**3.4 Free radical targets and the oxidative vicious circle**

134 Biomedical Science, Engineering and Technology

Fig. 4. The general mechanism leading to oxidative phosphorylation is as follows: high-

respiratory chain composed of four protein complexes (Complex I - IV) and two additional electrons carriers: ubiquinone (coenzyme Q, **Q**), a small molecule freely moving in the inner mitochondrial membrane layer, and cytochrome c (**Cyt c**), localized in the intermembrane space attached to the inner membrane. Part of the energy released in this process is used up in the action of proton pumps transporting protons (H+) from matrix to the intermembrane space. Across the inner membrane electrochemical gradient of protons is formed. Protons tend to return to the mitochondrial matrix and restore alignment of H+ concentration on both sides of the membrane. When they pass back through transmembrane protein complex – ATP synthase - the energy of their movement is used for the synthesis of ATP from ADP

Oxidative metabolism and ATP synthesis are closely associated with ROS generation in mitochondria. These organelles consume 80–90% of cell's oxygen during oxidative phosphorylation. The electron transport chain is the main source of ROS in functioning mitochondria. Approximately 0.2–2% of the oxygen taken up by a cell is converted by mitochondria to ROS. Superoxide (O2•−) is the main product of these transformations, and it is then converted to hydrogen peroxide (H2O2) by spontaneous dismutation or by superoxide dismutase (SOD). Glutathione peroxidase or catalase, in turn, convert hydrogen peroxide into water. If this change does not occur, in the presence of divalent cations H2O2 can undergo Fenton's reaction to produce even more harmful hydroxyl radical (•OH). Oxygen can be reduced to superoxide in one-electron step, theoretically, at each step of the respiratory chain, but in reality two major sites of superoxide generation are Complex I and Complex III (Paradies et al., 2010, as cited in Murphy, 2009). There is a considerable experimental support for two mechanisms of ROS production by complex I. The first one is

) derived from NADH and FADH2, are moving along the

energy electrons (marked as e-

and inorganic phosphate (**Pi**).

**3.3 Free radical generation by mitochondria** 

Mitochondria are continuously exposed to action of reactive oxygen species so they need to have a system that will prevent them against destructive effect of oxidative damage. In fact, mitochondria are equipped in complicated multi-leveled ROS defense network consisting of enzymes and non-enzymatic antioxidants. They contain a high concentration of glutathione, α-tocopherol and manganese-containing superoxide dismutase (MnSOD). The role of MnSOD is the dismutation process of superoxide radical to H2O2. The product of MnSOD reaction is detoxied by other enzymes, i.e. catalase, which converts H2O2 into O2 and H2O. Mitochondria possess also another system capable of efcient superoxide removal - the cytochrome c, which is than regenerated (oxidized) by its natural electron acceptor, cytochrome c oxidase (Complex IV). In intact mitochondria, superoxide may be efficiently scavenged by intramitochondrial antioxidant defences (Duchen, 2004). An imbalance between oxidants and antioxidants induces oxidative stress responsible for alteration of biomolecules and intracellular signaling pathways present in every cell (Edeas et al., 2010a). Mitochondria are a major source of ROS generation, but what is important, they are also its major target (Duchen, 2004). Mitochondrial membrane lipids, mainly long-chain polyunsaturated fatty acids (PUFAs), are also susceptible to oxidative stress. PUFAs are basal components of mitochondrial phospholipids. The sensitivity of PUFAs to oxidation increase with the increasing number of double bonds per fatty acid molecule. Peroxidation of membrane phospholipids causes alterations in their structure and consequently may disrupt organisation of the lipid bilayer. It contributes also to changes in membrane uidity and/or permeability, and causes changes in the mitochondrial membrane potential, in respiratory capacity and in oxidative phosphorylation. ROS are also responsible for alterations in proteins, which may manifest by changes in their structure, proteolytic susceptibility and spontaneous fragmentation. Oxidative damage especially affects the mitochondrial electron transport chain and, when the ETC enzymes stop working properly, the ROS production increases. This may result in the incomplete oxygen consumption, reduced production of ATP, and finally overproduction of ROS (Waldbaum & Patel, 2009).

## **4. Mitochondrial physiology in diabetes**

Mitochondria are provided with a variety of bioenergetic functions mandatory for the regulation of intracellular energy production. Alteration of bioenergetic activities may have drastic consequences on cellular function through the perturbation of energetic charge and balance of the cell (Fig. 5).

Fig. 5. The overall impact of the burden of hypergycaemia in diabetes on functioning of mitochondria.

Abnormalities of mitochondrial metabolism causing human disease have been recognised for more than 40 years. Numerous reports clearly indicate the association between mitochondrial dysfunction and diabetes. Nevertheless, some mechanisms of mitochondrial role in this pathology still requires further elucidation. Therefore different animal model studies are involved in the investigation of explaining these unknown mechanisms. There are several models of experimental diabetes that mimic two common types of diabetes. Streptozotocin-induced diabetes is a widely accepted animal model for type 1 diabetes, resulting from the inability of the pancreatic beta cells to produce insulin. For the research on type 2 or the insulin resistant state, resulting from the inefficient use of insulin by the tissues to regulate blood glucose concentration, some genetically manipulated animal models (e.g. Zucker fatty rats (ZFR), *ob/ob* (obese) mice, CP (corpulent) rats, GK (Goto-Kakizaki) rats, Akita mice) may be utilized (Srinivasan & Ramarao, 2007).

## **4.1 Mitochondrial dysfunction and diabetes type 1**

Streptozotocin (STZ) is a naturally occurring chemical that is particularly toxic to the insulinproducing β cells of the pancreas in mammals and is used to generate Type 1 diabetes in the experimental model. Animals with diabetes induced by STZ exhibit increased mitochondrial oxidative stress and dysfunction. Other agent, alloxan, a toxic glucose analogue, is also used in order to generate type 1 of diabetes. Alloxan selectively destroys insulin-producing cells in the pancreas when administered to rodents and many other animal species, and has been shown to cause also mitochondrial dysfunction. Alloxan-treated severe diabetic rats were shown to exhibit impaired mitochondrial phosphorylative activities and low mitochondrial oxidation– reduction states (Yamamoto et al., 1981). In one month old alloxan-diabetic animals the enzyme activity of the mitochondrial membrane marker, F0F1-ATPase, was found to be decreased. Insulin treatment caused hyperstimulation of the activity, whereas in late-stage diabetes the catalytic efficiency of the enzyme was increased and became decreased upon insulin treatment (Patel & Katyare, 2006).

Mitochondrial dysfunction in diabetic rats can be succinctly summarized into: decreased mitochondrial 3'-AMP forming enzyme activity, increased oxidative and nitrosative stress, decreased oxygen consumption, loss in mitochondrial transcriptional capacity, increased HMG-CoA synthase, increased levels of pyruvate and dicarboxylate transporters, increased degradation of ATPase, changes in phospholipid composition, increased pyruvate carboxylase activity, increased fatty acid beta oxidation and ultrastructure alterations.

### **4.2 Mitochondrial dysfunction and diabetes type 2**

136 Biomedical Science, Engineering and Technology

Fig. 5. The overall impact of the burden of hypergycaemia in diabetes on functioning of

Kakizaki) rats, Akita mice) may be utilized (Srinivasan & Ramarao, 2007).

**4.1 Mitochondrial dysfunction and diabetes type 1** 

Abnormalities of mitochondrial metabolism causing human disease have been recognised for more than 40 years. Numerous reports clearly indicate the association between mitochondrial dysfunction and diabetes. Nevertheless, some mechanisms of mitochondrial role in this pathology still requires further elucidation. Therefore different animal model studies are involved in the investigation of explaining these unknown mechanisms. There are several models of experimental diabetes that mimic two common types of diabetes. Streptozotocin-induced diabetes is a widely accepted animal model for type 1 diabetes, resulting from the inability of the pancreatic beta cells to produce insulin. For the research on type 2 or the insulin resistant state, resulting from the inefficient use of insulin by the tissues to regulate blood glucose concentration, some genetically manipulated animal models (e.g. Zucker fatty rats (ZFR), *ob/ob* (obese) mice, CP (corpulent) rats, GK (Goto-

Streptozotocin (STZ) is a naturally occurring chemical that is particularly toxic to the insulinproducing β cells of the pancreas in mammals and is used to generate Type 1 diabetes in the experimental model. Animals with diabetes induced by STZ exhibit increased mitochondrial oxidative stress and dysfunction. Other agent, alloxan, a toxic glucose analogue, is also used in order to generate type 1 of diabetes. Alloxan selectively destroys insulin-producing cells in the pancreas when administered to rodents and many other animal species, and has been shown to cause also mitochondrial dysfunction. Alloxan-treated severe diabetic rats were shown to exhibit impaired mitochondrial phosphorylative activities and low mitochondrial oxidation– reduction states (Yamamoto et al., 1981). In one month old alloxan-diabetic animals the enzyme activity of the mitochondrial membrane marker, F0F1-ATPase, was found to be decreased. Insulin treatment caused hyperstimulation of the activity, whereas in late-stage

mitochondria.

Type 2 diabetes is the most common metabolic disease in the world, and its prevalence much exceeds the prevalence of type 1 diabetes. Among different causes leading to diabetes the role of mitochondria is considered substantial. Disorders of the mitochondrial electron transport chain, overproduction of ROS and lipoperoxides or impairments in antioxidant defenses are encountered in type 2 diabetes. Increased ROS levels lead to generalized oxidative damage to all mitochondrial components. Moreover, it is well established that mitochondrial function is required for normal glucose-stimulated insulin secretion from pancreatic β cells. However, the studies in humans suggest that more subtle defects in mitochondrial function may also play a role in the pathogenesis of insulin resistance and type 2 diabetes (Fig. 6) (Luft, 1994).

Fig. 6. Relationships among hyperglycaemia, mitochondrial damage, oxidative burst and diabetic complications.

Some data support the hypothesis that insulin resistance in humans arises from defects in mitochondrial fatty acid oxidation, which in turn leads to increased intracellular fatty acid metabolites that disrupt insulin signaling (Petersen et al., 2003). Alternatively, the reduction in mitochondrial oxidative phosphorylation activity in insulin-resistant individuals could be due not to mitochondrial loss, but rather to a defect in mitochondrial function. This hypothesis is supported by muscle biopsy studies. In one such study, the activity of mitochondrial oxidative enzymes was found to be lower in type 2 diabetic subjects, and in another, the activity of mitochondrial rotenone-sensitive nicotinamide adenine dinucleotide oxidoreductase [NADH:O(2)] was found to be lower (Lowell & Shulman, 2005).

### **4.3 Alterations in cardiac mitochondria observed in diabetes**

Cardiovascular diseases are the predominant cause of death in patients with diabetes mellitus. Underlying mechanism for the susceptibility of diabetic patients to cardiovascular diseases still remains unclear. Elevated oxidative stress was detected in diabetic patients and in animal models of diabetes. Hyperglycaemia, oxidatively modified atherogenic lipoproteins, and advanced glycation end products act in a concerted action together with oxidative stress, and cumulatively contribute to progression of late diabetic complications. Mitochondrial dysfunction increases electron leak and the generation of ROS from the mitochondrial respiratory chain (MRC). High levels of glucose and lipids impair the activities of MRC complex enzymes. Furthermore, increased activity of NADPH oxidase (NOX), which generates superoxide from NADPH in cells, was detected in diabetic patients (Shen, 2010).

Because mitochondria constitute 20–30% of the cardiac myocytes, one of the potent causes for heart malfunctioning in diabetes is the impaired mitochondrial function and consequently the decreased ATP generation (Rolo & Palmeira 2006).

Many reports evidenced that diabetic hearts show impaired mitochondrial function, decreased ATP generation, decreased oxidative capacity, increased ROS, abnormal morphology, increased UCP-3 level, decreased mitochondrial calcium uptake and increased susceptibility to MPT induction (Fig. 5).

Distortions in cardiac mitochondrial bioenergetics are known to occur in both human types of diabetes and in models of diabetes in animals. Reduced mitochondrial calcium uptake was observed in heart mitochondria from STZ-treated rats. This was related to enhanced susceptibility to MPT induction rather than damage to the calcium uptake machinery. Interestingly, heart mitochondria from GK rats were less susceptible to the induction of MPT, showing larger calcium accumulation before the overall loss of mitochondrial impermeability. Different approaches of antioxidant administration in GK rats (vitamin E or coenzyme Q10) showed no success in reversing the diabetic phenotype (Oliveira et al., 2003).

Diabetic heart failure may be causally associated with alterations in cardiac energy metabolism. Fuel selection and capacity for ATP production in the normal and failing heart are dictated by several metabolic regulatory events at the level of gene expression. Decline in the capacity for ATP, as caused by progressive impairment of mitochondrial function, is a gradual step in the progression to heart failure of any cause. Fetal heart depends on glucose and the adult heart on glucose and fatty acids. The switch between fatty acid oxidation and glucose in the adult heart leads to a healthy metabolic situation (Huss & Kelly, 2005). In the insulin-resistant and diabetic heart, fatty acid oxidation is increased and glucose utilisation

Some data support the hypothesis that insulin resistance in humans arises from defects in mitochondrial fatty acid oxidation, which in turn leads to increased intracellular fatty acid metabolites that disrupt insulin signaling (Petersen et al., 2003). Alternatively, the reduction in mitochondrial oxidative phosphorylation activity in insulin-resistant individuals could be due not to mitochondrial loss, but rather to a defect in mitochondrial function. This hypothesis is supported by muscle biopsy studies. In one such study, the activity of mitochondrial oxidative enzymes was found to be lower in type 2 diabetic subjects, and in another, the activity of mitochondrial rotenone-sensitive nicotinamide adenine dinucleotide

Cardiovascular diseases are the predominant cause of death in patients with diabetes mellitus. Underlying mechanism for the susceptibility of diabetic patients to cardiovascular diseases still remains unclear. Elevated oxidative stress was detected in diabetic patients and in animal models of diabetes. Hyperglycaemia, oxidatively modified atherogenic lipoproteins, and advanced glycation end products act in a concerted action together with oxidative stress, and cumulatively contribute to progression of late diabetic complications. Mitochondrial dysfunction increases electron leak and the generation of ROS from the mitochondrial respiratory chain (MRC). High levels of glucose and lipids impair the activities of MRC complex enzymes. Furthermore, increased activity of NADPH oxidase (NOX), which generates superoxide from NADPH in cells, was detected in diabetic patients

Because mitochondria constitute 20–30% of the cardiac myocytes, one of the potent causes for heart malfunctioning in diabetes is the impaired mitochondrial function and

Many reports evidenced that diabetic hearts show impaired mitochondrial function, decreased ATP generation, decreased oxidative capacity, increased ROS, abnormal morphology, increased UCP-3 level, decreased mitochondrial calcium uptake and increased

Distortions in cardiac mitochondrial bioenergetics are known to occur in both human types of diabetes and in models of diabetes in animals. Reduced mitochondrial calcium uptake was observed in heart mitochondria from STZ-treated rats. This was related to enhanced susceptibility to MPT induction rather than damage to the calcium uptake machinery. Interestingly, heart mitochondria from GK rats were less susceptible to the induction of MPT, showing larger calcium accumulation before the overall loss of mitochondrial impermeability. Different approaches of antioxidant administration in GK rats (vitamin E or coenzyme Q10) showed no success in reversing the diabetic phenotype

Diabetic heart failure may be causally associated with alterations in cardiac energy metabolism. Fuel selection and capacity for ATP production in the normal and failing heart are dictated by several metabolic regulatory events at the level of gene expression. Decline in the capacity for ATP, as caused by progressive impairment of mitochondrial function, is a gradual step in the progression to heart failure of any cause. Fetal heart depends on glucose and the adult heart on glucose and fatty acids. The switch between fatty acid oxidation and glucose in the adult heart leads to a healthy metabolic situation (Huss & Kelly, 2005). In the insulin-resistant and diabetic heart, fatty acid oxidation is increased and glucose utilisation

oxidoreductase [NADH:O(2)] was found to be lower (Lowell & Shulman, 2005).

**4.3 Alterations in cardiac mitochondria observed in diabetes** 

consequently the decreased ATP generation (Rolo & Palmeira 2006).

susceptibility to MPT induction (Fig. 5).

(Shen, 2010).

(Oliveira et al., 2003).

is diminished. Long-term consequence of fatty acid oxidation is mitochondrial dysfunction. A number of mechanisms may be responsible for enhanced fatty acid utilisation in type 2 diabetic hearts, such as increased fatty acid uptake into the cell and mitochondria, increased UCP-3 expression, and stimulation of peroxisome proliferator-activated receptor-α (PPARα) (Fig. 7) (Rolo & Palmeira, 2006).

Fig. 7. The role of cardiac mitochondria in the development of heart failure in a course of diabetes.

Diabetes-associated metabolic disorders may cause the mitochondrial dysfuntion and upregulation of NOX in the cardiovascular system, which lead to increased ROS production and oxidative stress in vasculature and blood circulation. ROS may directly oxidize or indirectly regulate molecules related to atherosclerosis and thrombosis. Mitochondrial NOX, or its regulators may be considered as potential drug targets for the prevention and/or treatment of diabetic cardiovascular complications.
