**4.2. Unifying hypothesis: the role of mitochondrial oxidants**

With the growth of our knowledge about glucose-induced cellular damage and the various molecules and pathways involved in the process, the pathomechanism of glucose-induced damage has become inexplicable. In an effort to explain the puzzling complexity of the cellular events, Michael Brownlee introduced a unifying hypothesis in which he placed the events in an integrating linear model [26]. In the unifying mechanism, mitochondrial superoxide generation is placed in center stage followed by all other ROS-producing pathways as secondary events. As the contribution of mitochondrial energy production seems negligible in endothelial cells, this proposition was a striking novelty at first, but it renders the series of events logically based on a wealth of scientific results. First of all, the unifying framework assumes that the main ROS-producing mechanisms implicated in hyperglycemic cellular damage are interrelated and a common pathway is responsible for their activation [75]. Secondly, the overload of glycolysis rather occurs as a single downstream perturbation of metabolism that leaves behind glycolytic intermediates than by multiple blockades of glycolytic enzymes in response to excess glucose input. Thus, inhibition of a downstream step of glucose catabolism in the mitochondria might be responsible for the activation of the ROS-producing shunt pathways in the cytoplasm. The observation that prevention of mitochondrial superoxide generation inhibits the cytoplasmic ROS production pathways (PKC activation, sorbitol accumulation and AGE production) also supported the assumption that mitochondrial damage precedes the glycolytic impairment [83].

The exact nature of hyperglycemic perturbation of mitochondrial metabolism remains enigmatic, and it is still debatable whether superoxide itself or the steps leading to its increased production is the triggering event of glucose-induced damage. High TCA flux and elevated glycolytic pyruvate input were detected in hyperglycemia and these may serve as inducers of mitochondrial ROS production but might also be the consequences of dysfunctional OXPHOS [83]. Various pharmacological interventions that reduce the mitochondrial ROS production effectively inhibit the hyperglycemic damage [26, 38, 75]. Higher flux through the electron transport chain is expected to reduce the accumulation of glycolytic intermediates and prevent the activation of oxidative stress pathways but only some of the interventions increased the electron transport (e.g. uncoupling agents and proteins), whereas others (e.g. antioxidants) did not change it or severely reduced it (complex II inhibition). Also, the increased electron flow may induce a proportional rise in superoxide generation by the electron transport chain if electron leakage is unaffected. Furthermore, endothelial cells, in which the mitochondrial DNA is selectively depleted (rho zero cells) and lacks a functional electron transport chain, fail to activate PKC, the polyol and hexosamine pathways and they do not produce AGEs, though their mitochondrial metabolism is impaired and they are expected to accumulate glycolytic intermediates [26]. These observations led to the proposition that mitochondrial superoxide generated by the electron transport chain is responsible for the initiation of hyperglycemic endothelial damage [26, 83, 84].

#### **4.3. The mechanism of glucose-induced mitochondrial superoxide generation**

where it becomes activated via phosphorylation in response to cellular stress and translocates to the mitochondria. The active p66SHC diverts a fraction of the mitochondrial electron flow between complexes III and IV to produce ROS instead of water and is involved in the opening of the permeability transition pore during apoptosis. In hyperglycemia, p66SHC may function as a shunt pathway if complex IV activity is impaired. The activity of p66SHC is also regulated by acetylation: it is a direct target of SIRT1 and diminished SIRT1 activity increases the acetylation and activity of p66SHC in hyperglycemia [82]. Furthermore, acetylation of p66SHC promotes the phosphorylation-mediated activation of the protein, and since the acetylation-resistant p66SHC isoform partially protects against the vascular impairment, it may play a pathogenic role in diabetic vascular dysfunction. The linkage to SIRT1 and the protection associated with the loss of p66SHC suggest that p66SHC make a substantial contribution to oxidative stress in diabetes and it may represent the key target

With the growth of our knowledge about glucose-induced cellular damage and the various molecules and pathways involved in the process, the pathomechanism of glucose-induced damage has become inexplicable. In an effort to explain the puzzling complexity of the cellular events, Michael Brownlee introduced a unifying hypothesis in which he placed the events in an integrating linear model [26]. In the unifying mechanism, mitochondrial superoxide generation is placed in center stage followed by all other ROS-producing pathways as secondary events. As the contribution of mitochondrial energy production seems negligible in endothelial cells, this proposition was a striking novelty at first, but it renders the series of events logically based on a wealth of scientific results. First of all, the unifying framework assumes that the main ROS-producing mechanisms implicated in hyperglycemic cellular damage are interrelated and a common pathway is responsible for their activation [75]. Secondly, the overload of glycolysis rather occurs as a single downstream perturbation of metabolism that leaves behind glycolytic intermediates than by multiple blockades of glycolytic enzymes in response to excess glucose input. Thus, inhibition of a downstream step of glucose catabolism in the mitochondria might be responsible for the activation of the ROS-producing shunt pathways in the cytoplasm. The observation that prevention of mitochondrial superoxide generation inhibits the cytoplasmic ROS production pathways (PKC activation, sorbitol accumulation and AGE production) also supported the assumption that mitochondrial damage

The exact nature of hyperglycemic perturbation of mitochondrial metabolism remains enigmatic, and it is still debatable whether superoxide itself or the steps leading to its increased production is the triggering event of glucose-induced damage. High TCA flux and elevated glycolytic pyruvate input were detected in hyperglycemia and these may serve as inducers of mitochondrial ROS production but might also be the consequences of dysfunctional OXPHOS [83]. Various pharmacological interventions that reduce the mitochondrial ROS production effectively inhibit the hyperglycemic damage [26, 38, 75]. Higher flux through the electron transport chain is expected to reduce the accumulation of glycolytic intermediates and prevent

**4.2. Unifying hypothesis: the role of mitochondrial oxidants**

188 Endothelial Dysfunction - Old Concepts and New Challenges

precedes the glycolytic impairment [83].

of SIRT1.

Mitochondria produce superoxide nonenzymatically via multiple respiratory complexes in the electron transport chain and enzymatically via the mitochondrial xanthine oxidase [85– 87]. The nonenzymatic production of superoxide occurs when a single electron is directly transferred to oxygen by prosthetic groups of the respiratory complexes or by reduced coenzymes that act as soluble electron carriers. The electron transport chain may leak electrons to oxygen and it is the main source of superoxide in hyperglycemia. Mitochondrial monoamine oxidase (MAO) and p66SHC also produce H<sup>2</sup> O2 within the mitochondria that may contribute to oxidative stress in hyperglycemia [88].

Molecular oxygen is biradical; it has two unpaired electrons in the outer orbitals, which makes it chemically reactive. In the ground state, the unpaired electrons are arranged in the triplet state, and as a result of spin restrictions, molecular oxygen is not highly reactive: it can only react with one electron at a time. If one of the unpaired electrons is excited and changes its spin (oxygen goes from the triplet state to the short-lived singlet state), it will become a powerful oxidant that is highly reactive [86]. The reduction of oxygen by one electron at a time produces superoxide (O<sup>2</sup> •−) anion that might be converted to hydrogen peroxide (either spontaneously or through a reaction catalyzed by superoxide dismutase), which may be fully reduced to water or partially reduced to hydroxyl radical (OH•). In addition, superoxide may react with other radicals including nitric oxide (NO•) and form peroxynitrite (ONOO•−), another very powerful oxidant. The respiratory components are thermodynamically capable of transferring one electron to oxygen and form superoxide in the highly reducing environment of the mitochondria, since the standard reduction potential of oxygen to superoxide is −0.160 V and the respiratory chain incorporates components with standard reduction potentials between −0.32 V (NAD(P)H) and +0.39 V (cytochrome a<sup>3</sup> in Complex IV) [86].

In the respiratory chain, electrons move along the electron transport chain going from donor to acceptor molecules until they are transferred to molecular oxygen (the standard reduction potential of oxygen/H<sup>2</sup> O couple is +0.82 V), while the generated free energy is used to synthesize ATP from ADP and inorganic phosphate. Respiratory Complex I transfers electrons from NADH and Complex II from FADH<sup>2</sup> to coenzyme Q (CoQ, ubiquinone), which is the substrate of Complex III. Complex III transfers electrons from reduced CoQ to cytochrome C, which is used by Complex IV to reduce oxygen into water. The step-by-step transfer of electrons allows the free energy to be released in small increments. The energy released as electrons flow through the respiratory chain is converted into a H<sup>+</sup> gradient through the inner mitochondrial membrane: protons are transported from the mitochondrial matrix to the intermembrane space (by Complexes I, III and IV) and a proton concentration gradient forms across the inner mitochondrial membrane [89]. Since the mitochondrial outer membrane is freely permeable to protons, the pH of the mitochondrial matrix is higher (the proton concentration is lower) than that of the intermembrane space and the cytosol. An electric potential (mitochondrial membrane potential) of 140–160 mV is formed across the inner membrane by pumping of positively charged protons outward from the matrix, which becomes negatively charged [90]. Thus, free energy released during the oxidation of NADH or FADH<sup>2</sup> is converted to an electric potential and a proton concentration gradient—collectively, the protonmotive force—and this energy is used by ATP synthase (Complex V) for ATP generation via the chemiosmotic coupling [91]. While the majority of oxygen molecules are used for water formation during the above processes, superoxide is generated at an estimated rate of 0.1–2% of oxygen consumption under normal respiration (State 3) and physiological operation of the respiratory chain [85, 88].

The electron transport chain may produce superoxide by multiple mechanisms but electron leakage before Complex III is suspected to represent the main source of superoxide in hyperglycemic endothelial cells [26, 83]. Complexes I and III are the respiratory complexes that are capable to produce large amounts of superoxide under certain conditions (**Figure 2**). Complex I may produce superoxide by two mechanisms: (1) the reduced flavin mononucleotide (FMN) center can transfer electrons to oxygen instead of CoQ when the NADH/NAD<sup>+</sup> ratio is high (and the CoQ binding site is blocked or the CoQ pool is mostly reduced) and (2) by reverse electron transfer (RET) from the CoQ binding site if there is high electron supply from Complex II and the electrons are forced back to Complex I instead of proceeding to Complex III (by a reduced CoQ pool and high proton-motive force) [85, 92]. In Complex III, superoxide is produced from the semiquinone anionic state of CoQ (semiubiquinone) by directly reacting with oxygen instead of completing the Q-cycle [85, 93]. Reduced CoQ diffuses through the bilipid layer of the membrane to its binding site in Complex III and transfers the electrons to the iron-sulfur protein (Rieske protein) in two steps that produce a semiquinone intermediate state of CoQ after the first electron transfer, which is the source of superoxide. In the presence of respiratory inhibitors, Complex I may produce the highest amount of superoxide, especially through RET, but the contribution of Complexes I and III to superoxide production is unknown in healthy mitochondria [86]. Superoxide is also produced in the matrix by other enzymes that interact with the NADH pool and by enzymes connected to the inner membrane CoQ pool. These include α-ketoglutarate dehydrogenase that may produce superoxide if its substrate (α-ketoglutarate) concentration and the NADH/NAD<sup>+</sup> ratio increase in the matrix. In the membrane, α-glycerophosphate dehydrogenase may produce superoxide partly via RET and Complex II, which transfer electrons from succinate to CoQ, is also suspected to generate some superoxide [85].

In hyperglycemic endothelial cells, the increased production of superoxide originates from the reduced CoQ pool before Complex III [75, 83]. The high electron donor input from glycolysis and the TCA cycle may increase the membrane potential and inhibit the electron transfer at Complex III, thus increase the concentration of reduced and free radical intermediates of CoQ. Superoxide generation may occur as direct 'leakage' of electrons to oxygen, as a result of the longer half-life of CoQ intermediates in the lipid bilayer and bound to Complex III or via RET through Complex I. Superoxide generation is also promoted by the increased membrane potential and proton concentration gradient through the inner membrane [31, 35, 83, 94]. Superoxide production was found to increase exponentially above 140 mV with the increase of the mitochondrial membrane potential [95]. Since with the generation of each superoxide molecule one electron is lost compared to the number of protons, superoxide production per se may increase the membrane potential and the proton gradient or might be responsible for the maintenance of the elevated membrane potential. Furthermore, the proton and charge transfer of Complexes III and IV are disproportional since Complex III picks up two protons from the matrix side of the inner membrane (the negatively charged N-face) and releases four

**Figure 2.** Oxidant production by the mitochondrial electron transport chain. CoQ: coenzyme Q, ubiquinone; Cyt C:

p66SHC: 66-kDa Src homology 2 domain-containing protein; SQR: sulfide:quinone oxidoreductase and UCP: uncoupling

O2

: hydrogen peroxide; MnSOD: manganese-dependent

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 191

•−: superoxide, ONOO•−: peroxynitrite; PARP: poly(ADP-ribose) polymerase;

: flavin adenine dinucleotide; H<sup>2</sup>

cytochrome C; FAD+/FADH<sup>2</sup>

protein.

superoxide dismutase; NO•: nitric oxide; O<sup>2</sup>

substrate of Complex III. Complex III transfers electrons from reduced CoQ to cytochrome C, which is used by Complex IV to reduce oxygen into water. The step-by-step transfer of electrons allows the free energy to be released in small increments. The energy released as

mitochondrial membrane: protons are transported from the mitochondrial matrix to the intermembrane space (by Complexes I, III and IV) and a proton concentration gradient forms across the inner mitochondrial membrane [89]. Since the mitochondrial outer membrane is freely permeable to protons, the pH of the mitochondrial matrix is higher (the proton concentration is lower) than that of the intermembrane space and the cytosol. An electric potential (mitochondrial membrane potential) of 140–160 mV is formed across the inner membrane by pumping of positively charged protons outward from the matrix, which becomes negatively

charged [90]. Thus, free energy released during the oxidation of NADH or FADH<sup>2</sup>

verted to an electric potential and a proton concentration gradient—collectively, the protonmotive force—and this energy is used by ATP synthase (Complex V) for ATP generation via the chemiosmotic coupling [91]. While the majority of oxygen molecules are used for water formation during the above processes, superoxide is generated at an estimated rate of 0.1–2% of oxygen consumption under normal respiration (State 3) and physiological operation of the

The electron transport chain may produce superoxide by multiple mechanisms but electron leakage before Complex III is suspected to represent the main source of superoxide in hyperglycemic endothelial cells [26, 83]. Complexes I and III are the respiratory complexes that are capable to produce large amounts of superoxide under certain conditions (**Figure 2**). Complex I may produce superoxide by two mechanisms: (1) the reduced flavin mononucleotide (FMN) center can transfer electrons to oxygen instead of CoQ when the

reduced) and (2) by reverse electron transfer (RET) from the CoQ binding site if there is high electron supply from Complex II and the electrons are forced back to Complex I instead of proceeding to Complex III (by a reduced CoQ pool and high proton-motive force) [85, 92]. In Complex III, superoxide is produced from the semiquinone anionic state of CoQ (semiubiquinone) by directly reacting with oxygen instead of completing the Q-cycle [85, 93]. Reduced CoQ diffuses through the bilipid layer of the membrane to its binding site in Complex III and transfers the electrons to the iron-sulfur protein (Rieske protein) in two steps that produce a semiquinone intermediate state of CoQ after the first electron transfer, which is the source of superoxide. In the presence of respiratory inhibitors, Complex I may produce the highest amount of superoxide, especially through RET, but the contribution of Complexes I and III to superoxide production is unknown in healthy mitochondria [86]. Superoxide is also produced in the matrix by other enzymes that interact with the NADH pool and by enzymes connected to the inner membrane CoQ pool. These include α-ketoglutarate dehydrogenase that may produce superoxide if its substrate

membrane, α-glycerophosphate dehydrogenase may produce superoxide partly via RET and Complex II, which transfer electrons from succinate to CoQ, is also suspected to gener-

(α-ketoglutarate) concentration and the NADH/NAD<sup>+</sup>

ratio is high (and the CoQ binding site is blocked or the CoQ pool is mostly

gradient through the inner

ratio increase in the matrix. In the

is con-

electrons flow through the respiratory chain is converted into a H<sup>+</sup>

190 Endothelial Dysfunction - Old Concepts and New Challenges

respiratory chain [85, 88].

ate some superoxide [85].

NADH/NAD<sup>+</sup>

**Figure 2.** Oxidant production by the mitochondrial electron transport chain. CoQ: coenzyme Q, ubiquinone; Cyt C: cytochrome C; FAD+/FADH<sup>2</sup> : flavin adenine dinucleotide; H<sup>2</sup> O2 : hydrogen peroxide; MnSOD: manganese-dependent superoxide dismutase; NO•: nitric oxide; O<sup>2</sup> •−: superoxide, ONOO•−: peroxynitrite; PARP: poly(ADP-ribose) polymerase; p66SHC: 66-kDa Src homology 2 domain-containing protein; SQR: sulfide:quinone oxidoreductase and UCP: uncoupling protein.

In hyperglycemic endothelial cells, the increased production of superoxide originates from the reduced CoQ pool before Complex III [75, 83]. The high electron donor input from glycolysis and the TCA cycle may increase the membrane potential and inhibit the electron transfer at Complex III, thus increase the concentration of reduced and free radical intermediates of CoQ. Superoxide generation may occur as direct 'leakage' of electrons to oxygen, as a result of the longer half-life of CoQ intermediates in the lipid bilayer and bound to Complex III or via RET through Complex I. Superoxide generation is also promoted by the increased membrane potential and proton concentration gradient through the inner membrane [31, 35, 83, 94]. Superoxide production was found to increase exponentially above 140 mV with the increase of the mitochondrial membrane potential [95]. Since with the generation of each superoxide molecule one electron is lost compared to the number of protons, superoxide production per se may increase the membrane potential and the proton gradient or might be responsible for the maintenance of the elevated membrane potential. Furthermore, the proton and charge transfer of Complexes III and IV are disproportional since Complex III picks up two protons from the matrix side of the inner membrane (the negatively charged N-face) and releases four protons to the intermembrane space side (positively charged P-face), whereas Complex IV abstracts four protons from the matrix and releases two protons to the intermembrane space per transfer of two electrons. Thus, Complex III transfers four protons but only two positive charges, whereas Complex IV transfers two protons and four positive charges [89, 96], which may lead to an increase in the membrane potential if there is a mismatch between the activity of the two complexes. Also, while it is possible to generate considerably higher membrane potential than the physiological value, since the proton-motive force is sufficient to generate about 240 mV, the proton permeability of biological membranes increases above 130 mV; thus, the higher values are associated with energy loss [95]. To optimize the energy efficiency, OXPHOS is tightly regulated by the ATP concentration (or ATP/ADP ratio) in the matrix: high ATP concentration in the matrix allosterically inhibits Complex IV of the respiratory chain and decreases the mitochondrial membrane potential [97]. Complex IV has a low reserve capacity and it may represent the major controlling site of respiration and mitochondrial ATP synthesis [95]. This immediate regulation is supplemented by the phosphorylation-mediated regulation of respiratory complexes, which transmit the extramitochondrial and extracellular stimuli to adapt OXPHOS to stress conditions [95]. Phosphorylation sites were detected in all respiratory complexes and there is a growing list of stress factors that may induce phosphorylation of the complexes or mitochondrial hyperpolarization that might be associated with the adaptive process. This is how inflammatory cytokines may affect superoxide generation in diabetes.

and only survive for less than 3 weeks [101]. Mutations associated with reduced activity of MnSOD accelerate diabetic nephropathy and neuropathy [102–104]. On the other hand, overexpression of MnSOD prevents hyperglycemic injury in endothelial cells, suggesting that the respiratory chain is the source of oxidants in hyperglycemia [26, 83]. The amount of superoxide produced by the respiratory chain may not be excessively higher in hyperglycemia, since the overexpression of the MnSOD can efficiently scavenge the oxidants or low amounts of mitochondria-targeted antioxidants are able to neutralize ROS in hyperglycemia

In cells exposed to hyperglycemia, mitochondrial ROS production activates various mechanisms to reduce the oxidant production. This includes immediate responses that may control the mitochondrial potential in the short term and also longer term responses that may protect against the increase of the mitochondrial potential, but these mostly reduce the energy efficiency of OXPHOS. Hyperglycemia and ROS production activate the uncoupling proteins in the mitochondrial inner membrane that allow higher proton transfer from the intermembrane space to the matrix without coupled ATP production [32, 33]. This activity not only reduces the mitochondrial membrane potential but also decreases the amount of ATP generated in the

Hyperglycemia also increases the consumption of hydrogen sulfide, an inorganic substrate of

may provide electrons to CoQ without the additional protons, it can reduce the mitochondrial

that is used in small quantities or function as a buffer to control the mitochondrial potential.

These immediate reactions are supplemented with the morphological changes of mitochondria. Mitochondria are dynamically changing organelles in the cells: they may form long tubes that cross the whole length of the cell or short rods that are as long as wide or any length in between. Mitochondria continuously change their shape by fusion (elongation) and fission (fragmentation) and they move along microtubular tracks within the cells. This process is believed to help maintain functional mitochondria; it allows rapid redistribution of mitochondrial proteins and may help the elimination of dysfunctional parts or proteins. Hyperglycemia stimulates the fission of mitochondria that can reduce the mitochondrial membrane potential but also helps dissociate the respiratory complexes and decrease the chance of assembly of various proteins within a complex [110–114]. Altogether, it results in partly assembled respiratory complexes and higher superoxide production that will reduce the energy efficiency of mitochondria [98, 99]. Mitochondrial fission is a later process induced by high glucose exposure, and it occurs only after the superoxide production is induced.

S oxidation

S, and

S may represent an alternative energy source

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 193

S pool and the plasma concentration of H<sup>2</sup>

S in the mitochondria [94, 108, 109].

the mitochondria that can act as an endogenous electron donor [105–107]. Since H<sup>2</sup>

**5. Mechanism of damage: cell damaging responses to ROS** 

[26, 38, 83].

mitochondria.

**production in hyperglycemia**

potential and promote ATP synthesis; thus, H<sup>2</sup>

Hyperglycemia reduces the mitochondrial H2

it may deplete the buffering capacity of H<sup>2</sup>

Hyperglycemia-induced mitochondrial superoxide production is a functional change of the respiratory chain; no difference is detectable in the assembly or the relative amounts of the respiratory complexes in the early phases of the injury [26, 35]. At later stages, changes in the expression or assembly of some components of the respiratory chain may occur and these are typically associated with impaired functionality [98, 99]. The glucose-induced changes in the mitochondrial superoxide production are reversible: normalization of the membrane potential suppresses the ROS production in endothelial cells [26, 35, 83, 94, 100]. While elevated mitochondrial membrane potential is detectable in endothelial cells exposed to high glucose concentration, the overexpression of either uncoupling protein 1 (UCP1) or uncoupling protein 2 (UCP2) normalizes the membrane potential and reduces the ROS production [26, 35, 83]. The function of UCP2 is regulated by ROS itself: the proton conductance of the protein is controlled by glutathionylation, and if ROS is present, it increases the proton leakage, whereas in the absence of ROS, the channel closes, thus this feedback may control the mitochondrial potential and the ROS production simultaneously [32, 33]. Furthermore, hydrogen sulfide donors that normalize the mitochondrial potential by electron supplementation via sulfide:quinone oxidoreductase (SQR) also inhibit the superoxide generation induced by hyperglycemia [94, 100].

The mitochondrial matrix possesses antioxidant enzymes to defend against oxidative damage. Manganese-dependent superoxide dismutase (MnSOD, also known as superoxide dismutase 2 (SOD2)) is the mitochondrial enzyme that neutralizes superoxide produced by the respiratory chain and converts it to H2 O2 . Since functional mitochondria constantly produce ROS, it is necessary to scavenge oxygen radicals. The importance of MnSOD is underlined by the fact that MnSOD-deficient mice exhibit extensive mitochondrial injury and only survive for less than 3 weeks [101]. Mutations associated with reduced activity of MnSOD accelerate diabetic nephropathy and neuropathy [102–104]. On the other hand, overexpression of MnSOD prevents hyperglycemic injury in endothelial cells, suggesting that the respiratory chain is the source of oxidants in hyperglycemia [26, 83]. The amount of superoxide produced by the respiratory chain may not be excessively higher in hyperglycemia, since the overexpression of the MnSOD can efficiently scavenge the oxidants or low amounts of mitochondria-targeted antioxidants are able to neutralize ROS in hyperglycemia [26, 38, 83].
