TRIPLE PLAY: PROMOTING NEUROVASCULAR LONGEVITY WITH NICOTINAMIDE, WNT, AND ERYTHROPOIETIN IN DIABETES MELLITUS

Summary

Oxidative stress is a principal pathway for the dysfunction and ultimate destruction of cells in the neuronal and vascular systems for several disease entities, non promoting the ravages of oxidative stress to any less of a degree than diabetes mellitus. Diabetes mellitus is increasing in incidence as a result of changes in human behavior that relate to diet and daily exercise and is predicted to affect almost 400 million individuals worldwide in another two decades. Furthermore, both type 1 and type 2 diabetes mellitus can lead to significant disability in the nervous and cardiovascular systems, such as cognitive loss and cardiac insufficiency. As a result, innovative strategies that directly target oxidative stress to preserve neuronal and vascular longevity could offer viable therapeutic options to diabetic patients in addition to more conventional treatments that are designed to control serum glucose levels. Here we discuss the novel application of nicotinamide, Wnt signaling, and erythropoietin that modulate cellular oxidative stress and offer significant promise for the prevention of diabetic complications in the nervous and vascular systems. Essential to this process is the precise focus upon diverse as well as common cellular pathways governed by nicotinamide, Wnt signaling, and erythropoietin to outline not only the potential benefits, but also the challenges and possible detriments of these therapies. In this way, new avenues of investigation can hopefully bypass toxic complications, or at the very least, avoid contraindications that may limit care and offer both safe and robust clinical treatment for patients.

1. Introduction

Diabetes mellitus (DM) is a significant health concern in the clinical population [1]. The disease is present in at least 16 million individuals in the United States and more than 165 million individuals worldwide [2]. Furthermore, by the year 2030, it is predicted that more than 360 million individuals will be afflicted with DM [3]. At least 80 percent of all diabetics have type 2 DM that is increasing in incidence as a result of changes in human behavior that relate to diet and daily exercise [4]. Although type 1 insulin-dependent diabetes mellitus accounts for only 5–10 percent of all diabetics [5], it is increasing in adolescent minority groups [6]. Of potentially greater concern is the incidence of undiagnosed diabetes that consists of impaired glucose tolerance and fluctuations in serum glucose that can increase the risk for the development of DM [7]. Individuals with impaired glucose tolerance have a greater than two times the risk for the development of diabetic complications than individuals with normal glucose tolerance [8].

Both acute and long-term occurrence of type 1 and type 2 DM can result in complications of the neuronal and vascular systems. For example, DM can impair vascular integrity and alter cardiac output [9] that eventually diminishes the capacity of sensitive cognitive regions of the brain leading to functional impairment and dementia [10–12]. Disease of the nervous system can become the most debilitating complications for DM and affect sensitive cognitive regions of the brain, such as the hippocampus that modulates memory function, resulting in significant functional impairment and dementia [13]. DM also has been found to increase the risk for vascular dementia in elderly subjects [12, 14] as well as potentially alter the course of Alzheimer’s disease. Although some studies have found that diabetic patients may have less neuritic plaques and neurofibrillary tangles than non-diabetic patients [15], contrasting work suggests a modest adjusted relative risk of Alzheimer’s disease in patients with diabetes as compared with those without diabetes to be 1.3 [16]. Furthermore, costs to care for cognitive impairments resulting from diabetes that can mimic Alzheimer’s disease can approach $100 billion a year [17–19]

2. The role of oxidative stress in DM

Closely tied to the development of insulin resistance and the complications of DM in the nervous and vascular systems is the presence of cellular oxidative stress and the release of reactive oxygen species [5]. Oxidative stress occurs as a result of the development of reactive oxygen species that consist of oxygen free radicals and other chemical entities. Oxygen consumption in organisms, or at least the rate of oxygen consumption in organisms, has intrigued a host of investigators and may have had some of its original origins with the work of Pearl. Pearl proposed that increased exposure to oxygen through an increased metabolic rate could lead to a shortened life span [20]. Subsequent work by multiple investigators has furthered this hypothesis by demonstrating that increased metabolic rates could be detrimental to animals in an elevated oxygen environment [21]. When one moves to more current work, oxygen free radicals and mitochondrial DNA mutations have become associated with oxidative stress injury, aging mechanisms, and accumulated toxicity for an organism [22].

Oxidative stress represents a significant mechanism for the destruction of cells that can involve apoptotic neuronal and vascular cell injury [23–25]. In fact, it has recently been shown that genes involved in the apoptotic process are replicated early during processes that involve cell replication and transcription, suggesting a much broader role for these genes than originally anticipated [26]. Apoptotic induced oxidative stress in conjunction with processes of mitochondrial dysfunction can contribute to a variety of disease states such as diabetes, ischemia, general cognitive loss, Alzheimer’s disease, and trauma [10, 27–30]. Oxidative stress can lead to apoptosis in a variety of cell types that involve neurons, endothelial cells (ECs), cardiomyocytes, and smooth muscle cells through multiple cellular pathways [28, 31–35].

Membrane phosphatidylserine (PS) externalization is an early event during cell apoptosis [36, 37] and can become a signal for the phagocytosis of cells [25, 38, 39]. The loss of membrane phospholipid asymmetry leads to the externalization of membrane PS residues and assists microglia to target cells for phagocytosis [33, 40–43]. This process occurs with the expression of the phosphatidylserine receptor (PSR) on microglia during oxidative stress [11, 44], since blockade of PSR function in microglia prevents the activation of microglia [41, 45]. As an example, externalization of membrane PS residues occur in neurons during anoxia [46–48], nitric oxide exposure [49, 50], and during the administration of agents that induce the production of reactive oxygen species, such as 6-hydroxydopamine [51]. Membrane PS externalization on platelets also has been associated with clot formation in the vascular system [52].

The cleavage of genomic DNA into fragments [53–55] is considered to be a later event during apoptotic injury [56]. Several enzymes responsible for DNA degradation have been differentiated and include the acidic, cation independent endonuclease (DNase II), cyclophilins, and the 97 kDa magnesium - dependent endonuclease [10, 57]. Three separate endonuclease activities are present in neurons that include a constitutive acidic cation-independent endonuclease, a constitutive calcium/magnesium-dependent endonuclease, and an inducible magnesium dependent endonuclease [58, 59].

During oxidative stress, mitochondrial membrane transition pore permeability also is increased [33, 60–62], a significant loss of mitochondrial NAD⁺ stores occurs, and further generation of superoxide radicals leads to cell injury [42, 63]. In addition, mitochondria are a significant source of superoxide radicals that are associated with oxidative stress [10, 17]. Blockade of the electron transfer chain at the flavin mononucleotide group of complex I or at the ubiquinone site of complex III results in the active generation of free radicals which can impair mitochondrial electron transport and enhance free radical production [11, 57]. Furthermore, mutations in the mitochondrial genome have been associated with the potential development of a host of disorders, such as hypertension, hypercholesterolemia, and hypomagnesemia [64, 65]. Reactive oxygen species also may lead to the induction of acidosis-induced cellular toxicity and subsequent mitochondrial failure [27]. Disorders, such as hypoxia [66], diabetes [67, 68], and excessive free radical production [59, 69, 70] can result in the disturbance of intracellular pH.

In disorders such as DM, elevated levels of ceruloplasmin have been suggested to represent increased reactive oxygen species [71] and acute glucose fluctuations have been described as a potential source of oxidative stress [72]. Elevated serum glucose also has been shown to lead to increased production of reactive oxygen species in ECs, but prolonged duration of hyperglycemia is not necessary to lead to oxidative stress injury, since even short periods of hyperglycemia can generate reactive oxygen species in vascular cells [73]. Recent clinical correlates support these experimental studies to show that acute glucose swings in addition to chronic hyperglycemia can trigger oxidative stress mechanisms during type 2 DM, illustrating the importance for therapeutic interventions during acute and sustained hyperglycemic episodes [72].

The maintenance of cellular energy reserves and mitochondrial integrity also becomes a significant factor in DM [74]. During DM, fatty acid accumulation leads to both the generation of reactive oxygen species and mitochondrial DNA damage [75]. A decrease in the levels of mitochondrial proteins and mitochondrial DNA in adipocytes has been correlated with the development of type 2 DM [76]. In addition, insulin resistance in the elderly has been linked to fat accumulation and reduction in mitochondrial oxidative and phosphorylation activity [77, 78].

3. Innovative strategies for neurovascular protection during DM

Possible pathways that may decrease neuronal and vascular longevity during DM are broad in scope and involve multiple precipitating factors. Yet, oxidative stress-induced cellular signaling is believed to be one significant factor responsible for cell injury that initially is set in motion following hyperglycemia. For example, studies shave shown that administration of insulin or insulin growth factor at concentrations that were insufficient to reverse hyperglycemia could nevertheless reduce oxidative stress injury to cells and maintain mitochondrial inner membrane potential [1, 5]. As a result, innovative strategies that directly target the reduction of oxidative stress toxicity to neuronal and vascular cells could offer viable therapeutic options to patients with DM in addition to more conventional treatments that are targeted to control serum glucose levels.

4. Nicotinamide, a precursor for the coenzyme β-nicotinamide adenine dinucleotide (NAD⁺)

As the amide form of niacin or vitamin B₃, nicotinamide plays a critical role in cellular metabolism and can offer significant neuronal and vascular cell protection during a wide range of disorders that include DM. Nicotinamide is the precursor for the coenzyme β-nicotinamide adenine dinucleotide (NAD⁺) and is essential for the synthesis of nicotinamide adenine dinucleotide phosphate (NADP⁺) [42, 64]. Nicotinamide and nicotinic acid can be obtained either through synthesis in the body, such as in the liver, or through a dietary source that is rapidly absorbed through the gastrointestinal epithelium. Once nicotinamide is obtained in the body, it is utilized to synthesize NAD⁺ [11].

In clinical studies for DM, oral nicotinamide protects β-cell function, prevents clinical disease in islet-cell antibody-positive first-degree relatives of type-1 DM [79], and in combination therapy with insulin can reduce HbA_1c levels [80]. Potentially relevant to diabetic patients with renal failure, nicotinamide also has been shown to reduce intestinal absorption of phosphate and prevent the development of hyperphosphatemia and progressive renal dysfunction [81]. In animal and cell culture studies, nicotinamide also can maintain normal fasting blood glucose in animals with streptozotocin-induced diabetes [82, 83], reduce peripheral nerve injury during elevated glucose [84], lead to the remission of type 1 DM in mice with acetyl-l-carnitine [85], and can inhibit oxidative stress pathways that lead to apoptosis [63, 86–89].

Nicotinamide can have protean endocrine effects in the body [90] and derive its protective capacity through a number of cellular pathways. In addition to the neuroprotective attributes of nicotinamide [91–93], one potential pathway to consider for the protective capacity of nicotinamide in DM involves the maintenance of vascular integrity [11, 42, 64]. For example, nicotinamide can protect the function of the blood brain barrier [94, 95], influence arteriolar dilatation and blood flow [96], potentially lead to decreased atherosclerotic plaque through inhibition of poly(ADP-ribose) polymerase [97], and promote platelet production through megakaryocyte maturation [98]. Nicotinamide also can maintain EC viability during reactive oxygen species exposure [42, 87, 99, 100]. Nicotinamide is believed to be responsible for the preservation of cerebral [101] and endocardial [102, 103] ECs during models of oxidative stress [102, 103]. Interestingly, during periods such as ischemia and oxidative stress, acidosis-induced cellular toxicity may ensue [27] and lead to subsequent mitochondrial failure [104]. Yet, nicotinamide cannot prevent cellular injury during intracellular acidification paradigms [62].

An alternative mechanism for nicotinamide may require the maintenance of the mitochondrial membrane potential (ΔΨ_m) to protect cells from injury (Figure 1). Nicotinamide can preserve mitochondrial NAD-linked respiration and block the depolarization of the mitochondrial membrane [62, 87]. Interestingly, nicotinamide appears to act directly at the level of mitochondrial membrane pore formation to prevent cytochrome c release [62, 87].

Figure 1. Nicotinamide, Wnt signaling, and erythropoietin employ diverse as well as common pathways to foster cellular longevity.

As illustrated in cell schematic with nucleus, erythropoietin (EPO) and the EPO receptor (EPOR) can increase cellular longevity through protein kinase B (Akt), the forkhead transcription factor family member FOXO3a, glycogen synthase kinase-3β (GSK-3β), nuclear factor-κB (NF-κB), and Bcl-x_L. Similar to EPO, nicotinamide modulates the activity of FOXO3a along with 14-3-3 protein and can maintain cellular integrity and prevent inflammatory activation of microglia that ultimately can lead to apoptosis through the maintenance of mitochondrial membrane potential (ΔΨ_m), the release of cytochrome c, and the prevention of caspase activation. Wnt signaling begins with Frizzled (FZD) receptors resulting in the activation of Dishevelled followed by the inhibition of glycogen synthase kinase (GSK-3β). The suppressed GSK-3β along with other Wnt signaling complexes prevents phosphorylation (p) of β-catenin and leads to the accumulation of β-catenin. β-catenin enters into cellular nucleus and contributes to the formation of Lef/Tcf lymphocyte enhancer factor/T cell factor (Lef/Tcf) and β-catenin complex that may cooperate with factors activated by other signaling pathways resulting in cellular proliferation, differentiation, survival and apoptosis through the induction of target nuclear gene transcription. Interconnected pathways with Wnt, nicotinamide, and EPO involve IκB kinase (IKK), IκB, inhibitors of apoptotic protein (IAPs), GSK-3β, NF-κB, mitochondrial membrane potential (ΔΨ_m), and cytochrome c. Ultimately these pathways converge upon early apoptotic injury with phosphatidylserine (PS) exposure and later apoptotic DNA degradation.

Nicotinamide also can prevent inflammatory cell demise through the maintenance of membrane asymmetry, activation of protein kinase B (Akt), and the inhibition of cytokine release [11, 42, 64]. Nicotinamide blocks membrane PS externalization during a variety of insults that involve anoxia, free radical exposure, and oxygen-glucose deprivation [62, 87, 99]. Nicotinamide regulates membrane PS exposure and microglial activation through activation of Akt, a central pathway for cytoprotection [92]. Phosphorylation of Akt leads to its activation and protects cells against genomic DNA degradation and membrane PS exposure [45, 61, 105]. Up-regulation of Akt activity during multiple injury paradigms, such as vascular and cardiomyocyte ischemia [106, 107], free radical exposure [45, 108], N-methyl-D-aspartate toxicity [109], hypoxia [110, 111], β-amyloid toxicity [112–114], DNA damage [31, 41, 110, 115], metabotropic receptor signaling [38, 116, 117], cell metabolic pathways [42, 63], and oxidative stress [31, 33, 41] increases cell survival. Cytoprotection through Akt also can involve control of inflammatory cell activation [33, 41, 61], transcription factor regulation [118], maintenance of mitochondrial membrane potential (ΔΨ_m), prevention of cytochrome c release [45, 61, 105], and blockade of caspase activity [45, 61, 110]

In addition to targeting the activity of membrane PS exposure and microglial activation, nicotinamide inhibits several pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, interleukin-8, tissue factor, and tumor necrosis factor-α (TNF-α) [119–122]. Nicotinamide also can alter major histocompatibility complexes [123], inhibit intracellular adhesion molecule expression [124], and modulate the production of TNF in vascular cells [123] that may be responsible for the ability of nicotinamide to reduce demyelination in models of multiple sclerosis [125]. However, translation of these experimental studies to clinical efficacy appears to require further work, since some studies show that oral nicotinamide administration following endotoxin challenge in healthy volunteers did not demonstrate a significant effect upon serum cytokine levels [126].

Other studies suggest that nicotinamide also may utilize pathways of the mammalian forkhead transcription factor family that oversees processes that can involve cell metabolism, hormone modulation, and apoptosis [1, 127, 128]. The first member of this family was the Drosophila melanogaster gene Fork head. Since this time, greater than 100 forkhead genes and 19 human subgroups are known to exist that extend from FOXA to FOXS [128]. The forkhead box (FOX) family of genes is characterized by a conserved forkhead domain commonly noted as a “forkhead box” or a “winged helix” as a result of the butterfly-like appearance on X-ray crystallography [129] and nuclear magnetic resonance [130]. All Fox proteins contain the 100-amino acid winged helix domain, but it should be noted that not all winged helix domains are Fox proteins [131].

Of the forkhead transcription factors, FOXO3a is one member that exemplifies the ability to function as a versatile component during normal physiology as well as during disorders such as DM [128]. In relation to the designation of human Fox proteins, all letters are capitalized, otherwise only the initial letter is listed as uppercase. FOXO3a appears to be involved in several pathways responsible for cell metabolism, DM onset, and diabetic complications [57, 117, 132, 133]. A clinical study of 734 individuals that examined all exons of the FOXO genes FOXO1a, FOXO3a, and FOXO4 found one promoter single nucleotide polymorphism in the 5′ flanking region of FOXO3a that displayed a significant association with body mass index such that the highest body mass index was present in individuals homozygous for this allele [134]. Although other studies have reported that haplotype analyses of FOXO1a rather than FOXO3a in individuals is associated with higher HbA_1c levels to suggest evidence of at least an association with disorders of glucose intolerance, FOXO3a haplotypes also have been associated with an increased risk for stroke [135]. In addition, the human immunodeficiency virus (HIV) -1 accessory protein Vpr has been reported to contribute to insulin resistance in HIV patients by interfering with Foxo3a signaling with protein 14-3-3 [136].

In regards to experimental work for DM, administration of a high-fat diet in animals that lead to hyperinsulinemic insulin-resistant obesity was associated with an increased expression of Foxo3a [137]. Some studies have suggested that Foxo3a may be beneficial during elevated glucose exposure and DM. For example, interferon-gamma driven expression of tryptophan catabolism by cytotoxic T lymphocyte antigen 4 may activate Foxo3a to protect dendritic cells from injury in nonobese diabetic mice [138]. Yet, the role of forkhead transcription factors can vary among different cell types and tissues. For example, mice overexpressing FOXO1 in skeletal muscle suffer from reduced skeletal muscle mass and poor glycemic control [139]. Additional investigations have linked diabetic nephropathy to Foxo3a by demonstrating that phosphorylation of Foxo3a increases in rat and mouse renal cortical tissues two weeks after the induction of diabetes by streptozotocin [140]. Furthermore, enteric neurons can be protected from hyperglycemia by glial cell line-derived neurotrophic factor that can affect Akt signaling and prevent Foxo3a activation and nuclear translocation [141]. Interestingly, the ability of Akt to also inhibit pyruvate dehydrogenase kinase-4 expression, a protein that conserves gluconeogenic substrates during DM, requires the inhibition of Foxo3a activity [142].

As a result, FOXO3a has emerged as an important target for DM. Akt can phosphorylate FOXO3a and inhibit its activity to sequester FOXO3a in the cytoplasm by association with 14-3-3 proteins [118, 136, 143–145]. In the absence of inhibitory Akt1 phosphorylation, FOXO3a is activate, can translocate to the nucleus, and controls a variety of functions that involve cell cycle progression, cell longevity, and apoptosis [1, 11, 146]. Control of FOXO3a is considered to be a viable therapeutic target for agents such as metabotropic glutamate receptors [116], neurotrophins [147], and cytokines such as erythropoietin (EPO) [118] to increase cell survival. Given the potential treatment advantages of nicotinamide in DM, it should be of interest that nicotinamide may be cytoprotective through two separate mechanisms of post-translational modification of Foxo3a. Nicotinamide can not only maintain phosphorylation of Foxo3a and inhibit its activity, but also preserve the integrity of the Foxo3a protein [92] to block Foxo3a proteolysis that can yield potentially pro-apoptotic amino-terminal (Nt) fragments [148].

5. Cysteine-rich glycosylated Wnt proteins

Wnt proteins are secreted cysteine-rich glycosylated proteins that can be dependent upon Akt signaling and oversee embryonic cell proliferation, differentiation, survival, and death [44, 149–151]. More than eighty target genes of Wnt signaling pathways have been demonstrated in human, mouse, Drosophila, Xenopus, and zebrafish. This representation encompasses several cellular populations, such as neurons, cardiomyocytes, endothelial cells, cancer cells, and pre-adipocytes [152, 153]. In addition, at least nineteen of twenty-four Wnt genes that express Wnt proteins have been identified in the human.

In general, all Wnt signaling pathways are initiated by interaction of Wnt proteins with Frizzled (FZD) receptors and the binding of the Wnt protein to the FZD transmembrane receptor in the presence of the co-receptor LRP-5/6 [154] (Figure 1). Once Wnt protein binds to the FZD transmembrane receptor and the co-receptor LRP-5/6, this is followed by recruitment of dishevelled, a cytoplasmic multifunctional phosphoprotein [153, 155, 156]. The Wnt-Frizzled transduction pathway plays a significant role in the control of the pattern of the body axis as well as the development and maturation of the central nervous system [157, 158], cardiovascular system [159–162], and the limbs [163]. During embryological development, alternations of the Wnt-FZD pathway can lead to abnormal morphogenesis in animal models [158, 164, 165] and congenital defects in humans [166–168]. In mature tissues, the Wnt-FZD pathway is involved in the self-renewal of pluripotent embryonic stem cells [169], bone formation [170], and may be responsible for the maintenance of many normal tissues [171–174] as well as cellular senescence [175]. Other studies have revealed that dysfunction of the Wnt-FZD pathway can lead to neurodegenerative disorders, such as Alzheimer’s disease [149, 176–179] and heart failure [44, 180–182].

Wnt signaling can prevent cell injury through β-catenin/Tcf transcription mediated pathways [183] and against c-myc induced apoptosis through cyclooxygenase-2 and Wnt induced secreted protein [184]. However, more recent work has linked Wnt cytoprotection in neuronal and vascular cells with more unconventional pathways of Wnt that involve Akt. For example, neuronal cell differentiation that is dependent upon Wnt signaling and trophic factor induction is blocked during the repression of Akt activity [185] and Wnt differentiation of cardiomyocytes does not proceed without Akt activation [186]. Soluble secreted FZD-related proteins, which can modulate Wnt signaling, also employ Akt for cardiac tissue repair [187]. Reduction in tissue injury through Wnt signaling during pressure overload cardiac hypertrophy is linked to Akt activation [182] and the benefits of cardiac ischemic preconditioning appear to rely upon Akt [181]. In the neuronal system, Wnt over-expression can independently increase the phosphorylation and the activation of Akt to promote neuronal protection [149]. Inhibition of the phosphatidylinositol 3-kinase (PI 3-K) pathway or gene silencing of Akt expression prevents Wnt from blocking apoptotic injury and microglial activation [149].

Abnormalities in the Wnt signaling pathways, such as with transcription factor 7-like 2 gene, may impart increased risk for type 2 DM in some populations [188–190] as well as have increased association with obesity [191]. Additional work has described the expression of Wnt5b in adipose tissue, the pancreas, and the liver in diabetic patients, suggesting a potential regulation of adipose cell function [192]. Clinical observations in patients with coronary artery disease and the combined metabolic syndrome with hypertension, hyperlipidemia, and DM have observed impaired Wnt signaling through a missense mutation in LRP-6 [193]. Experimental studies in mice that develop hyperglycemia through a high fat diet also demonstrate increased expression of some Wnt family members, such as Wnt3a and Wnt7a [194]. Yet, intact Wnt family members may offer glucose tolerance and increased insulin sensitivity [195] as well as protect glomerular mesangial cells from elevated glucose induced apoptosis [196]. Animals that over-expressed Wnt10b and were placed on a high-fat diet had a reduction in bodyweight, hyperinsulinemia, triglyceride plasma levels, and improved glucose homeostasis [197].

These clinical and experimental investigations for the Wnt pathway suggest a potential protective cellular mechanism for Wnt during DM. Recent in vitro studies demonstrate that the Wnt1 protein is necessary and sufficient to provide cellular protection during elevated glucose exposure [150]. Administration of exogenous Wnt1 protein can significantly prevent apoptotic EC injury during elevated glucose exposure. Interestingly, this protection by Wnt1 can be regulated by the growth factor and cytokine EPO [132, 133, 198]. Through the Wnt pathway, EPO may offer an attractive therapy to maintain proper cellular metabolism and mitochondrial membrane potential (ΔΨ_m) during conditions of oxidative stress and DM. In cell culture and animal studies, EPO is cytoprotective during elevated glucose [150] and has the capacity to prevent the depolarization of the mitochondrial membrane that also affects the release of cytochrome c [105, 106, 110]. With the Wnt pathway, EPO maintains the expression of Wnt1 during elevated glucose exposure and prevents loss of Wnt1 expression that would normally occur in the absence of EPO during elevated glucose. In addition, blockade of Wnt1 with a Wnt1 antibody can neutralize the protective capacity of EPO, illustrating that Wnt1 is a critical component in the cytoprotection of EPO during elevated glucose exposure [150].

Interestingly, Wnt also can protect cells during oxidative stress [152] and other toxic injuries such as β-amyloid toxicity [152] through the modulation of glycogen synthase kinase-3β(GSK-3β) and β-catenin [149]. Inhibition of GSK-3β activity can increase cell survival during oxidative stress and, as a result, GSK-3β is considered to be a therapeutic target for some neurodegenerative disorders [10, 179, 199, 200]. GSK-3β also may influence inflammatory cell survival [32] and activation [201]. In regards to metabolic disease, inactivation of GSK-3β by small molecule inhibitors or RNA interference prevents toxicity from high concentrations of glucose and increases rat beta cell replication, suggesting a possible target of GSK-3β for pancreatic beta cell regeneration [202]. Clinical applications for Wnt that involve GSK-3β are attractive [203], especially in concert with EPO. For example, both the potential benefits of EPO to improve cardiovascular function in diabetic patients [204, 205] and the positive effects of exercise to improve glycemic control during DM [206] appear to rely upon the inhibition of GSK-3β activity. EPO blocks GSK-3β activity [39, 150, 207] and combined with exercise may offer synergistic benefits, since physical exercise also has been shown to phosphorylate and inhibit GSK-3β activity [208].

6. Erythropoietin, a cytokine and growth factor

EPO is a 30.4 kDa glycoprotein with approximately fifty-percent of its molecular weight derived from carbohydrates [133]. As a growth factor and cytokine, EPO is considered to be ubiquitous in the body [1, 5], since it can be detected in the breath of healthy individuals [209]. EPO also may provide developmental cognitive support in humans with the recent observations that elevated EPO concentrations during infant maturation have been correlated with increased Mental Development Index scores [210]. Although EPO is currently approved for the treatment of anemia, the role of EPO has become far more reaching beyond the need for erythropoiesis in other organs and tissues, such as the brain, heart, and vascular system [45, 110, 118, 211–213]. It is the discovery of EPO and the EPO receptor (EPOR) in the nervous and vascular systems that has resulted in a heightened level of interest and enthusiasm for the potential clinical applications of EPO, such as in Alzheimer’s disease, cardiac insufficiency [214, 215], and cardiac transplantation [216, 217]. In the nervous system, the major sites of EPO production and secretion are in the hippocampus, internal capsule, cortex, midbrain, cerebral ECs, and astrocytes [218, 219]. Further work has revealed several other organs as secretory tissues for EPO that include peripheral ECs [220], myoblasts [221], insulin-producing cells [222], and cardiac tissue [133, 223].

As a strong cytoprotectant against oxidative, stress EPO can enhance the survival of a number of cells in the nervous system [132, 133, 224]. In cells that involve the brain or the retina, EPO can prevent injury from hypoxic ischemia [45, 110, 225–227], excitotoxicity [228, 229], infection [230], free radical exposure [61, 105, 229], amyloid exposure [112], staurosporine [231], and dopaminergic cell injury [232]. In addition, administration of EPO also represents a viable option for the prevention of retinal cell injury during glutamate toxicity [233] and glaucoma [234]. Systemic application of EPO also can improve functional outcome and reduce cell loss during spinal cord injury [235, 236], traumatic cerebral edema [237], cortical trauma [238], and epileptic activity [211, 239]. In direct relation to the potential protective cerebral effects of EPO, enhanced survival by EPO also extends to afford protection of the neurovascular unit during cerebral vascular disease [17, 240]. In addition, EPO can protect sensitive hippocampal neurons from both focal and global ischemic brain injury [227, 241]. Systemic administration of EPO also represents a viable option for several other disorders. EPO administration for retinal cell injury can protect retinal ganglion cells from apoptosis [242], EPO can improve functional outcome and reduce lipid peroxidation during spinal cord injury [243], and EPO can maintain autoregulation of cerebral blood flow, reverse basilar artery vasoconstriction, and enhance neuronal survival and functional recovery following subarachnoid hemorrhage [244].

EPO also plays a significant role in the cardiovascular system [132, 133] and in the renal system [245] to limit injury from oxidative stress that ultimately can affect the function of the nervous system. For example, in patients with anemia, EPO administration can increase left ventricular ejection fraction and stroke volume [246]. More recent studies have shown that patients with acute myocardial infarction have increased plasma EPO levels within seven days of a cardiac insult, suggesting a possible protective response from the body [247]. In addition, EPO administration in patients with anemia and congestive heart failure can improve exercise tolerance, renal function, and left ventricular systolic function [215, 248]. Tightly integrated with cardiac performance, pulmonary function also is believed to be enhanced during EPO administration, especially in the setting of ischemic reperfusion injury of the lung [249]. Serum levels of EPO also may function as a biomarker for cardiovascular injury [250]. Work from experimental studies illustrates that EPO plays a critical role in the vascular and renal systems with the maintenance of erythrocyte [251] and podocyte [252] integrity, regulates the survival of ECs [61, 110], and may act as a powerful endogenous protectant during cardiac injury [253].

In light of the fact that antioxidants during elevated glucose concentrations can block free radical production and prevent the production of advanced glycation end-products known to produce reactive oxygen species and oxidative stress during DM [254], EPO may offer an attractive alternative therapy to maintain proper cellular metabolism and mitochondrial membrane potential (ΔΨ_m) during DM (Figure 1). In clinical studies with DM, plasma EPO is often low in diabetic patients with anemia [255] or without anemia [256]. Furthermore, the failure of these individuals to produce EPO in response to a declining hemoglobin level suggests an impaired EPO response in diabetic patients [257]. Yet, increased EPO secretion during diabetic pregnancies may represent the body’s attempt at endogenous protection against the complications of DM [258]. Similar to the potential protective role of insulin [259], EPO administration has been shown both in diabetics as well as non-diabetics with severe, resistant congestive heart failure to decrease fatigue, increase left ventricular ejection fraction, and significantly decrease the number of hospitalization days [205]. In studies that examine the toxic effects of elevated glucose upon vascular cells, EPO is protective and prevents early apoptotic membrane PS exposure and late DNA degradation at concentrations that are clinically relevant [110] to cellular protection in patients with cardiac or renal disease [260, 261].

Also relevant to cellular metabolism and DM management, cellular protection by EPO is closely tied to Akt and the maintenance of mitochondrial membrane potential (ΔΨ_m) to prevent cell injury and the subsequent induction of the apoptotic cascades [110, 118, 211]. Similar to nicotinamide, EPO may require other substrates of the Akt pathway, such as the forkhead transcription factor FOXO3a to prevent cell injury. FOXO3a interfaces with several pathways that regulate cellular lifespan [146] and function to control neoplastic growth [262]. In relation to cell survival, EPO controls the phosphorylation and degradation of Foxo3a to retain it in the cytoplasm through binding to 14-3-3 protein and foster vascular cell protection during oxidative stress [118].

Cytoprotection by EPO also is mediated through the activation of nuclear factor-κB (NF-κB) tied to Akt. NF-κB proteins are composed of several homo- and heterodimer proteins that can bind to common DNA elements. It is the phosphorylation of IκB proteins by the IκB kinase (IKK) and their subsequent degradation that lead to the release of NF-κB for its translocation to the nucleus to initiate gene transcription [263]. Dependent upon Akt controlled pathways, the transactivation domain of the p65 subunit of NF-κB is activated by IKK and the IKKκ catalytic subunit to lead to the induction of protective anti-apoptotic pathways [264]. Increased expression of NF-κB during injury can occur in cells, such as in inflammatory microglial cells [32, 112, 265] and in neurons [266]. NF-κB represents a critical pathway that is responsible for the activation of inhibitors of apoptotic proteins (IAPs), the maintenance of Bcl-x_L expression, [27, 267], and protection against cell injury during oxidative stress [112]. EPO employs NF-κB to prevent apoptosis through the enhanced expression and translocation of NF-κB to the nucleus to elicit anti-apoptotic gene activation [39, 112, 268, 269].

7. Controversies and challenges for clinical applications

Application of innovative agents and pathways can offer great promise to extend neuronal and vascular cell longevity during conditions such as DM, but treatments that depend upon nicotinamide, Wnt signaling, or EPO may stray from perfection for some patients and ultimately lead to disease progression or other consequences. For example, nicotinamide has been reported to have diverse biological roles that include cellular lifespan reduction. Prolonged exposure to nicotinamide in some studies can lead to impaired β-cell function and reduction in cell growth [270, 271]. Nicotinamide also may inhibit P450 and hepatic metabolism [272] and play a role in the progression of Parkinson’s disease if cellular compartmentation is abruptly changed [273]. Under other conditions, nicotinamide has been described as an agent that limits cell growth and promotes cell injury. Nicotinamide in the presence of transforming growth factor β-1 can block hepatic cell proliferation and lead to apoptosis with caspase 3 activation [274]. During moderate temperature hyperthermia or carbogen breathing, nicotinamide also can result in enhanced solid tumor radiosensitivity and assist with tumor load reduction [275]. In addition, nicotinamide offers cellular protection in millimole concentrations against oxidative stress, but in relation to cell longevity, lower concentrations of nicotinamide can function as an inhibitor of sirtuins that are necessary for the promotion of increased lifespan in yeast and metazoans [11, 276, 277]. Interestingly, it has been postulated that sirtuins may prevent nicotinamide from assisting with DNA repair by altering the accessibility of DNA damaged sites for repair enzymes [278]. Given the intimate and inverse relationship with nicotinamide and cell longevity, alternative approaches for the protection of neuronal and vascular cells during DM may be required that may involve the tight modulation of intracellular nicotinamide accumulation.

If one examines the Wnt pathway, Wnt signaling can either facilitate or prevent apoptosis depending upon the environmental stimuli. For example, Wnt proteins can enhance apoptosis within rhombomeres 3 and 5 in the developing hindbrain and in limb buds during vertebrate limb development to control growth of the hindbrain and limbs [279–281]. Wnt signaling also has been closely linked to tumorigenesis for a number of years [44, 282]. Furthermore, in studies that involve DM, neuronal disorders, or vascular disease, it is not consistently clear whether mutations in genes of the Wnt pathway or alterations in protein expression of the Wnt pathway components during these disorders confer protective or detrimental effects.

With annual sale revenues in the United States for EPO reported to approach 9 billion dollars [283], adverse effects or lack of efficacy during treatment with EPO are also becoming increasingly evident. Some cardiac injury experimental models do not consistently demonstrate a benefit with EPO [284] and elevated plasma levels of EPO independent of hemoglobin concentration can be associated with increased severity of disease in individuals with congestive heart failure [285] or contribute to vascular stenosis with intima hyperplasia [286]. Other adverse conditions associated with EPO can include increased incidence of thrombotic vascular effects, elevation in mean arterial pressure, and increased metabolic rate and blood viscosity [133, 287]. The potential progression of cancer has been another significant concern raised with EPO administration [288, 289]. Not only has both EPO and its receptor been demonstrated in tumor specimens, but under some conditions EPO expression has been suggested to block tumor cell apoptosis through Akt [290], enhance tumor progression, increase metastatic disease, [291], decrease survival in cancer patients [292], and negate the effects of radiotherapy by assisting with tumor angiogenesis [293]. In consideration of the possible tumor promoting ability of EPO [294], a number of competing factors must be considered that include the possible benefits of EPO administration in patients with cancer that involve the synergistic effects of EPO with chemotherapeutic modalities [295, 296], potential protection against chemotherapy tissue injury [297], and the treatment of cancer-related anemia.

For innovative therapeutic strategies to effectively and safely work against the complications of DM and address effective approaches to extend neuronal and vascular cell longevity, future studies that involve basic and clinical research must carefully and systematically address both the potential benefits and the detriments of new therapies. Critical to this process is the targeted focus upon intricate and often common cellular pathways governed by potential strategies, such as nicotinamide, Wnt signaling, and EPO to overcome the present challenges and controversies of existing or developing therapies. As a result, enthusiasm for new therapeutic agents to preserve neuronal and vascular longevity during debilitating conditions such as DM will continue to grow at an exponential pace and avoid clinical complications to offer patients the best available care.