Download Prevention of Mitochondrial Oxidative Damage as a

Prevention of Mitochondrial Oxidative Damage as a
Therapeutic Strategy in Diabetes
Katherine Green, Martin D. Brand, and Michael P. Murphy
Hyperglycemia causes many of the pathological consequences of both type 1 and type 2 diabetes. Much of this
damage is suggested to be a consequence of elevated
production of reactive oxygen species by the mitochondrial respiratory chain during hyperglycemia. Mitochondrial radical production associated with hyperglycemia
will also disrupt glucose-stimulated insulin secretion
by pancreatic ␤-cells, because pancreatic ␤-cells are
particularly susceptible to oxidative damage. Therefore, mitochondrial radical production in response to
hyperglycemia contributes to both the progression and
pathological complications of diabetes. Consequently,
strategies to decrease mitochondrial radical production
and oxidative damage may have therapeutic potential.
This could be achieved by the use of antioxidants or by
decreasing the mitochondrial membrane potential. Here,
we outline the background to these strategies and discuss
how antioxidants targeted to mitochondria, or selective
mitochondrial uncoupling, may be potential therapies for
diabetes. Diabetes 53 (Suppl. 1):S110 –S118, 2004
O
xidative damage due to hyperglycemia contributes to the microvascular pathology of diabetes
that occurs particularly in the retina, renal
glomerulus, and peripheral nerves, causing
blindness, renal failure, and peripheral neuropathy (1– 4).
Although the death of ␤-cells that underlies type 1 diabetes
is probably due to an autoimmune response, the particular
susceptibility of ␤-cells to oxidative damage from reactive
oxygen species (ROS) produced during inflammation may
be a predisposing factor (5,6). Supporting this, the streptozotocin and alloxan models of diabetes in rodents use
ROS production to kill ␤-cells, and oxidative damage to
␤-cells during hyperglycemia may contribute to the progression of the disorder (7,8). The association between
hyperglycemia and oxidative damage has been noted for
some time with various sources proposed for the under-
From the Medical Research Council Dunn Human Nutrition Unit, Cambridge,
U.K.
Address correspondence and reprint requests to Dr. Michael P. Murphy,
MRC Dunn Human Nutrition Unit, Wellcome Trust/MRC Bldg., Hills Rd.,
Cambridge CB2 2XY, U.K. E-mail: [email protected].
Received for publication 20 March 2003 and accepted 30 May 2003.
M.P.M. is a paid consultant for Antipodean Biotechnology.
This article is based on a presentation at a symposium. The symposium and
the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier.
⌬␮H⫹, proton electrochemical potential gradient; AGE, advanced glycation
end product; DCF, dichlorofluorescein; DNP, 2,4-dinitrophenol; GSIS, glucosestimulated insulin secretion; MitoVit E, mitochondria-targeted derivative of
␣-tocopherol; MnSOD, manganese superoxide dismutase; ROS, reactive oxygen species; UCP, uncoupling protein.
© 2004 by the American Diabetes Association.
S110
lying ROS (1,2). Recently, it has been suggested that
increased mitochondrial ROS production during hyperglycemia may be central to much of the pathology of diabetes
(3,9). Furthermore, because ␤-cell mitochondria play a
central role in glucose-stimulated insulin secretion (GSIS),
damage to ␤-cell mitochondria will attenuate this response
(7). Therefore, mitochondrial ROS production and oxidative damage may contribute to the onset, progression, and
pathological consequences of both type 1 and type 2
diabetes. Here, we outline how mitochondrial oxidative
damage occurs, consider the mechanisms by which it may
contribute to the pathophysiology of diabetes, and discuss
potential therapeutic strategies to prevent it.
MITOCHONDRIAL OXIDATIVE DAMAGE
Metabolism strips electrons from fatty acids, sugars, and
amino acids and accumulates them on the soluble electron
carrier NADH and on protein-bound FADH2 (Fig. 1). The
electrons are then passed down the mitochondrial respiratory chain to drive ATP synthesis by oxidative phosphorylation. As the electrons move down the potential energy
gradient from NADH/FADH2 to oxygen, the redox energy
is conserved by pumping protons across the inner membrane to build up a proton electrochemical potential
gradient (⌬␮H⫹). This gradient, composed of a substantial
membrane potential and a smaller pH gradient, is used by
the ATP synthase to make ATP, which is then mostly
exported to the cytoplasm to carry out work. Protons can
also reenter the mitochondrial matrix through nonspecific
leak pathways and via proteins such as uncoupling proteins (UCPs), which may catalyze an inducible proton
transport activity in the inner membrane. In both cases,
redox energy is dissipated as heat rather than being used
to make ATP.
Mitochondrial ROS production. The mitochondrial respiratory chain is the major site of ROS production within
the cell. Superoxide is thought to be produced continually
as a byproduct of normal respiration through the oneelectron reduction of molecular oxygen (Fig. 1) (10,11).
Superoxide itself damages iron sulfur center– containing
enzymes such as aconitase (12) and can also react with
nitric oxide to form the damaging oxidant peroxynitrite,
which is more reactive than either precursor (13). Nitric
oxide diffuses easily into mitochondria and may also be
produced there (14). The mitochondrial enzyme manganese superoxide dismutase (MnSOD) converts superoxide
to hydrogen peroxide, which, in the presence of ferrous or
cuprous ions, forms the highly reactive hydroxyl radical,
which damages all classes of biomolecules. The availability of free iron and copper within mitochondria is uncerDIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
K. GREEN, M.D. BRAND, AND M.P. MURPHY
FIG. 1. Mitochondrial oxidative damage. The mitochondrial respiratory chain (top) passes electrons from the electron carriers NADH and FADH2
through the respiratory chain to oxygen. This leads to the pumping of protons across the mitochondrial inner membrane to establish a proton
electrochemical potential gradient (⌬␮Hⴙ), negative inside: only the membrane potential (⌬␺m) component of ⌬␮Hⴙ is shown. The ⌬␮Hⴙ is used
to drive ATP synthesis by the F0F1ATP synthase. The exchange of ATP and ADP across the inner membrane is catalyzed by the adenine nucleotide
transporter (ANT) and the movement of inorganic phosphate (Pi) is catalyzed by the phosphate carrier (PC) (top left). There are also proton leak
pathways that dissipate ⌬␮Hⴙ without formation of ATP (top right). The respiratory chain also produces superoxide (O2䡠ⴚ), which can react with
and damage iron sulfur proteins such as aconitase, thereby ejecting ferrous iron. Superoxide also reacts with nitric oxide (NO) to form
peroxynitrite (ONOOⴚ). In the presence of ferrous iron, hydrogen peroxide forms the very reactive hydroxyl radical (䡠OH). Both peroxynitrite
and hydroxyl radical can cause extensive oxidative damage (bottom right). The defenses against oxidative damage (bottom left) include MnSOD,
and the hydrogen peroxide it produces is degraded by glutathione peroxidase (GPX) and peroxiredoxin III (PRX III). Glutathione (GSH) is
regenerated from glutathione disulfide (GSSG) by the action of glutathione reductase (GR), and the NADPH for this is in part supplied by a
transhydrogenase (TH).
tain, although the reaction of superoxide with the iron
sulfur center in aconitase releases ferrous iron (12). Consequently, mitochondrial superoxide production initiates a
range of damaging reactions through the production of
superoxide, hydrogen peroxide, ferrous iron, hydroxyl
radical, and peroxynitrite, which can damage lipids, proteins, and nucleic acids (15). Mitochondrial function is
particularly susceptible to oxidative damage, leading to
decreased mitochondrial ATP synthesis, cellular calcium
dyshomeostasis, and induction of the mitochondrial permeability transition, all of which predispose cells to necrosis or apoptosis (15).
DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
Mitochondrial antioxidant defenses. Mitochondria
have a range of defenses against oxidative damage (Fig. 1).
The antioxidant enzyme MnSOD converts superoxide to
hydrogen peroxide (16). The mitochondrial isoform of
glutathione peroxidase and the thioredoxin-dependent enzyme peroxiredoxin III both detoxify hydrogen peroxide
(17); alternatively, hydrogen peroxide can diffuse from the
mitochondria into the cytoplasm. The mitochondrial glutathione pool is distinct from that in the cytosol and is
maintained in its reduced state by a mitochondrial isoform
of glutathione reductase (17). This enzyme requires
NADPH, which is produced within mitochondria by the
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MITOCHONDRIAL OXIDATIVE DAMAGE AND DIABETES
NADP-dependent isocitrate dehydrogenase and through a
⌬␮H⫹-dependent transhydrogenase (17). Within the mitochondrial phospholipid bilayer, the fat-soluble antioxidants vitamin E and Coenzyme Q both prevent lipid
peroxidation, while Coenzyme Q also recycles vitamin E
and is itself regenerated by the respiratory chain (18). The
mitochondrial isoform of phospholipid hydroperoxide glutathione peroxidase degrades lipid peroxides within the
mitochondrial inner membrane (17). There are also a
range of mechanisms to repair or degrade oxidatively
damaged lipid, protein, and DNA (19).
Mitochondrial ROS production in vivo. Many diverse
pro-oxidant and antioxidant processes are ongoing in
mitochondria. Oxidative damage occurs whenever the
ROS produced by mitochondria evade detoxification, and
the steady-state level of oxidative damage depends on the
relative rates of damage accumulation, repair, and degradation (15,20). That mitochondrial ROS production occurs
at all times is suggested by mice lacking MnSOD, which die
within a few days of birth (21), while those lacking the
cytosolic isoform Cu,ZnSOD survive (22). Further evidence of mitochondrial ROS production under normal
conditions is the efflux of hydrogen peroxide from intact
mitochondria and from perfused organs, suggesting that
mitochondria produce superoxide, which is then converted to hydrogen peroxide in vivo (11). There is also
evidence that, under certain conditions, mitochondrial
DNA and protein accumulate greater oxidative damage in
vivo than the rest of the cell (19).
Complex III produces large amounts of superoxide
when inhibited by antimycin, which stabilizes a
ubisemiquinone radical at ubiquinol binding site o (10).
This ubisemiquinone radical transfers a single electron to
oxygen to form superoxide on the outside of the mitochondrial inner membrane (10,23). Complex I produces superoxide from NADH when it is inhibited by rotenone by a
⌬␮H⫹-independent mechanism (24,25). Complex I also
generates superoxide from ubiquinol when there is a
sufficiently large ⌬␮H⫹ to drive reverse electron transport
through complex I in intact mitochondria (23,26). In this
case, superoxide is produced on the matrix side of the
inner membrane, and its generation is inhibited by rotenone or an uncoupler (23). The maximum rate of superoxide production by antimycin-inhibited complex III is
generally greater than that of complex I, which has led
some to assume that the situation in vivo is similar.
However, in the absence of antimycin, superoxide production by complex III is minimal (23), and it seems probable
that in vivo complex I is the major source of superoxide
through reversed electron transport (23,24,26) and possibly also from forward electron transport (25).
Many other enzymes associated with mitochondria can
also produce superoxide or hydrogen peroxide, but even
though their contribution to ROS formation in vivo is
unclear, the current tacit assumption that only complexes
I and III produce ROS may have to be reassessed. Even so,
some conclusions about ROS formation by the respiratory
chain are possible (23). Older ideas that mitochondrial
ROS production is a simple function of the rate of oxygen
consumption, with faster respiration linked to a greater
rate of ROS production, have been abandoned. Instead, we
now recognize that mitochondrial ROS production is faS112
vored by high levels of reduction of the respiratory electron carriers, particularly the Coenzyme Q pool, and by a
large ⌬␮H⫹. These conditions favor superoxide production
from complex I by enhancing reverse electron transport
and may also act by increasing the lifetime of the semiquinone radical at the o site in complex III. Furthermore,
because the rates of the nonenzymatic reactions of oxygen
with radical intermediates to form superoxide are proportional to the local oxygen concentration, a high local
oxygen concentration will also favor superoxide production (27). All of the conditions that favor superoxide
production occur when mitochondria are respiring but not
making ATP (state 4). In contrast, when mitochondria are
actively making ATP (state 3), the lower ⌬␮H⫹, increased
oxidation of electron carrier pools, and lower local oxygen
concentration will decrease superoxide production.
Uncoupling and mitochondrial ROS production. As an
elevated ⌬␮H⫹ favors superoxide production, limiting the
magnitude of this gradient under state 4 conditions should
decrease superoxide production (28). Artificial uncouplers
such as 2,4-dinitrophenol (DNP) make the inner membrane permeable to protons, thereby lowering the ⌬␮H⫹,
which accelerates respiration and consequently oxidizes
the electron carrier pools (28,29). Uncouplers are typically
lipophilic weak acids for which both the protonated and
unprotonated forms are lipid soluble, thus enabling them
to catalyze proton movement through the phospholipid
bilayer by lowering the activation energy for proton leak.
Even low concentrations of artificial uncouplers have been
shown to lower the rate of superoxide production by
mitochondria (28).
In the absence of uncouplers, the mitochondrial inner
membrane is still slightly permeable to protons, but intriguingly this permeability increases dramatically at high
⌬␮H⫹ (state 4) compared with when the mitochondria are
making ATP (state 3) (30 –32). The increased leakiness of
the mitochondrial inner membrane to protons may be to
minimize superoxide production in state 4 by limiting the
maximum ⌬␮H⫹ (28). Although the mechanism of this
proton leak is not certain, it is quite distinct from the
thermogenic inducible proton leak catalyzed by UCP1
(33). This protein is confined to brown adipose tissue
mitochondria, and its function is primarily thermogenic
(33). There is considerable interest in two close homologues of UCP1 called (perhaps misleadingly) UCP2 and
UCP3, which are also found in the mitochondrial inner
membrane (33). UCP2 mRNA is widely expressed, but
significant activity of the protein appears to be restricted
to the cells of the immune system, white adipose tissue,
stomach, intestine, lung, kidney, and pancreatic ␤-cells
(34 –36), whereas UCP3 activity is confined to skeletal
muscle and brown adipose tissue (33). Because of their
homology to UCP1, these “novel UCPs” were initially
assumed to catalyze a proton leak through the mitochondrial inner membrane, but it is now clear that they do not
contribute to the basal proton conductance of the mitochondrial inner membrane (33,37). Instead, they appear to
catalyze a specific increase in proton conductance only
when activated by superoxide (35) or other activators
(38). The expression of these UCPs increases in response
to elevated mitochondrial oxidative stress (34), and one
possible function of these proteins is to lower the mitoDIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
K. GREEN, M.D. BRAND, AND M.P. MURPHY
FIG. 2. A possible model for the induction of mitochondrial ROS production by hyperglycemia. High concentrations of glucose lead to an increase
in the level of reducing equivalents such as NADH and FADH2 within mitochondria. This occurs through the increased uptake of reducing
equivalents from the cytoplasm by various mitochondrial redox shuttles (MS) and by increased uptake of pyruvate by the pyruvate transporter
(PT). Together these processes lead to an elevated proton electrochemical potential gradient, reduced electron carriers, and increased ROS
production by the respiratory chain. Decreasing the membrane potential (⌬␺m) by artificial uncouplers or by activating UCPs may prevent ROS
production. Superoxide activation of UCPs may trigger a feedback loop to lower ⌬␺ and ROS production as shown. Antioxidants can also degrade
ROS. Because it is not certain that hyperglycemia increases mitochondrial ROS production solely by increasing the supply of reducing equivalents
to mitochondria, a hypothetical direct stimulation of ROS production and oxidative damage outside mitochondria, and perhaps inside, in response
to hyperglycemia is also shown.
chondrial membrane potential when superoxide is generated under state 4 conditions. Echtay et al. (35) have
proposed a simple feedback cycle in which mitochondrial
oxidative stress acutely and chronically upregulates the
proton translocating activity of UCPs to lower the ⌬␮H⫹
and thus decrease superoxide production (Fig. 2). However, until the physiological role(s) of UCP2 and UCP3 are
fully clarified, the significance of the interaction of ROS
with UCPs in vivo will remain uncertain (33).
A further issue to consider is the role of UCP2 within
pancreatic ␤-cells. Glucose sensing by pancreatic ␤-cells
uses ATP as a coupling factor between glucose metabolism and insulin secretion (outlined in Fig. 3). Mitochondria in ␤-cells contain UCP2, and because uncoupling
protein activity lowers the ⌬␮H⫹, it is also a means of
attenuating GSIS (Fig. 2). The islets of UCP2(⫺/⫺) mice
exhibit markedly higher ATP levels and improved GSIS,
demonstrating that endogenous ␤-cell UCP2 is activated
and that this impairs GSIS (36). Furthermore, the hyperDIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
glycemia of ob/ob mice (a model of type 2 diabetes) is
significantly reduced by ablation of UCP2. This supports
the principle that reducing UCP2 activity in ␤-cells is a
valid means of treating diabetes (36), but also indicates
that while uncoupling and activation of UCP can reduce
ROS production in peripheral tissue, it may disrupt GSIS in
␤-cells.
MITOCHONDRIAL OXIDATIVE DAMAGE IN DIABETES
Much of the long-term pathology of diabetes occurs as a
consequence of persistent hyperglycemia. Four consequences of hyperglycemia of particular pathological relevance (1) are the formation, auto-oxidation, and interaction
with cell receptors of advanced glycation end products
(AGEs); activation of various isoforms of protein kinase C;
induction of the polyol pathway; and increased hexosamine
pathway flux. Many of these pathways have long been
associated with elevated oxidative stress (1), for example,
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MITOCHONDRIAL OXIDATIVE DAMAGE AND DIABETES
FIG. 3. Role of ␤-cell mitochondria in sensing increases in plasma glucose and inducing insulin secretion. Elevated plasma glucose leads to an
increase in the cytoplasmic concentration due to uptake though a glucose transporter (GLUT2). This increases the supply of pyruvate to the
mitochondrial tricarboxylic acid cycle (TCA), which leads to an increase in the NADH/NAD ratio, an elevated mitochondrial membrane potential
(⌬␺m), and increased ATP synthesis. The greater cytosolic ATP/ADP ratio inhibits the plasma membrane ATP-dependent Kⴙ (KATP) channel,
leading to depolarization of the plasma membrane potential (⌬␺p) and influx of calcium, which drives the release of insulin into the plasma. Thus,
the activity of ␤-cell mitochondria is central to GSIS. The proposed activation of UCP2 within ␤-cell mitochondria by superoxide is indicated.
through auto-oxidation of AGEs or through the action of the
polyol pathway depleting cytosolic NADPH and thereby
decreasing the cellular glutathione/glutathione disulfide ratio.
Recently, a hypothesis was proposed that suggests that all
these processes are a consequence of overproduction of
superoxide by the mitochondrial respiratory chain during
hyperglycemia (3,9,39). This is thought to occur because
hyperglycemia increases the flow of electrons to the respiratory chain by maintaining large mitochondrial NADH/NAD
and FADH2/FAD ratios under conditions of high ⌬␮H⫹ (Fig.
3). Thus, mitochondria in many tissues would spend more
time under state 4–like conditions of low respiration rate,
high ⌬␮H⫹, and reduced electron carriers, all of which favor
superoxide formation. However, it should be noted that the
validity of this link between hyperglycemia and increased
mitochondrial superoxide production has not yet been demonstrated. Indeed, in isolated hepatocytes, simply increasing
the glucose concentration does not increase ⌬␮H⫹, mitochondrial respiration rate, or cytosolic NADH/NAD ratio;
instead, most of the excess glucose is converted to glycogen
(40). Consequently, the link between hyperglycemia and
increased mitochondrial superoxide production may turn out
to be due to some other interaction with mitochondria, not
mediated directly by the redox state of electron carriers.
The evidence for increased mitochondrial ROS producS114
tion during hyperglycemia comes from experiments on
cultured endothelial cells, where raising the glucose concentration from 5 to 30 mmol/l increased cytosolic ROS
production, as measured by the rate of oxidation of
dichlorodihydrofluorescein to dichlorofluorescein (DCF)
(9). The reaction between hydrogen peroxide and dichlorodihydrofluorescein is catalyzed by intracellular peroxidases; thus, an increase in cellular DCF oxidation is
consistent with elevated mitochondrial superoxide production forming hydrogen peroxide, which then diffuses to
the cytoplasm. DCF oxidation was disrupted by overexpression of mitochondrial MnSOD, suggesting that the
proximal ROS produced was superoxide within the mitochondrial matrix. However, it is unclear why overexpressing MnSOD abolished the DCF oxidation signal, because
MnSOD should convert the superoxide to hydrogen peroxide, which should then efflux from the mitochondria and
enhance the DCF signal. This finding suggests that the
nature of the ROS being measured in these experiments
remains uncertain. DCF oxidation was also blocked by
inhibitors of mitochondrial pyruvate uptake and of succinate dehydrogenase, but not by rotenone, suggesting that
reverse electron transport was not involved. ROS production was also blocked by overexpression of UCP1, indicating ⌬␮H⫹ dependence. The increase in mitochondrial ROS
DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
K. GREEN, M.D. BRAND, AND M.P. MURPHY
production associated with hyperglycemia also led to the
activation of the redox-sensitive cytosolic transcription
factor NF␬B, the formation of AGEs, and the activation of
protein kinase C, all of which were prevented by blocking
mitochondrial ROS production with MnSOD, respiratory
inhibitors, or UCP1 (9).
The model that arose from these studies is that an
increase in mitochondrial ROS in response to hyperglycemia is the proximal defect that leads to most of the other
pathological consequences of hyperglycemia. There is no
doubt this view will prove too simplistic and will have to
be extended to accommodate other sites of ROS production (2). Nevertheless, this is a major new insight that
suggests how preventing the production of superoxide by
mitochondria, or increasing its rate of decomposition by
antioxidants, may block many of the pathological consequences of hyperglycemia. In addition, ␤-cell mitochondria
are essential for GSIS but are susceptible to oxidative
damage during hyperglycemia that suppresses GSIS (7,8),
thus contributing to the progression of the disease through
a vicious cycle in which hyperglycemia causes oxidative
damage, which in turn disrupts the ability of ␤-cells to
respond to elevated blood glucose, leading to further
hyperglycemia (2,7).
In summary, increased mitochondrial ROS production
during hyperglycemia may be a major factor in the pathology of diabetes. This suggests that therapeutic strategies
to limit mitochondrial radical production during hyperglycemia and to counteract their damaging effects may be
useful complements to conventional therapies designed to
normalize blood glucose.
ANTIOXIDANT THERAPIES
Because oxidative damage is part of the pathophysiology
of diabetes, there is interest in determining whether antioxidants can decrease this damage (1). Too few largescale double-blind trials on the use of antioxidants in
diabetes have been carried out for conclusions (1). However, a few small-scale trials have suggested the efficacy of
the natural antioxidants ␣-tocopherol (vitamin E), ascorbate (vitamin C), Coenzyme Q, and ␣-lipoic acid, although
in other trials, the efficacy of ascorbate and ␣-tocopherol
were ambiguous (1,2). Because these natural antioxidants
can be given at high doses and have shown some efficacy
in other degenerative diseases, there is a strong rationale
for trialing them in diabetes (1,41), even though the uptake
and distribution to tissues of hydrophobic natural antioxidants such as Coenzyme Q is often poor (42). In addition,
many other artificial antioxidants are being developed,
such as mimetics of SOD or peroxidase, that may be more
potent than natural antioxidants and also have improved
bioavailability, pharmacokinetics, and stability (43,44).
Because these artificial antioxidants are novel drugs (vs.
natural antioxidants), it will be more demanding to initiate
clinical trials. Both the natural and artificial antioxidants
distribute throughout the body, with only a small proportion reaching the mitochondria, where much of the oxidative damage associated with hyperglycemia may occur.
Mitochondria-targeted antioxidants in vivo. Because
mitochondrial oxidative damage is thought to be critical in
the pathophysiology of diabetes, antioxidants that accumulate within mitochondria may offer more protection
DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
than untargeted antioxidants. As a first step toward testing
this hypothesis, a strategy has been developed to deliver
antioxidants to mitochondria by covalent attachment to
the triphenylphosphonium cation through an alkyl chain
(Fig. 4) (45,46). The delocalized positive charge of these
lipophilic cations enables them to permeate lipid bilayers
easily and to accumulate several hundred–fold within
mitochondria, due to the large membrane potential (⌬␺m;
⫺150 to ⫺170 mV, negative inside; Fig. 4). The plasma
membrane potential (⌬␺p; ⫺30 to ⫺60 mV, negative inside;
Fig 4) also drives their accumulation from the extracellular fluid into cells, from where they are further concentrated within mitochondria. Because the natural antioxidants vitamin E and Coenzyme Q are thought to protect
mitochondria from oxidative damage in vivo, mitochondria-targeted derivatives of these molecules were first
developed (Fig. 4). Experiments in vitro showed that the
mitochondria-targeted derivative of ␣-tocopherol (MitoVit
E) and the mitochondria-targeted ubiquinone were rapidly
and selectively accumulated by isolated mitochondria and
by mitochondria within isolated cells (47– 49). Importantly, the accumulation of these antioxidants by mitochondria protected them from oxidative damage far more
effectively than untargeted antioxidants, suggesting that
the accumulation of antioxidants within mitochondria
does increase their efficacy. Most interestingly, these compounds were several hundred–fold more effective at preventing cell death in fibroblasts from Friedreich Ataxia
patients (49a). Because cell death in this model is due to
endogenous mitochondrial oxidative damage (50), it is
suggested that the accumulation of antioxidants by mitochondria within cells blocks mitochondrial oxidative damage and that their uptake into mitochondria makes them
far more effective than untargeted antioxidants.
Mitochondria-targeted antioxidants in vivo. If these
mitochondria-targeted molecules are to have therapeutic
potential in diabetes, then they must be taken up selectively by mitochondria in vivo. Because alkyltriphenylphosphonium cations pass easily through lipid bilayers by
noncarrier-mediated transport, they should be taken up by
the mitochondria of all tissues, in contrast to hydrophilic
compounds, which rely on the tissue-specific expression
of carriers for uptake (41). Mice were fed mitochondriatargeted antioxidants for several weeks, leading to stable
steady-state concentrations within all tissues assessed,
including the brain, heart, liver, and kidneys (51). Uptake
was reversible, as shown by the rapid clearance of the
simple lipophilic cation methyltriphenylphosphonium
from all organs when oral administration stopped (51).
That these compounds can enter the bloodstream and
distribute to tissues in their intact active form was shown
by solvent extraction of the brain, heart, and liver of mice
fed MitoVit E, followed by mass spectrometry (51). These
data are consistent with the following pharmacokinetic
model: following absorption from the gut into the bloodstream, orally administered mitochondria-targeted antioxidants are taken up into all tissues by nonmediated
movement through the lipid bilayer of the plasma membrane, assisted by the plasma membrane potential. From
the cytosol, most of the lipophilic cations are taken up into
mitochondria, driven by the large membrane potential.
After several days of feeding, the cation concentration
S115
MITOCHONDRIAL OXIDATIVE DAMAGE AND DIABETES
FIG. 4. Mitochondria-targeted antioxidants. A generic mitochondria-targeted antioxidant is shown constructed by the covalent attachment of an
antioxidant moiety (X) to the lipophilic triphenylphosphonium cation. This molecule is accumulated 5- to 10-fold into the cytoplasm driven by the
plasma membrane potential (⌬␺p) and then further accumulated into the mitochondria 100- to 500-fold driven by the mitochondrial membrane
potential (⌬␺m). The structures on the bottom are of two targeted antioxidants, MitoVit E and mitochondria-targeted Coenzyme Q (MitoQ).
within mitochondria comes to a steady-state distribution
with circulating blood levels. At this point, the mitochondrial concentration will be several hundred–fold higher
than that in the bloodstream. Because the mitochondrial
pool of compound is in dynamic equilibrium, once feeding
stops, the accumulated cations will re-equilibrate back
into the bloodstream and be relatively rapidly excreted.
The levels of methyltriphenylphosphonium and MitoVit
E that accumulated in mouse tissues in vivo after feeding
were in the range of 5–20 nmol/g wet wt, or about 5–20
␮mol/l in the tissue (51). Because these compounds accumulate within mitochondria, the intramitochondrial concentration will be about millimolar. These concentrations
are likely to be in the therapeutically effective range,
because mitochondria-targeted antioxidants prevented oxidative damage to isolated mitochondria at 1–2.5 ␮mol/l
(48,49). Because these compounds will be further accumulated into cells, similar protective effects were found by
S116
incubating cultured cells with 500 nmol/l to 1 ␮mol/l
mitochondria-targeted antioxidants (49,52). Therefore,
oral delivery of well-tolerated doses of mitochondriatargeted antioxidants can deliver potentially therapeutic
concentrations to mitochondria in vivo. Their efficacy at
preventing oxidative damage to mitochondria in vivo is
now being tested in mouse models of mitochondrial
oxidative damage.
Uncoupling to prevent oxidative damage. In addition
to inactivating ROS by antioxidants, another strategy is to
decrease mitochondrial ROS production in diabetes by
lowering the ⌬␮H⫹. This could be done using low doses of
artificial uncouplers to slightly lower the ⌬␮H⫹ and thus
decrease mitochondrial ROS production (29). The uncoupler DNP has been used extensively in the past to treat
obesity in humans, with surprisingly good results (29).
However, unregulated administration, abuse, and the very
narrow therapeutic window between efficacy and toxicity
DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
K. GREEN, M.D. BRAND, AND M.P. MURPHY
led to its abandonment, although it is still used unofficially
as a slimming agent (29). It should be possible to make a
safe uncoupler for human use, although the challenge is to
make one that is mild enough to decrease mitochondrial
ROS production without significantly affecting mitochondrial ATP production, and to make these molecules tissue
specific to reduce the possibility of compromising ATP
production in nervous and cardiac tissue. A further complication is that decreasing the membrane potential of
␤-cell mitochondria would make insulin secretion less
responsive to plasma glucose levels, which might be
counterproductive. Indeed, it has been reported that DNP
treatment of animals causes hyperglycemia (53).
An alternative way to decrease ⌬␮H⫹ may be activating
the inducible proton leak catalyzed by endogenous proteins such as the UCPs. However, whereas some molecules such as superoxide and retinoids do appear to
activate UCPs (35,38), the development of drugs designed
to modulate this function will probably require better
understanding of the physiological roles and molecular
mechanisms of UCPs.
CONCLUSIONS
It seems probable that mitochondrial radical production
and consequent oxidative damage contribute to the progression and pathophysiology of diabetes. Consequently,
therapeutic strategies to decrease ROS production, or to
intercept the ROS once formed, should be explored. Two
approaches suggest themselves: selective uncoupling of
mitochondria, and antioxidants. Whereas the development
of uncoupling strategies is not imminent, the time is upon
us to test antioxidant therapies in diabetes. As well as
protecting peripheral tissues from hyperglycemia-induced
oxidative damage, antioxidants may have the additional
benefit of improving GSIS, both by preventing the damage
to ␤-cells and possibly by blocking the proposed ROS
activation of UCP2 in ␤-cells. Whereas suitable uncouplers
should also protect peripheral tissues, there is the danger
that they would have counterproductive effects on ␤-cells,
rendering insulin secretion less responsive to plasma
glucose.
A first step in developing antioxidant therapies is to give
large doses of natural antioxidants such as vitamin E,
␣-lipoic acid, or Coenzyme Q to see if this approach has
potential. The advantage of natural antioxidants is their
safety and that large oral doses are well tolerated. However, in other degenerative diseases, very large doses have
been required to see beneficial effects, possibly because of
their poor bioavailability and distribution within the body
and the cell (42). If natural antioxidants show efficacy in
large-scale patient trials, then it may be more effective to
develop artificial antioxidants with improved potency,
bioavailability, and pharmacokinetics. Among these, a
case can be made for testing mitochondria-targeted antioxidants. To date, mitochondria-targeted versions of Coenzyme Q and vitamin E have been made and can be
administered safely to mice (51). Experiments are now
underway to see if these and other mitochondria-targeted
antioxidants show efficacy against the pathologies associated with hyperglycemia in cell and rodent models of
diabetes.
DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004
ACKNOWLEDGMENTS
We thank Meredith Ross and John Todd for helpful
comments on the manuscript.
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