**4.1. Glucose-induced oxidative stress pathways**

recent studies also found independent associations with the postprandial peaks [49]. These results highly suggest the action of secondary mediators that are rather induced by the fluctuations in blood glucose (glycemic variability) than by an absolute increase. Experimental models confirmed that glycemic swings caused at least as severe tissue damage as constant hyperglycemia, and persistence of high-glucose memory was postulated in cells and animals that were exposed to normoglycemic conditions following a hyperglycemic exposure [50– 52]. Endothelial cells when returned to normal glucose concentration after exposure to high glucose showed increased ROS production and activation of poly(ADP-ribose) polymerase (PARP) even a week following the normalization of the glucose level and in this respect they showed similar characteristics to cells maintained at high glucose [52]. The persistence of oxidative stress in endothelial cells *in vitro* confirms that 'glucose memory' is an inherent feature of these cells. It also means that once hyperglycemia activates the various ROS-producing pathways they continue to produce oxidants for multiple days or weeks in endothelial cells even if the glucose level is fully normalized. Oxidative stress is the key feature of the changes induced by hyperglycemia, and 'metabolic memory' is another term used that refers to the characteristic metabolic changes [51]. The length of high glucose memory is unknown in humans but it is suspected to last longer than *in vitro* because (1) inflammatory pathways are also involved and (2) the response is not limited by the life cycle of single cells but it is pos-

Blockade of the early changes has been confirmed to prevent or slow down the progression of complications but the reversal at a later phase may not be achieved by glycemic control [53]. Benefits of intensive glucose control can be detected after 3 years of treatment if no retinopathy or mild disease is present at the start of the treatment strategy in type 1 diabetes [54]. The importance of blocking the glucose-induced damage early on in type 2 diabetes has been confirmed by the results of the United Kingdom Prospective Diabetes Study (UKPDS) [41]. On the other hand, there is little benefit of strict glucose control if established cardiovascular disease is already present at the start of the treatment regimen [53]. Similarly, in diabetic rats, a 6-month long period of good glycemic control following 2 months of poor glycemic control results in significantly reduced progression, whereas no benefit is observed on retinopathy if good glycemic control was started after 6 months of poor glycemic control: both nitrosative stress and tissue damage were similarly advanced as with 12 months of poor glycemic control [55, 56]. These suggest that the processes started by hyperglycemia may be partially reversed if normoglycemia follows a shorter period of high glucose exposure. It is still unclear whether the detrimental effects of transient hyperglycemia is buffered within the individual cells or it is the entire population of endothelial cells that compensate for the changes and the reason why progressive damage occurs following an extended hyperglycemic period is the loss of

The mechanism of high glucose memory is still obscure and little is known about the pathways involved. Hyperglycemia modifies the metabolism of the cells and is suspected to induce various downstream pathways or molecules that are responsible for maintaining the

sibly carried over to multiple cell generations.

184 Endothelial Dysfunction - Old Concepts and New Challenges

the renewal capacity of the cells.

**3.2. Downstream molecules responsible for the damage**

Changes in glucose metabolism are presumed to be directly responsible for provoking oxidant production in endothelial cells. Endothelial cells predominantly use glucose as energy source and rely on glycolysis to generate ATP molecules [14]. Glycolytic flux exceeds the rate of oxidative phosphorylation (OXPHOS) by two orders of magnitude in endothelial cells *in vitro* and similar ratio is suspected *in vivo* [62, 63]. The contribution of fatty acid oxidation to energy production is thought to be negligible in capillary endothelial cells, though endothelial cells take up fatty acids and transport them to the neighboring cells, thus play important role in transendothelial fatty acid delivery [64]. Since the function of capillary endothelial cells is to deliver oxygen and fuel sources to other cells in tissues, they do not consume much oxygen or store energy but preserve them to other perivascular cells. Thus, excess glucose is not converted to glycogen for storage in endothelial cells, but is pushed toward glycolysis [65]. Glutamine is a further energy source in endothelial cells via glutaminolysis that directly produces one GTP molecule (that can be converted to ATP) and further five ATP molecules from NADH<sup>+</sup> and FADH2 via OXPHOS. Glutamine is the most abundant amino acid in the plasma, and glutaminolysis is a valuable energy source if glycolytic output is low since it feeds alpha-ketoglutarate to the TCA cycle and similarly produces lactate (or pyruvate). However, all the energy producing steps in glutaminolysis occur in the mitochondria via the TCA cycle and OXPHOS and mitochondrial impairment may affect the energy efficacy of glutaminolysis [66].

The metabolic balance between glycolysis and OXPHOS is controlled by nutrients (the ATP and NADH output) via Sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) in the cells. SIRT1 is a NAD<sup>+</sup> -dependent histone deacetylase enzyme that regulates energy homeostasis via gene expression changes induced by deacetylating a variety of histone proteins, transcription factors and coregulators [67]. The activity of SIRT1 is primarily controlled by NAD+ abundance and NAD+ /NADH ratio. AMPK is a master sensor of the energy level in the cells: it detects the cellular ATP concentration and is activated by a decrease in the ATP level. There is a complex interplay between AMPK and SIRT1: the two enzymes indirectly activate each other. Activated SIRT1 deacetylates LKB1 (tumor suppressor liver kinase B1) that phosphorylates and activates AMPK [68, 69], whereas AMPK activates SIRT1 by increasing the NAD+ /NADH ratio either by inducing the NAD<sup>+</sup> biosynthesis enzyme NamPRT (nicotinamide phosphoribosyltransferase) or by a NamPRT-independent mechanism [70]. Thus, caloric restriction activates both AMPK and SIRT1, whereas both enzymes are suppressed if energy sources are abundant like in hyperglycemia [71, 72]. In caloric restriction, SIRT1 deacetylates and activates peroxisome proliferation activating receptor-γ (PPAR-γ) coactivator 1-α (PGC-1) and forkhead box O1 protein 1 (FOXO1) and leads to glucose sparing: suppressed glycolysis and increased mitochondrial activity and they also activate gluconeogenesis [73, 74]. On the other hand, in hyperglycemia, the activity of AMPK and SIRT1 is suppressed and it results in enhanced glycolysis, inhibition of gluconeogenesis and decreased mitochondrial biogenesis and OXPHOS [73].

Overload of glycolysis and the pentose phosphate pathway are the initial steps that trigger alternate pathways of glucose metabolism (**Figure 1**). Prior perturbation of mitochondrial metabolism (TCA cycle overload and impaired OXPHOS) is highly possible since inhibition of mitochondrial superoxide generation prevents the activation of the above pathways but the exact mechanism that initiates these events is unknown [26]. The high glycolytic input and low OXPHOS capacity may gradually block the main metabolic steps and shunt the metabolism to alternative pathways. These include the methylglyoxal, hexosamine and polyol pathways: dihydroxyacetone phosphate (DHAP) is diverted to the methylglyoxal pathway and leads to protein kinase C (PKC) activation, and fructose-6-phosphate (F6P) increases the flux through the hexosamine pathway and excess glucose enters the polyol pathway when converted to sorbitol [66, 75]. Suppressed expression of the gluconeogenetic enzyme glucose-6-phosphate dehydrogenase (G6PDH) prevents shunting of glucose to the pentose phosphate pathway that further increases the glycolytic load [76, 77]. All these processes lead to ROS production and the generation of advanced glycation end products (AGEs), the products of nonenzymatic glycation and oxidation of proteins and lipids that accumulate in diabetes. AGEs signal through the receptor of AGE (RAGE), a cell surface receptor that is also activated by the damage-associated molecular patterns (DAMP) HMGB1 (high-mobility group box 1) and S100 proteins [78]. RAGE activates nuclear factor kappa B (NF-κB) and controls several inflammatory genes, thus links hyperglycemia to inflammation. Since RAGE itself is upregulated by NF-κB, inflammation is maintained by positive feedback in hyperglycemia as AGEs, the ligands are continuously produced.

Interestingly, hyperglycemia induces a long-lasting suppression in SIRT1 and AMPK activity in endothelial cells: the activity of both enzymes remains low weeks after the normalization of glucose level following a week long hyperglycemia [68]. Thus, SIRT1 and AMPK have been implicated in glucose memory since restoration of their activity reduces the ROS production

**Figure 1.** Hyperglycemia-induced ROS-producing pathways in the cytoplasm. AGEs: advanced glycation end-products; G6PDH: glucose-6-phosphate dehydrogenase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSH/GSSG:

/NADH: nicotinamide adenine dinucleotide; NADP+/NADPH: nicotinamide

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 187

One further molecule that possibly takes part in the maintenance of oxidative stress in hyperglycemic endothelial cells is p66SHC (66-kDa Src homology 2 domain-containing protein) [79]. p66SHC is induced by hyperglycemia and it contributes to oxidative stress and endothelial damage. Genetic ablation of p66SHC reduces the oxidative stress in diabetic animals, protects against vascular dysfunction and blocks the progression of nephropathy [79, 80]. p66SHC is a redox enzyme that associates with 70 kDa heat shock protein (Hsp70) and localizes within the intermembrane space in the mitochondria. Upon oxidative stress, p66SHC is released from the complex and transfers electrons from the electron transfer chain

(from cytochrome c specifically) to oxygen and produces hydrogen peroxide (H<sup>2</sup>

Under basal conditions, p66SHC is also present as an inactive enzyme in the cytoplasm

O2 ) [81].

and PARP activity in the cells.

reduced/oxidized glutathione; NAD<sup>+</sup>

adenine dinucleotide phosphate and ROS: reactive oxygen species.

glutaminolysis occur in the mitochondria via the TCA cycle and OXPHOS and mitochondrial

The metabolic balance between glycolysis and OXPHOS is controlled by nutrients (the ATP and NADH output) via Sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) in the

stasis via gene expression changes induced by deacetylating a variety of histone proteins, transcription factors and coregulators [67]. The activity of SIRT1 is primarily controlled by

cells: it detects the cellular ATP concentration and is activated by a decrease in the ATP level. There is a complex interplay between AMPK and SIRT1: the two enzymes indirectly activate each other. Activated SIRT1 deacetylates LKB1 (tumor suppressor liver kinase B1) that phosphorylates and activates AMPK [68, 69], whereas AMPK activates SIRT1 by increasing

tinamide phosphoribosyltransferase) or by a NamPRT-independent mechanism [70]. Thus, caloric restriction activates both AMPK and SIRT1, whereas both enzymes are suppressed if energy sources are abundant like in hyperglycemia [71, 72]. In caloric restriction, SIRT1 deacetylates and activates peroxisome proliferation activating receptor-γ (PPAR-γ) coactivator 1-α (PGC-1) and forkhead box O1 protein 1 (FOXO1) and leads to glucose sparing: suppressed glycolysis and increased mitochondrial activity and they also activate gluconeogenesis [73, 74]. On the other hand, in hyperglycemia, the activity of AMPK and SIRT1 is suppressed and it results in enhanced glycolysis, inhibition of gluconeogenesis and decreased

Overload of glycolysis and the pentose phosphate pathway are the initial steps that trigger alternate pathways of glucose metabolism (**Figure 1**). Prior perturbation of mitochondrial metabolism (TCA cycle overload and impaired OXPHOS) is highly possible since inhibition of mitochondrial superoxide generation prevents the activation of the above pathways but the exact mechanism that initiates these events is unknown [26]. The high glycolytic input and low OXPHOS capacity may gradually block the main metabolic steps and shunt the metabolism to alternative pathways. These include the methylglyoxal, hexosamine and polyol pathways: dihydroxyacetone phosphate (DHAP) is diverted to the methylglyoxal pathway and leads to protein kinase C (PKC) activation, and fructose-6-phosphate (F6P) increases the flux through the hexosamine pathway and excess glucose enters the polyol pathway when converted to sorbitol [66, 75]. Suppressed expression of the gluconeogenetic enzyme glucose-6-phosphate dehydrogenase (G6PDH) prevents shunting of glucose to the pentose phosphate pathway that further increases the glycolytic load [76, 77]. All these processes lead to ROS production and the generation of advanced glycation end products (AGEs), the products of nonenzymatic glycation and oxidation of proteins and lipids that accumulate in diabetes. AGEs signal through the receptor of AGE (RAGE), a cell surface receptor that is also activated by the damage-associated molecular patterns (DAMP) HMGB1 (high-mobility group box 1) and S100 proteins [78]. RAGE activates nuclear factor kappa B (NF-κB) and controls several inflammatory genes, thus links hyperglycemia to inflammation. Since RAGE itself is upregulated by NF-κB, inflammation is maintained by positive feedback in hyperglycemia as AGEs,


/NADH ratio. AMPK is a master sensor of the energy level in the

biosynthesis enzyme NamPRT (nico-

impairment may affect the energy efficacy of glutaminolysis [66].

/NADH ratio either by inducing the NAD<sup>+</sup>

cells. SIRT1 is a NAD<sup>+</sup>

abundance and NAD+

186 Endothelial Dysfunction - Old Concepts and New Challenges

mitochondrial biogenesis and OXPHOS [73].

the ligands are continuously produced.

NAD+

the NAD+

**Figure 1.** Hyperglycemia-induced ROS-producing pathways in the cytoplasm. AGEs: advanced glycation end-products; G6PDH: glucose-6-phosphate dehydrogenase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSH/GSSG: reduced/oxidized glutathione; NAD<sup>+</sup> /NADH: nicotinamide adenine dinucleotide; NADP+/NADPH: nicotinamide adenine dinucleotide phosphate and ROS: reactive oxygen species.

Interestingly, hyperglycemia induces a long-lasting suppression in SIRT1 and AMPK activity in endothelial cells: the activity of both enzymes remains low weeks after the normalization of glucose level following a week long hyperglycemia [68]. Thus, SIRT1 and AMPK have been implicated in glucose memory since restoration of their activity reduces the ROS production and PARP activity in the cells.

One further molecule that possibly takes part in the maintenance of oxidative stress in hyperglycemic endothelial cells is p66SHC (66-kDa Src homology 2 domain-containing protein) [79]. p66SHC is induced by hyperglycemia and it contributes to oxidative stress and endothelial damage. Genetic ablation of p66SHC reduces the oxidative stress in diabetic animals, protects against vascular dysfunction and blocks the progression of nephropathy [79, 80]. p66SHC is a redox enzyme that associates with 70 kDa heat shock protein (Hsp70) and localizes within the intermembrane space in the mitochondria. Upon oxidative stress, p66SHC is released from the complex and transfers electrons from the electron transfer chain (from cytochrome c specifically) to oxygen and produces hydrogen peroxide (H<sup>2</sup> O2 ) [81]. Under basal conditions, p66SHC is also present as an inactive enzyme in the cytoplasm 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 of SIRT1.

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

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

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

O2

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

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 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

thesize ATP from ADP and inorganic phosphate. Respiratory Complex I transfers electrons

O couple is +0.82 V), while the generated free energy is used to syn-

within the mitochondria that may contribute

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 189

•−) anion that might be converted to hydrogen

to coenzyme Q (CoQ, ubiquinone), which is the

in

endothelial damage [26, 83, 84].

oxidase (MAO) and p66SHC also produce H<sup>2</sup>

electron at a time produces superoxide (O<sup>2</sup>

from NADH and Complex II from FADH<sup>2</sup>

Complex IV) [86].

potential of oxygen/H<sup>2</sup>

to oxidative stress in hyperglycemia [88].
