK
reaction and oxidative phosphorylation (42). Notwithstanding,
MPT can be directly induced by mitochondrial ROS and it is
conceivable that the protective role of mt-CK activity against
MPT would occur through reduction of ROS generation by
keeping ADP phosphorylation (43). Based on the fact that
mt-HK activity exerts a key role on regulation of ROS generation in neurons, in the present work we propose that induction
of mt-CK activity by Cr would play an even more important
preventive antioxidant function by promoting re-cycling of
mitochondrial ADP during hyperglycemia.
EXPERIMENTAL PROCEDURES
Chemicals—ADP, ATP, NAD⫹, glucose, glucose 6-phosphate, 2-deoxyglucose (2-DOG), [3H]2-DOG, fatty acid-free
bovine serum albumin, succinate, rotenone, antimycin A,
atractyloside, safranine O, FCCP, Cr, Ap5A, polyornitine, RPMI
1640 medium, MM-CK, horseradish peroxidase, catalase,
G6PDH from Leuconostoc mesenteroides, were all purchased
from Sigma. Amplex Red was purchased from Invitrogen; Percoll was from Amersham Biosciences. Hydrogen peroxide was
from Merck (Germany). Neurobasal medium was from Invitrogen. CM-H2DCFDA and JC-1 were obtained from Molecular
Probes (Eugene, OR). All other reagents were analytical grade.
Animals and Mitochondrial Isolation—Adult male Wistar
rats weighting 200 –230 g were fed overnight prior to killing by
decapitation. Mitochondria were isolated from brain (RBM)
and liver (RLM) by conventional differential centrifugation as
described previously with small modifications (25, 44). Protein
was determined by the Lowry (45) method using bovine serum
albumin as standard. All experiments were carried out in a
standard respiration buffer containing 10 mM Tris-HCl, pH 7.4,
0.32 M mannitol, 8 mM inorganic phosphate, 4 mM MgCl2, 0.08
mM EDTA, 1 mM EGTA, 0.2 mg/ml fatty acid-free bovine
serum albumin, and 50 ␮M Ap5A (25).
Determination of Mitochondrial Hexokinase (mt-HK) Activity from Rat Brain—The radiochemical enzymatic activity
assay for mt-HK activity was determined based on a previously
described method with minor modifications (46). In this assay
we used the respiration buffer plus 4.5 ␮M rotenone, 10 mM
succinate, and 1 mM 2-DOG, after 0, 2, 5, 10, and 15 min of
incubation in a final volume of 50 ␮l. Briefly, HK activity was
measured by the selective adsorption of [3H]2-DOG-6P on
DE81 paper discs using [3H]2-DOG as substrate. The reaction
was stopped with 50 ␮l of ice-cold ethanol and dropped onto
DE81 Whatman filters. The filters were washed with 10 ml of
distilled water 10 times. The discs were dried and radioactivity
was counted in a liquid scintillation counter.
Determination of Mitochondrial Creatine Kinase (mt-CK)
Activity from Rat Brain—For this assay was used 0.1– 0.15
mg/ml of the mitochondrial protein and the activity of mt-CK
was determined by NADH formation following the absorbance
at 340 nm at 37 °C. The assay medium contained: 50 mM TrisHCl, pH 7.4, 10 mM glucose, 5 mM MgCl2, 2 mM ADP, 1 mM
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ment of drug therapies against these conditions. In support to
this view, it has been well documented that creatine (Cr) exerts
powerful protective effects in models of Huntington disease,
Parkinson disease, amyotrophic lateral sclerosis, as well as in in
vitro models of glutamate and ␤-amyloid toxicity (12, 16 –19).
Moreover, neuronal ATP depletion is a feature of neurodegenerative diseases and the proposed mechanism for Cr protection has been attributed to a build-up of phosphocreatine
(PCr) stores, which increase the efficiency of ATP regeneration (18, 19).
Hyperglycemia in animal and in vitro models of diabetes is
associated with both enhanced production as well as decreased
scavenging of ROS, leading to a cellular oxidative stress condition and impaired mitochondrial function, which ultimately
leads to O2. overproduction by the mitochondrial electron
transport chain (4, 5, 15, 20, 21). One of the hypotheses raised to
explain the establishment of oxidative stress conditions in
hyperglycemia is that excess glucose leads to an oversupply of
electrons in the mitochondrial electron transport chain, resulting in mitochondrial membrane (⌬⌿m) hyperpolarization and
ROS formation (12, 22). Glucose toxicity in chronic hyperglycemia is especially important in tissues where glucose uptake is
independent of insulin such as hepatocytes, endothelial, epithelial, and immune cells as well as the cells from the central and
peripheral nervous systems (4, 5, 20 –24). In this regard, our
group recently demonstrated that mitochondrial associated
hexokinase (mt-HK) activity plays a central role on preventing
mitochondrial ROS generation through steady-state ADP recycling in rat brain (25). However, when neurons were exposed
to high glucose levels, it was observed that an increase not only
in ROS production but also in the intracellular levels of glucose
6-phosphate (Glc-6-P) inhibits mt-HK activity impairing ADP
re-cycling through inner mitochondrial membrane. Thus, ADP
re-cycling enzymes would play a preventive antioxidant role in
mitochondria by keeping lower ⌬⌿m and ROS levels.
A search over other putative enzymes that would contribute
to the ADP re-cycling mechanism revealed that brain has high
levels of mitochondrial creatine kinase (mt-CK), which is
located in the intermembranal space of mitochondria (26 –33).
CK (EC 2.7.3.2) comprise a group of isoenzymes that catalyze
the following reversible reaction: Mg䡠ATP ⫹ Cr 7 PCr ⫹
Mg䡠ADP ⫹ H⫹. This enzyme performs a pivotal physiological
role in high energy consuming tissues, by acting as an energy
buffering and transport system between the sites of ATP production and consumption by ATPases (34, 35). The mt-CKs
form octamers assembled as four dimers, but only the
octameric form can interact with both inner and outer mitochondrial membranes through the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC)
(36). The mt-CK activity couples the oxidative phosphorylation
and mitochondrial PCr production by catalyzing the conversion of Cr to PCr at expenses of the intramitochondrially produced ATP. The PCr is exported to the cytosol, whereas the
produced ADP is pumped back to the mitochondrial matrix via
ANT, thus stimulating oxidative phosphorylation (37–39). In
fact, Cr is an excellent stimulant for mitochondrial respiration
during PCr generation (31, 40, 41). Besides its role on energy
metabolism it has recently been demonstrated that activation
Mitochondrial Kinases Prevent ROS Formation
DECEMBER 8, 2006 • VOLUME 281 • NUMBER 49
cence microscope using a standard filter for green fluorescence
(B2 FITC blue filter combination: excitation 465– 495 nm;
dichroic filter 505 nm; emission 515–555 nm) and a ⫻40 objective (Nikon Plan Fluor ELWD DM; N.A. 0.6; W.D. 3.7-2.7 mm;
PH2) at fixed exposure times. Fluorescence quantification was
determined by using the Adobe® Photoshop software.
Image Analysis—The JC-1 red/green ratio was determined
from each selected region of the cell culture, a set of two 24-bit
RGB bitmap images was acquired (green and red fluorescence
respectively) and both images were split into their corresponding three color channels. Images of green JC-1 fluorescence
contained information in their green channel and images of red
JC-1 fluorescence in the red channel only, which were extracted
for further processing. The resulting two 8-bit images of red
and green fluorescence intensity (denoted as red and green
image) were exported separately by selecting them individually
and subjecting them to further calculation of the integration
image. A routine has been written in LabVIEW software
(National Instruments, Austin, TX) and each image set was
subjected to the following steps. 1) Loading of the green and red
8-bit bitmap image (defining red and green fluorescence levels
in the range 0 –255 for each pixel). 2) Determination of the
background signal in each image: the program allows for the
selection of a region of interest in the image. Using this option
one selects a region free of any cellular structures in the image.
The determined two average signals of the same region of interest for the red and green image are used for later background
subtraction and the detected maximum background signal constitutes the threshold for background pixel removal. 3) For calculation of a division image, all background pixels of the two
images determined by the previous step were excluded from
analysis by the according threshold selection (as well as overexposed pixels with value 255 if present). If a pixel is excluded in
the red image, the corresponding pixel in the green image is
automatically also excluded, even if it would pass its own
threshold settings and vice versa, a procedure ensuring that
only pixel sets, which contain fluorescence signal in both
images, are analyzed. From each of these pixels the average
background is subtracted and the corresponding pixels of both
images are divided by each other, resulting in a pixel wise division image, which allows for the visualization of the red/green
ratio over the image. Automatically the division image is analyzed by calculation of the pixel average, standard deviation,
minimum and maximum, and the plot of the histogram and
probability distribution of pixel values with determination of
mode, skewness, and kurtosis of the histogram.
Statistical Analysis—Data were plotted with GraphPad
Prism 4.0 software (GraphPad) and analyzed by analysis of variance and a posteriori Tukey’s test. p values ⬍0.05 were considered statistically different.
RESULTS
mt-HK Activity Regulates Mitochondrial ROS Generation in
RBM—Recently our group demonstrated that mt-HK plays a
preventive antioxidant role in RBM through ADP re-cycling
(25). Our first step was to investigate the role of mt-HK on
regulation of the ⌬⌿m in RBM (Fig. 1A). Induction of mt-HK
activity by 2-DOG after ADP-induced state 3 led to a persistent
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NAD⫹, 5 units/ml yeast hexokinase, and 1 unit/ml G6PDH.
The reaction started when 5 mM PCr was added.
Oxygen Uptake Measurements—Oxygen uptake was measured in an oxymeter fitted with a water-jacket Clark-type electrode (Yellow Springs Instruments Co., model 5300). The RBM
and RLM (0.15– 0.25 mg/ml) were incubated with 1.5 ml of the
standard respiration buffer described above.
Determination of Mitochondrial Membrane Potential
(⌬⌿m)—The fluorescence signal changes of the cationic dye
safranine O was monitored as previously described (25, 47).
Data are reported as arbitrary fluorescence units. Other additions are indicated in the figure legends.
Determination of Mitochondrial Hydrogen Peroxide Generation—Mitochondrial release of H2O2 was determined by the
Amplex Red oxidation method (48). Mitochondria (0.15 mg of
protein/ml) were incubated in the standard respiration buffer
supplemented with 10 ␮M Amplex Red and 4 units/ml horseradish peroxidase. Fluorescence was monitored at excitation
and emission wavelengths of 563 (slit 5 nm) and 587 nm (slit 5
nm), respectively. Calibration was performed by the addition of
known quantities of H2O2. Other additions are indicated in the
figure legends. In all experiments, small variations in the levels
of H2O2 formation were observed with different preparations,
but the overall pattern of response to different modulators was
not changed.
Cortex Cell Cultures—Cortices from 14-day-old Wistar rat
embryos were dissected and cultured as previously described
(25, 49). Fluorescence images were acquired on a Nikon Eclipse
TE 300 inverted light microscope (Nikon, Kanagawa, Japan),
equipped with a Nikon CCD camera DXM 1200 controlled by
Nikons image acquisition software ACT-1 and an Osram mercury lamp HBO103W/2 for epi-illumination.
Intracellular Determination of ⌬⌿m in Neuronal Cells—To
investigate the effects of glucose and Cr on the ⌬⌿m, several
aliquots of RPMI 1640 medium were supplemented with different solutions to achieve the following final concentrations: 10
or 40 mM glucose, or 40 mM glucose ⫹ 30 mM 2-DOG, or 40 mM
glucose ⫹ 5 ␮M FCCP, or 40 mM glucose ⫹ 5 mM Cr. The cells
were incubated 25 min at 37 °C with 4% CO2 and then the
medium was supplemented with the dye, JC-1 (50), to achieve a
final concentration of 5 ␮g/ml and the cells were incubated an
additional 15 min. After that, each coverslip was washed and
examined under the epifluorescence microscope using two
standard filter combination sets for green and red fluorescence
(B2 FITC blue filter combination: excitation 465– 495 nm;
dichroic filter 505 nm; emission 515–555 nm; and G-1B green
filter combination; excitation 541–551 nm; dichroic filter 565
nm; emission 590 nm long-pass) and a ⫻40 objective (Nikon
Plan Fluor ELWD DM; N.A. 0.6; W.D. 3.7-2.7 mm; PH2) at
fixed exposure times.
Intracellular Determination of ROS in Neuronal Cells—After
96 h of culture, neurobasal medium was replaced with RPMI
1640 medium, supplemented with 2 ␮M CM-H2DCFDA to
assess intracellular ROS formation. To investigate the effects of
glucose, several aliquots of medium were supplemented with
different solutions as described in the legend to Fig. 8. The cells
were incubated during 40 min at 37 °C and 4% CO2 and, after
that the cells were washed and examined under the epifluores-
Mitochondrial Kinases Prevent ROS Formation
FIGURE 2. H2O2 production, oxygen consumption, and membrane potential (⌬⌿m) in RBM are regulated
by hexose phosphorylation by mt-HK. A represents the effect of glucose phosphorylation using low concentrations of ATP formed by RBM on H2O2 generation, oxygen consumption, and ⌬⌿m. The reactions were
measured using the respiration buffer described under “Experimental Procedures” with 0.2 mg/ml Percollpurified RBM. The arrows indicate sequential additions of: Suc, 10 mM succinate; ADP, 0.15 mM ADP; Glc, 5 mM
glucose; and FCCP, 5 ␮M FCCP. The trace ⫺Glc indicates that glucose was omitted from the reaction. B represents the effect of 2-DOG phosphorylation using a high concentration of ATP added to the medium on H2O2
generation, oxygen consumption, and on ⌬⌿m in RBM. The arrows indicate sequential additions as described
for A, except that ATP was used instead of ADP and 2-DOG replaces Glc: ATP, 1 mM ATP; 2-DOG, 10 mM 2-DOG.
The trace ⫺2-DOG represents that 2-DOG was omitted from the reaction. C shows a control in which addition
of 1000 units of catalase removed H2O2 formed in state 4 respiration. The temperature was 28 °C. Similar results
were obtained with at least five different independent mitochondrial preparations.
37364 JOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 1. Glucose 6-phosphate induces ⌬⌿m hyperpolarization and stimulates H2O2 production when
mt-HK is phosphorylating 2-deoxyglucose in RBM. A shows ⌬⌿m measured with safranine O. Numbers
indicates the amount of Glc-6-P added to a final concentration of: 1, 10 ␮M; 2, 25 ␮M; 3, 50 ␮M; 4, 200 ␮M, and 5,
350 ␮M. The dotted line represents the ⌬⌿m measurement in which a single dose of 300 ␮M Glc-6-P was added.
B, the mt-HK activity was measured, as described under “Experimental Procedures,” by the amount of [3H]2DOG-6-P formed from [3H]2-DOG phosphorylation using ATP synthesized by oxidative phosphorylation carried out by RBM (open circles). The H2O2 formation was measured in parallel reactions by the Amplex Red
fluorescence method (closed circles) using 15 ␮M Amplex Red and 5 units/ml horseradish peroxidase. The
increases in ⌬⌿m induced by Glc-6-P are depicted as closed triangles. The reaction was measured using respiration buffer plus 4.5 ␮M rotenone, 10 mM succinate, 5 mM 2-DOG, 0.15 mM ADP, and 0.5 mg/ml Percoll-purified
RBM. The reaction time was 10 min at the temperature of 28 °C. Similar results were obtained with at least five
different independent mitochondrial preparations.
depolarization of ⌬⌿m, which was
progressively reversed by Glc-6-P.
ROS production in RBM is inversely
related to the mt-HK activity using
2-DOG and intramitochondrially
generated ATP as substrates (Fig.
1B). This occurs because Glc-6-P
inhibits the mt-HK activity, thus
increasing ⌬⌿m and H2O2 generation, due to the blockage of ADP
recycling through inner mitochondrial membrane. These findings are
in agreement with our previous
observation in RBM (25). In addition, it is important to note that the
increments in ⌬⌿m are strictly correlated with the inhibition of
mt-HK activity (Fig. 1B, open circle
and closed triangles), but the threshold to increase H2O2 generation is
higher than those observed for ⌬⌿m
(Fig. 1B, closed circles). This observation is in accordance with Korshunov and co-workers (11), which
demonstrated that large changes in
H2O2 generation occurs only within
a small range of ⌬⌿m values near
the maximum. Moreover, due to its
localization, mt-HK is ready to use
both intra- and extramitochondrial
sources of ATP. mt-HK activation
by glucose and intramitochondrially generated ATP leads to a stimulation of oxygen consumption,
decrease in the ⌬⌿m, and reduction
in H2O2 generation (Fig. 2A). However, as previously described by our
group, the effects of glucose in isolated RBM are transient, due to an
increase in Glc-6-P accumulation
and further inhibition of mt-HK.
Thus, to overcome these effects, we
evaluated the same parameters mentioned above in conditions where the
mt-HK would be fully activated, by
using 2-DOG and an external source
of ATP. 2-DOG-6P, the product of
this reaction has less ability to block
mt-HK activity and consequently
allows full activation of mt-HK led to
a permanent stimulation of oxygen
consumption rate, persistent ⌬⌿m
dissipation, and total blockage of
H2O2 generation (Fig. 2B).
mt-CK Activity Also Regulates
Mitochondrial H2O2 Generation in
RBM—Besides mt-HK, it is very
well known that a large portion of
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blockage of H2O2 generation was
achieved when limiting amounts
(0.2 mM) of Cr was added, resulting
in a subsequent increase in H2O2
production after all Cr is converted
to PCr (Fig. 3A, trace 2). A simultaneous transitory acceleration in respiration rate and a decrease in ⌬⌿m
of RBM were also observed after
inclusion of the limiting Cr concentration (data not shown). These
observations indicate that the electron transport chain responds to Cr
immediately when the [ATP]/
[ADP] ratio is high. All three parameters analyzed were not affected by
Cr and ATP supplementation in
RLM, which are devoid in mt-CK
activity (Fig. 3C).
H2O2 Generation in RBM Is Controlled by the Steady-state Kinetics
of Mitochondrial Kinases—In an
attempt to evaluate whether regulation of mitochondrial H2O2 generation by either mt-HK or mt-CK
activities obeys the steady-state
kinetics of these two enzymes, we
next measured ROS production
using different amounts of substrates (glucose, 2-DOG, or Cr) in
two ATP concentrations (0.15 and
1.0 mM). Fig. 4 shows that activation
FIGURE 3. H2O2 production, oxygen consumption, and membrane potential (⌬⌿m) in RBM are regulated of mt-kinases through their subby Cr phosphorylation by mt-CK. A represents the effect of Cr phosphorylation using a high concentration of strates reduces H O formation in a
2 2
ATP added to the medium on H2O2 generation, oxygen consumption, and ⌬⌿m in RBM. The reactions were
measured using the respiration buffer as described under “Experimental Procedures” with 0.2 mg/ml Percoll- substrate concentration-dependent
purified RBM. The arrows indicate the sequential additions of: Suc, 10 mM; ATP, 1 mM; Cr, 10 mM; and FCCP, 5 ␮M. manner. Interestingly, at 0.15 mM
The trace ⫺Cr represents that the Cr was omitted from the reaction. Trace 1 shown the effect of ATP on H2O2
ATP (open circles), the effect of difgeneration in the absence (⫺Cr) or presence (⫹Cr) of 10 mM Cr. Trace 2 shows the effect of different concentrations of Cr (arrow) on H2O2 generation in the presence of 1 mM ATP. The dashed line represents the rate of ferent concentrations of glucose on
H2O2 generation after addition of 0.2 mM Cr. Panel B represents the effect of Cr phosphorylation using a low H O generation exhibited a bipha2 2
concentration of ATP formed by RBM on ROS generation, oxygen consumption, and ⌬⌿m. The arrows indicate
the sequential additions of: Suc, 10 mM; ADP, 0.15 mM; Cr, 10 mM; and FCCP, 5 ␮M. C shows the effect of Cr sic pattern, reducing the initial rate
supplementation on H2O2 generation, oxygen consumption, and ⌬⌿m in RLM. The arrows indicate the sequen- of H2O2 production at low glucose
tial additions of: Suc, 10 mM; ATP, 1 mM ⫹ Cr ⫽ 5 mM; ADP, 1 mM; and Atrac, 0.1 mM (only in ⌬⌿m). In the O2 concentrations, in the 40 –90 ␮M
consumption trace of RLM the additions of ADP were: 0.15 and 0.35 mM. The dotted line in C means the basal
rate of H2O2 production in RLM. The temperature was 28 °C. Similar results were obtained with at least five range and reaching the lowest value
different independent mitochondrial preparations.
near 5 mM glucose. On the other
hand, H2O2 production was only
the total creatine kinase activity is compartmentalized in RBM modestly affected in higher glucose concentrations (above
between VDAC and ANT, supporting ADP recycling (51). Sev- 10 mM) (Fig. 4A, open circles). A possible explanation for this
eral lines of evidence have shown that Cr supplementation pre- effect would be that, above 10 mM glucose, the ratio of [Glcvented induction of mitochondrial permeability transition and 6-P]/[ATP] was high, causing competition between the sugROS formation (52, 53). As an attempt to gain insight into other ar-phosphate and the ATP for the catalytic site of the mt-HK
mechanisms involved in ADP recycling in mitochondria, in (54). Nevertheless, the general effect of 1.0 mM ATP (closed
subsequent experiments we investigated whether mt-CK acti- circles), on H2O2 production was more pronounced than at
vation by Cr would also affect physiological functions of RBM. 0.15 mM ATP (open circles). Also, at 1.0 mM ATP, the inhiFig. 3A shows that mt-CK activation by Cr is able to impair bition of ROS production was detected with lower amounts
H2O2 production of RBM by simultaneously accelerating the of glucose, 2-DOG, or Cr than in 0.15 mM ATP (Fig. 4, closed
oxygen consumption rate and decreasing ⌬⌿m. Identical circles).
When mt-HK was activated by the glucose analog 2-DOG,
results were obtained when Cr and low (0.15 mM) or high
(1 mM) ATP were used (Fig. 3B). Interestingly, a transient a similar pattern of inhibition of H2O2 formation was
Mitochondrial Kinases Prevent ROS Formation
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ATP concentration (Fig. 4C). At
1.0 mM ATP, the half-maximum
inhibition of H2O2 formation for
Cr was detected near 100 ␮M,
which is in the same concentration
range of the estimated Km for the
mammalian ubiquitous mt-CK
activity found in humans or mouse
(58). The half-maximal inhibition
of H2O2 production at lower ATP
concentrations (0.15 mM) was
FIGURE 4. H2O2 generation in RBM is controlled by the steady-state kinetics of mt-kinases. The initial rates observed at a much higher Cr conof ROS production were measured using the respiration buffer described under “Experimental Procedures” centration, near 700 ␮M (Fig. 4C,
with 0.2 mg/ml Percoll-purified RBM induced by 10 mM succinate. For mt-HK (A and B) or mt-CK (C) reactions,
different concentrations of glucose, 2-DOG, or Cr were added before the inclusion of 1 mM ATP (closed circles) open circles). This difference in
or after the conversion of 0.15 mM ADP to ATP by RBM (open circles). The transition to a state 3-like respiration apparent affinity of mt-CK for Cr
was started by the activation of mt-kinase substrates and the decreased initial rate was monitored at 28 °C.
is due in part to the low ATP conSimilar results were obtained with at least five different independent mitochondrial preparations.
centration present in the medium,
which tends to increase the apparent Km value for Cr owing of the synergistic properties of
mt-CK (56, 58).
The Equilibrium of mt-CK Reaction Sets the Rate of H2O2
Production in Isolated RBM—The previous experiments (Figs.
1 and 2) demonstrated that when mt-HK activity is directed to
Glc-6-P or 2-DOG-6-P formation, the production of H2O2 is
lowered (25). Similar results were obtained when mt-CK activity uses Cr and ATP to form PCr (Fig. 3). Nevertheless, the
reaction catalyzed by CK is reversible and this property is central to cellular energy buffering (59). The interplay between the
mitochondrial and cytosolic CK isoenzymes allows the maintenance of high local [ATP]/[ADP] ratios in the vicinity of cellular
ATPases for a maximal ⌬G of ATP hydrolysis, whereas in the
mitochondrial matrix, relatively low [ATP]/[ADP] ratios are
found stimulating the oxidative phosphorylation (59). Thus, in
Fig. 5, we evaluated whether changes in the [PCr]/[Cr] ratio
would modulate H2O2 generation due to influences on the
[ATP]/[ADP] ratios by RBM. Setting the total amount of [PCr]
⫹ [Cr] at 7 mM, it was observed that at a very low ratio, [PCr]/
FIGURE 5. Initial rate of H2O2 production depends on the [PCr]/[Cr] ratio.
The initial rate of H2O2 production was measured in respiration buffer as [Cr] (close to zero; i.e. 7 mM Cr), the ROS production was
described under “Experimental Procedures” with 0.15 mg/ml Percoll-purified completely abolished. When the PCr/Cr ratio approaches 2,
RBM induced by 10 mM succinate. Different ratios of [PCr]/[Cr] were obtained
the rate of H2O2 generation is close to 50% of maximum,
varying the concentration of either PCr or Cr from 0 to 7 mM. The total creatine
whereas
the maximum rate is detected at a [PCr]/[Cr] ratio
pool ([PCr] ⫹ [Cr]) was maintained at 7 mM. The reaction mixture was incubated with different [PCr]/[Cr] ratios and a steady rate of H2O2 production was of 7 (i.e. 7 mM PCr).
obtained. After this period, 0.4 mM ATP was added to activate mt-CK, inducing
Microcompartmentation of mt-CK Regulates H2O2 Generaa state 3-like respiration.
tion—To evaluate the effect of the specific location and mt-CK
activity levels on H2O2 production by RBM, we investigated the
achieved, but the concentration range of 2-DOG necessary effect of an externally added cytosolic MM-CK on H2O2 generto reach the lowest rates was higher (500 – 600 ␮M) than for ation in RLM. This was proposed based on the fact that liver
glucose (40 –90 ␮M) (Fig. 4B). These values are in agreement mitochondria is almost devoid of CK activity and as ADP recywith the expected concentration ranges of the HK activities cling plays an important role on mitochondrial H2O2 generausing these sugars as substrates (55).
tion, any changes in ADP levels due to the externally added
The kinetics of CK can be explained as a random-order, cytosolic MM-CK activity would impact H2O2 production (Fig.
rapid equilibrium kinetic mechanism (56). The result shown 6). In fact, the CK activity levels in RLM are much lower than in
in Fig. 4C indicates that mt-CK catalyzing the forward reac- RBM (Fig. 6A and Ref. 60). In Fig. 6B it was shown that H2O2
tion (i.e. in the direction of PCr formation) is able to substan- production by RLM was also inhibited by activation of the cytotially lower the rate of H2O2 production at the physiologi- solic MM-CK reaction working in the PCr formation (Fig. 6B,
cally relevant Cr concentration ranges in brain (1–5 mM Cr) closed triangles). Noteworthy is that even using an order of
(57). Similarly to the mt-HK, inhibition of H2O2 generation magnitude more cytosolic MM-CK activity levels, as those
performed with mt-CK was also achieved regardless of the present in native RBM, the rate of H2O2 production was not
Mitochondrial Kinases Prevent ROS Formation
fully inhibited, suggesting that localization of the mitochondrial isoform of CK in brain plays an important preventive antioxidant role.
Activation of mt-CK Reduces H2O2 Generation in Conditions
Mimicking Hyperglycemia in RBM—Several lines of evidence
show that, in hyperglycemic conditions, there is an overshooting of intracellular Glc-6-P levels due to GLUT and HK activities in a variety of cell types (25, 61– 63). We have previously
demonstrated that accumulation of Glc-6-P levels in neurons
promotes mitochondrial H2O2 production as mt-HK-dependent ADP recycling becomes impaired (25). As the experiments
presented in Figs. 2 and 3 show that activation of either mt-HK
or mt-CK consumes the ⌬⌿m and causes a decrease in H2O2
generation, we evaluated whether activation of mt-CK would
release ADP recycling from the impairment observed in hyperglycemic conditions where mt-HK was inhibited (high Glc-6P). Fig. 7 (trace 1) shows that after addition of succinate, there is
a progressive and steady accumulation of H2O2 that was transiently blocked by 0.2 mM ADP. In agreement with our previous
experiments, when 2-DOG was added to the assay medium, a
sustained blockage of H2O2 production was achieved by 0.2 mM
ADP (Fig. 7, trace 2), which was only reversed when the mt-HK
was inhibited by 1 mM Glc-6-P inclusion. Interestingly, ROS
generation due to interference with ADP recycling performed
by mt-HK was reversed when 5 mM Cr was added after Glc-6-P
inclusion, causing an immediate fall in H2O2 production (Fig. 7,
trace 3). Moreover, this result also indicates that the ATP pool
formed by oxidative phosphorylation is promptly available for
mt-CK even when mt-HK is inhibited by Glc-6-P (Fig. 7, trace
3). Finally, when both kinases substrates, 2-DOG and Cr, were
present in the assay medium, we observed a sustained blockage
of H2O2 production upon 0.2 mM ADP addition (Fig. 7, trace 4),
which was unaffected by mt-HK inhibition by 1 mM Glc-6-P. It
is also important to note that the addition of 2-DOG and Cr to
the reaction medium did not modify the rate of H2O2 formation
DECEMBER 8, 2006 • VOLUME 281 • NUMBER 49
FIGURE 7. mt-CK activity prevents H2O2 generation due to inhibition of
mt-HK. H2O2 production was measured in respiration buffer as described
under “Experimental Procedures” with 0.2 mg/ml Percoll-purified RBM
induced by succinate. Trace 1 shows the effect of ADP on H2O2 production. In
trace 2, the reaction medium contained 10 mM 2-DOG before the start of the
reaction with succinate. The dashed line indicates the time course of the reaction in the absence of Glc-6-P. Trace 3 shows H2O2 production as in trace 2, but
after the addition of Glc-6-P (G6P), it was added to Cr. In trace 4, the reaction
medium contained 10 mM 2-DOG and Cr before the start of the reaction with
succinate. The arrows indicate the sequential additions of: 10 mM Suc, 0.2 mM
ADP, 1 mM Glc-6-P, and 5 mM Cr. The figure shows a representative experiment. Similar results were obtained with at least four different independent
mitochondrial preparations.
after the addition of succinate (Fig. 7, trace 4), suggesting that
both substrates do not have intrinsic antioxidant properties
under our assay conditions. These results indicate that mt-CK
is able to support ADP recycling, thus reducing H2O2 formation, even when mt-HK activity is impaired due to an accumulation of Glc-6-P in hyperglycemic conditions.
mt-CK Activity Prevents ⌬⌿m Hyperpolarization and ROS Formation in Hyperglycemic Embryonic Rat Cortical Neurons—
It has been reported that hyperglycemia induces intracellular
ROS formation in different cell cultures (4, 20 –22) that, in cortical neurons, is related to an impairment of ADP recycling
through mt-HK (25). If this possibility was correct, this would
imply that in hyperglycemic conditions the intracellular ⌬⌿m
would be increased causing an elevation in ROS production due
to an imbalance of the mt-HK activity. To confirm this, primary
cultures of embryonic rat cortical neurons were incubated in
different concentrations of glucose (10 or 40 mM) with or without the mitochondrial kinases substrates (2-DOG or Cr) and
the evaluated intracellular ⌬⌿m measured by the fluorescence
ratio of JC-1 and ROS levels by the CMH2-DCFDA fluorescence. Indeed, we found a pronounced increase in the intracellular levels of ⌬⌿m in 40 mM glucose, whereas simultaneous
addition of 40 mM glucose ⫹ 30 mM 2-DOG did not cause mitochondrial hyperpolarization (Fig. 8A). Likewise, activation of
mt-CK in hyperglycemia, by previous incubation with 5 mM Cr,
prevented the increase in ⌬⌿m levels, showing a similar effect
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37367
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FIGURE 6. Localization of mt-CK activity is important to control H2O2 production. A, mt-CK activity was measured in Percoll-purified RBM (open bar) or
RLM (closed bar). In B, the initial rate of ROS production was measured in
respiration buffer described under “Experimental Procedures” with 0.3
mg/ml Percoll-purified RBM (open circle) or 0.5 mg/ml RLM (closed triangles)
induced by 10 mM succinate. The reaction mixture of RLM was supplemented
with increasing amounts of exogenous cytosolic MM-CK (closed triangles)
plus 5 mM Cr and a steady rate of H2O2 production was obtained. After this
period, 0.4 mM ATP was added to activate mt-CK, inducing a state 3-like respiration. The maximal rate of H2O2 generation (state 4 respiration) in mt-CKdepleted RLM or RBM was used as 100%. Bars represent mean ⫾ S.E. of four
independent preparations and similar results were obtained with at least four
different independent mitochondrial preparations.
Mitochondrial Kinases Prevent ROS Formation
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NADH availability to the electron
transport chain (4, 5, 20–22, 65). In
previous work (25), our group showed
that ADP re-cycling through the
inner mitochondrial membrane, performed by mt-HK, plays an essential preventive antioxidant role by
decreasing ⌬⌿m. In a high glucose
medium condition, we observed an
accumulation of intracellular Glc-6-P
(25), indicating that excess pyruvate
has reached the mitochondria to
be oxidized. However, as Glc-6-P
FIGURE 8. Mitochondrial hyperpolarization and ROS formation induced by high glucose concentrations inhibits mt-HK, this would disare prevented by mt-CK activity in rat cortical neurons. A, ⌬⌿m was measured by the ratio between red/ rupt ADP recycling, as demongreen fluorescence of the probe JC-1 of cultured cortical neurons from 14-day-old Wistar rat embryos incubated with RPMI medium containing 10 mM (10 Glc) or 40 mM (40 Glc) glucose; 40 mM Glc plus 30 mM 2-DOG (40 strated previously (25), favoring
Glc 30 DOG); 40 mM Glc plus 5 ␮M FCCP (40 Glc FCCP); and 40 mM Glc plus 5 mM Cr (40 Glc 5 Cr). B, quantification the increase in ⌬⌿m. Thus, both
of green fluorescence microscopy images (arbitrary fluorescence intensity (AUF)) of CM-H2DCFDA staining increased pyruvate oxidation and
reflecting the intracellular ROS levels. The glucose and Cr concentrations are the same as for A. When antimycin
A was used (Ant A) it was added to a final concentration of 5 ␮M. Data represent mean ⫾ S.E. corresponding to the impairment of mitochondrial
four different experiments. The fluorescence microscopy images utilized to quantify ⌬⌿m and ROS formation ADP re-cycling, due to a blockage of
in A and B are available as supplemental figures S1–S2.
mt-HK activity, would lead to ⌬⌿m
hyperpolarization and reduce electo 5 ␮M FCCP addition (Fig. 8A, see also supplemental materials tron flux in the electron transport chain, inducing ROS generation. In the present work we demonstrate that the activities of
Fig. 1).
Regarding the cellular ROS formation, we noticed that 40 mM mitochondrial kinases (mt-HK and mt-CK) in RBM contribute
glucose in the medium increased ROS levels as indicated by to regulation of ROS generation through the ADP re-cycling
CMH2-DCFDA fluorescence, which was reduced by simultane- mechanism. Particularly, this mechanism is relevant in high
ous addition of 5 mM Cr (Fig. 8B, see also supplemental mate- glucose medium, where the mt-HK is not fully active (Figs. 1, 2,
rials Fig. 2). This result raises the possibility that the presence of 4, 8, and 9 and Ref. 25) but the impairment of ROS generation
Cr per se in the cells may be acting as an antioxidant scavenger. can be achieved by mt-CK activity. In fact in RLM, which is
To test this hypothesis, we simulated a pro-oxidant condition devoid of mt-CK, Cr had no effect on ⌬⌿m, O2 consumption, or
by adding 2.5 ␮M antimycin A, an inhibitor of the electron H2O2 generation (Fig. 3C), suggesting that the preventive antitransport chain at complex III and a known powerful inducer of oxidant role of Cr depends on the presence of mt-CK. Thus, the
superoxide formation, to the low glucose medium (10 mM). As effects observed in Fig. 8A (see also Ref. 25), indicating that
expected, antimycin A caused a significant raise in ROS forma- 2-DOG decreases cellular ⌬⌿m and prevents ROS generation,
tion that was not affected by the presence of 5 mM Cr, indicating could be interpreted either by decreasing pyruvate oxidation,
that the observed effect of Cr on intracellular ROS generation because 2-DOG competes for glucose phosphorylation by
was not derived from scavenger activity of this molecule (Fig. mt-HK, or by activating the ADP re-cycling through the ANT8B, see also supplemental materials Fig. 2). Taken together, the VDAC complex. In our previous work (25), we could not disresults presented in Fig. 8 indicate that activation of rat brain tinguish clearly between these two possibilities. However, the
mt-CK in hyperglycemia is sufficient to avoid hyperpolariza- main contribution of the present work is that when pyruvate
tion, preventing electron leakage and ROS formation due to a oxidation is high (high glucose medium) the activation of ADP
steady-state ADP recycling through the inner mitochondrial re-cycling by mt-CK activity, induced by Cr, is sufficient to
reduce both the ⌬⌿m and ROS generation. These observations
membrane in embryonic rat cortical neurons.
can be concluded based on experiments where Cr supplemenDISCUSSION
tation abolishes ROS generation in high glucose medium (Figs.
Hyperglycemia is associated with several metabolic dysfunc- 3, 7, and 8). One would speculate that Cr might cause an inhitions, such as diabetes and sepsis (22, 23), and excessive glucose bition of glucose metabolism resulting in a decrease in both
can be harmful to tissues (3–5). ROS production by mitochon- ⌬⌿m and ROS production. Based on the literature, we suggest
dria through the electron transport chain is a causal link that Cr does not change the glycolytic flux and so the pyruvate
between high glucose and the main pathways responsible for levels. Conversely, conditions where glucose metabolism are
hyperglycemic damage (4, 8, 20, 21). Even a short-term expo- impaired would reduce pyruvate oxidation in mitochondria,
sure of neurons to hyperglycemia produces oxidative damage avoiding ⌬⌿m hyperpolarization and ROS generation. Noteand apoptosis in nervous cells and these effects can be pre- worthy, if Cr directly decreased glucose metabolism, we would
vented by antioxidants (5). Although the mechanism of glucose expect a reduction in ROS production independently from the
toxicity is not completely understood, it has been proposed that CK activity. However, several reports showed that Cr has no
increased pyruvate oxidation would stimulate mitochondrial inhibitory effects on glucose metabolism in skeletal muscle and
respiratory chain and O2. radicals production by an excess of brain, because: 1) dietary supplementation of Cr to rats caused
Mitochondrial Kinases Prevent ROS Formation
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It is known that several neurological disorders, with different primary defects, often converge to display similar impairments in cellular
energy metabolism in the brain (33).
In these instances, the intracellular
ATP concentration is decreased,
resulting in cytosolic accumulation
of Ca2⫹ and ROS formation. A common feature among these disorders
is the impairment of brain Cr
metabolism, i.e. decrease in total Cr
and PCr concentration, CK activity,
and/or Cr transporter content (67,
68). In cultured rat neurons, as well
as in astrocytes, Cr protected
against glutamate, ␤-amyloid, and
3-nitropropionic acid toxicity (18,
19, 69). Furthermore, reduced neuronal damage and ROS formation
FIGURE 9. Schematic representation of the proposed mechanism by which mt-CK regulates oxygen con- were observed when cultures were
sumption, ⌬⌿m, and ROS production in mitochondria during hyperglycemic conditions. In brain and
other tissues, HK is bound to the outer mitochondrial membrane through an association with the VDAC. The administered with Cr at least 6 h
octameric form of mt-CK localizes in the intermembrane space, through an association to VDAC and ANT. The before 3-hydroxyglutarate treatfigure represents mitochondria under a hyperglycemic condition and Cr supplementation, in which mt-HK is
inhibited by Glc-6-P accumulation (Fig. 1 and Ref. 25) but ADP re-cycling is maintained by mt-CK activity, ment (53). In isolated mitochondria,
regulating oxygen consumption, ⌬⌿m, and ROS generation. Bold arrows and solid lines indicate a high flux of the inhibition of the MPT by CK
metabolites, whereas gray dashed lines represent low flux. Numbers represent the complexes of respiratory substrates seems to be an important
electron chain. UQ, ubiquinone; Cyt c, cytochrome c; SOD, superoxide dismutase; GPx, glutathione peroxidase.
blocker of both necrotic and apoptotic cell death (42, 52). Although Cr
no changes in basal nor insulin-glucose uptake in rats (64). 2) protected the brain against malonate-induced hydroxyl radical
Brain glycolytic flux is enhanced in hyperglycemia, not affecting generation, due to increased high energy phosphate reserves
PCr to Cr conversion under ischemia (65). 3) Stimulation in (17), a direct involvement of Cr with cellular and mitochondrial
brain functional activity led to an increase of glycolytic flux, ROS generation was not yet established. Therefore, the neuroparallel to PCr utilization and Cr accumulation (66). Thus, we protection of Cr against Huntington disease could involve the
cannot support the hypothesis that decrease of ⌬⌿m and ROS partial restoration of neuronal ROS homeostasis mediated by
production in our conditions (at 40 mM glucose ⫹ 5 mM Cr) is mt-CK activity. The involvement of mt-CK as modulator of the
due to reduced pyruvate levels (Fig. 8). Also, succinate-induced MPT by Cr was challenged by data showing that Cr still exerts
ROS generation in isolated brain mitochondria is not affected neuroprotective effects in mt-CK knock-out mice, suggesting
by Cr (Figs. 3B and 7) and is drastically reduced when ADP that these effects are not mediated by mt-CK to inhibit the MTP
re-cycling is performed by mt-CK activation through ATP (70). However, these data may be explained by the fact that
addition, simulating a state 3 respiration. These observations enough levels of cytosolic CK activity were found in mt-CK
reinforce the concept that the preventive antioxidant role of Cr knock-out mice to support Cr phosphorylation. The cytosolic
is mediated by mt-CK activation, allowing the ADP re-cycling. isoform would compensate the absence of the mitochondrial
The possibility that Cr itself would be exerting scavenger anti- enzyme assuring the maintenance of Cr phosphorylation and
oxidant effects seems not to be the case, as state 4 H2O2 gener- ADP production, supporting the reduction of ROS generation
ation in isolated brain mitochondria (Figs. 3B and 7) and neu- (Fig. 6B and Ref. 71). Supporting this idea is that the double
ronal ROS generation induced by antimycin A (Fig. 8B) were knock-out mice lacking both isoforms of CK caused cognitive
both unaffected by Cr supplementation. Altogether, these data dysfunctions and spatial learning. Interestingly, the absence of
support the notion that mitochondrial ROS production the mt-CK isoform caused a compensatory increase in the
induced by high glucose oxidation can be related to the rate of PCr/Cr ratio induced by Cr supplementation in both cortex and
ADP re-cycling. Therefore, any event that regulates, directly or cerebellum (70). Together with our results, the change in the
indirectly, the electron flux, would modulate ROS generation. PCr/Cr ratio in the mt-CK knock-out mice indicates that the
In this way, Nishikawa and colleagues (4) showed that pharmaco- cytosolic CK would be sufficient to allow mitochondrial ADP
logical uncoupling or overexpression of uncoupling proteins in recycling. A common explanation of the protective effects of Cr
hyperglycemia abrogated ROS generation. Taking into account preventing or ameliorating the features of neurodegenerative
this information, the main contribution of the present work is not diseases is based on the improvement of the energy charge of
only to support the Nishikawa proposal but also to suggest that the neural cells, evaluating the PCr/Cr ratio. Thus, based in our
regulation of ⌬⌿m through mitochondrial kinase activation leads results, we propose in Fig. 9 that mt-CK controls mitochondrial
ROS generation through the ADP cycling system. This mechato impairment of hyperglycemia-induced oxidative stress.
Mitochondrial Kinases Prevent ROS Formation
Acknowledgments—We express our gratitude to Dr. Roger Castilho
(Unicamp, SP, Brasil) for valuable contributions and helpful discussions as well as for the kind supply of Amplex Red. We also thank Dr.
Leopoldo de Meis for the laboratory facilities.
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