Experimental Neurology 196 (2005) 112 – 119
www.elsevier.com/locate/yexnr
CoQ10 therapy attenuates amyloid h-peptide toxicity in brain
mitochondria isolated from aged diabetic rats
Paula I. Moreira a, Maria S. Santos a, Cristina Sena b, Elsa Nunes b,
Raquel Seiça b, Catarina R. Oliveira c,*
a
Center for Neuroscience and Cell Biology, Department of Zoology-Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra, Portugal
b
Center for Neuroscience and Cell Biology, Institute of Physiology-Faculty of Medicine, University of Coimbra, 3004-517 Coimbra, Portugal
c
Center for Neuroscience and Cell Biology, Institute of Biochemistry-Faculty of Medicine, University of Coimbra, 3004-517 Coimbra, Portugal
Received 26 April 2005; revised 12 July 2005; accepted 18 July 2005
Available online 29 August 2005
Abstract
Using brain mitochondria isolated from 20-month-old diabetic Goto – Kakizaki rats, we evaluated the efficacy of CoQ10 treatment against
mitochondrial dysfunction induced by Ah1 – 40. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (RCR
and ADP/O ratio), transmembrane potential (DCm), repolarization lag phase, repolarization and ATP levels and the capacity of mitochondria
to produce hydrogen peroxide. We observed that 4 AM Ah1 – 40 induced a significant decrease in the RCR and ATP content and a significant
increase in hydrogen peroxide production. CoQ10 treatment attenuated the decrease in oxidative phosphorylation efficiency and avoided the
increase in hydrogen peroxide production induced by the neurotoxic peptide. These results indicate that CoQ10 treatment counteracts brain
mitochondrial alterations induced by Ah1 – 40 suggesting that CoQ10 therapy can help to avoid a drastic energy deficiency that characterizes
diabetes and Alzheimer’s disease pathophysiology.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Amyloid h-peptide; Brain; Coenzyme Q10; Diabetes; Mitochondria; Neuroprotection
Introduction
Type 2 diabetes accounts for about 90% of the existing
cases of diabetes and is characterized by defects in both
insulin action and secretion (Gavin et al., 1997). Many
studies demonstrated that diabetes produces molecular,
cellular, morphological and behavioral changes in the
central nervous system (CNS) (Biessels et al., 2002).
Diabetes is often associated with mitochondrial diseases
characterized by defects in the mitochondrial genome
(Gerbitz et al., 1995). Mitochondria play a central role in
the development of type 2 diabetes by regulating energy
balance and the generation of reactive oxygen species
(ROS) (Wallace, 1999; Lowell and Shulman, 2005).
* Corresponding author. Fax: +351 239 822776.
E-mail address: [email protected] (C.R. Oliveira).
0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2005.07.012
Defects in energy metabolism are a consistent feature of
AD-affected brain. In Alzheimer’s disease (AD), a major
imbalance between glucose and oxygen consumption has
been found in the incipient stage, whereas in the advanced
stage, both glucose and oxygen consumption are diminished
(Hoyer, 1991). This could be a consequence of deviant
insulin action or brain insulin receptor function, which can
affect brain energy metabolism (Hoyer, 1991). These
abnormalities in insulin metabolism may account for the
pathological changes (formation of senile plaques and
neurofibrillary tangles) found in Alzheimer’s disease (Frolich et al., 1998; Hoyer, 2002).
Several lines of evidence suggest that amyloid deposition
in the brain contributes to neuronal degeneration in AD
(Selkoe, 2001) just as amyloid formation in the pancreas is
believed to contribute to h-cell loss in type 2 diabetes
(Höppener et al., 2000). Hoyer (1998) argues that in lateonset AD, a disturbance in the control of neuronal glucose
P.I. Moreira et al. / Experimental Neurology 196 (2005) 112 – 119
metabolism consequent to impaired insulin signaling
strongly resembles the pathophysiology of type 2 diabetes
in nonneural tissue. Furthermore, several epidemiological
studies have established that diabetes increases the risk of
dementia in general (Hassing et al., 2004) and AD in
particular (Leibson et al., 1997; Ott et al., 1999).
The observation that oxidative stress is increased in AD
and diabetes has generated the notion that antioxidants and
other regulators of oxidative stress may protect against
oxidative damage. Coenzyme Q is a potent antioxidant and
free radical scavenger as well as membrane stabilizer (Beyer
and Ernster, 1990). Besides its antioxidant properties,
coenzyme Q has an important function in mitochondrial
bioenergetics. It participates as a cofactor of dehydrogenases
in the transport of electrons and protons as well as in ATP
production (Mitchell, 1991; Crane and Navas, 1997).
Several animal models are available for experimental
investigation of type 2 diabetes. One of those models is
the Goto –Kakizaki (GK) rat: a nonobese, spontaneously
diabetic animal (Serradas et al., 1998) produced by selective
breeding of Wistar rats and first characterized by Goto and
Kakizaki (Goto and Kakizaki, 1981).
Previous data from our laboratory have shown that
diabetes-related mitochondrial dysfunction is exacerbated
by aging and/or by the presence of the neurotoxic Ah
peptides (Moreira et al., 2003). In this line, this study was
aimed to evaluate the efficacy of coenzyme Q10 (CoQ10)
treatment against the mitochondrial dysfunction induced by
the neurotoxic agent Ah1 – 40. For this purpose, we used
brain mitochondria isolated from 20-month-old GK rats and
the following mitochondrial parameters were examined:
respiratory indexes (RCR and ADP/O ratio), transmembrane potential, lag phase of repolarization, repolarization
and ATP levels and the production of hydrogen peroxide.
Studies in an aged animal model of type 2 diabetes may
identify a key metabolic feature common to that disorder
and late-onset AD.
Materials and methods
Materials
Ah1 – 40 was obtained from Bachem AG (Bubendorf,
Germany). Protease (Subtilisin, Carlsberg) type VIII,
CoQ10 and soybean oil were obtained from Sigma
(Portugal). Digitonin was obtained from Calbiochem. All
the other chemicals were of the highest grade of purity
commercially available.
113
access to water and powdered rodent chow (diet C.R.F. 20,
Charles River, France). Glucose tolerance tests were used to
select GK rats for study. GK rats were randomly divided
into three groups: group 1: soybean oil (vehicle solution) (2
ml/kg i.p.), group 2: CoQ10 (20 mg/kg i.p.) and group 3:
GK rats without treatment. Animals were injected each 48 h
during 7 weeks. Experiments were conducted 24 h after the
last injection. Adhering to procedures approved by the
Institutional Animal Care and Use Committee, the animals
were sacrificed by cervical displacement and decapitation.
Biochemical analysis
Blood glucose was determined immediately after animals
sacrifice by a glucose oxidase reaction, using a glucometer
(Glucometer-Elite, Bayer) and compatible reactive tests.
Hemoglobin A1C (HbA1c) levels were determined through
ionic change chromatographic assay (Abbott Imx Glicohemoglobin, Abbott Laboratories, Portugal). Levels of urinary
8-hydroxydeoxyguanosine (8-OHdG) were determined
using a commercially available enzyme-linked immunosorbent assay (Bioxytech 8-OHdG-EIA Kit, Oxis Health
Products, Portland, USA).
Isolation of brain mitochondria
Brain mitochondria were isolated from male Wistar and
GK rats by the method of Rosenthal et al. (1987), with
slight modifications, using 0.02% digitonin to free mitochondria from the synaptosomal fraction. In brief, a rat was
decapitated, and the whole brain minus the cerebellum was
rapidly removed, washed, minced and homogenized at 4-C
in 10 ml of isolation medium (225 mM mannitol, 75 mM
sucrose, 5 mM Hepes, 1 mM EGTA, 1 mg/ml bovine serum
albumin, pH 7.4) containing 5 mg of the bacterial protease.
Single brain homogenates were brought to 30 ml with the
isolation medium and then centrifuged at 2000g for 3
min. The pellet, including the fluffy synaptosomal layer,
was resuspended in 10 ml of the isolation medium
containing 0.02% digitonin and centrifuged at 12,000g
for 8 min. The brown mitochondrial pellet without the
synaptosomal layer was then resuspended again in 10 ml of
medium and recentrifuged at 12,000g for 10 min. The
mitochondrial pellet was resuspended in 300 Al of
resuspension medium (225 mM mannitol, 75 mM sucrose,
5 mM Hepes, pH 7.4). Mitochondrial protein was determined by the biuret method calibrated with bovine serum
albumin (Gornall et al., 1949).
Membrane potential (DWm) measurements
Animals and experimental protocol
Male GK and control Wistar rats with 20 months of age
were housed in our animal colony (Laboratory Research
Center, University Hospital, Coimbra, Portugal). They were
maintained under controlled light and humidity with free
The mitochondrial transmembrane potential was monitored by evaluating the transmembrane distribution of TPP+
(tetraphenylphosphonium) with a TPP+-selective electrode
prepared according to Kamo et al. (1979) using a Ag/AgCl2
electrode as reference.
114
P.I. Moreira et al. / Experimental Neurology 196 (2005) 112 – 119
Reactions were carried out in a chamber with magnetic
stirring in 1 ml of reaction medium (100 mM sucrose, 100
mM KCl, 2 mM KH2PO4, 5 mM Hepes, 10 AM EGTA, pH
7.4) supplemented with 3 AM TPP+. The experiments were
started by adding 5 mM succinate to mitochondria in
suspension at 0.8 mg protein/ml. After a steady-state
distribution of TPP+ had been reached (ca. 2 min of
recording), Ca2+ was added and DCm recorded. Membrane
potential was estimated from the decrease of TPP+ concentration in the reaction medium as described elsewhere
(Moreno and Madeira, 1991). Homogenates were incubated
with 4 AM Ah1 – 40 for 5 min before succinate addition.
were incubated at 37-C with 10 mM succinate in 1.5 ml of
phosphate buffer, pH 7.4, containing 0.1 mM EGTA, 5 mM
KH2PO4, 3 mM MgCl2, 145 mM KCl, 30 mM Hepes, 0.1
mM homovalinic acid and 6 U/ml horseradish peroxidase in
the presence or absence of 4 AM Ah1 – 40. The incubation
was stopped at 15 min with 0.5 ml of cold 2 M glycine
buffer containing 25 mM EDTA and NaOH, pH 12. The
fluorescence of supernatants was measured at 312 nm as
excitation wavelength and 420 nm as emission wavelength.
The rate of H2O2 production was calculated using a standard
curve of H2O2.
Statistical analysis
Mitochondrial respiration
Results are presented as mean T SEM of the indicated
number of experiments. Statistical significance was determined using the one-way ANOVA test for multiple
comparisons, followed by the post hoc Tukey –Kramer test.
A P value < 0.05 was considered significant.
Oxygen consumption of isolated mitochondria was
monitored polarographically with a Clark oxygen electrode
(Estabrook, 1967) connected to a suitable recorder in a 1 ml,
thermostated, water-jacketed closed chamber, with magnetic
stirring. The reactions were carried out at 30-C in 1 ml of
the reaction medium with 0.8 mg protein. Homogenates
were incubated with 4 AM Ah1 – 40 for 5 min before
succinate addition.
Results
CoQ10 treatment decreases glycemia levels
Analysis of ATP
To confirm diabetes mellitus in GK rats glycemia,
glycated hemoglobin, body weight and the urinary levels of
8-OHdG were determined (Table 1). The percentage of
glycated hemoglobin (HbA1C), glycemia and levels of 8OHdG were significantly higher in GK rats (66.38% T 4.89,
158.75% T 10.52, 172.74% T 12.1, respectively) when
compared with control animals. However, body weight was
significantly lower in diabetic animals (49.54% T 2.18) when
compared with control animals. CoQ10 as well as soybean oil
(vehicle solution) induced a slight improvement in glycemia
levels (17.45% T 1.01 and 13.05% T 2.83, respectively) when
compared to GK rats without treatment. However, both
CoQ10 and soybean oil did not induce any significant
alteration in body weight, HbA1C and 8-OHdG levels when
compared to GK rats without treatment (Table 1).
At the end of the DCm experiments, each mitochondrial
suspension was rapidly centrifuged at 14,000 rpm for 6 min
with 0.3 M perchloric acid. The supernatants were neutralized
with 10 M KOH in 5 M Tris and centrifuged at 14,000 rpm for
5 min. The resulting supernatants were assayed for ATP by
separation in a reverse-phase high performance liquid
chromatography. The chromatography apparatus was a
Beckman-System Gold, consisting of a 126 Binary Pump
Model and 166 Variable UV detector controlled by a
computer. The detection wavelength was 254 nm, and the
column was a Lichrospher 100 RP-18 (5 Am) from Merck. An
isocratic elution with 100 mM phosphate buffer (KH2PO4)
pH 6.5 and 1.0% methanol was performed with a flow rate of
1 ml/min. The required time for each analysis was 6 min.
Measurement of H2O2 production
CoQ10 avoids a drastic decrease in respiratory control
ratio (RCR) induced by the amyloid b-peptide
The rate of hydrogen peroxide (H2O2) production was
measured fluorimetrically using a modification of the
method described by Barja (1999). Briefly, mitochondria
The RCR is defined as the ratio between the states 3
(consumption of oxygen in the presence of substrate and
Table 1
Characterization of Wistar control and GK diabetic rats
Wistar
GK
GK vehicle
GK CoQ10
Glycemia (mg/dl)
HbA1C (%)
114.9 T
297.3 T
258.5 T
245.4 T
5.01
8.34
7.87
7.52
28.1
16.1***
7.6**
19.1**
T
T
T
T
0.08
0.33***
0.06***
0.35***
Body weight (g)
760.9
383.9
387.7
398.6
T
T
T
T
28.0
11.4***
3.32***
9.03***
8OHdG (ng/ml)
141.6
386.2
303.2
303.8
T
T
T
T
18.8
35.9***
49.9***
60.3***
Data shown represent mean T SEM from 6 animals for each condition studied. ***P < 0.001; **P < 0.01, statistically significant when compared with Wistar
control rats.
P.I. Moreira et al. / Experimental Neurology 196 (2005) 112 – 119
ADP) and 4 (consumption of oxygen after ADP has been
consumed) of respiration. At basal conditions, no
significant alterations were induced by CoQ10 treatment
(Fig. 1A). The presence of 4 AM Ah1 – 40 induced a
significant decrease in all the conditions tested, however,
less pronounced in diabetic animals treated with both
CoQ10 (15.09% T 2.03) and soybean oil (17.12% T
1.01) than in GK rats without treatment (19.63% T 0.24),
when compared to GK rats without treatment and in the
absence of the neurotoxic peptide (Fig. 1A).
ADP/O ratio, an indicator of oxidative phosphorylation
efficiency, is expressed by the ratio between the amount of
ADP added and the oxygen consumed during state 3
respiration.
We did not observe any significant alteration in this
respiratory parameter induced by CoQ10 treatment and/or
the presence of 4 AM Ah1 – 40 (Fig. 1B).
115
CoQ10 treatment prevents the decrease in oxidative
phosphorylation efficiency induced by amyloid b-peptide
DCm is fundamental for the phenomenon of oxidative
phosphorylation, the conversion of ADP to ATP via ATP
synthase. Mitochondrial respiratory chain pumps H+ out of
the mitochondrial matrix across the inner mitochondrial
membrane. The H+ gradient establishes an electrochemical
potential (Dp) resulting in a pH (DpH) and a voltage
gradient (DCm) across the mitochondrial inner membrane.
We observed that neither CoQ10 treatment nor Ah1 – 40
exposure induced any significant alteration in the DCm as
well as in the repolarization lag phase (Table 2). In this
study, repolarization lag phase is defined as the time
required for mitochondria to phosphorylate the ADP added
to the reaction medium. Concerning the repolarization level
(capacity of mitochondria to re-establish the DCm, after
ADP phosphorylation), we observed that the presence of
Ah1 – 40 induced a significant decrease of this parameter in
GK rats without treatment when compared with GK rats at
basal conditions (9.55% T 0.97) as well as when compared
with GK rats treated with CoQ10 and soybean oil (10.92% T
0.08 and 11.64% T 0.95, respectively) (Table 2). Mitochondria isolated from diabetic animals treated with soybean oil
and CoQ10 did not suffer any significant alteration of this
parameter when exposed to the neurotoxic peptide.
At basal conditions, CoQ10 and soybean oil did not
induce any significant alteration on ATP content of brain
mitochondria. However, the presence of Ah1 – 40 induced a
significant decrease of this parameter in mitochondria
isolated from diabetic rats without treatment (10.65% T
3.7) when compared with GK rats at basal condition.
However, CoQ10 treatment prevented the decrease in ATP
levels induced by Ah1 – 40 (Table 2).
CoQ10 treatment prevents the increase in hydrogen
peroxide levels induced by amyloid b-peptide
Fig. 1. Effect of CoQ10 treatment and h-amyloid exposure on
mitochondrial respiratory indexes. (A) Respiratory control ratio (RCR),
(B) ADP/O ratio. Freshly isolated brain mitochondria (0.6 mg) in 1 ml
of the standard medium supplemented with 2 AM rotenone were
energized with 5 mM succinate. Isolates were incubated with 4 AM
Ah1 – 40 for 5 min, at 30-C, before mitochondria energization. #P <
0.05, statistically significant when compared to GK rats. $$P < 0.01;
$P < 0.05 statistically significant when compared with GK rats treated
with the vehicle solution. &&&P < 0.001; &&P < 0.01, statistically
significant when compared with GK rats treated with CoQ10. Data
shown represent mean T SEM from 6 animals for each condition
studied.
The production of H2O2 by mitochondria gives an
indication of the propensy of mitochondria to originate
and/or exacerbate oxidative stress. We observed that
diabetic rats mitochondria exposed to Ah1 – 40 produced a
significantly higher level of H2O2 when compared with
brain mitochondria isolated from CoQ10 treated animals in
the presence (127.15% T 2.5) or absence (83.68% T 4.4) of
Ah1 – 40 indicating that CoQ treatment avoids the increase of
H2O2 production (Fig. 2).
Discussion
Neuron viability and defense against neurodegenerative
injury can be achieved by targeting mitochondrial function
to reduce oxidative stress, increase mitochondrial defense
mechanisms or promote energetic metabolism. In this study,
we observed that GK rats submitted to CoQ10 treatment
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P.I. Moreira et al. / Experimental Neurology 196 (2005) 112 – 119
Table 2
Effect of CoQ10 treatment and h-amyloid exposure on mitochondrial transmembrane potential (DCm), repolarization level, repolarization lag phase and ATP
content of brain mitochondria isolated from diabetic rats
DCm (
Wistar
GK
GK vehicle
GK CoQ10
GK + Ah1 – 40
GK vehicle + Ah1 – 40
GK CoQ10 + Ah1 – 40
178.1
180.5
182.0
180.7
165.5
170.9
180.3
T
T
T
T
T
T
T
mV)
3.09
3.35
2.87
2.89
3.58
3.19
3.21
Repolarization level (
147.7
152.8
156.0
155.1
138.2
145.2
143.6
T
T
T
T
T
T
T
2.23
2.14
2.96
3.50
3.62#,*,$,&&
3.49
3.61
mV)
Repolarization lag phase (min)
0.668
0.676
0.613
0.629
0.721
0.670
0.726
T
T
T
T
T
T
T
0.043
0.056
0.038
0.027
0.075
0.039
0.067
ATP content (nmol/mg)
318.0
309.8
296.1
293.2
276.8
231.5
294.2
T
T
T
T
T
T
T
14.72
18.85
16.81
12.08
7.40#
19.70*
12.28
Freshly isolated brain mitochondria (0.6 mg) in 1 ml of the reaction medium supplemented with 3 AM TPP+ and 2 AM rotenone were energized with 5 mM
succinate. *P < 0.05 statistically significant when compared with Wistar rats. #P < 0.05 statistically significant when compared with GK rats. $P < 0.05
statistically significant when compared with GK rats treated with the vehicle solution. &&P < 0.01 statistically significant when compared with GK rats treated
with CoQ10. Data shown represent mean T SEM from 6 animals for each condition studied.
possess brain mitochondria more resistant to Ah1 – 40
neurotoxicity.
The characterization of diabetes mellitus in GK rats was
performed by determining glycemia, glycated hemoglobin
(HbA1C), urinary levels of 8-OHdG and body weight. A
significant increase in glycated hemoglobin, glycemia and
8-OHdG levels (DNA oxidation) was observed in GK rats
when compared with control animals (Table 1). A considerable amount of evidence suggests that oxidative stress
may play an important role in the pathogenesis and
complications of diabetes (Baynes, 1991, 1995; Seghrouchni et al., 2002). Different mechanisms can contribute
to the enhanced oxidative stress in diabetic patients, in
particular in subjects with poor glycemic control and
hypertriglyceridemia (Kitahara et al., 1980; Armstrong and
Al-Awadi, 1991; Leinonen et al., 1997; Collins et al., 1998;
Fig. 2. Effect of CoQ10 treatment and h-amyloid exposure on H2O2
production. Freshly isolated brain mitochondria were incubated at 0.2 mg
protein/ml under standard conditions as described in Materials and
methods. *P < 0.05 statistically significant when compared with Wistar
rats. #P < 0.05 statistically significant when compared with GK rats. $$P <
0.01 statistically significant when compared with GK rats in the presence of
4 AM Ah1 – 40. Data shown represent mean T SEM from 6 animals for each
condition studied.
Hinokio et al., 1999). Increased urinary excretion and higher
levels of 8-OHdG in mononuclear leukocyte DNA have
been found in types 1 and 2 diabetic patients (Dandona et
al., 1996; Leinonen et al., 1997; Hinokio et al., 1999).
Studies with the comet assay have shown increased levels of
DNA breakage in peripheral blood cells of diabetic patients
with poor glycemic control, but not in patients with normal
glycemia (Collins et al., 1998; Anderson et al., 1998).
Furthermore, we observed that GK rats present a lower body
weight when compared with control animals (Table 1). The
pathogenesis of diabetes in the GK rat involves an impaired
insulin secretion, insulin resistance, an abnormal glucose
metabolism as well as an impaired ontogenetic development
of pancreatic islet cells. However, in contrast to many other
rodent models of type 2 diabetes, GK rats do not become
obese and do not develop hyperlipidemia (for review see
Janssen et al., 2004).
Accumulating evidence suggests that mitochondrial
dysfunction is intimately associated with diabetes and AD
pathophysiology. Studies from our laboratory showed that
Ah inhibits the respiratory chain complexes and reduces
ATP levels in PC12 cells (Pereira et al., 1998, 1999). We
also reported that Ah exacerbates the Ca2+-induced opening of the mitochondrial permeability transition in isolated
brain mitochondria, without inducing the permeability per
se (Moreira et al., 2001, 2002). In addition, we have
demonstrated that a functional mitochondria is required for
Ah-induced neurotoxicity, as investigated using U+ and U0
mitochondrial DNA depleted cells (Cardoso et al., 2001).
Interestingly, several antioxidants (vitamin E, idebenone
and reduced glutathione), melatonin and nicotine showed
protective effects by maintaining the mitochondrial membrane potential, improving the activity of the respiratory
complexes and the cellular energetic levels (Cardoso et al.,
2001). Recently, Lustbader et al. (2004) reported that Ah
interacts with Ah-binding dehydrogenase (ABAD) in
mitochondria obtained from brains of AD patients and
transgenic mice, which suggests that ABAD is a direct
molecular link from Ah to mitochondrial toxicity. Furthermore, we found that type 2 diabetes-related mitochondrial dysfunction is exacerbated by aging and/or by the
P.I. Moreira et al. / Experimental Neurology 196 (2005) 112 – 119
presence of the neurotoxic Ah peptides (Moreira et al.,
2003). Recently, Lowell and Shulman (2005) discussed the
emerging evidence supporting the potentially unifying
hypothesis that the prominent features of type 2 diabetes
are caused by mitochondrial dysfunction. In accordance
with our previous results, we observed that the presence of
4 AM Ah1 – 40 induces a significant decrease in the RCR
(Fig. 1A) and ATP content (Table 2) and a significant
increase in H2O2 production (Fig. 2). Recently, Takuma et
al. (2005) reported that neurons cultured from transgenic
mice with targeted overexpression of a mutant form of
amyloid precursor protein and ABAD display spontaneous
S
generation of H2O2 and superoxide anion (O2 ), and
decreased ATP, as well as subsequent release of cytochrome c from mitochondria and induction of caspase-3like activity followed by DNA fragmentation and loss of
cell viability.
Coenzyme Q (CoQ) alterations have been suggested to
be involved in diabetes (Jameson, 1991; McCarthy, 1999)
and in neurodegenerative diseases such as Huntington’s
disease (HD) (Koroshetz et al., 1997), Parkinson’s disease
(PD) (Ebadi et al., 2001), Alzheimer’s disease (AD) (Edlund
et al., 1992) and amyotrophic lateral sclerosis (Sohmiya et
al., 2005).
Under nonpathological processes, the capability of tissues
to synthesize CoQ apparently decreases during aging (Kalen
et al., 1989); however, it has been reported that CoQ
biosynthesis can be affected by dietary supplementation
(Kwong et al., 2002). Supplementation with CoQ10 or
analogues has shown benefits in neurodegenerative processes
such as PD (Sharma et al., 2004), HD (Beal and Shults, 2003)
and AD (Gutzmann and Hadler, 1998), and also in
mitochondrial disorders (Sobreira et al., 1997; Di Giovanni
et al., 2001).
In this study, we choose the intraperitoneal administration of CoQ10, instead of CoQ10 supplemented chow,
because this experimental procedure allows us to control the
exact amount of CoQ10 administered to each animal.
Recently, Fernández-Ayala et al. (2005) reported that
HL-60 cells incorporate exogenously added CoQ10 into
mitochondria increasing complex I + III and complex II +
III activities. Although, we did not observe any statistical
difference in mitochondrial content of CoQ10 in the three
experimental groups (data not shown), the improvement of
mitochondrial function can be explained by the better
performance of CoQ10 as mitochondrial electron carrier.
The primary role of coenzyme Q is to transfer electrons
between redox components of the electron transport chain,
to create a proton gradient across the inner mitochondrial
membrane and drive ATP formation (Ebadi et al., 2001).
Besides its role in electron transfer reactions, CoQ10 is a
powerful antioxidant that has been shown to be protective
against oxidative stress (Singh et al., 1998; Hoppe et al.,
1999; Tomasetti et al., 1999). In this line, McCarthy et al.
(2004) reported that pre-incubation of SHSY-5Y cells with
water-soluble CoQ10 inhibits ROS production induced by
117
paraquat, a nonselective herbicide. Pretreatment with
CoQ10 also significantly reduced the number of apoptotic
cells and DNA fragmentation. Furthermore, CoQ10 was
able to inhibit mitochondrial ROS generation and inner
mitochondrial depolarization induced by paraquat. Furthermore, it has been shown that supplementing the diet of rats
with CoQ10 does not increase ROS production in mitochondria (Lass and Sohal, 2000; Kwong et al., 2002) but
increases plasma membrane protection against oxidative
stress (Gomez-Diaz et al., 2003), and extends life span
(Quiles et al., 2004). In accordance, our results show that
CoQ treatment avoids the increase in H2O2 production (Fig.
2) and the decrease in oxidative phosphorylation efficiency
(Table 2) induced by Ah1 – 40. Recently, a CoQ10 binding
site has been proposed to be located on the permeability
transition pore where it may inhibit its opening and thus
prevent collapse of mitochondrial membrane potential
(Papucci et al., 2003).
We observed that the effects of the vehicle solution
(soybean oil) and CoQ10 were similar concerning glycemia
(Table 1), RCR (Fig. 1) and repolarization levels (Table 2)
suggesting that the effects of both components (soybean oil
and CoQ10) are not synergistic nor potentiated since their
coadministration did not increase the protection observed.
The protective effect exerted by the soybean oil can be
explained by the fact that it is a triglyceride derived from
soybean that is rich in C-18 unsaturated components,
tocopherols (specially g-tocopherol, a-tocopherol is present
in modest amounts), ubiquinones and other antioxidants
such as bioflavonoids and aromatic compounds (Cabrini et
al., 2001; Lee et al., 2000). Several studies in animals and
humans have shown that the consumption of soybean has
beneficial effects in a variety of diseases including diabetes
and obesity (Bhathena and Velasquez, 2002) and neurodegenerative disorders (Kim et al., 2000).
In conclusion, our results show that CoQ10 treatment
exerts a partial protection against Ah1 – 40-induced mitochondrial dysfunction. Given the importance of mitochondria as
primary source of oxidative stress in AD and diabetes, the use
of CoQ10 may be useful. However, the broad occurrence of
both diseases, the nonregenerative nature of the CNS and the
fact that AD diagnosis often does not occur until late in
disease progression, suggest that the ideal antioxidant should
be used as prophylactic treatment in aged population. Due to
their low toxicity, low cost and their ability to target the
earliest sources of oxidative stress, CoQ treatment alone or in
combination with other antioxidants is particularly attractive.
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