**3. Anabolism and catabolism. Redox‐sensitive enzymes**

Prooxidant reactions play an important role in cellular signaling and redox regulation of met‐ abolic processes such as immune defense, growth, and apoptosis among others. The increase in the ROS production requires antioxidant strategies to prevent a potential oxidative damage to cellular components. The imbalance of prooxidants and antioxidants leading to cell dam‐ age and tissue injury may cause oxidative stress. Oxidative stress is common in organs and tissues with high metabolic and energy turnover, including skeletal and heart muscle, blood cells, and liver [14].

It has been recognized as the interplay between energy metabolism and ROS to make pos‐ sible the homeostasis in the liver physiology. As it was mentioned above, redox couples as NADH/NAD+ , FADH2 /FAD+ , and GSH/GSSG are involved in the donation and acceptance of electrons in a variety of reactions. NADPH is a key cofactor for many enzymatic reactions in the metabolism, and it is considered as one of the main regulator of the redox potential (**Figure 3**). Its production is required for the regeneration of GSH in mitochondria for scaveng‐ ing mitochondrial ROS through glutathione reductase and peroxidase systems [15]. NADP+ is synthesized from NAD+ by NAD+ kinase, whereas NADPH is derived from NADH by three major enzymes in the mitochondrial matrix: NAD(P)+ transhydrogenase, NADP+ ‐dependent isocitrate dehydrogenase (IDH) and malic enzyme and by other three cytosolic enzymes in cytosol: glucose‐6‐phosphate dehydrogenase (G6PD), 6‐phosphogluconate dehydrogenase in the pentose phosphate pathway (PPP), and the malic enzyme.

and vascular smooth muscle cells, in hypoxic condition there is an increase in mitochondrial ROS production from complex III; however, the exact mechanism is not well understood.

In liver and muscle cells, angiotensin II signaling also promotes higher production of mito‐ chondrial ROS, which are necessary to activate downstream responses such as mitogen‐acti‐

When an organism is infected primordial T‐cells rapidly proliferate, and differentiate into effector T‐cells, and mitochondria contribute to this activation through the production of ROS. This has been analyzed adding antioxidants to mice after a viral infection and showing that they exhibited a depressed immune system. Data suggest that ROS play an important

A fundamental characteristic of cancerous cells is their uncontrolled proliferation. It has been

antioxidants have been detected, maybe to implement a protective cellular response. The final equilibrium allows a high rate of ROS synthesis concomitant with a transformed functional

ROS are responsible of oncogenes activation and/or loss of tumor suppressors enhancing

Mutations in mitochondrial DNA result in ROS increase that has been associated to a great variety of human cancers. One example is the mutation in the gene of NADH dehydrogenase in the mitochondrial complex I, which promotes an elevation of ROS production. The pro‐ oxidant stimulus leads to the proliferation of several human and mouse cell lines, as well as tumor formation in rodents. Interestingly, this condition could be rescued by the reconstitu‐

Prooxidant reactions play an important role in cellular signaling and redox regulation of met‐ abolic processes such as immune defense, growth, and apoptosis among others. The increase in the ROS production requires antioxidant strategies to prevent a potential oxidative damage to cellular components. The imbalance of prooxidants and antioxidants leading to cell dam‐ age and tissue injury may cause oxidative stress. Oxidative stress is common in organs and tissues with high metabolic and energy turnover, including skeletal and heart muscle, blood

It has been recognized as the interplay between energy metabolism and ROS to make pos‐ sible the homeostasis in the liver physiology. As it was mentioned above, redox couples as

O2

; in addition, elevated levels of

role in T‐cell activation, proliferation and adaptive immune function [12].

**3. Anabolism and catabolism. Redox‐sensitive enzymes**

observed that they generate high levels of ROS, especially H2

tion of the wild‐type enzymatic activity [13].

vated protein kinases (MAPK) [11].

160 Redox - Principles and Advanced Applications

**2.2. Immune system**

**2.3. Cancer**

phenotype.

mitogenic signaling.

cells, and liver [14].

One of the mechanisms for generating cytosolic NADPH from mitochondrial oxidations implies substrates shuttles. This is the case of the shuttle mechanism that involves NADP+ ‐ dependent IDHs present in both, mitochondrial and extramitochondrial spaces, whereas NAD+ ‐dependent IDH is solely mitochondrial. IDHs catalyze oxidative decarboxylation of iso‐ citrate to α‐ketoglutarate in the tricarboxylic acid cycle (TCA), in a NAD+ or NADP+ ‐dependent manner producing NADH or NADPH, respectively. NADPH may be transported by the iso‐ citrate‐2‐ketoglutarate shuttle by the cytosolic IDH in which liver has a high enzymatic activ‐ ity. NADP+ ‐dependent IDH is induced by ROS and controls the mitochondrial redox balance. A decreased expression of NADP+ ‐dependent IDH importantly elevates ROS generation, lipid peroxidation and DNA fragmentation; consequently, a significant reduced ATP level is associated to the mitochondrial damage, whereas overexpression of NADP+ ‐dependent

**Figure 3.** Redox cycles linking metabolic and antioxidant pathways. Glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), isocitrate dehydrogenase (IDH), glucose‐6‐phosphate dehydrogenase (G6PD), α‐ ketoglutarate dehydrogenase (α‐KGDH), sirtuin (SIRT), reduced glutathione (GSH), oxidized glutathione (GSSG).

IDH protects from ROS‐induced damage [16]. Thus, both cytosolic and mitochondrial IDHs play important roles in cellular defense against oxidative damage by providing NADPH needed for the generation of GSH [17, 18] (**Figure 2**). Likewise, NADP+ ‐dependent IDH is inactivated by GSSG‐dependent glutathionylation leading to enzyme inactivation, followed by GSH‐dependent reactivation, suggesting an alternative modification to the redox regula‐ tion of IDHs [19].

Among enzymes sensitive to redox state is α‐ketoglutarate dehydrogenase (α‐KGDH). α‐ KGDH is a mitochondrial enzyme of the TCA that catalyzes the conversion of α‐ketoglu‐ tarate to succinyl‐CoA, producing NADH that supplies electrons for the respiratory chain (**Figure 2**). α‐KGDH is considered a component of the mitochondrial antioxidant system and a key sensor of redox status [20]. It is sensitive to ROS and its inhibition impact significantly in the energetic deficit induced by oxidative stress; α‐KGDH can also generate ROS by its catalytic action regulated by the NADH/NAD+ ratio [21]. A reversible inhibition of α‐KGDH is obtained by glutathionylation. Since α‐KGDH controls supply of reducing equivalents gen‐ erated by the TCA, the redox regulation of α‐KGDH would control energy production in response to oxidative stress.

Another source of NADPH required for detoxification of free radicals and peroxides is the activity of G6PD, the rate‐limiting enzyme of the PPP (**Figure 2**). It has an important role for cell growth by providing NADPH for redox balance [22] and its expression is induced due to oxidative stress [23], whereas the reduction of G6PD sensitizes cells to oxidative stress [24]. In humans, mutants of G6PD alleles are associated with hemolytic anemia, while mutants in mouse embryonic stem cells by targeted homologous recombination have shown that G6PD is essential to protect cells against even mild oxidative stress whereas the null mutant is lethal. A high‐carbohydrate, fat‐free diet promotes an increase in hepatic G6PD activity [25], but polyunsaturated fatty acids content of the diet decreases its activity. Besides the effect at activ‐ ity level, gene expression is also altered. Inhibition of G6PD gene expression is caused by polyunsaturated fatty acids but not by saturated or monounsaturated fatty acids [26]. In the liver of young Zucker obese fa/fa rats, G6PD expression and activity are increased prior to the onset of diabetes type 2, which seems to be contributing factors for the induction of oxidative stress [27].

Furthermore, human G6PD is negatively regulated by acetylation on a phylogenetically con‐ served lysine 403 becoming unable to form active dimers and consequently the loss of activ‐ ity. The exposure to extracellular oxidative stimuli promotes reduced G6PD acetylation due to deacetylating activity of sirtuins (SIRTs) (**Figure 2**). The inhibition of SIRT2 increases cel‐ lular susceptibility to oxidative stress. SIRT2 deletion leads to a higher level of lysine 403 acetylation and impaired activity of G6PD, whereas the addition of SIRT2 rescues the cell death induced by the deletion [28].

In mammals, seven members of SIRT family are known; they have diverse subcellular local‐ ization and activity. SIRT1, SIRT6 and SIRT7 are nuclear, SIRT6 is associated with heterochro‐ matic regions and SIRT7 with nucleoli, SIRT2 is in cytosol, and SIRT3–5 are mitochondrial. SIRT1 shows a potent NAD+ ‐dependent deacetylase activity on lysine 16 of histone H4 that could promote the formation of heterochromatin, as well as on lysine 382 of p53, while SIRT6 and SIRT7 lack deacetylase activity [29]. Several proteins of this family regulate lifespan in diverse organisms and in human cells.

IDH protects from ROS‐induced damage [16]. Thus, both cytosolic and mitochondrial IDHs play important roles in cellular defense against oxidative damage by providing NADPH

inactivated by GSSG‐dependent glutathionylation leading to enzyme inactivation, followed by GSH‐dependent reactivation, suggesting an alternative modification to the redox regula‐

Among enzymes sensitive to redox state is α‐ketoglutarate dehydrogenase (α‐KGDH). α‐ KGDH is a mitochondrial enzyme of the TCA that catalyzes the conversion of α‐ketoglu‐ tarate to succinyl‐CoA, producing NADH that supplies electrons for the respiratory chain (**Figure 2**). α‐KGDH is considered a component of the mitochondrial antioxidant system and a key sensor of redox status [20]. It is sensitive to ROS and its inhibition impact significantly in the energetic deficit induced by oxidative stress; α‐KGDH can also generate ROS by its

is obtained by glutathionylation. Since α‐KGDH controls supply of reducing equivalents gen‐ erated by the TCA, the redox regulation of α‐KGDH would control energy production in

Another source of NADPH required for detoxification of free radicals and peroxides is the activity of G6PD, the rate‐limiting enzyme of the PPP (**Figure 2**). It has an important role for cell growth by providing NADPH for redox balance [22] and its expression is induced due to oxidative stress [23], whereas the reduction of G6PD sensitizes cells to oxidative stress [24]. In humans, mutants of G6PD alleles are associated with hemolytic anemia, while mutants in mouse embryonic stem cells by targeted homologous recombination have shown that G6PD is essential to protect cells against even mild oxidative stress whereas the null mutant is lethal. A high‐carbohydrate, fat‐free diet promotes an increase in hepatic G6PD activity [25], but polyunsaturated fatty acids content of the diet decreases its activity. Besides the effect at activ‐ ity level, gene expression is also altered. Inhibition of G6PD gene expression is caused by polyunsaturated fatty acids but not by saturated or monounsaturated fatty acids [26]. In the liver of young Zucker obese fa/fa rats, G6PD expression and activity are increased prior to the onset of diabetes type 2, which seems to be contributing factors for the induction of oxidative

Furthermore, human G6PD is negatively regulated by acetylation on a phylogenetically con‐ served lysine 403 becoming unable to form active dimers and consequently the loss of activ‐ ity. The exposure to extracellular oxidative stimuli promotes reduced G6PD acetylation due to deacetylating activity of sirtuins (SIRTs) (**Figure 2**). The inhibition of SIRT2 increases cel‐ lular susceptibility to oxidative stress. SIRT2 deletion leads to a higher level of lysine 403 acetylation and impaired activity of G6PD, whereas the addition of SIRT2 rescues the cell

In mammals, seven members of SIRT family are known; they have diverse subcellular local‐ ization and activity. SIRT1, SIRT6 and SIRT7 are nuclear, SIRT6 is associated with heterochro‐ matic regions and SIRT7 with nucleoli, SIRT2 is in cytosol, and SIRT3–5 are mitochondrial.

could promote the formation of heterochromatin, as well as on lysine 382 of p53, while SIRT6

‐dependent deacetylase activity on lysine 16 of histone H4 that

‐dependent IDH is

ratio [21]. A reversible inhibition of α‐KGDH

needed for the generation of GSH [17, 18] (**Figure 2**). Likewise, NADP+

tion of IDHs [19].

162 Redox - Principles and Advanced Applications

catalytic action regulated by the NADH/NAD+

response to oxidative stress.

stress [27].

death induced by the deletion [28].

SIRT1 shows a potent NAD+

SIRT1 is the homolog of Sir2, a protein with an important role in longevity in yeast, and with an important role in mammalian development, metabolic regulation and modulation of cel‐ lular stress response and survival by acting on p53, NF‐κB signaling and FoxO transcription factors [29]. SIRT1 is an important metabolic regulator that orchestrates hepatic gluconeogen‐ esis and lipid metabolism, suggesting that it can play an important role in the developing of metabolic and age‐related diseases. The effects of SIRT1 are mediated through the induction of antioxidant proteins, SOD2, nuclear respiratory factor 1 (NRF1) [30], catalase, peroxire‐ doxins 3 and 5, thioredoxin 2, thioredoxin reductase 2, and uncoupling protein 2 (UCP‐2) via formation of forkhead box/peroxisome proliferator‐activated receptor gamma coactivator 1‐α complex (FoxO3a/PGC‐1α) [31] (**Figure 3**).

Increasing ROS level modulates activity of SIRT1. Likewise, SIRT1 activity is regulated by AMPK, which is a redox‐sensing enzyme, and a metabolic gauge by increasing cellular NAD+ synthesis [32]. AMPK is activated by high AMP/ATP ratio and is sensitive to glutathionyl‐ ation by action of H2 O2 and NO that oxide reactive thiol residues [33]. Oxidative stress also induces S‐glutathionylation of SIRT1 and reduces NAD+ level, thus inhibiting SIRT1 activity [34]. SIRT1 has an important role in the liver glucose metabolism. Ablation of SIRT1 in liver induces lipid accumulation by upregulating lipogenic genes expression and reducing β‐oxi‐ dation, whereas overexpression of SIRT1 protects against hepatic steatosis induced by high‐ fat diet [35]. SIRT1 regulates positively β‐oxidation through the activation of nuclear receptors peroxisome proliferator‐activated receptor α (PPARα) that regulates gene expression of lipid catabolic genes.

Peroxisomes are oxidative subcellular organelles for H2 O2 , fatty acids, and cholesterol. PPARs are lipophilic ligand‐activated transcription factors that belong to nuclear hormone receptor superfamily with three subtypes: PPARα, PPARγ, and PPARβ/δ [36]. PPARα is mainly pres‐ ent in the liver, playing a relevant role in the fasting response. Additionally, PPARα contrib‐ utes to protection from oxidative stress by upregulating expression of genes of the chaperone and proteasome families, with consequences in protein folding and degradation of damaged proteins [37]. In cancerous cells, PPARα leads to increased peroxisome proliferation and production of ROS contributing to DNA damage [38]. Oxidized lipids are produced during oxidative stress and are natural endogenous PPARγ ligands suggesting a role in oxidative stress response. PPARγ upregulates the expression of antioxidant and prooxidant genes such as catalase, SOD2, GPx3, eNOS and mitochondrial uncoupling protein 2 (UCP‐2), whereas downregulates cyclooxygenase‐2 (COX‐2) and iNOS [39].

Handling of cellular oxidative stress involves several metabolic enzymes that are originally described as part of other metabolic pathways. Most of these enzymes reduce free‐radical production and protect cell from injury. The loss of control in the events that coordinates redox homeostasis contributes to oxidative damage, metabolic diseases and progression of degenerative diseases associated to aging. Understanding the mechanisms involved in these events will contribute to improve the quality of life during physiological or pathological conditions.
