The brain has a high energy demand and, although it represents only 2% of body weight, it accounts for 20% of total body oxygen consumption. This energy requirement is largely driven by neuronal request of energy to maintain the ion gradients across the plasma membrane, which are critical for the generation of action potentials. This intense energy requirement is continuous; even brief periods of oxygen or glucose deprivation result in neuronal death. Diabetes mellitus leads to functional and structural changes in the brain, which appear to be most pronounced in the elderly. Furthermore, increased age is associated with insulin resistance. Increasing data support the idea that mitochondrial function declines with aging and in age-related diseases such as diabetes and AD.

Normal glucose metabolism is required for the performance of cognitive functions, and impairments in glucose metabolism might contribute to cognitive dysfunction. Imaging

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 35

DNA. Minor modifications of the nucleic acid bases are repaired through base excision repair involving DNA glycosylase and AP endonuclease, which are located in nuclei and mitochondria. The progression of AD is associated with the diminished expression of these DNA repair enzymes (Nakabeppu et al., 2004). The accumulation of the oxidatively damaged nucleic acids and proteins likely exceeds the limit of cellular repair and detoxification mechanisms and leads to the onset or progression of diabetic and neurological pathologies. In general, accumulation of oxidatively damaged proteins, lipids, and nucleic acids correlates with the onset of age-related diseases, especially in diabetes and

Oxidative stress and lipid peroxidation seem to be able to induce Abeta accumulation: studies in a mouse model of AD have demonstrated that brain lipid peroxidation increases before that Abeta levels increase (Pratico et al., 2001) and that the onset of Abeta deposition is associated with an increase in the level of RNS (Apelt et al., 2004). Further evidence supporting this hypothesis has been obtained from studies of a mouse model of AD in which mutations in the genes encoding APP and presenilin 1 cause an increase in Abeta42 production. In these animals, lipid and protein peroxidation is evident at the disease onset (Matsuoka et al., 2001). In a triple-transgenic animal model of AD, in which mice develop Abeta plaques, tangles and cognitive defects, a decrease in antioxidant capacity and an increase in lipid peroxidation were noted before the development of AD pathology (Resende et al., 2008). Oxidative stress seems to affect APP either directly, by increasing APP levels, or indirectly, by modulating APP processing, and both mechanisms could increase levels of Abeta. Studies in transgenic mice and postmortem brain tissue from patients with AD suggest also that an increase in Abeta production leads to a rise in the production of ROS and that oxidative stress occurs early in the development of the disease.

Several studies are consistent with the view that diabetes-related mitochondrial dysfunction is exacerbated by aging and/or by the presence of neurotoxic agents, such as Abeta. This suggests that diabetes and aging are risk factors for the neurodegeneration induced by this peptide. Mitochondrial dysfunction could be one of the common underlying mechanisms explaining the association between diabetes and AD. Mitochondrial dysfunction and the resulting energy deficit trigger the onset of neuronal degeneration and death. Mitochondria serve also as high capacity Ca2+ sink, which allows them to follow the changes in cytosolic Ca2+ loads and helps in maintaining cellular Ca2+ homeostasis, required for normal neuronal function (Rizzuto et al., 2000). Conversely, excessive Ca2+ uptake inside mitochondria has been shown to increase ROS production, inhibit ATP synthesis, release cytochrome C, and induce mitochondrial permeability transition (Brustovetsky et al., 2002). The mitochondrial permeability transition (MPT) is defined as the sudden increase of inner mitochondrial membrane permeability to solutes of molecular mass lower than 1500 Da (Bernardi et al., 1994). Strong evidence now exists that the MPT is due to the opening of a nonselective megachannel (estimated to be 2–3 nm in diameter). Because the chemiosmotic theory is based on the impermeability of the inner mitocondrion membrane to solutes that are not specifically transported, MPT would collapse the mitochondrial membrane potential (ΔΨm) and uncouple the electron transport system from the production of ATP. Additionally, MPT results in mitochondrial swelling and can lead to the release of proapoptotic proteins.

**4. The mitochondrial dysfunction**

studies have revealed that patients with AD and individuals at risk of developing this disease typically have reductions in glucose metabolism in temporal and parietal brain regions and hippocampus (Garrido et al., 2002). Moreover, compared to healthy individuals, patients with AD often have increased plasma insulin levels and/or a decreased cerebrospinal fluid (CsF)-to-plasma insulin ratio. These findings indicates that glucose metabolism and insulin signaling are important in normal brain function. The negative effect of impaired glucose metabolism on cognitive functioning can be caused by an increase in oxidative stress that is associated with mitochondrial dysfunction.

Mitochondria are essential subcellular organelles for generating the energy that fuels normal cellular functioning. At the same time, the mitochondria have a strategic task because, depending on environmental factors, they can decide whether to continue the healthy life of the cell or to terminate it by apoptosis activation. Mitochondria are essential for neuronal function because the limited glycolytic capacity of these cells makes them highly dependent on aerobic oxidative phosphorylation for their energetic needs. However, oxidative phosphorylation is a major source of endogenous free radicals. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in vivo through both enzymatic and non-enzymatic routes. ROS include hydrogen peroxide, hydroxyl radical, superoxide ion and singlet oxygen, products of normal cellular respiration. The hydroxyl radical is a highly reactive species and has a relatively short life time. It is abundantly produced in the mitochondria during respiration cycles and reacts with the proteins, lipids, and nucleic acids during their production. Peroxynitrous acid is one of the major RNS found intracellularly and it is involved in the rapid nitration of aromatic residues of proteins, such as tyrosine, to give 3-nitrotyrosine, which may alter the protein structure. Further it is also a marker of oxidative stress (Smith et al., 1997). Under normal conditions, antioxidant defenses can counteract oxidative stress damage. In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes redox imbalanced, leading to the activation of a stress-sensitive intracellular signaling pathway and, in extreme conditions, to apoptosis. Increased oxidative damage and impaired antioxidant defenses are prominent both in the onset of AD and diabetes (Smith et al., 1996; Evans et al. 2002). Abnormal glucose metabolism can also increase the production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). This overproduction of free radicals can exhaust the antioxidant capacity of the cell and lead to a condition known as oxidative stress, which is a hallmark of both T1D and T2D and a contributing factor to diabetic neuropathy (Russell et al., 2008; Vincent et al., 2004). Oxidative stress is not only associated with diabetes and its complications, but has been linked to insulin resistance, the subnormal response to a given amount of insulin (West, 2000).

ROS-induced and RNS-induced protein modifications and/or lipid peroxidations result in cell damage that can lead to cell death, and they are increased in patients with diabetes or AD compared with healthy controls (Pratico et al., 2004). Brain and cerebrospinal fluid (CSF) levels of lipid peroxidation biomarkers, including malondialdehyde and 4-hydroxynonenal (two highly toxic products generated in part by lipid oxidation and ROS), are both higher in individuals with AD and diabetes mellitus than in healthy people (Reddy et al., 2009; Slatter et al., 2000). Furthermore, levels of oxidized proteins are increased in the frontal and parietal lobes and in the hippocampus of patients with mild cognitive impairment compared with healthy controls, indicating that oxidative damage might occur early in the development of AD (Butterfield et al., 2007). ROS, as mentioned earlier, are also involved in the damage of DNA. Minor modifications of the nucleic acid bases are repaired through base excision repair involving DNA glycosylase and AP endonuclease, which are located in nuclei and mitochondria. The progression of AD is associated with the diminished expression of these DNA repair enzymes (Nakabeppu et al., 2004). The accumulation of the oxidatively damaged nucleic acids and proteins likely exceeds the limit of cellular repair and detoxification mechanisms and leads to the onset or progression of diabetic and neurological pathologies.

Oxidative stress and lipid peroxidation seem to be able to induce Abeta accumulation: studies in a mouse model of AD have demonstrated that brain lipid peroxidation increases before that Abeta levels increase (Pratico et al., 2001) and that the onset of Abeta deposition is associated with an increase in the level of RNS (Apelt et al., 2004). Further evidence supporting this hypothesis has been obtained from studies of a mouse model of AD in which mutations in the genes encoding APP and presenilin 1 cause an increase in Abeta42 production. In these animals, lipid and protein peroxidation is evident at the disease onset (Matsuoka et al., 2001). In a triple-transgenic animal model of AD, in which mice develop Abeta plaques, tangles and cognitive defects, a decrease in antioxidant capacity and an increase in lipid peroxidation were noted before the development of AD pathology (Resende et al., 2008). Oxidative stress seems to affect APP either directly, by increasing APP levels, or indirectly, by modulating APP processing, and both mechanisms could increase levels of Abeta. Studies in transgenic mice and postmortem brain tissue from patients with AD suggest also that an increase in Abeta production leads to a rise in the production of ROS and that oxidative stress occurs early in the development of the disease.

#### **4. The mitochondrial dysfunction**

34 Topics in the Prevention, Treatment and Complications of Type 2 Diabetes

studies have revealed that patients with AD and individuals at risk of developing this disease typically have reductions in glucose metabolism in temporal and parietal brain regions and hippocampus (Garrido et al., 2002). Moreover, compared to healthy individuals, patients with AD often have increased plasma insulin levels and/or a decreased cerebrospinal fluid (CsF)-to-plasma insulin ratio. These findings indicates that glucose metabolism and insulin signaling are important in normal brain function. The negative effect of impaired glucose metabolism on cognitive functioning can be caused by an increase

Mitochondria are essential subcellular organelles for generating the energy that fuels normal cellular functioning. At the same time, the mitochondria have a strategic task because, depending on environmental factors, they can decide whether to continue the healthy life of the cell or to terminate it by apoptosis activation. Mitochondria are essential for neuronal function because the limited glycolytic capacity of these cells makes them highly dependent on aerobic oxidative phosphorylation for their energetic needs. However, oxidative phosphorylation is a major source of endogenous free radicals. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in vivo through both enzymatic and non-enzymatic routes. ROS include hydrogen peroxide, hydroxyl radical, superoxide ion and singlet oxygen, products of normal cellular respiration. The hydroxyl radical is a highly reactive species and has a relatively short life time. It is abundantly produced in the mitochondria during respiration cycles and reacts with the proteins, lipids, and nucleic acids during their production. Peroxynitrous acid is one of the major RNS found intracellularly and it is involved in the rapid nitration of aromatic residues of proteins, such as tyrosine, to give 3-nitrotyrosine, which may alter the protein structure. Further it is also a marker of oxidative stress (Smith et al., 1997). Under normal conditions, antioxidant defenses can counteract oxidative stress damage. In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes redox imbalanced, leading to the activation of a stress-sensitive intracellular signaling pathway and, in extreme conditions, to apoptosis. Increased oxidative damage and impaired antioxidant defenses are prominent both in the onset of AD and diabetes (Smith et al., 1996; Evans et al. 2002). Abnormal glucose metabolism can also increase the production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). This overproduction of free radicals can exhaust the antioxidant capacity of the cell and lead to a condition known as oxidative stress, which is a hallmark of both T1D and T2D and a contributing factor to diabetic neuropathy (Russell et al., 2008; Vincent et al., 2004). Oxidative stress is not only associated with diabetes and its complications, but has been linked to insulin resistance, the subnormal response to a given amount of insulin (West,

ROS-induced and RNS-induced protein modifications and/or lipid peroxidations result in cell damage that can lead to cell death, and they are increased in patients with diabetes or AD compared with healthy controls (Pratico et al., 2004). Brain and cerebrospinal fluid (CSF) levels of lipid peroxidation biomarkers, including malondialdehyde and 4-hydroxynonenal (two highly toxic products generated in part by lipid oxidation and ROS), are both higher in individuals with AD and diabetes mellitus than in healthy people (Reddy et al., 2009; Slatter et al., 2000). Furthermore, levels of oxidized proteins are increased in the frontal and parietal lobes and in the hippocampus of patients with mild cognitive impairment compared with healthy controls, indicating that oxidative damage might occur early in the development of AD (Butterfield et al., 2007). ROS, as mentioned earlier, are also involved in the damage of

in oxidative stress that is associated with mitochondrial dysfunction.

2000).

Several studies are consistent with the view that diabetes-related mitochondrial dysfunction is exacerbated by aging and/or by the presence of neurotoxic agents, such as Abeta. This suggests that diabetes and aging are risk factors for the neurodegeneration induced by this peptide. Mitochondrial dysfunction could be one of the common underlying mechanisms explaining the association between diabetes and AD. Mitochondrial dysfunction and the resulting energy deficit trigger the onset of neuronal degeneration and death. Mitochondria serve also as high capacity Ca2+ sink, which allows them to follow the changes in cytosolic Ca2+ loads and helps in maintaining cellular Ca2+ homeostasis, required for normal neuronal function (Rizzuto et al., 2000). Conversely, excessive Ca2+ uptake inside mitochondria has been shown to increase ROS production, inhibit ATP synthesis, release cytochrome C, and induce mitochondrial permeability transition (Brustovetsky et al., 2002). The mitochondrial permeability transition (MPT) is defined as the sudden increase of inner mitochondrial membrane permeability to solutes of molecular mass lower than 1500 Da (Bernardi et al., 1994). Strong evidence now exists that the MPT is due to the opening of a nonselective megachannel (estimated to be 2–3 nm in diameter). Because the chemiosmotic theory is based on the impermeability of the inner mitocondrion membrane to solutes that are not specifically transported, MPT would collapse the mitochondrial membrane potential (ΔΨm) and uncouple the electron transport system from the production of ATP. Additionally, MPT results in mitochondrial swelling and can lead to the release of proapoptotic proteins. Importantly, Ca2+, Pi, oxidative stress, and low inner membrane potential promote the onset

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 37

plaques and NFTs in patients with AD. Furthermore, hippocampal neurons from patients with this neurodegenerative disease contain Abeta-positive, AGE-positive and RAGE positive granules. (Sasaki et al., 2001). Whether the modifications of Abeta and tau by AGEs are a primary or secondary event in AD is a controversial topic. Nevertheless, AGEs are widely accepted to be active participants in the progression of AD, since AGE-induced glycation of Abeta and tau protein has been shown to cause the Abeta aggregation and the formation of NFTs, respectively (Ledesma et al., 1994). Moreover, diabetic mice with cognitive impairments exhibit increased RAGE expression in neurons and glia compared with wild-type control mice (Toth et al. 2006), and in one clinical study, AGE immunostaining was increased in postmortem brain slices from patients with AD and

The question of whether AGEs are the cause or consequence of the pathology is not clear, although there is likely a primary role of oxidative stress in both pathologies. However, it should be pointed out that glycoxidation and oxidative stress are mutually dependent and reinforce each other. Thus, while the sources of oxidative stress may widely differ in diabetes and AD, and while a number of AGEs accumulate in both conditions, other AGEs

Given the importance of mitochondria as the primary source of oxidative stress in AD and diabetes, the use of antioxidants may also be useful. However, the broad occurrence of both diseases, the non-regenerative nature of the CNS and the fact that AD diagnosis often does not occur until late in the disease progression, suggest that the ideal antioxidant should be used as a prophylactic treatment for the aged population. Oxidative stress is one of the earliest events in the neurological and pathological changes of AD, while the effects of oxidative stress are manifested in the slow accumulation of AGEs in diabetes. Thus, antioxidant therapy in combination with AGE inhibitor therapy may be effective approaches for AD and diabetes-related complications. Oxidative stress leads to irreversible protein aggregation and consequent neuronal degeneration in AD (Liu et al., 2007). Advanced lipoxidation products, such as HNE, bind to phosphorylated tau protein to form paired helical filaments, accelerating the formation of neurofibrillary tangles. Oxidative stress also results in the covalent crosslinking of tau filaments to form large aggregates that are resistant to proteolytic cleavage. Larbig and coworkers reported a series of inhibitors for tau protein aggregation (Larbig et al., 2007). Remarkably, thiazolium-based compounds, which are also AGE inhibitors and potentially useful for diabetic therapy, are effective inhibitors of

Extensive studies of pharmacological interventions based on biological antioxidants have been carried out both for AD and diabetes (Lee et al., 2010; Maritim et al., 2002). Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Other antioxidants include lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid; vitamins B1, B2, B6 and B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C, with vitamin E, inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metals involved in some reactions in lipid peroxidation.

diabetes compared with non-diabetic patients with AD (Girones et al., 2004).

**6. Antioxidant therapy in Alzheimer's disease and diabetes**

found in diabetes have yet to be characterized in AD.

tau aggregation.

of MPT, whereas cyclosporin A, Mg2+, ADP, and the existence of a high membrane potential oppose the onset (Bernardi et al., 1994).

Increasing data support the idea that mitochondrial function declines with aging and in agerelated diseases, such as diabetes and AD (Calabrese et al., 2001). Some data show the existence of an age-related impairment of the respiratory chain and an uncoupling of oxidative phosphorylation in brain mitochondria isolated from Goto-Kakizaki (GK) rats, as model of T2D (Moreira et al., 2003). Furthermore, aging exacerbates the decrease in the energetic levels promoted by diabetes. The maintenance of oxidative phosphorylation capacity is extremely important in the brain since a large amount of the energy required for the normal functioning of neurons is provided by mitochondria. Moreover, the CNS requires a large amount of ATP for the transmission of impulses along the neural pathway, thus indicating that mitochondrial function impairment can result in neurodegeneration and loss in neuronal metabolic control (Calabrese et al., 2001).

#### **5. Advanced glycation end products (AGE)**

Abnormal glucose metabolism and oxidative stress contribute to the formation of advanced glycation end products (AGE). This process occurs through the Maillard reaction or "nonenzymatic browning", a complex series of reactions between reducing carbohydrates with lysine side chains and N-terminal amino groups of proteins. This process initially leads to rather labile Schiff bases which as a rule rearrange to the more stable Amadori products. The Amadori compounds are slowly degraded, in complex reaction pathways via dicarbonyl intermediates, to a plethora of compounds (Ledl & Schleicher, 1990) designated summarily as "advanced glycation end products" (AGEs); this overall reaction sequence proceeds both in vitro and in vivo. In long-lived tissue proteins, these chemical modifications accumulate with age and may contribute to pathophysiologies associated with aging and long-term complications of diabetes and atherosclerosis (Lederer & Klalber, 2000).

Practically, AGEs comprise a heterogeneous group of molecules formed by irreversible, non-enzymatic reactions between sugars and the free amino groups of proteins, lipids and nucleic acids. Auto-oxidation of glucose leads to the formation of oxygen radicals, which are intermediates in the AGE pathway and the predominant source of endogenous AGEs. AGEs may exist as protein cross-links or as modification of the side chains of a single protein, and significantly alter protein conformations leading to protein inactivation. Numerous AGEs have been isolated and characterized by spectroscopic analysis after cleavage from the protein backbones. AGEs involving protein cross-links include: pentosidine, a dimer of arginine and lysine; methylglyoxal-lysine dimer (MOLD), a dimer of two lysine residues; and methylglyoxal-derived imidazolium cross-link (MODIC) and glyoxal-derived imidazolium cross-link (GODIC), dimers of arginine and lysine residues. Examples of AGEs resulting from the single protein modification are pyrraline and Nε -(carboxymethyl)lysine (CML), the lysine-residue modified products, and argpyrimidine, an arginine-residue modified protein. Although many other AGEs, including the hydroimidazolone adduct MG-H1, have been characterized in diabetes, some of them have common occurrence in AD (Rabbani et al., 2008).

The formation and accumulation of AGEs occur during normal aging; however, these processes are exacerbated in patients with diabetes and the binding of AGE to its receptor (receptor for AGEs or RAGE) induces a series of biological processes that cause further diabetic complications (Singh et al., 2001). AGE immunoreactivity is present in both Abeta

of MPT, whereas cyclosporin A, Mg2+, ADP, and the existence of a high membrane potential

Increasing data support the idea that mitochondrial function declines with aging and in agerelated diseases, such as diabetes and AD (Calabrese et al., 2001). Some data show the existence of an age-related impairment of the respiratory chain and an uncoupling of oxidative phosphorylation in brain mitochondria isolated from Goto-Kakizaki (GK) rats, as model of T2D (Moreira et al., 2003). Furthermore, aging exacerbates the decrease in the energetic levels promoted by diabetes. The maintenance of oxidative phosphorylation capacity is extremely important in the brain since a large amount of the energy required for the normal functioning of neurons is provided by mitochondria. Moreover, the CNS requires a large amount of ATP for the transmission of impulses along the neural pathway, thus indicating that mitochondrial function impairment can result in neurodegeneration and

Abnormal glucose metabolism and oxidative stress contribute to the formation of advanced glycation end products (AGE). This process occurs through the Maillard reaction or "nonenzymatic browning", a complex series of reactions between reducing carbohydrates with lysine side chains and N-terminal amino groups of proteins. This process initially leads to rather labile Schiff bases which as a rule rearrange to the more stable Amadori products. The Amadori compounds are slowly degraded, in complex reaction pathways via dicarbonyl intermediates, to a plethora of compounds (Ledl & Schleicher, 1990) designated summarily as "advanced glycation end products" (AGEs); this overall reaction sequence proceeds both in vitro and in vivo. In long-lived tissue proteins, these chemical modifications accumulate with age and may contribute to pathophysiologies associated with aging and long-term

Practically, AGEs comprise a heterogeneous group of molecules formed by irreversible, non-enzymatic reactions between sugars and the free amino groups of proteins, lipids and nucleic acids. Auto-oxidation of glucose leads to the formation of oxygen radicals, which are intermediates in the AGE pathway and the predominant source of endogenous AGEs. AGEs may exist as protein cross-links or as modification of the side chains of a single protein, and significantly alter protein conformations leading to protein inactivation. Numerous AGEs have been isolated and characterized by spectroscopic analysis after cleavage from the protein backbones. AGEs involving protein cross-links include: pentosidine, a dimer of arginine and lysine; methylglyoxal-lysine dimer (MOLD), a dimer of two lysine residues; and methylglyoxal-derived imidazolium cross-link (MODIC) and glyoxal-derived imidazolium cross-link (GODIC), dimers of arginine and lysine residues. Examples of AGEs resulting from the single protein modification are pyrraline and Nε -(carboxymethyl)lysine (CML), the lysine-residue modified products, and argpyrimidine, an arginine-residue modified protein. Although many other AGEs, including the hydroimidazolone adduct MG-H1, have been characterized in diabetes, some of them have common occurrence in AD

The formation and accumulation of AGEs occur during normal aging; however, these processes are exacerbated in patients with diabetes and the binding of AGE to its receptor (receptor for AGEs or RAGE) induces a series of biological processes that cause further diabetic complications (Singh et al., 2001). AGE immunoreactivity is present in both Abeta

oppose the onset (Bernardi et al., 1994).

loss in neuronal metabolic control (Calabrese et al., 2001).

complications of diabetes and atherosclerosis (Lederer & Klalber, 2000).

**5. Advanced glycation end products (AGE)**

(Rabbani et al., 2008).

plaques and NFTs in patients with AD. Furthermore, hippocampal neurons from patients with this neurodegenerative disease contain Abeta-positive, AGE-positive and RAGE positive granules. (Sasaki et al., 2001). Whether the modifications of Abeta and tau by AGEs are a primary or secondary event in AD is a controversial topic. Nevertheless, AGEs are widely accepted to be active participants in the progression of AD, since AGE-induced glycation of Abeta and tau protein has been shown to cause the Abeta aggregation and the formation of NFTs, respectively (Ledesma et al., 1994). Moreover, diabetic mice with cognitive impairments exhibit increased RAGE expression in neurons and glia compared with wild-type control mice (Toth et al. 2006), and in one clinical study, AGE immunostaining was increased in postmortem brain slices from patients with AD and diabetes compared with non-diabetic patients with AD (Girones et al., 2004).

The question of whether AGEs are the cause or consequence of the pathology is not clear, although there is likely a primary role of oxidative stress in both pathologies. However, it should be pointed out that glycoxidation and oxidative stress are mutually dependent and reinforce each other. Thus, while the sources of oxidative stress may widely differ in diabetes and AD, and while a number of AGEs accumulate in both conditions, other AGEs found in diabetes have yet to be characterized in AD.

#### **6. Antioxidant therapy in Alzheimer's disease and diabetes**

Given the importance of mitochondria as the primary source of oxidative stress in AD and diabetes, the use of antioxidants may also be useful. However, the broad occurrence of both diseases, the non-regenerative nature of the CNS and the fact that AD diagnosis often does not occur until late in the disease progression, suggest that the ideal antioxidant should be used as a prophylactic treatment for the aged population. Oxidative stress is one of the earliest events in the neurological and pathological changes of AD, while the effects of oxidative stress are manifested in the slow accumulation of AGEs in diabetes. Thus, antioxidant therapy in combination with AGE inhibitor therapy may be effective approaches for AD and diabetes-related complications. Oxidative stress leads to irreversible protein aggregation and consequent neuronal degeneration in AD (Liu et al., 2007). Advanced lipoxidation products, such as HNE, bind to phosphorylated tau protein to form paired helical filaments, accelerating the formation of neurofibrillary tangles. Oxidative stress also results in the covalent crosslinking of tau filaments to form large aggregates that are resistant to proteolytic cleavage. Larbig and coworkers reported a series of inhibitors for tau protein aggregation (Larbig et al., 2007). Remarkably, thiazolium-based compounds, which are also AGE inhibitors and potentially useful for diabetic therapy, are effective inhibitors of tau aggregation.

Extensive studies of pharmacological interventions based on biological antioxidants have been carried out both for AD and diabetes (Lee et al., 2010; Maritim et al., 2002). Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Other antioxidants include lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid; vitamins B1, B2, B6 and B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C, with vitamin E, inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metals involved in some reactions in lipid peroxidation.

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 39

pseudostem on hyperglycemia and AGEs in streptozotocin-induced diabetic rats. The results indicate that fructosamine and AGEs formed during diabetes were inhibited in

However, the results of clinical trials of antioxidant therapy are not clear because of duration of treatment as well as recruitment of patients at different stages of the diseases. In spite of inconsistency in the conclusions of clinical trials on the beneficial effects of antioxidants on these pathologies, epidemiological studies indicate that antioxidants may reduce the risk of their insurgence. It is suggested that a combination of antioxidants might be of greater potential benefit, especially if these agents work in different cellular compartments or have complementary activity (e.g. Vitamins E, C, and ubiquinone). If oxidative stress plays as important a role in AD and diabetes pathologies as the literature suggests, regular intake of antioxidants may be beneficial much before any sign or symptoms of the disease are visible.

**7. Insulin resistance, Tau hyperphosphorylation and the amyloid cascade**

be an early marker of AD (Baker et al., 2011) (Figure 2).

In addition to being a modulator of food intake and energy homoeostasis, insulin is also an important neurothrophic factor. It modulates brain activity, particularly for such high glucose demanding functions such as memory. As mentioned above, T2D is associated with cognitive impairment. This form of diabetes is characterized by insulin resistance, hyperinsulinemia and impaired insulin signaling. Insulin resistance is the common link of the components of the much invoked metabolic syndrome (a cluster of high adiposity, abnormal glucose level, dyslipidemia, hypertension and high inflammation) and it is known to cause common diseases such as stroke, heart disease, and cancer. Given the aging of the population and the epidemic of elevated insulin resistance, evidenced by the rise in elevated adiposity, prediabetes, and diabetes, it is alarming that insulin resistance could contribute to AD. Many epidemiologic studies have found an association of elevated adiposity, insulin resistance, and T2D with cognitive impairment and dementia (Baker et al., 2011). However, there are several important questions to be addressed for investigators studying the relation of insulin resistance and AD. For example is not clear if the association between insulin resistance and AD is causal. However, one of the links found is that defects in brain insulin signaling have been reported in AD and it has been proposed that insulin resistance could

Fig. 2. Insulin deficency can lead to Abeta plaques formation and Tau hyperphos-phorylation

treated groups when compared with the diabetic group (Bhaskar et al., 2011).

Vitamins A, C, and E are interesting antioxidant molecules because are diet-derived and directly detoxify free radicals. They also interact in recycling processes to generate reduced forms of the vitamins. Tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is generated, is recycled by glutathione. Under some conditions, these vitamins also foster toxicity by producing pro-oxidants. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet oxygen and protects membranes from lipid peroxidation (Weber et al., 1997). A deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. There have been conflicting reports about vitamin E levels in diabetic animals and human subjects. Plasma and/or tissue levels of vitamin E are reported to be unaltered, increased, or even decreased by diabetes (Asayama et al., 1994).

Some AGE inhibitors and AGE crosslink breakers have been utilized as potential therapies. Some attention to AGE inhibitors was focused on aminoguanidine, which blocks electrophilically activated 1,3-dicarbonyl compounds, the precursors of AGEs (Thomas et al. 2005; Webster et al., 2005). This compound was not approved by the US Food and Drug Administration due to adverse side effects in diabetic patients during Phase III clinical trials, and the search to find alternatives continues. Pyridoxamine (vitamin B6, pyridorin) and thiamin pyrophosphate have been proposed as potential alternatives to aminoguanidine (Ahmed et al., 2007; Androver at al., 2008). Furthermore, these compounds are also good metal ion chelators and attenuate oxidative stress. N-acetylcysteine and lipoic acid act as inhibitors through attenuation of oxidative stress. While the AGE-inhibitory effect of these compounds is not clearly understood, a similar mechanism may operate in the case of AGEs. Carnosine, homocarnosine, and related compounds are potentially suitable as AGE inhibitors although further studies are needed to prove their efficacy in diabetes and AD (Reddy et al., 2005). Carnosine protects superoxide dismutase, catalase, and -crystallin from non-enzymatic glycation and protein crosslinking (Hipkiss, 2007). OPB-9195 inhibits AGE formation, especially pentosidine and N-(carboxymethyl)lysine (CML), apparently through carbonyl trapping and metal ion chelation (Wada et al., 2001). Thiazolium-based compounds such as alagebrium chloride (ALT-711) and N-phenyacyl-1,3-thiazolium bromide (PTB) are effective AGE crosslink breakers, and are potentially useful drugs for diabetes and AD (Susic, 2007). It should, however, be pointed out that the mechanisms of the action of the latter compounds are not clearly understood. In addition to their action as crosslink breakers of AGEs derived from 1,2-dicarbonyl compounds, they may also act as antioxidants through chelation of transition metal ions. The development of these drugs as therapeutics thus depends on the detailed understanding of their mechanisms of action. An alternative strategy involves removal of AGEs through the soluble receptors for AGEs (sRAGEs). Poor glycemic control in diabetes results in decreased concentrations of sRAGES, and upon insulin treatment, significant improvements in the levels of sRAGEs were observed, with concomitant decrease in AGEs (Devangelio et al., 2007). Treatment of diabetic patients with rosiglitazone, a 2,4-thiazolidine dione derivative, results in increase of plasma sRAGEs, comparable to controls (Tan et al., 2007). Significant amounts of plasma sRAGEs are also produced when angiotensin converting enzyme inhibitors (ACEi; e.g., perindopril) were used for the treatment of diabetes (Tan et al., 2006). However, the protective effect of sRAGEs has been questioned recently as their level are much higher in experimental animal models than those found in vivo, suggesting they may be only markers of inflammation (Humpert et al., 2007). Following the trend in using natural antioxidants, a recent paper has examined the effects of banana (Musa sp. var. elakki bale) flower and

Vitamins A, C, and E are interesting antioxidant molecules because are diet-derived and directly detoxify free radicals. They also interact in recycling processes to generate reduced forms of the vitamins. Tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is generated, is recycled by glutathione. Under some conditions, these vitamins also foster toxicity by producing pro-oxidants. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet oxygen and protects membranes from lipid peroxidation (Weber et al., 1997). A deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. There have been conflicting reports about vitamin E levels in diabetic animals and human subjects. Plasma and/or tissue levels of vitamin E are reported to be unaltered, increased, or even decreased by diabetes (Asayama et al., 1994). Some AGE inhibitors and AGE crosslink breakers have been utilized as potential therapies. Some attention to AGE inhibitors was focused on aminoguanidine, which blocks electrophilically activated 1,3-dicarbonyl compounds, the precursors of AGEs (Thomas et al. 2005; Webster et al., 2005). This compound was not approved by the US Food and Drug Administration due to adverse side effects in diabetic patients during Phase III clinical trials, and the search to find alternatives continues. Pyridoxamine (vitamin B6, pyridorin) and thiamin pyrophosphate have been proposed as potential alternatives to aminoguanidine (Ahmed et al., 2007; Androver at al., 2008). Furthermore, these compounds are also good metal ion chelators and attenuate oxidative stress. N-acetylcysteine and lipoic acid act as inhibitors through attenuation of oxidative stress. While the AGE-inhibitory effect of these compounds is not clearly understood, a similar mechanism may operate in the case of AGEs. Carnosine, homocarnosine, and related compounds are potentially suitable as AGE inhibitors although further studies are needed to prove their efficacy in diabetes and AD (Reddy et al., 2005). Carnosine protects superoxide dismutase, catalase, and -crystallin from non-enzymatic glycation and protein crosslinking (Hipkiss, 2007). OPB-9195 inhibits AGE formation, especially pentosidine and N-(carboxymethyl)lysine (CML), apparently through carbonyl trapping and metal ion chelation (Wada et al., 2001). Thiazolium-based compounds such as alagebrium chloride (ALT-711) and N-phenyacyl-1,3-thiazolium bromide (PTB) are effective AGE crosslink breakers, and are potentially useful drugs for diabetes and AD (Susic, 2007). It should, however, be pointed out that the mechanisms of the action of the latter compounds are not clearly understood. In addition to their action as crosslink breakers of AGEs derived from 1,2-dicarbonyl compounds, they may also act as antioxidants through chelation of transition metal ions. The development of these drugs as therapeutics thus depends on the detailed understanding of their mechanisms of action. An alternative strategy involves removal of AGEs through the soluble receptors for AGEs (sRAGEs). Poor glycemic control in diabetes results in decreased concentrations of sRAGES, and upon insulin treatment, significant improvements in the levels of sRAGEs were observed, with concomitant decrease in AGEs (Devangelio et al., 2007). Treatment of diabetic patients with rosiglitazone, a 2,4-thiazolidine dione derivative, results in increase of plasma sRAGEs, comparable to controls (Tan et al., 2007). Significant amounts of plasma sRAGEs are also produced when angiotensin converting enzyme inhibitors (ACEi; e.g., perindopril) were used for the treatment of diabetes (Tan et al., 2006). However, the protective effect of sRAGEs has been questioned recently as their level are much higher in experimental animal models than those found in vivo, suggesting they may be only markers of inflammation (Humpert et al., 2007). Following the trend in using natural antioxidants, a recent paper has examined the effects of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and AGEs in streptozotocin-induced diabetic rats. The results indicate that fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group (Bhaskar et al., 2011).

However, the results of clinical trials of antioxidant therapy are not clear because of duration of treatment as well as recruitment of patients at different stages of the diseases. In spite of inconsistency in the conclusions of clinical trials on the beneficial effects of antioxidants on these pathologies, epidemiological studies indicate that antioxidants may reduce the risk of their insurgence. It is suggested that a combination of antioxidants might be of greater potential benefit, especially if these agents work in different cellular compartments or have complementary activity (e.g. Vitamins E, C, and ubiquinone). If oxidative stress plays as important a role in AD and diabetes pathologies as the literature suggests, regular intake of antioxidants may be beneficial much before any sign or symptoms of the disease are visible.

#### **7. Insulin resistance, Tau hyperphosphorylation and the amyloid cascade**

In addition to being a modulator of food intake and energy homoeostasis, insulin is also an important neurothrophic factor. It modulates brain activity, particularly for such high glucose demanding functions such as memory. As mentioned above, T2D is associated with cognitive impairment. This form of diabetes is characterized by insulin resistance, hyperinsulinemia and impaired insulin signaling. Insulin resistance is the common link of the components of the much invoked metabolic syndrome (a cluster of high adiposity, abnormal glucose level, dyslipidemia, hypertension and high inflammation) and it is known to cause common diseases such as stroke, heart disease, and cancer. Given the aging of the population and the epidemic of elevated insulin resistance, evidenced by the rise in elevated adiposity, prediabetes, and diabetes, it is alarming that insulin resistance could contribute to AD. Many epidemiologic studies have found an association of elevated adiposity, insulin resistance, and T2D with cognitive impairment and dementia (Baker et al., 2011). However, there are several important questions to be addressed for investigators studying the relation of insulin resistance and AD. For example is not clear if the association between insulin resistance and AD is causal. However, one of the links found is that defects in brain insulin signaling have been reported in AD and it has been proposed that insulin resistance could be an early marker of AD (Baker et al., 2011) (Figure 2).

Fig. 2. Insulin deficency can lead to Abeta plaques formation and Tau hyperphos-phorylation

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 41

it degrades Abeta peptide, thus reducing the rate of aggregation and the plaque formation (Qiu et al., 1998). IDE levels have been reported to be decreased in the brains of AD patients (Cook et al., 2003). It has also been hypothesized that hyperinsulinemia in people with prediabetes and T2D effectively sequesters IDE, reducing Abeta peptide degradation. This would increase levels of Abeta, and promote many of the pathological features associated with Alzheimer's disease. Supporting this model, the affinity for the binding of insulin to IDE is much greater than that for the Abeta (Qiuet al., 1997). In patients with Alzheimer's disease, IDE expression in the hippocampus is substantially reduced, with regards to controls, in particular among patients with the APOEvar epsilon4 genotype. This latter observation could explain the potential interaction between diabetes and the APOEvar epsilon4 genotype in multiplying the risk of dementia (Cook et al., 2003). Curiously, although the presence of the APOEvar epsilon4 is associated with an increased incidence of Alzheimer's disease, it seems that insulin resistance is only a significant risk factor for AD in those patients without APOEvar epsilon4 (Craft et al., 1998). Subjects with AD without the APOEvar epsilon4 also had improved memory scores when they had hyperinsulinemia, which was not the case for people with at least one APOEvar epsilon4 allele (Craft et al.,

However, unexpectedly, recent clinicopathological studies have shown no evidence that the pathological hallmarks of AD, including amyloid plaque, were increased in the brains of diabetic patients. This suggests that T2D could affect the pathogenesis of AD through mechanisms other than modulation of Abeta metabolism even if the underlying

There is ongoing interest in defining mechanisms that govern insulin resistance and AD. The O subfamily of Forkhead/winged helix transcription factors (FOXO) plays important roles in regulating key physiological functions, including cell proliferation, cell differentiation, and survival together to cell cycle arrest and apoptosis. (Accili et al., 2004; Huang et al., 2007: van der Horst & Tindall, 2007). Thus, FOXO transcription factors are key players in cell death/life pathways. In addition, FOXO works in a complex way to regulate insulin signaling and glucose and lipid metabolism (Accili et al., 20004; Barthel et al., 2005). An additional layer of complexity exists in the transciptional activity of FOXO that is regulated by insulin through the phosphatidyl inositol 3 kinase (PI3K)/Akt signaling pathway. Both insulin and insulin-like growth factor-1 (IGF-1), through activation of their receptors, induce PI3K/Akt-dependent phosphorylation of FOXO, which facilitates its interaction with 14-3-3 protein, leading to nuclear exclusion and eventual ubiquitylationdependent proteasomal degradation (Matzusaki et al., 2006). In particular, in presence of insulin, activated Akt translocates to the nucleus where directly phosphorylates FOXO at distinct sites stimulating interaction with 14-3-3 protein (Greer and Brunet, 2005). This chaperone protein promotes the nuclear export and inhibits the nuclear import of FOXO proteins, driving the cells towards cell survival (van der Heide et al., 2004). In contrast, FOXO proteins, under conditions of oxidative stress, are phosphorylated by other protein kinases, including Mst1 and JNK, able to disrupt its interaction with 14-3-3, promoting FOXO nuclear translocation and thereby inducing cell death in neurons, thus opposing Akt's action (Sunayama et al., 2005). Thus, it is well established that Akt plays a key role in repressing FOXO transcriptional activity. Immediately upstream from FOXO, the activity of

mechanisms for this association remain largely unknown (Takeda et al., 2011).

**8. FOXO: A common biomarker for AD and T2D**

1999).

Insulin crosses the blood-brain barrier, and might even be produced locally in the brain, exerting its effects on cells by binding to a specific cell surface receptor. Insulin receptors are expressed throughout the CNS, expecially in the hippocampus and cortex, even if their function in the brain is not fully understood. Binding of insulin to its receptor activates the intrinsic tyrosine kinase activity of the cytoplasmic domain of the insulin receptor. This leads to autophosphorylation of tyrosine residues, which initiates several intracellular signaling cascades. In the brain, insulin influences the release and reuptake of neurotransmitters, and also appears to improve learning and memory (Zhao et al., 2004). The initial components of the insulin receptor signaling cascade in the brain are largely similar to those of the periphery. The downstream targets of the cascade are quite different, however, probably involving, among others, neuronal glutamate receptors (Zhao et al., 2004). Neurodegeneration and cognitive impairment in T2D and AD could be caused, in part, by damage to insulin receptor signaling (de la Monte & Wands, 2005). In fact, decreases in the sensitivity of such receptors are known to affect the expression and metabolism of Abeta and tau and impaired insulin receptor activity and hyperinsulinemia are observed in patients with AD and in animal models of this disease (Frolic et al., 1998). In addition, dysfunction of insulin receptor signaling is associated with impairments in Abeta oligomer clearance (Zhao, 2009) and increases the rate of NFT development (Lesort & Johnson, 2000). In fact, insulin transiently increases tau phosphorylation in primary cortical neurons, and hyperinsulinemia results in tau hyperphosphorylation in rat brains. Furthermore, insulin receptor substrate 2 knockout mice demonstrate typical pathological signs of T2D and have an increased number of NFTs in hippocampal neurons compared with control wild-type mice (Schubert, 2003). Thus, impaired insulin signaling could increase tau phosphorylation and cleavage. Insulin receptor signaling leads to the activation of two major signaling pathways, the mitogen-activated protein kinase (MAPK) pathway and the Akt signaling pathway. MAPK signaling is a required component of cell differentiation, cell proliferation and cell death, whereas Akt signaling is involved in the regulation of cell growth, cell proliferation, protein synthesis (via the mammalian target of rapamycin signaling pathway) and cell survival (through the inhibition of several proapoptotic agents).

Akt signaling induces the inhibition of glycogen synthase kinase-3β (GSK-3β) phosphorylates and, hence, inactivates glycogen synthase, a key enzyme in glycogenesis (Balaraman et al., 2006). Thus, under normal conditions, insulin signaling via the insulin receptor leads to GSK-3β inactivation, whereas insulin resistance leads to GSK-3β dephosphorylation and activation (Balaraman et al., 2006). The regulation of GSK-3β in the hippocampus and cortex changes in response to changes in glucose and insulin concentrations and in T2D an increase in GSK-3β activity might lead to insulin resistance by reducing glucose clearance (Lee & Kim, 2007). Increased GSK-3β activation might also lead to an elevation in Abeta production (resulting from a GSK-3β-mediated increase in presenilin 1 activity) and an increase in tau phosphorylation associated with NFT formation (Balaraman et al., 2006; Phiel et al., 2003). In contrast, inhibition of GSK-3β attenuates APP processing and inhibits hyperphosphorylated tau-associated neurodegeneration in cellculture and animal models of AD (Phiel et al., 2003).

Another important link between insulin resistance and the amyloid cascade may be related to the insulin degrading enzyme (IDE). This enzyme is a metalloprotease responsible for insulin degradation and is also the main enzyme responsible for Abeta degradation (Farris et al., 2003). IDE is secreted to the extracellular space by microglial cells in the brain, where

Insulin crosses the blood-brain barrier, and might even be produced locally in the brain, exerting its effects on cells by binding to a specific cell surface receptor. Insulin receptors are expressed throughout the CNS, expecially in the hippocampus and cortex, even if their function in the brain is not fully understood. Binding of insulin to its receptor activates the intrinsic tyrosine kinase activity of the cytoplasmic domain of the insulin receptor. This leads to autophosphorylation of tyrosine residues, which initiates several intracellular signaling cascades. In the brain, insulin influences the release and reuptake of neurotransmitters, and also appears to improve learning and memory (Zhao et al., 2004). The initial components of the insulin receptor signaling cascade in the brain are largely similar to those of the periphery. The downstream targets of the cascade are quite different, however, probably involving, among others, neuronal glutamate receptors (Zhao et al., 2004). Neurodegeneration and cognitive impairment in T2D and AD could be caused, in part, by damage to insulin receptor signaling (de la Monte & Wands, 2005). In fact, decreases in the sensitivity of such receptors are known to affect the expression and metabolism of Abeta and tau and impaired insulin receptor activity and hyperinsulinemia are observed in patients with AD and in animal models of this disease (Frolic et al., 1998). In addition, dysfunction of insulin receptor signaling is associated with impairments in Abeta oligomer clearance (Zhao, 2009) and increases the rate of NFT development (Lesort & Johnson, 2000). In fact, insulin transiently increases tau phosphorylation in primary cortical neurons, and hyperinsulinemia results in tau hyperphosphorylation in rat brains. Furthermore, insulin receptor substrate 2 knockout mice demonstrate typical pathological signs of T2D and have an increased number of NFTs in hippocampal neurons compared with control wild-type mice (Schubert, 2003). Thus, impaired insulin signaling could increase tau phosphorylation and cleavage. Insulin receptor signaling leads to the activation of two major signaling pathways, the mitogen-activated protein kinase (MAPK) pathway and the Akt signaling pathway. MAPK signaling is a required component of cell differentiation, cell proliferation and cell death, whereas Akt signaling is involved in the regulation of cell growth, cell proliferation, protein synthesis (via the mammalian target of rapamycin signaling pathway) and cell survival (through the inhibition of several

Akt signaling induces the inhibition of glycogen synthase kinase-3β (GSK-3β) phosphorylates and, hence, inactivates glycogen synthase, a key enzyme in glycogenesis (Balaraman et al., 2006). Thus, under normal conditions, insulin signaling via the insulin receptor leads to GSK-3β inactivation, whereas insulin resistance leads to GSK-3β dephosphorylation and activation (Balaraman et al., 2006). The regulation of GSK-3β in the hippocampus and cortex changes in response to changes in glucose and insulin concentrations and in T2D an increase in GSK-3β activity might lead to insulin resistance by reducing glucose clearance (Lee & Kim, 2007). Increased GSK-3β activation might also lead to an elevation in Abeta production (resulting from a GSK-3β-mediated increase in presenilin 1 activity) and an increase in tau phosphorylation associated with NFT formation (Balaraman et al., 2006; Phiel et al., 2003). In contrast, inhibition of GSK-3β attenuates APP processing and inhibits hyperphosphorylated tau-associated neurodegeneration in cell-

Another important link between insulin resistance and the amyloid cascade may be related to the insulin degrading enzyme (IDE). This enzyme is a metalloprotease responsible for insulin degradation and is also the main enzyme responsible for Abeta degradation (Farris et al., 2003). IDE is secreted to the extracellular space by microglial cells in the brain, where

proapoptotic agents).

culture and animal models of AD (Phiel et al., 2003).

it degrades Abeta peptide, thus reducing the rate of aggregation and the plaque formation (Qiu et al., 1998). IDE levels have been reported to be decreased in the brains of AD patients (Cook et al., 2003). It has also been hypothesized that hyperinsulinemia in people with prediabetes and T2D effectively sequesters IDE, reducing Abeta peptide degradation. This would increase levels of Abeta, and promote many of the pathological features associated with Alzheimer's disease. Supporting this model, the affinity for the binding of insulin to IDE is much greater than that for the Abeta (Qiuet al., 1997). In patients with Alzheimer's disease, IDE expression in the hippocampus is substantially reduced, with regards to controls, in particular among patients with the APOEvar epsilon4 genotype. This latter observation could explain the potential interaction between diabetes and the APOEvar epsilon4 genotype in multiplying the risk of dementia (Cook et al., 2003). Curiously, although the presence of the APOEvar epsilon4 is associated with an increased incidence of Alzheimer's disease, it seems that insulin resistance is only a significant risk factor for AD in those patients without APOEvar epsilon4 (Craft et al., 1998). Subjects with AD without the APOEvar epsilon4 also had improved memory scores when they had hyperinsulinemia, which was not the case for people with at least one APOEvar epsilon4 allele (Craft et al., 1999).

However, unexpectedly, recent clinicopathological studies have shown no evidence that the pathological hallmarks of AD, including amyloid plaque, were increased in the brains of diabetic patients. This suggests that T2D could affect the pathogenesis of AD through mechanisms other than modulation of Abeta metabolism even if the underlying mechanisms for this association remain largely unknown (Takeda et al., 2011).

#### **8. FOXO: A common biomarker for AD and T2D**

There is ongoing interest in defining mechanisms that govern insulin resistance and AD. The O subfamily of Forkhead/winged helix transcription factors (FOXO) plays important roles in regulating key physiological functions, including cell proliferation, cell differentiation, and survival together to cell cycle arrest and apoptosis. (Accili et al., 2004; Huang et al., 2007: van der Horst & Tindall, 2007). Thus, FOXO transcription factors are key players in cell death/life pathways. In addition, FOXO works in a complex way to regulate insulin signaling and glucose and lipid metabolism (Accili et al., 20004; Barthel et al., 2005). An additional layer of complexity exists in the transciptional activity of FOXO that is regulated by insulin through the phosphatidyl inositol 3 kinase (PI3K)/Akt signaling pathway. Both insulin and insulin-like growth factor-1 (IGF-1), through activation of their receptors, induce PI3K/Akt-dependent phosphorylation of FOXO, which facilitates its interaction with 14-3-3 protein, leading to nuclear exclusion and eventual ubiquitylationdependent proteasomal degradation (Matzusaki et al., 2006). In particular, in presence of insulin, activated Akt translocates to the nucleus where directly phosphorylates FOXO at distinct sites stimulating interaction with 14-3-3 protein (Greer and Brunet, 2005). This chaperone protein promotes the nuclear export and inhibits the nuclear import of FOXO proteins, driving the cells towards cell survival (van der Heide et al., 2004). In contrast, FOXO proteins, under conditions of oxidative stress, are phosphorylated by other protein kinases, including Mst1 and JNK, able to disrupt its interaction with 14-3-3, promoting FOXO nuclear translocation and thereby inducing cell death in neurons, thus opposing Akt's action (Sunayama et al., 2005). Thus, it is well established that Akt plays a key role in repressing FOXO transcriptional activity. Immediately upstream from FOXO, the activity of

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 43

Moreover, unpublished results indicate that insulin counteracts oxidative stress induced by amyloid beta by activation of Akt survival pathway. Akt traslocates from the cytoplasm to nucleus where phophorylates FOXO3a that, in turn, moves from the nucleus to the cytoplasm inhibiting, in this way the transcription of the FOXO-dependent genes. Since after Abeta-induced oxidative stress are usually activated pro-apoptotic genes, their trascriptional inhibition helps the survival program (Picone et al., 2011). Moreover, it has been suggested that since insulin signalling in the brain is known to decline with age, the outcome of the balance of different molecules, as Akt and FOXO, represents a risk factor for AD that is well suited for therapeutic intervention. By restoring the balance of molecules to favour neuron survival, new drugs, designed to specifically enhance CNS insulin signalling,

The present chapter highlights the overlap and the many points of intersection existing between T2D and AD. Insulin resistence in the CNS results in the dysregulation of multiple extracellular and intracellular signaling cascades and molecular mechanisms, which in turn could lead to decrease in neuronal and synaptic functions up to neurodegeneration. An understanding of how each molecular pathway intersects and affects the others is essential

The present work was supported by a Grant from the Italian Ministry of Universty and Scientific Research for Programs of Relevant National Interest (PRIN 2008 - prot. 20083Y34Y7) "*Development of a molecular strategy for the prevention of protein aggregation and*

Accili, D., Arden, K.C. (2004) FOXOs at the crossroads of cellular metabolism,

Adrover, M., Vilanova, B., Frau, J., Munoz, F. & Donoso, J. (2008) The pyridoxamine action

Ahmed, N., Thornalley, P.J. (2007) Advanced glycation endproducts: what is their relevance to diabetic complications? *Diabetes Obes. Metab.*, Vol. 9, pp. 233–245.

Antzutkin, O.N., Balbach, J.J., Leapman, R.D., Rizzo, N.W., Reed, J. & Tycko, R. (2000)

Apelt, J., Bigl, M., Wunderlich, P. & Schliebs, R. (2004) Aging-related increase in oxidative

on Amadori compounds: A reexamination of its scavenging capacity and chelating

Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of b-sheets in Alzheimer's b-amyloid fibrils. *Proc. Natl. Acad. Sci. USA*,

stress correlates with developmental pattern of betasecretase activity and betaamyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like

would provide a new and potentially significant class of AD therapeutics.

for the development of future drug intervention strategies for these pathologies.

differentiation, and transformation. *Cell*, Vol. 117, pp. 421–426

effect. Bioorg. Med. Chem., Vol. 16, pp. 5557–5569.

pathology. *Int. J. Dev. Neurosci.*, Vol. 22, pp. 475–484

**9. Conclusions**

**10. Acknowledgments**

**11. References**

*fibrillogenesis: a biophysical approach"*

Vol. 97, pp. 13045–13050.

Akt itself is governed by several protein kinases and phosphatases. Akt is activated by phosphorylation at Thr-308 within its catalytic domain by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and by phosphorylation at Ser-473 within a C-terminal hydrophobic motif by mammalian target of rapamycin (mTOR) (Stokoe et al., 1997; Sarbassov et al., 2005). Some studies show that phospho-Thr-308 and phospho-Ser-273 are dephosphorylated by protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and pleckstrin homology (PH) domain leucine rich repeat protein phosphatase (PHLPP), a member of the protein phosphatase 2C family (Gao et al., 2005). Akt regulates a variety of key physiological functions, and there is strong evidence suggesting that defective Akt signaling contributes to development of insulin resistance (Zdychova & Komers, 2005).

However, although it is clear that FOXO governs multiple events in the insulin signaling cascade, mediating both positive and negative effects, the underlying molecular mechanisms are unknown. Some evidence has been reported that FOXO3 activation is also able to increase basal levels of Akt phosphorylation and kinase activity thus it is capable to activating its own inhibitor, providing a feedback regulation (Ni et al., 2007) . Moreover, FOXO transcription factors are involved in both the insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and insulin resistence (Manolopoulos et al., 2010). Both insulin resistence and oxidative stress due to Abeta stimulus, may promote the transcriptional activity of FOXO proteins, resulting in hyperglycaemia and a further increased production of ROS.

The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of Abeta plaques and tau protein phosphorylation. Wnt inhibition may also result in a sustained activation of FOXO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, insulin resistence and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. Thus, it has been proposed that FOXO proteins may provide a potential molecular target for the treatment of both insulin resistence and AD (Manolopoulos et al., 2010). Recently it has been demostrated that insulin plays a protective role by inhibiting mithocondrial dysfunction and apoptosis activation triggered by Abeta oligomers (Di Carlo et al., 2010) (Figure 3).

Fig. 3. *Insulin recovers mitochondrial respiratory activity reduced by rAb42 oligomers.* LAN5 untreated (control), treated with rAb42 oligomers oligomers), and rAb42 oligomers and insulin (oligomers+insulin) were incubated with Mito Red and Hoechst 33258. The images of each sample were merged. Mitochondrial activity is indicated by red arrows, fragmentation of nuclei is indcate by blu arrows.

Moreover, unpublished results indicate that insulin counteracts oxidative stress induced by amyloid beta by activation of Akt survival pathway. Akt traslocates from the cytoplasm to nucleus where phophorylates FOXO3a that, in turn, moves from the nucleus to the cytoplasm inhibiting, in this way the transcription of the FOXO-dependent genes. Since after Abeta-induced oxidative stress are usually activated pro-apoptotic genes, their trascriptional inhibition helps the survival program (Picone et al., 2011). Moreover, it has been suggested that since insulin signalling in the brain is known to decline with age, the outcome of the balance of different molecules, as Akt and FOXO, represents a risk factor for AD that is well suited for therapeutic intervention. By restoring the balance of molecules to favour neuron survival, new drugs, designed to specifically enhance CNS insulin signalling, would provide a new and potentially significant class of AD therapeutics.

#### **9. Conclusions**

42 Topics in the Prevention, Treatment and Complications of Type 2 Diabetes

Akt itself is governed by several protein kinases and phosphatases. Akt is activated by phosphorylation at Thr-308 within its catalytic domain by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and by phosphorylation at Ser-473 within a C-terminal hydrophobic motif by mammalian target of rapamycin (mTOR) (Stokoe et al., 1997; Sarbassov et al., 2005). Some studies show that phospho-Thr-308 and phospho-Ser-273 are dephosphorylated by protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and pleckstrin homology (PH) domain leucine rich repeat protein phosphatase (PHLPP), a member of the protein phosphatase 2C family (Gao et al., 2005). Akt regulates a variety of key physiological functions, and there is strong evidence suggesting that defective Akt signaling contributes to development of insulin resistance (Zdychova & Komers, 2005). However, although it is clear that FOXO governs multiple events in the insulin signaling cascade, mediating both positive and negative effects, the underlying molecular mechanisms are unknown. Some evidence has been reported that FOXO3 activation is also able to increase basal levels of Akt phosphorylation and kinase activity thus it is capable to activating its own inhibitor, providing a feedback regulation (Ni et al., 2007) . Moreover, FOXO transcription factors are involved in both the insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and insulin resistence (Manolopoulos et al., 2010). Both insulin resistence and oxidative stress due to Abeta stimulus, may promote the transcriptional activity of FOXO proteins, resulting in

The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of Abeta plaques and tau protein phosphorylation. Wnt inhibition may also result in a sustained activation of FOXO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, insulin resistence and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. Thus, it has been proposed that FOXO proteins may provide a potential molecular target for the treatment of both insulin resistence and AD (Manolopoulos et al., 2010). Recently it has been demostrated that insulin plays a protective role by inhibiting mithocondrial dysfunction and apoptosis activation triggered by Abeta oligomers (Di Carlo

Fig. 3. *Insulin recovers mitochondrial respiratory activity reduced by rAb42 oligomers.* LAN5 untreated (control), treated with rAb42 oligomers oligomers), and rAb42 oligomers and insulin (oligomers+insulin) were incubated with Mito Red and Hoechst 33258. The images

of each sample were merged. Mitochondrial activity is indicated by red arrows,

fragmentation of nuclei is indcate by blu arrows.

hyperglycaemia and a further increased production of ROS.

et al., 2010) (Figure 3).

The present chapter highlights the overlap and the many points of intersection existing between T2D and AD. Insulin resistence in the CNS results in the dysregulation of multiple extracellular and intracellular signaling cascades and molecular mechanisms, which in turn could lead to decrease in neuronal and synaptic functions up to neurodegeneration. An understanding of how each molecular pathway intersects and affects the others is essential for the development of future drug intervention strategies for these pathologies.

#### **10. Acknowledgments**

The present work was supported by a Grant from the Italian Ministry of Universty and Scientific Research for Programs of Relevant National Interest (PRIN 2008 - prot. 20083Y34Y7) "*Development of a molecular strategy for the prevention of protein aggregation and fibrillogenesis: a biophysical approach"*

#### **11. References**

Alzheimer's Disease and Type 2 Diabetes: Different Pathologies and Same Features 45

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**3**

*Korea*

**Insulin Resistance and Alzheimer's Disease**

Alzheimer disease (AD) is known as a form of type III diabetes due to its similar cellular responses and pathogenesis. Insulin alters normal brain function and peripheral glucose metabolism, and conditions that are related to insulin dysregulation, such as obesity, diabetes mellitus, and cardiovascular disease, have potentially harmful effects on brain function. Many reports have demonstrated that insulin resistance increases age-related memory impairments and is a risk factor for AD. The molecular and cellular link between insulin resistance and AD, however, is unknown. We discuss the potential mechanisms of these metabolic disorders in the pathogenesis of AD. Glucose homeostasis is critical for energy maintenance, neurogenesis, neuronal survival, and synaptic plasticity, which are required for learning and memory. During insulin resistance, one develops reduced sensitivity to insulin, resulting in hyperinsulinemia, and this impairment in insulin signaling mediates the pathogenesis of AD, which manifests as brain inflammation, oxidative stress, alterations in amyloid beta (Aβ) levels, and cell death. Human and experimental animal studies have noted that drugs that modulate insulin resistance decrease the accumulation of Aβ in the brain and the cognitive impairments that are associated with AD. Therapeutic strategies that target the link between insulin resistance and AD might benefit the

Insulin is a peptide hormone that comprises 51 amino acids and is synthesized in and secreted from pancreatic β-cells in the islet of Langerhans (Huang et al., 2010). Its rapid release is generally triggered by increased levels of glucose in the blood, despite other mechanisms that release insulin; normal blood glucose levels range from 4.4 to 6.1 mmol/L

Of its many functions, the most significant effects of insulin are its promotion of cellular growth and differentiation, activation or repression of transcription, and regulation of protein kinases and phosphatases (Saltiel and Pessin, 2002). Defects in insulin signaling can affect many diseases, such as type 2 diabetes mellitus (T2DM), metabolic syndrome, and Alzheimer disease (AD). Although most studies have examined the function of insulin and the dysregulation of insulin secretion or insulin receptor signaling in peripheral tissue, it has

recently been reported to cause serious mental illness (Huang et al., 2010).

**1. Introduction**

development of future AD drugs.

**2. Insulin signaling**

**2.1 Introduction**

(82 to 110 mg/dL).

Sung Min Son, Hong Joon Shin and Inhee Mook-Jung

*Department of Biochemistry & Biomedical Sciences, Seoul National University College of Medicine, Seoul*

### **Insulin Resistance and Alzheimer's Disease**

Sung Min Son, Hong Joon Shin and Inhee Mook-Jung

*Department of Biochemistry & Biomedical Sciences, Seoul National University College of Medicine, Seoul Korea*

#### **1. Introduction**

52 Topics in the Prevention, Treatment and Complications of Type 2 Diabetes

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in pathophysiology of diabetes and its complications. *Physiol. Res.*, Vol. 54, pp. 1–16.

(2008) Insulin receptor dysfunction impairs cellular clearance of neurotoxic

Alzheimer disease (AD) is known as a form of type III diabetes due to its similar cellular responses and pathogenesis. Insulin alters normal brain function and peripheral glucose metabolism, and conditions that are related to insulin dysregulation, such as obesity, diabetes mellitus, and cardiovascular disease, have potentially harmful effects on brain function. Many reports have demonstrated that insulin resistance increases age-related memory impairments and is a risk factor for AD. The molecular and cellular link between insulin resistance and AD, however, is unknown. We discuss the potential mechanisms of these metabolic disorders in the pathogenesis of AD. Glucose homeostasis is critical for energy maintenance, neurogenesis, neuronal survival, and synaptic plasticity, which are required for learning and memory. During insulin resistance, one develops reduced sensitivity to insulin, resulting in hyperinsulinemia, and this impairment in insulin signaling mediates the pathogenesis of AD, which manifests as brain inflammation, oxidative stress, alterations in amyloid beta (Aβ) levels, and cell death. Human and experimental animal studies have noted that drugs that modulate insulin resistance decrease the accumulation of Aβ in the brain and the cognitive impairments that are associated with AD. Therapeutic strategies that target the link between insulin resistance and AD might benefit the development of future AD drugs.

#### **2. Insulin signaling**

#### **2.1 Introduction**

Insulin is a peptide hormone that comprises 51 amino acids and is synthesized in and secreted from pancreatic β-cells in the islet of Langerhans (Huang et al., 2010). Its rapid release is generally triggered by increased levels of glucose in the blood, despite other mechanisms that release insulin; normal blood glucose levels range from 4.4 to 6.1 mmol/L (82 to 110 mg/dL).

Of its many functions, the most significant effects of insulin are its promotion of cellular growth and differentiation, activation or repression of transcription, and regulation of protein kinases and phosphatases (Saltiel and Pessin, 2002). Defects in insulin signaling can affect many diseases, such as type 2 diabetes mellitus (T2DM), metabolic syndrome, and Alzheimer disease (AD). Although most studies have examined the function of insulin and the dysregulation of insulin secretion or insulin receptor signaling in peripheral tissue, it has recently been reported to cause serious mental illness (Huang et al., 2010).

Insulin Resistance and Alzheimer's Disease 55

Moreover, PKB phosphorylates the tuberous sclerosis complex 1 (TSC1)-TSC2 complex, relieving its inhibition of the mammalian target of rapamycin (mTOR) (Proud, 2006), which promotes protein synthesis through p70 ribosomal S6 kinase (p70S6k) and inhibition of eIF-4E binding protein (4E-BP1), and thereby governs ribosomal protein translation (Proud,

The brain is the primary consumer of glucose, requiring two-thirds of total circulating glucose daily (Peters, 2011); thus, the regulation of glucose homeostasis by insulin in the central nervous system (CNS) is paramount for normal brain function. Insulin, by a saturable transport process that is mediated by insulin receptor, can cross the blood-brain barrier (BBB), which governs the transduction between the CNS and peripheral tissues (Neumann et al., 2008). Although insulin is synthesized locally in the brain (Banks et al.,

IRs in the CNS are widely expressed throughout the brain in neurons and glial cells, at particularly high concentrations in the cerebral cortex, hippocampus, hypothalamus, and olfactory bulb (Neumann et al., 2008). They have disparate functions, molecular weights, and structures from IRs in peripheral organs (Heidenreich et al., 1983), but it is unknown whether the counter effects of insulin in the CNS compared with those in peripheral tissues are attributed to such differences. Insulin increases glucose and inhibits feeding in the CNS, but decreases glucose and stimulates feeding in peripheral tissue (Florant et al., 1991). A recent report observed that impaired brain insulin activity might affect unrestrained lipolysis that initiates and exacerbates type 2 diabetes mellitus in humans (Scherer et al.,

In humans, brain insulin enhances learning, memory, and, in particular, verbal memory (Benedict et al., 2004). Insulin signaling in the limbic system and hypothalamus is especially important for cognitive function (Zhao et al., 1999), independent of changes in peripheral glucose (Craft et al., 1999; Kern et al., 1999). These functions have been supported by evidence that insulin modulates the concentrations of neurotransmitters, which influence cognition, such as Ach (acetylcholine) (Kopf and Baratti, 1999). Insulin also alters synaptic plasticity by regulating the endocytosis of 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, which causes long-term depression (LTD) of excitatory synaptic transmission in the hippocampus and cerebellum (Huang et al., 2003; Man et al., 2000; Wang and Linden, 2000), and by enhancing N-methyl-D-aspartate (NMDA) receptor-mediated

Insulin-like growth factors are polypeptides that are similar in sequence to proinsulin (Clemmons, 2007). Insulin-like growth factor 1, which stimulates cell growth and proliferation, especially in nerve cells, binds to IGF receptor and IRs (Jones et al., 2009). IGF-1 functions similarly to insulin receptor. IGF-1, a tyrosine kinase, initiates signaling cascades

Deficiency of or irresponsiveness to IGF-1 causes not only growth failure but also dyslipidemia (Twickler et al., 2003), insulin resistance (Conti et al., 2002; Twickler et al.,

synaptic transmission at hippocampal CA1 synapses (van der Heide et al., 2005).

through IRS and enhances insulin activity (Clemmons, 2007).

1997; Devaskar et al., 1994; Rulifson et al., 2002), its function is unknown.

2006).

2011).

**2.4 Role of insulin in cognitive function**

**2.5 Insulin-like growth factor (IGF)**

**2.3 Insulin in the brain**

#### **2.2 General effects of insulin signaling**

Insulin increases glycogen and lipid synthesis in liver and muscle cells and simultaneously inhibits glycogenolysis and gluconeogenesis in the liver during feeding (Saltiel and Kahn, 2001). These actions are mediated by its binding to membrane-bound insulin receptors (IRs), members of the tyrosine kinase receptor family (Saltiel and Pessin, 2002). The binding of insulin to the alpha subunit of IR induces a conformational change, resulting in the autophosphorylation of tyrosine residues in the beta subunit of the receptor (Van Obberghen et al., 2001). This ligation leads to the subsequent tyrosine phosphorylation of insulin receptor substrates 1-4 (IRS1-4), Shc, Gab-1, Cbl, and APS (Saltiel and Pessin, 2002), and activates downstream cytoplasmic effectors, such as the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) (Cole et al., 2007; Plum et al., 2005).

During insulin-mediated glucose uptake, the phosphorylated residues of insulin receptor and IRS1, 2, and 3 are recognized by the Src homology 2 (SH2) domain (Bevan, 2001) of the p85 regulatory subunit of PI 3-kinase. The catalytic subunit of PI 3-kinase, p110, phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2), leading to the formation of Ptd(3,4,5)P3 (Lizcano and Alessi, 2002). Ptd(3,4,5)P3 then binds to the plasma membrane and associates with phosphoinositol-dependent kinase-1 (PDK-1) and induces the phosphorylation and activation of protein kinase B (PKB)/Akt, a serine-threonine kinase. Activated Akt enters the cytoplasm, where it evokes the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3), which promotes glycogen synthesis.

By activating PI3-kinase and AKT (Lizcano and Alessi, 2002), insulin affects the translocation of glucose transporter 4 (GLUT4) from intracellular stores to the plasma membrane: When insulin concentrations are low, GLUT4 exists in cytoplasmic vesicles. After insulin binds to its receptors, GLUT4-containing vesicles fuse with the plasma membrane, and the newly inserted GLUT4 takes up glucose efficiently. In parallel, there is a PI 3-kinase independent pathway that recruits GLUT4 to the plasma membrane, led by the phosphorylation of the proto-oncogene Cbl, which is associated with the adaptor protein c-Cbl-associated protein (CAP) (Saltiel and Kahn, 2001).

Insulin promotes the uptake and synthesis of fatty acids in the liver and prevents lipolysis by inhibiting the intracellular lipase that hydrolyzes triglycerides to release fatty acids. Although insulin promotes the synthesis of glycogen in the liver, further synthesis is suppressed when the liver is saturated with glycogen. Any additional glucose that is taken up is then shunted into pathways that lead to fatty acid synthesis (Saltiel and Kahn, 2001).

One study has indicated that lipid synthesis requires increased expression of the transcription factor steroid regulatory element-binding protein (SREBP)-1c (Shimomura et al., 1999), which is synthesized as a membrane protein in the endoplasmic reticulum (ER) (Sato, 2010). Insulin is believed to induce SREBP-1c proteolytic activity and, consequently, insulin-mediated lipogenesis (Sato, 2010). Further, insulin inhibits lipid metabolism by decreasing cellular concentrations of cAMP through activation of a cAMP-specific phosphodiesterase in adipocytes (Kitamura et al., 1999).

Insulin stimulates amino acid uptake into cells, inhibits protein degradation, and promotes protein synthesis (Saltiel and Kahn, 2001) by stimulating components of the translational machinery, including eukaryotic initiation factors (elFs) and eukaryotic elongation factors (eEFs) (Proud, 2006). This entails the activation of PKB, leading to the phosphorylation and inactivation of GSK3, which in turn promotes phosphorylation and inhibition of a guanine nucleotide exchange factor, eIF2B, to regulate the initiation of translation (Proud, 2006).

Moreover, PKB phosphorylates the tuberous sclerosis complex 1 (TSC1)-TSC2 complex, relieving its inhibition of the mammalian target of rapamycin (mTOR) (Proud, 2006), which promotes protein synthesis through p70 ribosomal S6 kinase (p70S6k) and inhibition of eIF-4E binding protein (4E-BP1), and thereby governs ribosomal protein translation (Proud, 2006).

#### **2.3 Insulin in the brain**

54 Topics in the Prevention, Treatment and Complications of Type 2 Diabetes

Insulin increases glycogen and lipid synthesis in liver and muscle cells and simultaneously inhibits glycogenolysis and gluconeogenesis in the liver during feeding (Saltiel and Kahn, 2001). These actions are mediated by its binding to membrane-bound insulin receptors (IRs), members of the tyrosine kinase receptor family (Saltiel and Pessin, 2002). The binding of insulin to the alpha subunit of IR induces a conformational change, resulting in the autophosphorylation of tyrosine residues in the beta subunit of the receptor (Van Obberghen et al., 2001). This ligation leads to the subsequent tyrosine phosphorylation of insulin receptor substrates 1-4 (IRS1-4), Shc, Gab-1, Cbl, and APS (Saltiel and Pessin, 2002), and activates downstream cytoplasmic effectors, such as the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK)

During insulin-mediated glucose uptake, the phosphorylated residues of insulin receptor and IRS1, 2, and 3 are recognized by the Src homology 2 (SH2) domain (Bevan, 2001) of the p85 regulatory subunit of PI 3-kinase. The catalytic subunit of PI 3-kinase, p110, phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2), leading to the formation of Ptd(3,4,5)P3 (Lizcano and Alessi, 2002). Ptd(3,4,5)P3 then binds to the plasma membrane and associates with phosphoinositol-dependent kinase-1 (PDK-1) and induces the phosphorylation and activation of protein kinase B (PKB)/Akt, a serine-threonine kinase. Activated Akt enters the cytoplasm, where it evokes the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3), which promotes glycogen synthesis. By activating PI3-kinase and AKT (Lizcano and Alessi, 2002), insulin affects the translocation of glucose transporter 4 (GLUT4) from intracellular stores to the plasma membrane: When insulin concentrations are low, GLUT4 exists in cytoplasmic vesicles. After insulin binds to its receptors, GLUT4-containing vesicles fuse with the plasma membrane, and the newly inserted GLUT4 takes up glucose efficiently. In parallel, there is a PI 3-kinase independent pathway that recruits GLUT4 to the plasma membrane, led by the phosphorylation of the proto-oncogene Cbl, which is associated with the adaptor protein c-

Insulin promotes the uptake and synthesis of fatty acids in the liver and prevents lipolysis by inhibiting the intracellular lipase that hydrolyzes triglycerides to release fatty acids. Although insulin promotes the synthesis of glycogen in the liver, further synthesis is suppressed when the liver is saturated with glycogen. Any additional glucose that is taken up is then shunted into pathways that lead to fatty acid synthesis (Saltiel and Kahn, 2001). One study has indicated that lipid synthesis requires increased expression of the transcription factor steroid regulatory element-binding protein (SREBP)-1c (Shimomura et al., 1999), which is synthesized as a membrane protein in the endoplasmic reticulum (ER) (Sato, 2010). Insulin is believed to induce SREBP-1c proteolytic activity and, consequently, insulin-mediated lipogenesis (Sato, 2010). Further, insulin inhibits lipid metabolism by decreasing cellular concentrations of cAMP through activation of a cAMP-specific

Insulin stimulates amino acid uptake into cells, inhibits protein degradation, and promotes protein synthesis (Saltiel and Kahn, 2001) by stimulating components of the translational machinery, including eukaryotic initiation factors (elFs) and eukaryotic elongation factors (eEFs) (Proud, 2006). This entails the activation of PKB, leading to the phosphorylation and inactivation of GSK3, which in turn promotes phosphorylation and inhibition of a guanine nucleotide exchange factor, eIF2B, to regulate the initiation of translation (Proud, 2006).

**2.2 General effects of insulin signaling**

(Cole et al., 2007; Plum et al., 2005).

Cbl-associated protein (CAP) (Saltiel and Kahn, 2001).

phosphodiesterase in adipocytes (Kitamura et al., 1999).

The brain is the primary consumer of glucose, requiring two-thirds of total circulating glucose daily (Peters, 2011); thus, the regulation of glucose homeostasis by insulin in the central nervous system (CNS) is paramount for normal brain function. Insulin, by a saturable transport process that is mediated by insulin receptor, can cross the blood-brain barrier (BBB), which governs the transduction between the CNS and peripheral tissues (Neumann et al., 2008). Although insulin is synthesized locally in the brain (Banks et al., 1997; Devaskar et al., 1994; Rulifson et al., 2002), its function is unknown.

IRs in the CNS are widely expressed throughout the brain in neurons and glial cells, at particularly high concentrations in the cerebral cortex, hippocampus, hypothalamus, and olfactory bulb (Neumann et al., 2008). They have disparate functions, molecular weights, and structures from IRs in peripheral organs (Heidenreich et al., 1983), but it is unknown whether the counter effects of insulin in the CNS compared with those in peripheral tissues are attributed to such differences. Insulin increases glucose and inhibits feeding in the CNS, but decreases glucose and stimulates feeding in peripheral tissue (Florant et al., 1991). A recent report observed that impaired brain insulin activity might affect unrestrained lipolysis that initiates and exacerbates type 2 diabetes mellitus in humans (Scherer et al., 2011).

#### **2.4 Role of insulin in cognitive function**

In humans, brain insulin enhances learning, memory, and, in particular, verbal memory (Benedict et al., 2004). Insulin signaling in the limbic system and hypothalamus is especially important for cognitive function (Zhao et al., 1999), independent of changes in peripheral glucose (Craft et al., 1999; Kern et al., 1999). These functions have been supported by evidence that insulin modulates the concentrations of neurotransmitters, which influence cognition, such as Ach (acetylcholine) (Kopf and Baratti, 1999). Insulin also alters synaptic plasticity by regulating the endocytosis of 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, which causes long-term depression (LTD) of excitatory synaptic transmission in the hippocampus and cerebellum (Huang et al., 2003; Man et al., 2000; Wang and Linden, 2000), and by enhancing N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission at hippocampal CA1 synapses (van der Heide et al., 2005).

#### **2.5 Insulin-like growth factor (IGF)**

Insulin-like growth factors are polypeptides that are similar in sequence to proinsulin (Clemmons, 2007). Insulin-like growth factor 1, which stimulates cell growth and proliferation, especially in nerve cells, binds to IGF receptor and IRs (Jones et al., 2009). IGF-1 functions similarly to insulin receptor. IGF-1, a tyrosine kinase, initiates signaling cascades through IRS and enhances insulin activity (Clemmons, 2007).

Deficiency of or irresponsiveness to IGF-1 causes not only growth failure but also dyslipidemia (Twickler et al., 2003), insulin resistance (Conti et al., 2002; Twickler et al.,

Insulin Resistance and Alzheimer's Disease 57

Insulin-degrading enzyme is the chief enzyme that degrades excess insulin and other substrates, including Aβ, a peptide that is implicated in the pathogenesis of AD (Wang et al., 2010). IDE knockout mice experienced decreased Aβ degradation, hyperinsulinemia, and hyperglycemia (Farris et al., 2003). As insulin levels rise, IDE expression increases to prevent the chronic activation of insulin (Zhao et al., 2004). Reduced IDE activity might contribute to the formation of senile plaques through the inability to degrade Aβ peptides (Jones et al., 2009).

Insulin resistance is a physiological condition in which one loses or reduces his sensitivity to insulin—that is, it becomes less effective at lowering glucose levels in the blood. There are several representative phenotypes of insulin resistance that are used to diagnose diabetes, such as hyperinsulinemia (elevated levels of insulin that are required to maintain normal glucose levels in the bloodstream) and hyperglycemia (elevated glucose levels in the bloodstream that result from the failure of insulin to mediate glucose uptake or insufficient production of insulin by β-cells). Thus, insulin resistance reduces glycogen storage in

High cortisol and uric acid levels and low vitamin D levels (Chiu et al., 2004; Vuorinen-Markkola and Yki-Jarvinen, 1994) are believed to be dietary conditions that precipitate insulin resistance. In addition, insulin resistance is closely related to obesity, hypertension, polycystic ovarian syndrome, dyslipidemia, and atherosclerosis (Saltiel and Pessin, 2002).

Advanced glycation end products (AGEs) production is a pathological phenotype that results from hyperglycemia (Jager et al., 2007). Activation of the jun-N-terminal kinase (JNK) pathway by the binding of AGEs to their receptor (RAGE) leads to serine phosphorylation of IRS proteins and disrupts the imbalance in glucose levels further (Han et al., 2011). In addition to excess AGE production, prolonged hyperglycemia activates the transcription factor nuclear factor kappa B (NF-kB), which regulates proinflammatory and antiapoptotic pathways by modulating the transcription of cytokine and antioxidant genes (Baeuerle and Baltimore, 1996); it also controls the expression of genes that mediate immune

Peripheral insulin resistance mediates the progression to hyperglycemia and affects other functions of insulin. In particular, it affects reduced uptake of circulating lipids and increased hydrolysis of stored triglycerides.  FFAs increase 3-fold above basal levels, reducing insulindependent skeletal muscle glucose uptake by 50% (Roden et al., 1996). This phenomenon, a direct cause of insulin resistance, induces inhibits serine phosphorylation of IRS and decreases its chances of binding to insulin receptor, further hampering insulin signaling

identified as a significant trigger of insulin resistance (Jones et al., 2009)

**2.6 Insulin-degrading enzyme (IDE)**

**3.1 Definition of insulin resistance**

muscle and fat cells and decreases hepatic glucose uptake.

**3.2 Peripheral effects of insulin resistance**

(Hotamisligil et al., 1996; Schinner et al., 2005).

responses and proliferation.

**3. Insulin resistance**

2003), and obesity (Rasmussen et al., 1995). According to recent longitudinal studies, these phenotypes are closely related to a high risk of neurodegenerative disorders, such as dementia and AD (Luchsinger et al., 2004; Ott et al., 1996; Ronnemaa et al., 2008). Similarly, mice in which IGF-1 has been deleted develop increased Aβ levels in the brain, suggesting that IGF-1 promotes the clearance of Aβ (Carro et al., 2002). Moreover, IGF-1 mediates transient site-selective increases in tau phosphorylation—hyperphosphorylated tau is a pathological hallmark of AD—in primary cortical neurons via GSK-3 pathways (Lesort and Johnson, 2000).

Fig. 1. Schematic diagram of the effects of peripheral insulin resistance on insulin, insulindegrading enzyme (IDE) and Aβ levels. Peripheral insulin resistance and hyperinsulinemia triggers an excess release of FFA from adipocytes to liver and muscles. A three-fold increase of FFAs reduce insulin-dependent skeletal muscle glucose uptake by 50% (Roden et al., 1996), thereby hampering further insulin signal transduction (Hotamisligil et al., 1996; Schinner et al., 2005). In addition, Increased peripheral FFA levels invoke elevations in TNFα in the periphery and in the CNS, which may result in increased accumulation of Aβ. Chronic elevations in plasma Aβ may result in increased transport of Aβ into the brain. In contrast, peripheral hyperinsulinemia decrease insulin transport into the CNS. Low insulin level in CNS, and low level of IDE may contribute to the formation of senile plaques via disability to degrade Aβ peptides (Jones et al., 2009), further promoting intraneuronal Aβ accumulation, which is the hallmark of AD. <modified from Craft's paper (Craft, 2007)>

#### **2.6 Insulin-degrading enzyme (IDE)**

Insulin-degrading enzyme is the chief enzyme that degrades excess insulin and other substrates, including Aβ, a peptide that is implicated in the pathogenesis of AD (Wang et al., 2010). IDE knockout mice experienced decreased Aβ degradation, hyperinsulinemia, and hyperglycemia (Farris et al., 2003). As insulin levels rise, IDE expression increases to prevent the chronic activation of insulin (Zhao et al., 2004). Reduced IDE activity might contribute to the formation of senile plaques through the inability to degrade Aβ peptides (Jones et al., 2009).

#### **3. Insulin resistance**

56 Topics in the Prevention, Treatment and Complications of Type 2 Diabetes

2003), and obesity (Rasmussen et al., 1995). According to recent longitudinal studies, these phenotypes are closely related to a high risk of neurodegenerative disorders, such as dementia and AD (Luchsinger et al., 2004; Ott et al., 1996; Ronnemaa et al., 2008). Similarly, mice in which IGF-1 has been deleted develop increased Aβ levels in the brain, suggesting that IGF-1 promotes the clearance of Aβ (Carro et al., 2002). Moreover, IGF-1 mediates transient site-selective increases in tau phosphorylation—hyperphosphorylated tau is a pathological hallmark of AD—in primary cortical neurons via GSK-3 pathways (Lesort and