THE LINK BETWEEN TYPE 3 DIABETES AND ALZHEIMER S DISEASE: MECHANISMS OF A NEURO- ENDOCRINE DISORDER

THE LINK BETWEEN TYPE 3 DIABETES AND ALZHEIMER S DISEASE: MECHANISMS OF A NEURO- ENDOCRINE DISORDER By Mohammad H. Almermesh, BSc. Presented To the Graduate Faculty of MCPHS University In partial fulfillment of the requirements for the degree of Master of Science under the supervision of Dr. David Albers May 2015 Thesis approved by the Advisory Committee: David Albers, PhD/Research Advisor Associate Professor of Pharmacology/Toxicology Ahmed Mehanna, PhD Professor of Medicinal Chemistry Mattia Migliore, PhD Associate Professor of Pharmacology/Toxicology Timothy J. Maher, PhD Professor of Pharmacology Associate Dean of Graduate Studies

THE LINK BETWEEN TYPE 3 DIABETES AND ALZHEIMER S DISEASE: MECHANISMS OF A NEURO-ENDOCRINE DISORDER Abstract A growing body of evidence supports the concept that type 3 diabetes (T3DM) is a neuroendocrine disease with abnormalities in brain glucose utilization and responsiveness to insulin and insulin-like growth factor (IGF) activation. In addition, deregulated glucose metabolism has a role in neurodegeneration and neuronal loss (usually accompanied by T3DM) along with multiple pathogenic factors including oxidative stress and mitochondrial dysfunction. Moreover, impaired insulin signaling can disturb both amyloid precursor protein (APP) processing and amyloid-beta (Aβ) clearance. This leads to increased neurotoxic effects of Aβ on neurons ending up with possible neurodegeneration and neuronal cell death. In the same context, the neuronal aging is characterized by decreased mitochondrial function and increased reactive oxygen species (ROS) production, which are associated with cognitive decline. Also, accelerated cognitive decline is associated with long-term complications. These complications are due to multifactorial factors including circulatory and metabolic considerations and recurrent hypoglycemia. Most of diabetic complications can be found in T3DM, but with increased severity. This review discusses the molecular, biochemical and cellular dysfunctions in both Alzheimer s disease (AD) and diabetes mellitus (DM) along with the causation of neuronal loss and cell death, and so called neurodegenerative disease. Also it explains the mechanism of the crosstalk of the attenuated phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K)/Akt pathway and the intensified activation of glycogen synthase kinase-3-beta (GSK-3 β) pathway, due to trophic factor resistance, which eventually cause abnormal hyperphosphorylated tau and impaired tau gene expression proceeding neurodegeneration. Also, it discusses the relationship between T3DM and AD based on the fact that both the processing of AβPP and the clearance of Aβ are attributed to impaired insulin signaling in the brain. Additionally, it focuses on the molecular mechanism of brain insulin resistance or T3DM that may involve either increased serine phosphorylation of insulin receptor substrate 1 (IRS-1) protein (i.e., IRS-1 inhibition) and/or elevated degradation of IRS protein as common pathological mechanisms. Moreover, this review emphasizes on the potential link between type 2 diabetes mellitus (T2DM) and AD that might have devastating impacts on public health or healthcare systems such as socio-economic burdens. Lastly, possible biomarkers and insulin-related therapeutic strategies are suggested to help improve the early detection, diagnosis, and treatment of T3DM or AD by slowing down their progressive nature or even halt their future complications. i

Acknowledgements All thanks to my almighty Allah for his uncountable blessings, love and support. Then, many thanks to my dad, Hajaj, and mom, Ayshah, as they brought me up when I was little. Then, my sincere thanks to my wife, Ebtesam, and to my two sons, Heatham and Heasham, for their unconditional love, care, support and patience. Also I would like to express my deep appreciation to my thesis supervisor, Dr. Albers, who helped me a lot in the lab and my writing of this thesis. Also special thanks to the respected committee: Dr. Ahmed Mehanna, Dr. Mattia Migliore, and to the Associate Dean of Graduate Studies Dr. Timothy J. Maher. Lastly, I would like to thank Dr. Anne Roberti and my fellow graduate students for their help and support in editing my thesis. March 2015 Mohammad H. Almermesh ii

Table of Contents Title Pages ABSTRACT... I ACKNOWLEDGEMENTS... II TABLE OF CONTENTS... III LIST OF FIGURES... V LIST OF ABBREVIATIONS... VI INTRODUCTION... 1 TYPE3 DIABETES MELLITUS... 4 TYPE 3 DIABETES AND GLUCOSE HOMEOSTASIS... 4 TYPE 3 DIABETES AND ENERGY METABOLISM... 7 Mitochondrial Dysfunction... 7 Oxidative Stress... 9 SHORT-TERM AND LONG-TERM COMPLICATIONS... 12 ALZHEIMER S DISEASE... 13 MECHANISMS OF PATHOLOGY... 14 TYPE 3 DIABETES AND COMMON ALZHEIMER S DISEASE - CELLULAR MECHANISMS LINKING... 16 IMPAIRED GLUCOSE METABOLISM IN AGING... 16 SHOULD ALZHEIMER S DISEASE BE CONSIDERED BRAIN DIABETES?... 19 BRAIN INSULIN RESISTANCE... 22 Brain Insulin Dysfunction and Impaired Insulin Signaling... 22 PUTATIVE MECHANISMS OF PATHOGENESIS... 24 Type 3 Diabetes and amyloid-beta toxicity... 24 Impaired Kinase activities and Tau Pathology Hypothesis... 26 Advanced Glycation End-Product and Amyloid-beta Neurotoxicity Hypothesis... 31 MOLECULAR MECHANISM OF INSULIN RECEPTOR SUBSTRATE-1INHIBITION... 33 iii

POTENTIAL TYPE 2 DIABETES AND ALZHEIMER S DISEASE LINK... 35 PERIPHERAL INSULIN RESISTANCE DISEASE STATES... 35 PERIPHERAL INSULIN RESISTANCE AND TYPE 2 DIABETES AS COFACTORS OF COGNITIVE IMPAIRMENT AND NEURODEGENERATION... 37 PERIPHERAL INSULIN RESISTANCE AND TYPE2 DIABETES AS CONTRIBUTOR FACTORS OF COGNITIVE IMPAIRMENT AND NEURODEGENERATION... 38 Vascular factors... 38 Peripheral Ceramides... 39 INFLAMMATORY AND PRO-INFLAMMATORY CAUSE OF PERIPHERAL INSULIN RESISTANCE... 41 PUBLIC HEALTH ISSUES... 43 POSSIBLE BIOMARKERS... 48 BRAIN GLUCOSE HYPOMETABOLISM... 48 ALTERED THIAMINE METABOLISM... 49 INSULIN SUBSRATE-1... 50 INSULIN DEGRADING ENZYME... 50 INSULIN-RELATED INTERVENTIONS... 51 INSULIN RELATED ANTIDIABETICS... 51 STEM CELLS TECHNOLOGY... 55 REFERENCES... 58 iv

List of Figures Figure 1. Effects of energy failure leading to excitotoxicity and oxidative stress.... 11 Figure 2. The diagram of multiple pathogenic cascades in Alzheimer s disease.... 15 Figure 3. Brain insulin resistance and A PP-A deposition and toxicity from.... 19 Figure 4. Scheme of the pathway mediating insulin-induced inactivation of GSK3... 27 Figure 5. GSK3B is associated with neuropathological mechanisms involved in Alzheimer s disease... 29 Figure 6. Common pathological processes in Alzheimer s disease (AD) and diabetes.... 32 Figure 7. Neuronal insulin signaling pathways... 33 Figure 8. The liver-brain axis of neurodegeneration in type 2 diabetes mellitus... 36 Figure 9 Insulin dysfunction might enhance Alzheimer s disease pathology through distinct mechanisms... 40 Figure 10. T2DM, brain insulin resistance, and AD have been linked in a number of different epidemiological, clinical, and animal studies.... 43 Figure 11. All diabetes drugs are likely to have indirect effects in the CNS by affecting circulating concentrations of glucose and insulin.... 52 v

LIST OF ABBREVIATIONS AD - Alzheimer s Disease ADDL - Aβ-Derived Diffusible Ligands ADOA - Autosomal Dominant Optic Atrophy AIDS - Acquired Immune Deficiency Syndrome Akt - Serine/Threonine-Specific Protein Kinase ALS - Amytrophic Lateral Sclerosis. APO -1 - Apoptosis Antigen 1 APOE 4 - Apolipoprotein E 4 Allele APP - Amyloid Precursor Protein ATP - Adenosine Triphosphate Aβ - Amyloid Beta AβPP - Amyloid-Beta Protein Precursor BBB - Blood-Brain Barrier C-Ab1 - Abl Protein Tyrosine Kinases CAP - Cbl-Associated Protein Cbl - Casitas b Lineage Lymphoma CD95 - Cluster of Differentiation 95 CDC42 - Cell Division Control Protein 42 CNS - Central Nervous System CREB - camp Response Element-Binding Protein vi

CSF - Cerebrospinal Fluid DM - Diabetes Mellitus DNA - Deoxyribonucleic Acid ER - Endoplasmic Reticulum ERK - Extracellular-Signal-Regulated Kinase FADD - Fas-Associated Protein With Death Domain FAS - Fas Cell Surface Death Receptor FBS - Fetal Bovine Serum GCK - Glucokinase GDM - Gestational Diabetes Mellitus GLUT - Glucose Transporter GLUT1 - Glucose Transporter 1 GLUT4 - Glucose Transporter Type 4 GSH - Glutathione GSK-3β - Glycogen Synthase Kinase-3-beta HD - Huntington Disease hesc - Embryonic Stem Cell HIV - Human Immunodeficiency Virus HNF4 - Hepatic Nuclear Factor 4 IDE Insulin Degrading Enzyme IGF - Insulin-like Growth Factor vii

IGFBP-1, 2, 3 - Insulin-like Growth Factor Binding Protein 1, 2, 3 IKK - IkBα Kinase INT - Iodophenyl Nitrophenyl Phenyl Tetrazolium Chloride IPF -1 - Insulin Promoter Factor 1 ipsc - Somatic Cells Induced Pluripotent Stem Cell IR - Insulin Receptors IRS - Insulin Receptor Substrate IPF-1 - Insulin Promoter Factor 1 IκBα - Nuclear Factor of Kappa Light Polypeptide Gene Enhancer In B-Cells Inhibitor, Alpha JNK - C-Jun N-terminal kinase LDH Lactate Dehydrogenase L-DOPA - L-Dihydroxyphenylalanine LHON - Leber s Hereditary Optic Neuropathy MAPK - Mitogen-Activated Protein Kinase MitDNA - Mitochondrial DNA MMP - Mitochondrial Membrane Permeability MODY - Maturity Onset Diabetes in the Young MPTP - 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydro-Pyridine NADH - Nicotinamide Adenine Dinucleotide NADPH - Nicotinamide Adenine Dinucleotide Phosphate ND4 - NADH Dehydrogenase Subunit 4 viii

NEUROD1 - Neurogenic Differentiation 1 NFT - Neurofibrillary Tangle NMDA - N-Methyl-D-aspartic Acid or N-Methyl-D-Aspartate NO. - Nitric Oxide -O2 - Superoxide OH. - Hydroxyl Ion OPA1 - Optic Atrophy 1 (autosomal dominant) PD - Parkinson s Disease PET - Positron Emission Tomography PI3K - Phosphatidylinositol-4, 5-Bisphosphate 3-Kinase PINK1 - PTEN-Induced Putative Kinase 1 PKR - Protein Kinase R PS1 - Presenilin-1 PSP - Progressive Supranuclear Palsy RIiP - Receptor Interacting Protein ROS - Reactive Oxygen Species SDAT - Sporadic Alzheimer s Disease SMAC/DIABLO - Diablo Homologue Mitochondrial Protein SNpc - Substantia Nigra Pars Compacta SOD - Superoxide Dismutase T1DM - Type 1 Diabetes Mellitus ix

T2DM - Type 2 Diabetes Mellitus T3DM - Type 3 Diabetes Mellitus TCA - Tricarboxylic Acid TNF - Tumor Necrosis Factor TNFR - Tumor Necrosis Factor Receptor TNF-α - Tumor Necrosis Factor Alpha TRADD - Tumor Necrosis Factor Receptor Type 1-Associated Death Domain Protein UPS - Ubiquitin Proteasome System Δψm - Mitochondrial Membrane Potential x

Introduction Diabetes mellitus (DM) is a complex, chronic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. 1 The chronic elevation of systemic sugar is associated with long-term problems including the progressive development of the specific complications of retinopathy with potential blindness, nephropathy that may lead to renal failure, and neuropathy with risk of foot ulcers, and amputation. 1 DM is a modern epidemic and a highly concerning global health care issue. 2 It is the most prevalent chronic endocrine disease, affecting an estimated 5% to 10% of the adult population in Western countries, Asia, Africa, Central America and South America. Additionally, the worldwide estimation of DM, in 2000, was 2.8% (171 million) as compared to the future estimation, in 2030, which would be 4.4% (366 million). 1 In addition, in 2012, the prevalence of DM for all age-groups in the US was estimated to be 29.1 million or 9.3% of the population had diabetes. 3 In 2010, undiagnosed people were 8.1 million cases out of the total population. 3 In the same context, the prevalence of seniors aged 65 years old or older was 11.8 million or 25% of either diagnosed or undiagnosed cases. 3 On the other hand, the new cases in 2012 were 1.7 million per a year. 3 Additionally, the pre-diabetic cases, in 2012, were 86 million Americans aged 20 or older. 3 In terms of mortality rate, DM remains the 7th leading cause of death in the United States; for example, in 2010, the death certificates with DM as the underlying cause of death were as high as 96,071. 3 Interestingly, the prevalence of DM is higher among men than women. The urban population of DM patients in developing countries may be doubled between 2000 and 2030. Additionally, people aged 65 years old tend to be at high risk for getting DM during their lifetime. 1 1

DM may be classified into four clinical categories: type1, type 2, type 3, and type 4. Type1 diabetes (T1DM) is mainly due to β-cell destruction, mostly leading to absolute insulin deficiency. The T1DM is known to affect cognitive functions in patients. It has later onset and is seen more in adults. It is due to autoimmune destruction of pancreatic β-cells leading to decrease or no production of insulin. Insulin represents the mainstay therapy with hypoglycemia as a major side effect representing 90% of all insulin treated patients. 4 However, the type2 diabetes (T2DM) is due to a progression of insulin secretary defect concomitantly with insulin resistance. It is mostly associated with aging. The pancreas can produce insulin, but cells are unable to use it. The early phase of T2DM is associated with overproduction of insulin to overcome insulin resistance. This leads to hyperinsulinaemia, and thus, in the longrun, the pancreas fails to produce enough insulin causing hyperglycemia and insulin deficiency as the main features of T2DM. 4 In addition, gestational diabetes mellitus (GDM) which sometimes is referred to as type 4 diabetes, is diagnosed during pregnancy and is not clearly overt diabetes. Also GDM is pregnancy associated (during the second or third trimester of pregnancy) and caused by insulin deficiency and hyperglycemia. 5 High systemic sugar levels affect both mother and fetus and necessitate treatment to overcome these complications. Regular physical activity with a suitable diet (behavioral therapy) or insulin are the possible treatment options that the pregnant women may have. Unfortunately, 5% to 10 % of women GDM may continue to have hyperglycemia post pregnancy. 3 These women may also be diagnosed usually with T2DM. Therefore, GDM is a risk factor for developing DM or T2DM or multiple incidents of GDM. Additionally, the children who were born of a mother with GDM may be at risk of developing obesity and DM in their future lives. 3 2

Moreover, established risk factors for GDM are advanced maternal age, obesity, and family history of DM (i.e. previous GDM). Therefore, the frequency of GDM usually reflects the frequency of T2DM in the underlying population. 6 Recent data have shown that GDM prevalence has increased by 10%-100% in several race/ ethnic groups during the past 20 years. 7 For example, in the US, Native Americans, Asians, Hispanics, and African-American women are at a higher risk for GDM. 8 In addition, a true increase in the prevalence of GDM might contribute to the continuous pattern of increasing DM and obesity. Therefore, coordinated efforts are necessary to change the increasing course of GDM and to prevent chronic DM in GDM patients and their children. 7 However, there is another pregnancy-associated diabetes, which is gestational diabetes insipidus. It is a rare complication of pregnancy characterized by development of symptoms over a few days. These symptoms include late onset of skin dehydration, weight loss, nausea, fatigue, and excessive thirst and urination. This complication occurs in 4 out of 100,000 pregnancies. 9 In addition, this complication is originated from increased degradation of arginine vasopressin (AVP) via an enzyme is called vasopressinase in the placenta. Additionally, vasopressinase is a cysteine aminopeptidase of molecular weight 330 kda. This enzyme is produced by placental trophoblasts during pregnancy. 10 Furthermore, this enzyme can be detected early during the 7 th week of pregnancy, peaking within 40 th week. However, it is undetectable beyond after the delivery of baby by 6 weeks. 11 Moreover, oral desmopressin is considered the drug of choice in patients with gestational diabetes insipidus. 12 Furthermore, a recently discovered form, type3 diabetes mellitus (T3DM), is a term recently used by scientists. These scientists have tried to define it as a metabolic syndrome that may lead to abnormalities linked to progressive brain insulin resistance with consequent 3

impairment of central insulin signaling processes, accumulation of neurotoxins, neuronal stress, and resulting in a course of neurodegeneration. 13 On the other hand, there are other specific types of DM that also have been verified and they seem to be due to other causes such as genetic defects in β-cell function, for example, maturity onset diabetes in the young (MODY). Moreover, genetic studies have defined a number of subtypes of MODY (i.e., MODY1-6). The cause of these six forms of MODY are as follow: mutations in the genes encoding hepatic nuclear factor 4 (HNF4), glucokinase (GCK), hepatic nuclear factor 1 alpha and 1 beta, insulin promoter factor 1 (IPF-1), and neurogenic differentiation 1 (NEUROD1). 14 Additional specific types of DM are attributed to genetic defects in insulin action and diseases of the exocrine pancreas such as cystic fibrosis, and drug- or chemical-induced diabetes; for instance, in the treatment of HIV/AIDS or after organ transplantation. 15 Type3 Diabetes Mellitus Type 3 Diabetes and Glucose Homeostasis There are two distinct features of adult neurons that make them vulnerable to either neuronal cell death (apoptosis) or a diseased state such as neurodegeneration or neuronal loss. The first feature is that fully differentiated (adult) neurons are permanently post-mitotic cells, which lack regenerative ability. 16 Therefore, when adult neurons are exposed to any cellular stresses such as lack of adenosine triphosphate (ATP) moieties or energy crisis, and/or oxidative stress, they either die or experience apoptosis, degenerate or cause neuronal degeneration and loss, and thus predispose neurodegenerative diseases. 16 The second important feature is that brain neurons or 4

tissues are highly demanding excitable cells, in which more than 40 % of the present ATP is used to keep neurons viable or alive. 17 The adult brain is only 2% of the total body weight, but it contains the most predominant energy metabolism in the human body. 18 Additionally, the glucose metabolism dysfunction increases the risk of cognitive impairment in the elderly. 18 This has been based on two assumptions. First, the brain is a high energy demanding and consuming organ. Second, glucose is the main source for energy. Therefore, this makes the brain vulnerable to impaired energy metabolism or energy crisis. 18 In general, the brain is fueled by glucose as a main source of energy. There are two sources of brain glucose that involve cortical glucose metabolism stimulation through basal insulin levels 19 and astrocytic glycogen conversion to glucose that is stimulated by the activation of glial β- adrenoceptors 19 (via insulin-mediated inhibition of neuronal norepinephrine uptake). The increase in glucose uptake is transported by insulin-sensitive glial glucose transporter type 1 (GLUT1) to the plasma membrane for neuronal use. 19 Therefore, the balanced cellular glucose transportation depends on astrocytes and glucose transporters (GLUTs) that expressed in the brain. 18 However, ketones may be considered an alternative source of brain energy in the case of impaired glucose metabolism that might take place in the brain. 18 In addition, cellular glucose metabolism consists of two processes including glucose transportation and intracellular glucose metabolism. The glucose transport is controlled by the insulin signaling pathway. Also the glucose transport plays a key role in regulating glucose transmembrane transportation. 20 Therefore, the crosstalk between central nervous system (CNS) insulin levels/ action and glucose metabolism is very important to be controlled to avoid any deleterious changes that might affect energy production and neuronal survival. 21 Moreover, a glucose 5

homeostasis defect might be important in the pathogenesis of T3DM due to impaired glucose uptake as a result of impaired glucose metabolism in the brain. The mechanisms that are involved in glucose transportation abnormalities include brain insulin resistance and intracellular glucose metabolic disturbance. These two abnormalities possibly may contribute to cerebral glucose hypometabolism in T3DM or the brain insulin resistance disease state. According to Liu Ying, decreased glucose transporters correlated to abnormal hyperphosphorylation of tau in neurodegenerative diseases. 22 Therefore, impairment of insulin signaling not only affects systemic glucose blood levels but also causes various degenerative processes or neuronal cell death or loss. 23 In addition, T3DM is characterized by insulin resistance in the brain tissues. This kind of resistance directly decreases insulin responsiveness and action through decreasing the gene expression of both insulin (as a polypeptide) and its corresponding receptors insulin receptors (IRs). Consequently, this leads to insulin deficiency and impaired glucose transport inside the neurons due decreased number of expressed GLUTs in the cell membrane. Furthermore, insulin resistance in the CNS correlates with insulin resistance in the periphery. Therefore, loss of responsiveness to insulin could make neurons more susceptible to neurotoxic insults due to their being devoid of protective effect of insulin. 24 Also insulin-resistant patients have many increased pathologic features such as apoptosis, neurodegeneration, and the resultant decline in cognition. 24 The desensitization of the neuronal insulin receptor in brain insulin resistance, similar to the process in T2DM, may play a key role in causing T3DM and its future complications 25. This phenomenon or abnormality a long with a decrease in brain insulin levels is known to induce 6

various disturbances including cellular glucose, acetylcholine, cholesterol, and ATP associated with abnormalities in membrane pathology and the formation of both amyloidgenic derivatives and hyperphosphorylated tau protein. 25 In the same context, hyperglycemia and hypoglycemia (i.e., T2DM) have a heavy impact on human brain health, especially cognitive functions. Besides, T2DM is a metabolic syndrome characterized by insulin resistance, which is also a pathological feature of neurodegeneration or neuro-endocrine disorder or T3DM. 20 Thus, glucose homeostasis plays a role in T3DM pathogenesis. Brain glucose uptake or metabolism is impaired in T3DM. Therefore, the combination of T2DM and neurodegenerative brain diseases may be considered, nowadays, as this new classification of diabetes, called T3DM or a neuro-endocrine disorder. Type 3 Diabetes and Energy Metabolism Mitochondrial Dysfunction The T3DM is associated with brain insulin resistance and insulin deficiency. Insulin deficiency can cause abnormal expression of genes such as insulin polypeptide and its relative receptors, and activation of kinases (e.g. GSK-3β). However, brain insulin resistance may impair energy metabolism leading to oxidative stress, the generation of reactive oxygen species (ROS), deoxyribonucleic acid (DNA) damage, mitochondrial dysfunction. These cellular abnormalities drive pro-apoptosis, pro-inflammatory, and pro-amyloid-beta (Aβ) cascades. 26 7

Genetic and environmental factors may affect both insulin sensitivity and mitochondrial function. Mitochondrial function is associated with insulin resistance in skeletal muscle and other tissues such as the liver, heart, fat, vessels and pancreas. Insulin resistance is responsible for common pathophysiologic etiology of chronic diseases. 27 The possible mechanism of mitochondrial dysfunction may be triggered by high levels of loaded free fatty acids (FFAs) or hyperglycemia. 28 As a result ROS increases and mitochondrial biogenesis goes down, causing mitochondrial dysfunction. Consequently, a reduction in β- oxidation and ATP production and a buildup of high ROS levels take place. This leads to many metabolic diseases such as insulin resistance, DM and cardiovascular diseases. 28 Thus, peripheral insulin resistance causes insulin deficiency in the whole body including the brain. This lack of brain insulin sensitivity or availability is responsible for the reduction of insulin polypeptide and gene expression in the brain and cerebrospinal fluid (CSF), and thus causing brain insulin resistance or T3DM. As the insulin/insulin-like growth factor (IGF) signaling is down, mitochondrial dysfunction prevails and worsens or disrupts the electron transport chain (ETC) functionality that ends with an accumulation of ROS and a reduction in the ATP species. 26 In addition, brain insulin resistance may lead to an impaired energy metabolism and this could further cause oxidative stress, generation of ROS, DNA damage and mitochondrial dysfunction. 18 In addition, glucose transportation abnormalities are due to insulin resistance and a deregulated intracellular metabolism. Besides, the glucose transport problem is due to mitochondrial dysfunction that occurs in neurodegeneration or neuronal cell death. 18 Additionally, glucose metabolism dysfunction increases the risk of cognitive impairment in the elderly. 18 The disturbance of signal transduction and enzymatic pathway required for energy metabolism and homeostasis and neuronal survival is attributed to abnormal structural and 8

functional neurons. This is triggered initially by the inhibition of the insulin/igf signaling that causes an increased oxidative stress, questionable mitochondrial dysfunction, and possible activation of pro-inflammatory cytokines. 18 Normally, the neuro-inflammatory responses are triggered by abnormal astrocytes and microglia through the activation of pro-inflammatory cytokines. These neuronal responses are characterized by an elevation of oxidative stress, increased organelle dysfunction and the activation of pro-apoptosis cascades. 26 As oxidative stress progresses, activation of the Glycogen synthase kinase-3-beta (GSK-3β) signaling predominates and increases phosphorylation of tau in the brain. This eventually leads to an increase in Aβ deposition and toxic fibril formation that ends with amyloid-β precursor protein (AβPP) toxicity, tau cytoskeletal pathology and neuroinflammation. Therefore, the role of the insulin/igf cascade activation is to reduce oxidative stress, protect organelles and suppress pro-apoptosis signaling. 26, 29 In addition, it can also inhibit AβPP gene expression and halt AB toxicity. Thus, trophic factor signaling is critical for the suppression of the neuroinflammatory responses as well as a reduction in Aβneurotoxicity. 26 Oxidative Stress Deregulated glucose metabolism has a role in neurodegeneration and neuronal loss, usually accompanied by T3DM, along with multiple pathogenic factors including oxidative stress and mitochondrial dysfunction. 18 Oxidative stress is an imbalance in free radical formation within a cell or an organism, most commonly in the form of ROS and/or reactive nitrogen species (RNS). These free radical species, such as superoxide anions, hydroxyl radicals, hydrogen peroxide, nitric 9

oxide, and peroxynitrite, come from many sources including impaired glucose metabolism, immune activation, ultra violet (UV) radiation, heme accumulation, and hypoxia. 30 In addition, failure of innate antioxidant mechanisms to compensate for accumulating insults such as mitochondrial dysfunction, DNA damage, misfolded proteins, and lipid peroxidation can trigger programmed cell death pathways. This has been linked to clinically relevant diseases including, diabetes, neurodegenerative disease, cardiovascular disease, asthma, and others. Furthermore, abnormal oxidative stress promotes the buildup of ROS and RNS. These harmful species may start to attack cellular macromolecules or components and organelles. They interact with DNA, ribonucleic acid (RNA), lipids and protein by forming adducts that may lead eventually to structural and functional abnormalities of the neurons. 31 In addition, there are other complications that originate from these non-functioning neurons; for example, a reduction in neurotransmitter function and neuronal plasticity, the loss of cell membrane functions, and an abnormal cytoskeleton with dystrophy and synaptic disconnection. 26 Moreover, mitochondria are major sites of ROS production, because ROS represent about 0.2% to 2% of the total oxygen taken by the cells. 28 These mitochondrial sites are mostly in complex-1(nadh CoQ reductase) and complex-3 (bc1 complex) regions. These complexes produce excess electrons that are provided to a mitochondrial respiratory chain and interact with oxygen species and yield superoxide free radicals. 28 These free radicals are normally neutralized by superoxide dismutase (SOD) to form hydrogen peroxide moieties. However, in the case of mitochondrial dysfunction or disease state, the proton gradient increases, at the same time, oxygen levels decrease causing high ROS levels that further cause cellular damage and mitochondrial dysfunction and this process becomes a vicious cycle. 27 10

On the other hand, the oxidative phosphorylation mechanism comes as a result of an important interaction between mitochondrial energy metabolism and oxidative damage. This system produces most of free radical in the cell. 32 Free radicals are augmented by the deactivation of the electron transport chain. 32 This mechanism involves uncoupling electron transfer which increases ROS. Free radicals are uncharged molecules (typically highly reactive and short-lived), having an unpaired valence electron, that gain electrons from adjacent molecules in order to be stable in the outer shield of their orbitals. 33 They oxidize cellular contents such as proteins, lipids and nucleic acids, leading to cellular damage and cell apoptosis or death. In addition, these free radicals include hydroxyl ion (OH.), superoxide (-O2) and nitric oxide (NO.) 33. Upon catalyzation of hydrogen peroxide via transitional metal ion such as iron, copper or zinc, the most reactive ROSs hydroxyl ion, is formed. This harmful ion interacts with cellular proteins, lipids and nucleic acids and causes damage to the sensitive mitochondrial DNA (mitdna), leading to oxidative stress and mitochondrial dysfunction 33. As a result, energy failure will exist, and thus neuronal loss 33 (Fig.2). Figure 1. Effects of energy failure leading to excitotoxicity and oxidative stress. 33 11

Short-Term and Long-Term Complications Central insulin resistance or brain insulin resistance, T3DM, is quite similar to T2DM in terms of its short- or long-term complications. Additionally, both T2DM and impaired fasting glucose were clearer in Alzheimer s disease (AD) patients than in non-ad individuals. 2 The early abnormalities, seen in brain insulin resistance, are associated with impaired cognitive function including memory loss, learning difficulties and dementia. 13 The DM and age-related neurodegeneration diseases (e.g., AD) usually compromise the trophic factor signaling in the brain 34. This suppressed pathway or attenuated trophic signaling occurs due to low insulin/insulinlike growth factor (IGF-1) brain concentrations and impaired receptors of insulin and IGF-1. 34 This affects gene expression leading to neurodegeneration/death, cognitive impairment, and long-term complications such as AD or other dementia. 34 Neuronal aging is characterized by decreased mitochondrial function and increased ROS production, which are associated with cognitive decline. Accelerated cognitive decline is associated with long-term complications. 13,35 These complications are due to the multifactorial factors including circulatory and metabolic considerations and recurrent hypoglycemia. 35 In addition, T2DM is a risk factor for dementia (both vascular dementia and AD), because it negatively impacts the brain structures and functions. 2 In general, most diabetic complications can be found in T3DM, but with increased severity. One of the most pronounced diabetic complication is diabetic retinopathy that is characterized by increased expression of monocyte chemoattractant protein 1 (MCP-1). 36 It has been found in vitreous humor samples of patients with retinal detachment (RD) and diabetic retinopathy. Also it has been found in the brain tissues of patients 12

with neurodegenerative diseases, including AD and multiple sclerosis. 37 A role for Aβ42 and tau in the pathogenesis of diabetic retinopathy is confirmed. 36 According to the Rotterdam study that used mini-mental state scores in an elderly population, a possible link exists between serum insulin levels and cognitive function. The researchers observed that raised insulin levels, as seen in response to insulin resistance, was associated with decreased cognitive function and dementia in women that is independent of other cardiovascular risk factors. 38 Alzheimer s disease AD is asymptomatic progressive multifactorial neurodegenerative disease. It involves abnormalities of molecular, biochemical and cellular dysfunctions. It usually leads to neuronal loss 4, 39 and cell death. Therefore it is called a neurodegenerative disease. AD is considered to be the most common neurodegenerative disease, which accounts for around 80% of dementia globally in elder patients. AD leads to progressive loss of mental, behavioral, and functional deterioration and the ability to learn. 40 The total estimated prevalence of AD is expected to be 13.8 million in 2050 41. Also it is expected, in 2050, there will be a million new cases per a year. Around 200,000 people younger than 65 years with AD represent the younger onset AD population 40. An estimated 5.2 million Americans of all ages of the US population had AD in 2014. 41 13

In aging developed countries, AD has become epidemic as it is an age-related disease. Dementia syndrome is associated with the elderly and represents a public health crisis. AD accounts for about 55 70% of adult - onset dementia in the industrialized world 42, and it is the fifth leading cause of death in Americans older than 65. 43 Death due to AD is mostly caused by infection or cardiopulmonary arrest. AD is a costly disease in the US ($100 billion annually). 44 AD is characterized by the lack of acetylcholine, which is a neurotransmitter in both the peripheral nervous system (PNS) and CNS. It is characterized by the presence of extracellular amyloid plaques deposits of Aβ and neurofibrillary tangles (NFTs) of tau in the AD brains 45, and thus synaptic and neuronal cell death. Additionally, insulin signaling dysregulation might contribute to AD risk by increasing Aβ aggregation and tau phosphorylation, and, consequently, NFT formation. 46 Moreover, hyperglycemia has been linked with poorer cognition, but not with the development of AD. 47 According to The Prospective Study of Pravastatin in the Elderly at Risk study (PROSPER), elderly patients with DM without dementia have accelerated progression of brain atrophy with significant consequences in cognition compared to subjects without DM. 39 Mechanisms of Pathology There are many contributing factors that may lead to the predisposition of AD. Neuronal cell death is the main cause of AD. There are different AD pathogenic mechanisms such as the beta-amyloid hypothesis, and the tau hyperphosphorylation hypothesis (Fig.2) However, the exact etiology is still unknown. 14

Figure 2. The diagram of multiple pathogenic cascades in Alzheimer s disease. 18 One of these mechanisms is the accumulation of Aβ (hard plaques) inside the neurons that may interfere with the ability of acetylcholine to effect synaptic transmission and initiate inflammatory processes. 48 As a result, ROS builds up forming free radicals that attack the cell membrane, mitochondria, lipids and proteins, causing neuronal cell apoptosis. 48 Also the Aβ affects directly the generation of ROS. Some studies show that Aβ can activate the calcium channels in cell membranes that enhance calcium trafficking leading to accumulation of calcium concentrations. 49,50 High intracellular calcium concentration may cause damage to the cell contents including mitochondrial dysfunction, inflammation and cell death or apoptosis. 49 Another mechanism that might result in AD is a chemical change in the tau protein. 51 This distortion of tau may cause microtubules paired with other tubules forming NFTs. 51 Consequently, 15

this may lead to the disintegration of tubules and blockage of neurotransmitters, and thus neuronal cell death. 51 The other possible mechanism is centered on glutamate cytotoxicity. 52 This toxicity is triggered by overstimulation of N-Methyl-D-aspartate (NMDR) receptors, in the neurons, causing calcium to penetrate neuronal cells. This causes persistent depolarization of the post-synaptic neurons resulting in formation of ROS, and thus cell apoptosis. 52 Type 3 Diabetes and Common Alzheimer s disease - Cellular Mechanisms Linking Impaired Glucose Metabolism in Aging Aging is defined as a gradual buildup of varied changes in the cells and tissues that hamper the overall function of the body. 53 It is an inevitable process witnessed in all life forms that is characterized by a gradual loss of physiological functions that sets the entire metabolic system to become disease prone and susceptible to death. 54 Nine major signs of aging have been reported including telomere erosion, epigenetic alterations, genomic instability, exhaustion of stem cells, cellular senescence, damaged proteostasis, dysfunctional intercellular communication and altered nutrient sensing along with mitochondrial function. 54 This plethora of changes that aging accompanies sets the stage for many diseases including neurodegenerative disorders. 55 In addition, postmortem brain studies have shown that there is a non-significant reduction in glucose transporter type 4 (GLUT4) expression in AD. 26 Additionally, deficits of the brain glucose utilization and energy are due to impaired GLUT4 trafficking between cytosol and the 16

plasma membrane. 26 However, impairments in cerebral glucose utilization and energy metabolism represent very early abnormalities that precede the initial phases of cognitive impairment. 26 Furthermore, glucose transportation is important for normal neuronal metabolism and energy that help maintain intact memory and cognition. 26 Decreased cerebral glucose utilization and reduced energy metabolism represent an early course of AD, prior to or with initial cognitive 26, 31 dysfunction, supporting the role of impaired insulin signaling in predisposing AD. However, T3DM is associated with endothelial cell dysfunction, which plays a key role in the development and progression of cardiovascular and other diseases. 56 In the same context, some researchers have pointed out that heart disease increases a patient s odds of developing AD. 57 They found that artery stiffness, a condition called atherosclerosis, is associated with the buildup of Aβ plaque in the brain, a hallmark of AD. Therefore, the process of vascular aging may predispose the brain to increased amyloid plaque buildup. Thus, plaque builds up with age and appears to worsen in those with stiffer arteries. 57 In the same context, aging tends to increase the risk of deteriorating systemic control of glucose utilization. Thus, it may increase the risk of declining brain glucose uptake in some brain regions 58. In addition, the glucose uptake process depends on glucose transport that is facilitated by the expression of GLUT4. 18 This beneficial expression is normally stimulated by insulin neuronal peptide that also controls the uptake and utilization of glucose. 18 This can be accomplished by rectifying the glucose trafficking from the cytosol to the plasma membrane through activated GLUT4 expression. 59, 60, 61 This has a positive impact on neuronal metabolism and energy that plays a key role in memory and cognition. As a person approaches old age, mitochondrial mutations and disrupted oxidative phosphorylation results in excessive production of ROS; these changes then set the stage for 17

myriad diseases related to aging 62. Thus, oxidative stress caused majorly by ROS marks the onset of many diseases, during the aging phase of one s life span. 63 In addition, T2DM is the most common chronic metabolic disorder in the elderly. 64 The possible mechanisms that are responsible for age-related glucose intolerance include reduced insulin sensitivity and reduced β-cell function. 64 Decreased β-cell function may be related to the toxic effects of excessive levels of glucose in the blood and mitochondrial dysfunction. Insulin resistance is related to mitochondrial dysfunction and increased intracellular muscle cell triglyceride. 64 Furthermore, the relationship between the brain glucose metabolism and aging in AD is characterized by a low bran glucose metabolism prior to the onset of a clinically measurable cognitive decline. 58, 65 This has been noticed in two groups of patients that include apolipoprotein E 4 allele (APOE-4) carriers and that have a maternal family history of AD. 58 This is supported by in vitro animal studies that have suggested that brain metabolism may precede, and thus contribute to a neuropathologic cascade causing cognitive deterioration in AD. 58 This may be attributed to defects in brain glucose transport, impaired glycolysis and mitochondrial dysfunction. (Fig.3) 18

Figure 3. Brain insulin resistance and A PP-A deposition and toxicity. 66 Should Alzheimer s Disease Be Considered Brain Diabetes? According to de la Monte et al., the term, T3DM, has been coined to account for the underlying abnormalities associated with AD-type neurodegeneration. 5 This is because the brain insulin resistance and deficiency play a critical role in affecting the AD brain. Therefore, it corresponds to a chronic insulin resistance and insulin deficiency state that is largely confined to the brain, but can overlap with T2DM. 5 In addition, the AD molecular and biochemical abnormalities superimpose those seen in T1DM and T2DM. 5 Thus, the research has supported that insulin/igf resistance may be considered critical to the progression of AD. Therefore, it has been proposed that T3DM represents a major pathogenic mechanism of AD neurodegeneration. 5 19

In the same context, Steen et al. has suggested that AD may represent a neuro-endocrine disorder that somewhat mimics diabetes. 67 Therefore, the scientist has proposed the term, T3DM, to reflect a new pathogenic mechanism of neurodegeneration. 67 In T3DM, unlike both T1DM and T2DM, the low insulin levels are confined to the brain only, which is due to decreased insulin responsiveness leading to abnormal brain function. However, in T2DM or T1DM, there is no enough insulin or none at all is produced to process glucose correctly or the body no longer responds to insulin that affects the functioning of the whole body. A Brown Medical School research team identified the possibility of a new form of DM in the brain after they found that insulin is produced by the brain as well as the pancreas. 5 Furthermore, AD is a kind of metabolic disease mediated by compromised brain insulin responsiveness, glucose utilization, and energy metabolism, leading to increased oxidative stress, inflammation, and magnified insulin resistance. 5, 68, 69 The reevaluation of the literature revealed that disarrangements in cerebral glucose utilization and energy metabolism resemble very early abnormalities that exist before cognitive impairment induction. This understanding has led to the concept that impaired insulin signaling has an important role in the predisposition of AD and the proposal that AD superimposes, T3DM. 5 On the other hand, some scientists at the University of Pennsylvania have found a clue that brain insulin resistance is linked to reduced central insulin and IGF-1 responsiveness as an early characteristic in AD and cognitive decline. 70 However, they prefer the term insulin-resistant insulin state over the term, T3DM. The reason is that neither hyperglycemia nor classical DM are consistently found in AD, but still there are some features of metabolic syndrome that represent up to two-thirds of AD cases. 70 20

Thus, brain insulin resistance seems to be an early and common problem in AD. 71 This phenomenon is accompanied by IGF-1 resistance that is linked to insulin receptor substrate 1 (IRS- 1) dysfunction. The IRS-1 abnormality is caused by Aβ oligomers and, at the same time, can promote cognitive decline that is independent of pure AD pathology. 71 All these points taken together, T3DM is quite similar to AD based on the fact that the AD brain is characterized by a scarcity of insulin and the resistance of its receptors that result in cognitive impairment, as it is critical for neurological signaling processes to commence. Recently, there is a growing body of evidence that considers AD as a brain diabetes. This is because AD in its basic form is a brain form of diabetes. 26 The proponents of this opinion have based this concept on the assumption that AD is associated with progressive brain insulin resistance in the absence of T2DM, obesity, or peripheral insulin resistance. 26 In addition, the postmortem studies have shown that different abnormalities (e.g., molecular, biochemical and signaling transduction) in AD are similar to those that occur in both T1DM and T2DM. 26 Furthermore, Steen et al. has studied postmortem brain tissues and found that there was a reduced CNS expression of genes encoding insulin, IGF-1, and IGF-2 and their corresponding receptors. 67 The strongest evidence has been shown in experimental animal studies. In these studies rats were injected by a pro-diabetic drug (Streptozotocin; STZ). These rats developed cognitive impairment with deficits in spatial learning and memory, brain insulin resistance and insulin deficiency and AD-type neurodegeneration, but not DM. 67 However, intraperitoneal (IP) or intravenous (IV) STZ injection causes DM with mild degrees of fatty liver and neurodegeneration. 26 These studies imply that when animals are exposed to a single pro-diabetic drug, their organs or tissues are prone to degeneration that is characterized by insulin signaling impairment, and energy metabolism impairment and accompanied by an 21

elevation in oxidative stress, mitochondrial dysfunction and cell apoptosis. 26 Therefore, the severity of the disease is controlled by dose and route of drug administration. 26 In the same direction, there is a new hypothesis that supports the concept that AD is the brain-type-diabetes. 34 This hypothesis is based on the followings: a decreased number or binding capacity of brain IRs in both AD patients and T2DM mouse models, an increased risk of T2DM in AD patients, i.e. mutual effect, and an accumulation of hyperphosphorylated tau in the CNS of T2DM mouse model with a disturbed IRS-2. 34 In addition, a growing body of research suggests that there may be a strong connection between foods and the risk of AD and dementia, by similar pathways that cause T2DM. 72 Insulin and leptin can cross the blood brain barrier (BBB) and exert their various brain actions. 72 Moreover, laboratory studies have shown that insulin and leptin have cognitive-enhancing properties. 72 Both hormones have a great impact on hippocampal synaptic function. 72 Also they have noticeable effects on neuronal morphology, activity-dependent synaptic plasticity, and glutamate trafficking processes. 72 Thus, dysfunction in the insulin and leptin signaling are associated with not only clear neurodegeneration abnormalities but also an increased risk of neurodegenerative disorders. 72 Brain Insulin Resistance Brain Insulin Dysfunction and Impaired Insulin Signaling In general, insulin resistance can cause abnormalities in glucose transportation by decreasing the gene expressions of GLUT4 transporters and/or insulin peptide. 73 Therefore, the 22

glucose transportation is controlled by the insulin signaling pathway, the normal function of astrocytes, and glucose transporters well-being 18. Thus, it impairs acutely the ability of cells to maintain energy homeostasis. In addition, AD is associated with brain insulin resistance and insulin deficiency. The trophic factor deficiency is associated with reduced levels of insulin polypeptide and gene expression in the brain and CSF. 74 Both insulin abnormalities are regulated by insulin/igf signaling and may lead eventually to pro-apoptosis, pro-inflammatory and pro-aβ cascades. 26 On the other hand, insulin deficiency is a companied with significant abnormalities in the expression of genes (e.g., choline acetyltransferase, tau, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) that support cholinergic/cognitive, neuronal cytoskeletal, and metabolic functions. All these important genes are suppressed in AD. 26 This implies that impaired insulin signaling may have an important role in the pathogenesis of AD or brain insulin resistance state or T3DM. However, the exact etiology of T3DM is still unknown. In addition, insulin resistance has been identified as a major risk factor for the onset of AD. 69 It is defined generally as reduced cellular responsiveness to insulin. It could promote AD onset by reducing brain insulin uptake and by raising brain levels of Aβ, tau-phosphorylation, oxidative stress, and pro-inflammatory cytokines, advanced glycation end-product (AGEs), dyslipidemia, and apoptosis. 71 Thus, insulin resistance or lack of insulin, in addition to affecting the blood glucose levels, it contributes to degenerative processes in the brain. 23 Furthermore, brain insulin resistance plays a critical role, with other contributors such as obesity, T2DM, dyslipidemia, and non-alcoholic steatohepatitis, in causing mild cognitive impairment, dementia and AD. 74 In addition, recent studies have shown that brain insulin resistance and deficiency are related to cognitive impairment. 75 Moreover, brain insulin resistance 23

has been identified by Talbot et al. 18, 71 of postmortem AD patients even in the absence of diabetes. Therefore, it has been concluded that impaired insulin signaling can disturb both the processing of AβPP and the clearance of Aβ. 26 (Fig. 3) Putative Mechanisms of Pathogenesis Type 3 Diabetes and Amyloid-Beta Toxicity According to Trivedi et al. there is a toxic cycle between continuous insulin exposure (i.e., in the case of brain insulin resistance) and Aβ accumulation inside the neurons. 76 As the neurons are exposed to excessive or undue insulin, Aβ accumulates largely inside these neurons. This toxic accumulation may cause further neuronal insulin resistance, and thus vicious cycle goes on. According to Farris et al., insulin degrading enzyme (IDE) regulates the levels of insulin, Aβ protein, and amyloid precursor protein (APP) intracellular domain in vivo. 76 This study showed that a rat model of T2DM of mutant IDE was associated with hyperinsulinaemia and glucose intolerance. Also IDE (-/-) knockout resulted in more than 50% decrease in Aβ degradation in both brain membrane fraction and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE (-/-) knockout mice also showed increased cerebral accumulation of endogenous Aβ, and had hyperinsulinaemia and glucose intolerance, hallmarks of T2DM and T3DM or brain insulin resistance. This implies that IDE hypofunction may underlie or contribute to some forms of T3DM and T2DM and provide a mechanism for the recently recognized association among hyperinsulinaemia, diabetes, and neurodegeneration or neuronal loss. 24

Therefore, in normal subjects, IDE reduces Aβ, regulates insulin and also degrades APP intracellular domain (AICD). This stops the AICD translocation to the nucleus and halts transcriptional process that is carried out by this domain. Thus, there is a regulatory relationship among insulin, IDE and Aβ. Thus, in the case of brain insulin resistance, insulin possibly fail to stimulate the clearance of Aβ, which permits its buildup inside the neurons causing neurodegeneration or neuronal loss, as hallmarks of T3DM or brain insulin resistance. 76 Therefore, the mitochondrial dysfunction may result in a severe energy deficiency and high degeneration effect of ROS in neurons and eventually neuronal death. 77 Furthermore, this implies that the energy crisis is the main underlying cause of neuronal loss or death in T3DM. There is a debate about T3DM, brain insulin resistance as to, whether it is a consequence or a cause of abnormal Aβ expression and protein processing. 26 In terms of the concept of T3DM being consequence, Aβ toxicity may cause insulin resistance in the brain. The Aβ disturbs insulin signaling by competing with insulin on its receptors, 78 reducing the surface expression of IRs (i.e., desensitizing or internalizing them), and reducing the insulin affinity to its relative receptors (i.e., reducing ligand binding affinity), 78 and interfering directly with phosphatidylinositol-4, 5- bisphosphate 3-kinase (PI3K)/Akt activation causing blockade of its signaling, leading to impaired survival signaling, increased activation of GSK-3β activity, and increased hyperphosphorylation of tau. The net effect is neurodegeneration and neuronal loss. 79 On the other hand, in terms of concept of T3DM being cause, the brain insulin resistance with oxidative stress and neuro-inflammation may cause Aβ accumulation. The studies that incorporate this concept claim that insulin stimulation may increase or accelerate trafficking of Aβ from the Golgi network to the plasma membrane. Therefore, insulin may activate Aβ extracellular excretion and, at the same time, inhibit its intracellular accumulation by activating its degradation 25

by IDE. 80 Thus, impaired insulin signaling can disturb both APP processing and Aβ clearance. 81 This leads to increased neurotoxic effects of Aβ on neurons ending up with possible neurodegeneration and neuronal cell death. Impaired Kinase Activities and Tau Pathology Hypothesis Previous research by Steen et al. revealed that many important components of CNS neurodegeneration that occur in AD are mediated by impaired insulin signaling in the brain. 67 In addition, the normal activation of insulin/igf signaling stimulates PI3K/Akt via c-ampdependent protein kinase leading to inactivation of GSK-3β activity. 82 This results in multiple molecular effects such as the synthesis of proteins involved in neuronal glucose metabolism, antiapoptotic mechanisms, and antioxidant defense. 82 Additionally, it has been reported that compromised insulin-activated Akt phosphorylation, attributed to chronic hyperinsulinaemia 83, decreases GSK-3β phosphorylation (i.e., activation). 82 Moreover, constitutively active Akt suppresses APP crossing and Aβ release via feedback deactivation of IRS and PI3K. For this reason, a good control of Akt signaling is very important for both amyloid and tau neuropathology in AD. 84 Furthermore, the insulin/igf resistance in AD brain can interfere with three signaling pathways such as PI3K Akt, Wnt/β-catenin and GSK-3β cascades. 26 It inhibits the PI3K/Akt signaling resulting in impaired tau gene expression that is responsible for tau pathology in AD. 85 Also insulin/igf resistance suppresses Wnt/β-catenin pathway via de-phosphorylation of β catenin leading to its low degradation by proteasome (ubiquitination). Therefore, this process reduces synaptic plasticity in the brain (i.e., Wnts signaling promote synaptic assembly by signaling to the 26

developing pre and postsynaptic compartments). 86 In addition, the insulin/igf resistance enhances the activation of GSK-3β activity that causes augmentation of misfolded and aggregated hyperphosphorylated tau proteins that worsens or disrupts the tau gene expression leading to AD pathogenesis. 87 Thus, the crosstalk of the attenuated PI3K/Akt pathway and the intensified activation of the GSK-3 β pathway, due to trophic factor resistance, will eventually cause abnormal hyperphosphorylated tau and impaired tau gene expression preceding AD disposition. 26 (Fig.4) Figure 4. Scheme of the pathway mediating insulin-induced inactivation of GSK3. 88 A GSK3 is an enzyme that is able to phosphorylate, and thereby inhibit, glycogen synthase, a key regulatory process in the synthesis of glycogen. Also it is important for many cellular signaling pathways. It regulates several transcription factors that control the expression of numerous genes. According to de la Monte et al., 2009, the GSK3β, mitogen-activated protein 27

kinase (MAPK) and cyclin-dependent kinases are responsible for tau phosphorylation. 89 There are two isoforms of this enzyme GSK-3α and GSK-3β. The GSK-3α promotes Aβ production by activating amyloid precursor protein (APP) γ-secretase activity, while GSK-3β is the main tau kinase responsible for tau hyperphosphorylation and the formation of NFTs. 84 Moreover, defective insulin signaling decreases both isoforms phosphorylation; and therefore, constitutively activates these kinases, and thus affects both Aβ and tau building up inside neurons. 84 However, it has been found that overexpression of a constitutively active GSK-3β promoted cell death, while its inhibition prevented apoptosis. In the case of impaired insulin/igf signaling, the PI3K/Akt activity is suppressed; consequently, this inactivation potentiates the GSK-3β activity, which in its turn, rectifies the tau gene expression and its phosphorylation to reach a state of hyperphosphorylation of tau and the formation of NFTs, promoting their misfolding and aggregation, and ending up with neuronal loss or AD. 5, 68, 18, 26 The tau pathology is associated with some complications such as less generation of normal soluble tau protein, relative accumulation of hyperphosphorylated insoluble fibrillar tau, worsening of cytoskeletal collapse, and neurite deterioration as well as synaptic disconnection. 26 On the other hand, the critical GSK-β activity inhibition regulates Wnt signaling by phosphorylating β-catenin leading to its degradation or ubiquitination via proteasome-mediated mechanism. 90 Thus, a sustained loss of Wnt signaling function may be involved in the Aβdependent neurodegeneration or neuronal loss observed in AD brains. 90 Therefore, trophic factor signaling is important for Wnt signaling activation. 91 (Fig.5) 28

Figure 5. GSK3B is associated with neuropathological mechanisms involved in Alzheimer s disease. 88 Furthermore, the activated GSK-3β enzyme is considered as a pro-apoptotic kinase as it promotes apoptosis by inhibiting pro-survival transcription factors, such as camp response element-binding protein (CREB) and heat shock factor-1 88, and facilitating pro-apoptotic transcription factors such as p53. 92 Another interesting kinase activity is MAPK as it is one of the factors that are involved in insulin resistance brain state, contributing to AD pathological alterations. The MAPK pathway has been found to be significantly activated in AD patients. This is correlated with increased neuroinflammation, elevated tau hyperphosphorylation and enhanced Aβ trafficking. 93 The other mechanisms that mediate the hyperphosphorylation of tau include cyclin-dependent kinase, Abl 29

protein tyrosine kinases (C-Ab1), and protein phosphatase 1 and 2 A, which are beyond the scope of this thesis. In addition, the neuronal cytoskeletal lesions are associated with dementia in AD, because they are characterized by neuronal fibrillary tangles and dystrophic neurites that aggregate as insoluble fibrillar tau. 67, 5, 93, 26 Thus, the tau accumulation is the most significant structural feature of dementia in AD. Generally, in AD, the microtubule-associated protein tau is phosphorylated by the activation of proline-directed kinases (e.g., GSK-3β enzyme). This makes tau selfaggregated and misfolded. These soluble tau oligomers disturb the normal cytoskeletal networks and axonal transport leading to synaptic disconnection, progressive neurodegeneration, and thus, neuronal loss or death. Furthermore, further accumulation of insoluble fibrillar tau, oxidative stress, and ROS generation are ultimate steps of hyperphosphorylated tau ubiquitination and ubiquitin proteasome system (UPS) dysfunction. The impacts of these abnormalities, at the cellular level of the AD brain, are neuronal apoptosis, mitochondrial dysfunction and necrosis. Similarly, the brain insulin/igf resistance can cause all the aforementioned cellular problems via inhibition of the downstream pro-growth and prosurvival signaling pathways as insulin/igf signaling activation reduces the tau gene expression and phosphorylation. 26 Cellular abnormalities that precede or accompany classic AD showed that cell loss was associated with increased activation of pro-death genes and signaling pathways that cause impaired energy metabolism, mitochondrial dysfunction, chronic oxidative stress, and cerebrovascular disease/cerebral hypoperfusion. 67 30

Advanced Glycation End-Product and Amyloid-beta Neurotoxicity Hypothesis Glucose metabolism abnormality, in the systemic circulation and brain, plays a dramatic role in predisposing most features of T2DM and AD. 13 Both diseases are characterized by an elevation of oxidative stress and production of advanced glycation end-products (AGEs). 13 These AGEs are reducing sugars that react with amino group of proteins to produce cross-linked complexes and unstable compounds. 94 They are found in the brain of diabetic patients, retinal vessels, peripheral nerves and kidney. In addition, they play two dangerous roles either coupled with free radicals that produce oxidative damage ending up with cellular injury or they modify plaques and NFTs. 94 They are located also inside NFTs consisting tau protein and senile plaque (Aβ protein), predisposing T2DM. 13 They have an established link with vascular dementia and possible link with AD. 18 Also diabetic patients produce AGEs; and therefore, they are at an increased risk of having AD. Additionally, T2DM accelerates AGEs production that may be 18, 94 considered as a causative factor for AD. On the other hand, AD is known to have abnormal expression and processing of AβPP that is characterized by accumulation of Aβ oligomeric fibrils or insoluble neurotoxic larger aggregated fibrils (plaques). Thus, increased AβPP gene expression and abnormal proteolysis (dysfunction of UPS) allow accumulation and aggregating of Aβ40 and Aβ42. Similarly, the incidence of familial AD is 10% with mutations in AβPP, presenilin 1 (PS1), and presenilin 2 (PS2) genes and inherited ApoE ε4 gene. 95 All these mutated genes increase the synthesis and deposition of Aβ peptides in the brain. 95 However, sporadic AD represents 90% of cases that also are characterized by Aβ accumulation and toxicity. 96 31

In general, an elevated AβPP gene expression with abnormal proteolysis leads to accumulation and aggregation of Aβ40 and Aβ42 (most toxic form of Aβ). (Fig.6) Figure 6. Common pathological processes in Alzheimer s disease (AD) and diabetes. 82 32

Molecular Mechanism of Insulin Receptor Substrate-1Inhibition IRS-1 is a signaling adapter protein that plays a key role in transmitting signals from the insulin and IGF-1 receptors to intracellular pathways PI3K / Akt and Erk / MAP kinase pathways. It also plays an important role in insulin signaling in the brain and may cause brain insulin resistance that occurs in AD. 71 Moreover, IRS proteins and PI3K isoforms (downstream effectors of insulin) have unique patterns of expression in the CNS that interfere with expression of the IRs. 97 The phosphorylation of the specialized adapter protein, IRS, at tyrosine residues, induces the activation of downstream pathways and regulates different range of biologic responses, including glucose transport, protein synthesis, mitogenesis, and cell survival. 98,99 (Fig.7) Figure 7. Neuronal insulin signaling pathways. 82 33

The family of IRS proteins consists of six similarly structured proteins (IRS 1 6) with different tissue distribution and functions in the conduction of the hormonal signal. 98 Different biological responses are due to different protein adaptors or IRSs. 100 For example, both IRS1 and IRS2 are important for muscle metabolism 100, meanwhile, IRS2 plays an important role in hypothalamic effects of insulin for growth regulation and energy homeostasis. 101 In the same context, (IRS4-/-) knockout mice have normal phenotypes except for reduced fertility. 102 These IRSs 1-6 have three downstream targets: phosphorylation and activation of PI3K/Akt and the mammalian target of rapamycin (mtor), controlling metabolic and transcriptional programming of cells at the nuclear level. 103 Furthermore, the IRS-1 and IRS-2 are the most common protein adaptors that control insulin action. 103 They undergo phosphorylation at serine sites causing their dissociation from IR and reduction in tyrosine phosphorylation sites; therefore, the balance between, for example, IRS-1pSer / IRS-1 ptyr determines the extent of insulin actions. 103 There is an accumulating body of evidence showing the importance of IRS-1 inhibition in AD pathology and the predisposition of peripheral insulin resistance, and thus T2DM. 103 Some studies have shown that activation of TNF-α signaling activates JNK that phosphorylates IRS-1 at serine position (IRS-1 pser), leading to the blockage of downstream insulin signaling and triggering peripheral insulin resistance or T2DM pathogenesis. Similarly, the Aβ oligomers activate the previous tumor necrosis factor-alpha (TNF-α)/ c-jun N-terminal kinase (c-jnk) signaling pathway leading to metabolic diseases. 103 The central role of IRS-1inhibition is supported by several pieces of evidence. Firstly, in the hippocampi of Cynomolgus monkeys 103,70, intracerebroventricular (ICV) injection of Aβ oligomers induced IRS-1 inhibition. Secondly, IRS-1 inhibition also was demonstrated in the brain 34

of transgenic mouse model of AD. 104 Thirdly, the postmortem studies of AD brains showed elevated IRS-1pSer and activated JNK. 103 To sum up, the molecular mechanism of brain insulin resistance or T3DM may involve either increased serine phosphorylation of IRS protein (IRS-1 inhibition) and/or elevated degradation of IRS protein. 105 These two mechanisms are observed in insulin resistance animal models and obese individuals lipid infused patients. 106 Thus, the possible mechanism of IRS-1 and 2 inhibition is mediated by the activation of Ser / Thr kinases that can phosphorylate IRS family members at multiple serine sites and inhibit interaction of IRSs with IRs leading to reduced tyrosine phosphorylation of IRSs, and eventually reduced activation of PI3K signaling. 107 Potential Type 2 Diabetes and Alzheimer s Disease Link Peripheral Insulin Resistance Disease States Peripheral insulin resistance is a physiological condition in which cells fail to respond to the normal actions of insulin. This leads to hyperglycemia and, in the long-run, hyperinsulinaemia that may downregulate BBB insulin receptors, leading to a reduction of insulin transport into the brain, and thus reduced cellular glucose uptake. 108 In addition, peripheral insulin resistance may lead to increased ceramides in the liver and in the peripheral blood. 29,109 Therefore, it may be considered as a key factor in developing brain insulin resistance, cognitive impairment and neurodegeneration. 26 Some studies have shown that parenteral ceramides are cytotoxic and can cause sustained impairments in spatial learning memory with neurodegeneration and brain insulin/igf resistance. 26 35

In addition, one of the complications of peripheral insulin resistance is hepatic dyslipidemic states. 29,110 In this abnormality, the ceramide levels are increased due to increased biosynthesis or reduced degradation of ceramides attributed to altered gene expression and enzymatic activity. 110 Thus, the net effect is high neurotoxic ceramide concentrations that may cause neuronal insulin resistance, oxidative stress and abnormalities that are similar to AD. 71 Therefore, peripheral insulin resistance without T2DM is a risk factor for AD. 71 Furthermore, it has been hypothesized that ceramides are toxic lipids generated by the liver with hepatic insulin resistance, and are caused by obesity, T2DM, metabolic syndrome, and chronic alcohol abuse, and low dose of nitrosamine, can mediate the neurotoxic effect in the brain via a liver-brain-axis, leading to neurodegeneration. 26 Experimental animals in which brain insulin receptor expression and function were inhibited showed cognitive impairment and neurodegeneration with characteristics that overlap with AD. 26 (Fig.8) Figure 8. The liver-brain axis of neurodegeneration in type 2 diabetes mellitus. 89 36

Peripheral Insulin Resistance and Type 2 Diabetes as Cofactors of Cognitive Impairment and Neurodegeneration It has been hypothesized that brain insulin resistance may cause neurodegeneration 26. This theory may be accepted, if it fulfils three conditions. 31 Firstly, peripheral insulin resistance states and T2DM can cause neurodegeneration including AD. Secondly, peripheral insulin resistance states and T2DM should serve as cofactors to predispose neurodegeneration associated with AD. Lastly, T2DM and AD should have the same disease process in different target organ and tissues. The first evidence that T2DM and other peripheral insulin resistance diseases serve as cofactors in causing neurodegeneration and AD is the epidemiological studies. They have indicated that patients with glucose intolerance, insulin secretion defect and T2DM have an increased risk of developing mild cognitive impairment (MCI) or AD-type dementia. 4,26 Additional supporting evidence includes the longitudinal studies that have shown that patients with T2DM, obesity (dyslipidemic disorder) are correlated with later development of MCI, dementia, or AD. 26 In addition, one study has shown that obesity with or without T2DM (systemic factor other than T2DM) leads to an increased risk of MCI, AD or other forms of neurodegeneration. 26, 111 The limitation here is that despite the fact that a high percentage of patients with MCI or dementia have T2DM, peripheral insulin resistance or obesity, the majority of patients with AD do not have these diseases. 26 On the other hand, postmortem human brain studies have demonstrated no significant 32, 112 increase in AD diagnosis among diabetics regardless of the presence of plaques and NFTs. Additionally, they found that peripheral insulin resistance was more common in elderly AD patients than with normal age individuals. 32,113 Also, they indicated that T2DM alone is not 37

sufficient to cause AD, based on the fact that, NFTs and dystrophic neurites are hallmarks of AD 4, 26 and correlate with the severity of dementia. Other experimental studies show that high fat diet mouse and rat models had obesity linked to T2DM that causes cognitive impairment with deficits in special learning and memory. 114 Also they found that experimental obesity concurrent with T2DM causes mild brain atrophy with brain insulin resistance, neuro-inflammation, oxidative stress, and abnormalities in cholinergic function. 5 However, the limitation is that the brain abnormalities are mild cases not sever ones that lack the most important structural lesions of AD such as NFTs. 115 Lastly, the observational studies showed that obesity or T2DM can be linked to cognitive impairment, mild brain atrophy, and a group of AD-type brain diseases, but they do not cause significant AD pathology. Therefore, the researchers suggest that T2DM, obesity, and other peripheral insulin resistance diseases serve as cofactors leading to the pathogenesis or progression of neurodegeneration. 5 Peripheral Insulin Resistance and Type2 Diabetes as Contributor Factors of Cognitive Impairment and Neurodegeneration Vascular Factors Based on the context that a similar degree of dementia occurs in moderate and severe AD, as well as chronic ischemic encephalopathy, the ischemic lesions and localized infarcts that characterize the ischemia are targeted by the AD neurodegeneration leading to white matter changes with extensive degradation of white matter fibers. 116 In addition, magnetic resonance 38

imaging (MRI) studies have shown that hippocampal and amygdalar targets of AD 26, 117 neurodegeneration increase with duration and the progression of T2DM. Peripheral Ceramides Ceramides are lipid signaling molecules that cause insulin resistance by two mechanisms activation of pro-inflammatory cytokines and suppression of the PI3KAkt signaling pathway (i.e., insulin resistance).59, 118 Consequently, these two mechanisms result in the accumulation of peripheral cytokines and induction of hepatic insulin resistance, respectively. 43,115 Further damage of the hepatic cells generates more toxic ceramides. The cytokines and the neurotoxic ceramides cross the BBB and cause brain insulin resistance, inflammation, energy failure, toxicity, and local production of toxic ceramides. This causes progressive neurodegeneration including AD. One study has suggested that cognitive impairment with brain insulin resistance and neurodegeneration are significantly correlated with fatty liver associated with insulin resistance rather than obesity or T2DM. 119 Therefore, it has been hypothesized that cognitive impairment is mediated by a liverbrain axis of neurodegeneration including obesity, T2DM, peripheral insulin resistance. 74 In addition, there is a link between T1DM and tau pathology. Insulin dysfunction might enhance AD pathology through distinct mechanisms. 4 It is manifested by T1DM as characteristic of insulin deficiency. This abnormality compromises the PI3K/Akt signaling and directly activates or rectifies GSK-3β activity that might lead to tau phosphorylation and NFT formation, which are the major histopathological hallmarks of AD 4 (Fig.9). Similar to T1DM, insulin dysfunction, manifested as T2DM, is characterized by insulin resistance, which also might be due to other mechanisms including inflammation or stress. Insulin resistance directly inhibits phosphatase 39

activity or indirectly affects the same activity by affecting the hypothermia leading to tau pathology, and thus AD pathogenesis. 4 (Fig.9) Figure 9. Insulin dysfunction might enhance Alzheimer s disease pathology through distinct mechanisms. 4 40

Inflammatory and Pro-inflammatory Cause of Peripheral Insulin Resistance Inflammation is an important feature of both DM and AD and may cause both diseases. 120,121 Metabolic disorders are commonly characterized by macrophages activation or infiltration and increased production of pro-inflammatory cytokines including an elevated TNF-α. For example, in obesity, there is an increased TNF-α overexpression in adipose tissues of obese individuals. 122 This leads to peripheral insulin resistance. 123 Similarly, AD usually features as elevated cytokines/chemokines, gliosis (microgliosis), and systemic increase of TNF- α (as an inflammatory mediator), and increased interleukin-6 (IL-6) and interleukin-1-beta (IL-1β). 122,124 Thus, in both brain and peripheral tissues, chronic inflammation is deleterious in causing progressive tissue damage in neurodegenerative diseases. 120 Some studies have shown that inflammation causes hypothalamic dysfunction in obesity. In addition, inflammation and endoplasmic reticulum (ER) stress are important pathogenic events in causing hypothalamic insulin resistance and peripheral insulin resistance in metabolic disorders. 125, 126 For example, animal models of T2DM and obesity have inflammation responses in the hypothalamus induced by activation of TNF- α and the IkBα kinase (IKK)-β / nuclear factor- KB pathway. In addition, the former pathway is an important mechanism causing pathogenesis of metabolic disorders. 127 Thus, the hippocampal dysfunction in AD and hypothalamic deregulation in obesity may share common inflammatory pathogenic pathways. 126 In the same context, activation of pro-inflammatory and cell stress signaling pathways impairs brain insulin signaling in AD. 128 Moreover, there are three major inflammatory/stress signaling pathways: ER stress, stress kinases IKK and PKR (double-stranded RNA dependent protein kinase R). 129 These pathways are triggered in case of peripheral insulin resistance via the 41

activation of TNF-α/c-JNK pathway. In T2DM, elevated levels of TNF-α trigger stress kinase phosphorylation of IRS-1 that blocks insulin signaling. However, in the brain, TNF- α mainly is secreted by microglial cells. The high concentration of TNF- α is often seen in microvessels and CSF in AD. 130 Therefore, impaired neuronal insulin signaling in AD is linked to pro-inflammatory signaling, based on the assumption that amyloid-beta oligomers(aβo) cause the inhibition of IRS- 1 through the activation of TNF- α/jnk pathway. 120,104 The common mechanisms that cause impaired peripheral insulin signaling in T2DM and brain insulin resistance in AD, are IKK, PKR, and ER stress that are elevated in AD. 131 These mechanisms are mediated by AβO-induced IRS-1 inhibition in the hippocampal neurons. 104 The stress kinase (IKK) is activated by TNF- α signaling. 104 The activated IKK causes peripheral insulin resistance and also mediates AβO-induced brain IRS-1 inhibition. 104 Therefore, IKK activation is an important signaling pathway because it can cause metabolic defects underlying T2DM. 104 Also it is involved in AD pathogenesis through IRS-1 inhibition activity. 104 Therefore, this pathway is a connecting point between peripheral and central insulin resistance. The other PKR is identified as a pathogen sensor and a regulator of insulin immune response against viral infection. 132 It regulates metabolic homeostasis with other inflammatory kinases. 133 This kinase is involved in AβO-induced brain insulin IRS-1 inhibition. 104 Thus, pro-inflammatory TNFα signaling and activation of cell stress mechanisms play critical roles in peripheral and central IRS- 1 inhibition and in neuronal dysfunction in AD. 121 (Fig.10) 42

Figure 10. T2DM, brain insulin resistance, and AD have been linked in a number of different epidemiological, clinical, and animal studies. 35 Public Health Issues Nowadays, both AD and DM have become epidemic including insulin resistance and prediabetes and T2DM. 26, 94, 134, 68,135 Over the past several decades, AD is the most common cause of dementia in North America. 26 The prevalence of DM for all age-groups is around 10% of the population. DM is the 7th leading cause of death in the United State. It is high among men than women, and women are mostly middle-aged. It seems to have doubled between 2000 and 2030, especially in the urban regions of developing countries. More importantly, people aged 65 years old tend to be at a high risk for getting DM during their lifetime. 1 (Table1) 43

Table 1. Potential Type 2 Diabetes and Possible Alzheimer s Disease Link. 3,136 Total 20 years or older By Age 20 44 45 64 65 years or older By Sex Men Women Number with diabetes (Millions) Percentage with diabetes (unadjusted) Number with AD (Millions) Percenta ge with AD Total 28.9 12.3-4.3 13.4 11.2 15.5 13.4 4.1 16.2 25.9 13.6 11.2 0.2 1 5 2 1.8 3.2 4 15 44 38 36 64 By Age 20-44 45-64 65-74 75-84 85 years or older By Sex Men Women 1 <65 years 2 >65 years 44

In the same context, AD and T2DM are the two conditions that affect a large number of people in industrialized countries. 137, 138 In addition, T2DM is a risk factor for AD, supporting the hypothesis that AD is, a T3DM, or brain insulin resistant state. This metabolic disorder constitutes an important public health concern in the 21st century, especially concerning T2DM. 135 Furthermore, T2DM and AD are coincidentally more prevalent with ageing. However, evidence suggests that patients with T2DM, with hallmarks of hyperinsulinaemia and insulin resistance, are at an increased risk of getting AD. 139 A large body of evidence suggests that the two diseases, T2DM and AD, are linked. 35 Also, T2DM and AD represent huge burdens on the healthcare system and society in the US and worldwide. In 2004, complications linked to DM were the sixth leading cause of death, while AD was seventh. A more recent estimate, has placed AD as the sixth-leading cause of death. 140 Moreover, what makes these metabolic disorders have a heavy socio-economic burden on healthcare systems are the increase of T2DM with age, the concurrence of DM risk factors such as obesity, a sedentary life, and hypertension, and long-term consequences of T2DM such as peripheral and autonomic neuropathy, stroke, retinopathy, cardiovascular disease, renal failure. Also DM is a costly disorder because the global burden is dramatically increased. The gradual silent growing onset and asymptomatic nature of DM makes most patients remain undiagnosed and at great risk of developing life-threatening vascular complications. Therefore, early diagnosis, prevention, and treatment can reduce the incidence of DM and slow down its progression. 1 For example, microvascular complications, affecting the microvasculature of the nerves, eyes and kidneys, are associated with increased morbidity. 1 In addition, these complications may compromise patients quality of life and may increase morbidity and mortality rates. 135 45

The economic and social costs of DM are huge on both health care services and through productivity loss. 1 In industrialized countries, 10 % or more of the total health budget is spent on the management of DM and its complcations. 1, 141 Moreover, the costs of productivity loss contribute to a huge impact on socio-economic burden. It includes disability and premature mortality such as workday absence, reduced performance during work, reduced productivity days for those not in the labor force, and reduced labor force participation. Besides, productivity loss exists in each age group across the whole population. However, it affects the middle-age group (25-54 years) most. 142 46

Table 2. Financial burden of type 2 diabetes and Alzheimer s disease Disease Country System or category affected T2DM 3 USA Total cost of diagnosed patients with 72% direct medical cost and 28% indirect cost due to productivity loss AD 4 USA Health Care costs on AD and other dementia The cost of caring for Alzheimer s patients in the U.S T2DM France Germany Italy UK Total direct costs of T2DM AD All Europe Aggregated costs of AD T2DM Worldwide Health expenditure on diabetes AD Worldwide The global cost of Alzheimer s and dementia Cost Year $ 245 billion 2012 143 $ 9.1 billion $220 billion per year 2012 136 2014 136 12.9 billion 2010 144 43.2 billion 2.34 billion 4.54 billion $ 238.64 billion 2010 145 $ 376 billion $ 490 billion 2010 146 2030 102 $605 billion 2014 136 3 Type 2 Diabetes Mellitus 4 Alzheimer s disease 47

Therefore, there is a need for a cost-effective studies and cost-effective analysis to identify new therapeutic strategies that reduce the disease cost efficiently, justify the additional cost of these interventions, and measure the quality of patients lives that are gained by using these interventions. Thus, the early diagnosis of this metabolic disorder or endocrine-metabolic disorder, T3DM, or AD, optimal glycemic control and a preemptive prevention of its complications, together with good therapeutic strategies, are researchers top priorities to slow down or halt its progression, or even stop it from becoming epidemic and reduce its socio-economic impact on our societies. Possible Biomarkers Brain Glucose Hypometabolism Neuroimaging has enabled researchers to study the relationship between brain energy metabolism and AD onset and progression. One in vivo approach is to use positron emission tomography (PET) using a tracer. 18 F-fluorodeoxyglucose (FDG) is the suitable tracer for glucose metabolism. 147 This is due to several advantages. First, it can be transported into the brain at the same rate as glucose. Second, it activates both glucose transport and subsequent phosphorylation in glycolysis. Third, it can be phosphorylated by hexokinase in the first step of glycolysis. The FDG works in AD as a biomarker by using a reference brain region to measure brain glucose uptake. This region is usually the cerebellum because of its low involvement in AD pathology. 58 By comparing different regions to the reference region, we can have complete picture of the glucose flow in different brain regions. 48

The FDG-PET studies in AD have shown consistent and progressive cerebral glucose metabolism reductions that correlates with symptoms severity. 18 Another study showed that compared to the control, AD patients show regional glucose metabolism impairment in the parietal-temporal lobe, posterior cingulate cortex and the frontal areas during disease progression. 18 Also the hippocampus, entorhinal cortex, parietal, temporal and posterior cingulate cortex were less affected brain regions. Additionally, there was a reduction of the local metabolic rate of glucose. Lastly, there was regional distribution of reduced synaptic activity and density in AD. To sum up, there is selectively regional hypometabolism match specific cognition impairment in AD patients. 18,148 The FDG-PET is an effective biomarker for identifying the presence of cognitive dysfunction-associated progressive neurodegeneration with 95% sensitivity and 79% specificity, and producing 89% high diagnostic accuracy compared to clinical measures at 77%. The clear evidence is that FDG-PET has the capacity of high specificity in differentiating AD from other types of dementia. 18 However, for better AD prediction and diagnosis, the combination of genetic risk (homozygous APoE 4) with cerebral metabolism rate of glucose (CMRglu) determined by FDG-PET, would be beneficial for AD prediction and diagnosis. 18 Altered Thiamine Metabolism The dysfunction of thiamine metabolism contributes to impair cerebral glucose metabolism leading to neuronal degeneration processes (with other pathogenic factors e.g., insulin resistance) ending up with cognitive deficits in AD patients. The coenzyme, thiamine diphosphate (TDP), has shown altered levels both in the blood and brain of AD patients. 149 Clinical and 49

experimental studies have confirmed that mitochondrial dysfunction can cause AD. 150 These studies also emphasize the important roles of mitochondrial dysfunction and impaired thiaminedependent processes in cerebral glucose hypometabolism of AD. Insulin Subsrate-1 A reduction of IRs and IRS-2 leads to a reduction in brain growth and increased tau phosphorylation. 18 Insulin resistance is characterized by a reduction of the following: IRS-1 expression, insulin receptor expression, insulin tyrosine kinase activity and PI3K activation, especially in the skeletal muscle and adipocytes. Therefore, IRS proteins play a key role in opposing brain insulin resistance adverse effects on brain tissues and peripheral tissues. As Aβ may trigger the internalization of neuronal IRs, it is possible that removal of IRs from the cell surface facilitates the phosphorylation of IRS-1 at seine sites (IRS-1 pser). 103 Therefore, IRS-1 may trace the activity of Aβ which is the main feature of AD. Insulin degrading enzyme A good biomarker should reflect an early pathological change before irreversible neuronal change occurs. One possible biomarker is IDE as a primary enzyme responsible for Aβ clearance in the brain. A large body of evidence suggests defective clearance mechanism may be a key factor in cerebral Aβ accumulation that is crucial to AD pathology. One study has shown that protein levels and the catalytic activity of IDE are reduced in AD brains as compared to controls. 76 In vivo studies have shown that IDE hypofunction increases chronic accumulation of cerebral Aβ. 76 Thus, 50

the addition of a new biomarker to a panel of established ones may help increase the sensitivity and specificity of a diagnostic blood test for AD or any neuroendocrine-metabolic brain disease or T3DM. Insulin-Related Interventions Insulin Related Antidiabetics An established body of reasonable evidence indicates a link between brain insulin resistance and the neurodegeneration that characterizes AD. 82, 85, 101, 109, 151 Therefore, insulin has physiological importance in terms of brain function. The ultimate novel goal of using antidiabetic drugs is to minimize any further damage to already compromised organs (e.g., brain) and eventually delaying or avoiding the development of long-term complications such as dementia, AD and T3DM. Glycemic control is of paramount priority to reduce, but yet not sufficient, the risk of T2D-associted long-term vascular and cardiovascular complications including AD or brain insulin resistance. Tight control of DM may be essential for APOE ε4 carriers since they may be more susceptible than others to get both diseases. 111 The Cardiovascular Health Study showed that DM 111, 152 in APOE ε4 allele carriers were at greater risk of cognitive decline than those non-carriers. However, there is a reduction of the sensitivity to insulin in AD patients who are not ApoE4- positive suggests that optimization of blood sugar levels may have therapeutic benefits. Therefore, maintaining healthy blood glucose control in middle-aged men prevents AD or T3DM. 51

In addition, peripheral insulin administration has been shown to improve brain atrophy, cognitive function and dementia severity. Therefore, insulin related antidiabetics are aimed to prevent or correct insulin abnormalities, and thus benefiting patients with age-related memory or cognitive impairments and AD. Therefore, the suggested treatment for AD, as a neuroendocrine disorder, or T3DM includes insulin related antidiabetic agents that target the metabolic pathways. Among the best antidiabetics that could have a great impact on treating AD or T3DM are exogenous insulin, amylin analogs, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, dipeptidyl peptidase 4 (DDP-4) inhibitors (Fig. 11). Figure 11. All diabetes drugs are likely to have indirect effects in the CNS by affecting circulating concentrations of glucose and insulin. 35 52