Version 2018-08-29 The GOP Research Project Possible Role of the Glucosylamine Oxidation Pathway in Alzheimer’s

Hexokinase Glucosylamine ATP ADP +1 NADPH Oxidation Non-enzymatic αVal1 Glucosylamine βVal1-NH2 Pathway glycation Pentose

+ αVal1-NH2 αVal1-Imine αVal1 Phosphate Glucose Glut1 Glucose βVal1 Glucosylamine + βVal1-NH βVal1-Imine βVal1 Fructosamine Non-enzymatic Pathway 2 DHA oxidation +1 NADPH βVal1 Glucosylamine βVal1-NH2 AA Gluconic acid + βVal1-NH2 Gluconic acid AA DHA

GSSG GSH Net Metabolic Result Glut1 PPP GOP DHA AA NADPH +2 0 Intracellular Ascorbate 0 +1 Intra/Extracellular Oxidized lipoprotein Reduced lipoprotein Gluconate 0 +1 Intra/Extracellular • ATP -1 0 H2O OH

Figure 1. The Glucosylamine Oxidation Pathway Chapter Links A. Introduction B. Biological Variation in HbA1c 1. The hemoglobin glycation index (HGI) 2. The glucosylamine oxidation pathway (GOP) 3. HGI reflects variation in GOP activity 4. A unifying GOP hypothesis C. Key Alzheimer’s Disease Observations 1. A high GA/HbA1c ratio is associated with increased risk for Alzheimer’s disease 2. A high GA/HbA1c ratio is evidence of a low HGI phenotype 3. Is variation in GOP activity associated with risk for Alzheimer’s disease? D. Alzheimer’s Disease Review 1. Alzheimer’s disease is a neurodegenerative disorder 2. Apolipoprotein E polymorphism is associated with risk for Alzheimer’s disease 3. ApoE4 is the ancestral isoform 4. ApoE4 is associated with both harms and benefits 5. exacerbates Alzheimer’s disease pathology 6. resistance and are associated with Alzheimer’s disease in non-diabetic subjects 7. Insulin degrading enzyme (IDE) hypofunction adversely affects beta amyloid 8. GLP-1 analogs and DPP4 inhibitors promote insulin activity and improve cognitive function 9. Is Alzheimer’s disease a condition of oxidative or reductive ? 10. RBC Glut1 transporter levels are elevated in both early and late onset Alzheimer’s disease 11. Low glutathione and vitamin C are characteristic of Alzheimer’s disease

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1. Low HGI and Alzheimer’s disease are both associated with and reductive stress 2. Low HGI and ApoE4 are both associated with lower risk for nephropathy in 3. Low HGI is associated with higher ApoE4 allele and genotype frequency 4. Can high RBC Glut1 transporter density mechanistically explain low HGI in Alzheimer’s disease? 5. Alzheimer’s disease and salsalate therapy are both associated with higher plasma pentosidine levels F. Summary 1. HGI is a comprehensive biomarker of systemic redox status 2. HGI and the antagonistic pleiotropy theory of aging 3. Future directions G. References

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A. Introduction The hemoglobin glycation index (HGI) identifies people who have (HbA1c) levels that are lower or higher than average compared to other people with similar glucose levels (1). As detailed below, we and others have reported that individuals with a high HGI phenotype (i.e. higher HbA1c than predicted by blood glucose) have greater risk for chronic vascular disease. In contrast, a recent report by Mukai et al. (2) suggests to us that people with Alzheimer’s disease (AD) have a low HGI phenotype (i.e. lower HbA1c than predicted by blood glucose). Based on both laboratory and clinical studies we proposed (3) that person-to-person and within-person variation in HGI can be mechanistically explained by variation in glucose flux through the glucosylamine oxidation pathway (GOP), a previously unrecognized pathway of glucose metabolism that our research suggests has a central role in the regulation of systemic redox homeostasis (Figure 1). Evidence of a mechanistic link between variation in GOP activity and AD could help identify new AD diagnostic criteria and novel interventions. B. Biological Variation in HbA1c B1. The hemoglobin glycation index (HGI) Home The HbA1c test is used clinically for the diagnosis and management of diabetes. The intended target of standardized HbA1c assays are hemoglobin molecules that are chemically modified by glucose on the N-terminal valine of the beta globin subunit (βVal1) in a stable closed chain fructosamine configuration. HGI measures the quantitative difference between an individual’s observed HbA1c and a predicted HbA1c determined by inserting a date-matched blood glucose concentration into a linear regression equation that describes the relationship between HbA1c and blood glucose in a population (1). Individuals with a low or high HGI phenotype have lower or higher than average HbA1c, respectively, compared to other people with similar blood glucose concentrations. We and others have reported that a high HGI phenotype is associated with greater risk for chronic vascular disease in non-diabetic (4-6), type 1 diabetic (7), prediabetic (8) and type 2 diabetic (9-12) subjects. B2. The glucosylamine oxidation pathway (GOP) Home The proposed GOP consists of a non-enzymatic glycation phase and a non-enzymatic oxidation phase (Figure 1). The glycation phase begins when glucose enters red blood cells (RBC) by facilitated diffusion via the Glut1 transporter and forms highly unstable open chain imines in a spontaneous reaction with the βVal1 amino group. Three things can then happen to the imine, it can 1) dissociate to the free amino group and glucose, 2) cyclize to the relatively more stable glucosylamine, or 3) cyclize to the much more stable fructosamine. The intramolecular rearrangements that form imines, glucosylamines and are reversible, with ring opening and closing occurring much more rapidly for glucosylamines than fructosamines. The oxidation phase begins when oxidized vitamin C (DHA, dehydroascorbic acid) enters RBC, not accidentally by the same Glut1 transporter as glucose, and spontaneously reacts with βVal1 glucosylamine in an irreversible oxidation-reduction reaction that produces gluconic acid, ascorbic acid (AA) and the free βVal1 amino group. Although the glycation and oxidation reactions are also thermodynamically favorable for the N-terminal valine of alpha globin (αVal1), the chemical formation of imines, glucosylamines and fructosamines, as well as glucosylamine oxidation, all occur more rapidly with the βVal1 amino group due to the catalytic activity of the histidine imidazole at position 2 of beta globin (βHis2) (3). B3. HGI reflects variation in GOP activity Home Kinetic GOP modeling studies (3) indicate that with glucose held constant, higher rates of βVal1 glucosylamine oxidation increase gluconic acid production, decrease the rate at which glucose carbon chains accumulate in the 3 Version 2018-08-29 The GOP Research Project

βVal1 fructosamine (HbA1c) compartment, and lower HGI. Based on these observations we propose that 1) population variation in HGI is primarily a consequence of person-to-person variation in βVal1 glucosylamine oxidation, and 2) low and high HGI are caused by higher and lower βVal1 glucosylamine oxidation, respectively. A key feature of the proposed GOP is that βVal1 glucosylamine oxidation is cooperatively/competitively influenced by the use of GSH in vitamin C recycling. From the GOP perspective, βVal1 fructosamine (HbA1c) level can be viewed as a complex quantitative trait that reflects the combined effects of multiple known and unknown genetic and environmental factors that collectively influence non-enzymatic βVal1 glucosylamine synthesis (supply) and non-enzymatic βVal1 glucosylamine oxidation (demand). As depicted in the model (Figure 1), chief among known factors that affect supply and demand are Glut1 transporter activity and the concentrations of glucose, βVal1 amino groups, DHA and reduced glutathione (GSH). Because of how HGI is calculated, it mathematically factors out the effect of blood glucose concentration (supply) on population variation in βVal1 fructosamine (HbA1c) levels. Consequently, if βVal1 fructosamine (HbA1c) level is a function of factors affecting βVal1 glucosylamine supply and demand, and HGI factors out the effect of βVal1 glucosylamine supply, then we can conclude that HGI is a biomarker of variation in βVal1 glucosylamine demand controlled for the effects of blood glucose concentration. Simply put, at any blood glucose concentration more glucose carbon chains will end up as βVal1 fructosamine (HbA1c) and HGI will be higher in individuals with lower βVal1 glucosylamine demand. In contrast, more glucose carbon chains will end up as gluconic acid and HGI will be lower in individuals with higher βVal1 glucosylamine demand. The fact that HGI tends to be consistent within individuals over long periods of time and over a wide range of blood glucose concentrations (13) suggests that βVal1 glucosylamine supply and demand increase proportionally with glucose concentration. B4. A unifying GOP hypothesis Home Based on our analysis of the analytical chemistry, biochemistry and clinical chemistry of hemoglobin glycation we proposed the following unifying GOP hypothesis (3): • Hypothesis 1: Natural selection found a way to use the unavoidable chemical reaction between glucose and hemoglobin amino groups to provide context-specific reducing power under the control of insulin and other factors that influence blood glucose concentration And this clinically important extension: • Hypothesis 2: Hyperglycemia evolved as a homeostatic adaptation that generates βVal1 glucosylamine reducing power in response to C. Key Alzheimer’s Disease Observations C1. A high GA/HbA1c ratio is associated with increased risk for Alzheimer’s disease Home Mukai et al. (2) reported that people with AD have higher glycated albumin (GA) to HbA1c ratios. The authors compared three biomarkers of glycemic control (HbA1c, GA and 1,5-anhydroglucitol) in a longitudinal study of 1187 Japanese people 65 years or older without dementia at baseline. There was no association between AD and HbA1c or 1,5-AG in the study population. However, the age and gender-adjusted incidence of AD among non- diabetic subjects was higher in individuals with higher GA/HbA1c ratios. A numerically but not statistically higher ratio was also observed in diabetic subjects with AD. The authors concluded that 1) a high GA/HbA1c ratio is associated with greater risk for AD regardless of glycemic status, and 2) the ratio may be a clinically useful biomarker for predicting incident AD.

4 Version 2018-08-29 The GOP Research Project C2. A high GA/HbA1c ratio is evidence of a low HGI phenotype Home Albumin is an extracellular protein and hemoglobin is an intracellular protein. A high GA/HbA1c ratio identifies people who have disproportionately higher GA levels compared to their HbA1c levels. The glycation gap (GG) is a GA-based index of biological variation in HbA1c that assesses disproportionality between GA (measure of extracellular glycation) and HbA1c (measure of intra-erythrocyte glycation). GG is calculated in exactly the same way as HGI except that GA replaces blood glucose concentration in the linear regression equation used to calculate an individual’s predicted HbA1c (14). We and others have shown that HGI and GG are positively correlated and thus more or less interchangeable indices of biological variation in HbA1c in human populations (15; 16). High HGI and high GG have both been associated with greater risk for chronic vascular disease. What’s important in this discussion is that a high GA/HbA1c ratio is the same as a low GG is the same as a low HGI. C3. Is variation in GOP activity associated with risk for Alzheimer’s disease? Home If a low HGI is equivalent to a high GA/HbA1c ratio, and a high GA/HbA1c ratio is associated with greater risk for AD (2), then it is logical to propose that: • Hypothesis 3: Alzheimer’s disease is associated with a low HGI phenotype Furthermore, if a low HGI phenotype is mechanistically explained by greater GOP activity (3) then it is also logical to propose that: • Hypothesis 4: Alzheimer’s disease is associated with greater GOP activity Chronic oxidative stress is a natural consequence of the characteristic slow, age-related decline in normal systems. Hyperinsulinemia and hyperglycemia are common traits in normal aging, type 2 diabetes and AD, leading some investigators to classify AD as “type 3” diabetes. From the GOP perspective, we propose that hyperglycemia evolved as a way to generate vitamin C reducing capacity in the form of βVal1 glucosylamine (3). More research is needed to determine if the apparent association between AD and low HGI is real and whether or not GOP activity has a cause or effect relationship with AD pathophysiology. D. Alzheimer’s disease review D1. Alzheimer’s disease is a neurodegenerative disorder Home Alzheimer’s disease (AD) is characterized by a progressive cognitive decline that afflicts over 46 million people worldwide (17). The neuropathological hallmarks of AD are 1) intracellular neurofibrillary tangles (NFT) which are paired helical filaments and straight filaments that are constituted of hyperphosphorylated tau protein, neuropil threads (NTs) and dystrophic neurites, and 2) extracellular deposits of beta amyloid, the major component of senile plaques in the (18-20). Despite decades of research, the genetic and metabolic basis of AD pathology remains poorly understood. The amyloid cascade hypothesis remains popular and maintains that increased beta amyloid is the root cause of both rare familial forms of AD and the more common sporadic forms (21). However, total beta amyloid plaque deposition correlates poorly with cognitive impairment or neuronal loss, and treatments designed to intervene in beta amyloid metabolism have not been successful. GWAS studies by Giri et al. (22) show that some variants are associated with early onset AD (EOAD, before age 65) and some with late onset AD (LOAD, over 65). EOAD is caused by rare and dominantly inherited mutations and has a strong genetic component. The three considered the main risk factors for EOAD are all related to beta amyloid production. LOAD is genetically more complex due to the involvement of more than 20 known genes and environmental factors that play a key role in the onset, progression and severity of the disease. The majority of genes associated with LOAD roughly cluster within three pathways 1) metabolism, 2) the inflammatory response, and 3) endocytosis (22). AD associated genes involved in lipid metabolism include 5 Version 2018-08-29 The GOP Research Project apolipoprotein E (ApoE) which is the major cholesterol carrier in the brain. AD associated genes involved in the inflammatory response include CR1 (also known as C3b/C4b receptor or CD35) a single-chain type I transmembrane glycoprotein comprising four main structural domains that is mainly expressed on erythrocytes, leukocytes, and splenic dendritic cells. AD associated genes involved in endocytosis and vesicle-mediated transport include BIN1 which has been implicated in synaptic vesicle endocytosis, intracellular amyloid precursor protein (APP) trafficking, immune response, apoptosis, and clathrin-mediated endocytosis. Drugs currently available for the treatment of AD do not stop the progression of the disease and offer only incremental improvements in symptoms (23). Much research is focused, however, on well-documented observations indicating that ApoE polymorphism and insulin resistance are both strongly associated with risk for Alzheimer’s disease and increased morbidity and mortality in elderly populations in modern industrialized societies. D2. Apolipoprotein E polymorphism is associated with risk for Alzheimer’s disease Home Apolipoproteins are structural components of lipoproteins that transport through the lymphatic and circulatory systems (24). ApoE is mainly produced in the liver and by macrophage peripherally, and by astrocytes in the central nervous system where it is the principal cholesterol carrier in the brain. There are three common ApoE isomers in human populations identified as E2, E3 and E4. ApoE3 (Cys112) and ApoE4 (Arg112) differ by only a single amino acid (25). The cysteine in ApoE3 contains a sulfhydryl side chain that might participate in oxidation-reduction reactions with glutathione and other thiols. The arginine in ApoE4 contains a guanidinium side chain that is protonated and positively charged at physiological pH and can thus interact with negatively- charged non-protein anions and carboxylates (26). The presence of the Arg moiety decreases the stability of the N-terminal domain (27) which enhances ApoE4 binding to triglyceride-rich lipoprotein particles in plasma and to beta amyloid deposits in the brain (28). The arginine side chain has a geometry and charge distribution that is able to form multiple hydrogen bonds and the guanidinium group promotes binding to negatively-charged groups on phosphates. Key structural and functional differences between ApoE4 and ApoE3 include variation in salt bridge interactions, protein stability, molten-like-globule propensity, and relative binding to HDL (higher in ApoE3) or LDL/VLDL (higher in ApoE4) (29). ApoE4 is the most robust known for premature cognitive decline, especially among people of European ancestry (30). Evidence suggests that the ApoE4 allele is a cognitive risk factor in both blacks and whites but has weaker effect in black people (31). Despite racial differences in ApoE4 frequency, resilience factors like literacy and other lifestyle factors are similar across races (30). ApoE4 has been associated with metabolic syndrome in some studies (32) but not others (33). Rasmussen et al. (34) reported that low plasma ApoE levels were associated with increased risk of dementia independent of ApoE genotype. D3. ApoE4 is the ancestral isoform Home ApoE4 is believed to be the evolutionarily oldest ApoE allele in the human genome and the one from which the E2 and E3 alleles were derived (35). Based on its functional properties and population distribution, Corbo and Scacchi (36) suggested that ApoE4 is a thrifty allele, i.e. a gene that enhances fat storage during periods of abundance to be used during periods of food shortage. Transgenic mice expressing the human ApoE4 gene exhibited an ApoE4-directed global metabolic shift toward lipid oxidation and enhanced thermogenesis (37). ApoE3 is the highest frequency allele in all current human populations with a tendency to be highest in regions with long-established agricultural economies like those surrounding the Mediterranean (36). Worldwide, ApoE4 frequency ranges from 3 to 40% and is higher in 1) northern latitudes (e.g. 22.7% in Finland vs. 5.2% in Sardinia), 2) indigenous populations who are or were recent hunter gatherers, and 3) populations characterized by darker skin (35). Eisenberg et al. (38) reported that ApoE4 frequencies show a curvilinear relationship with latitude, with 6 Version 2018-08-29 The GOP Research Project higher frequencies near the equator, decreasing to a nadir in mid-latitudes, and then increasing again closer to the poles. The authors concluded that because metabolic rates are elevated in both hot and cold environments, this pattern of ApoE4 frequency distribution across latitudes is consistent with the hypothesis that the ApoE4 allele was selected for in populations with elevated metabolic rates. This conclusion lends mixed support for an evolutionary link between past global temperatures and human ApoE allele distribution. D4. ApoE4 is associated with both harms and benefits Home Although ApoE4 has been convincingly associated with cognitive harms in elderly human populations (39) there are also reported instances where ApoE4 is beneficial. For example, Jiang et al. (40) reported that Chinese diabetes patients with higher ApoE4 allele or genotype frequencies had lower risk for nephropathy. In stark contrast to modern societies, higher ApoE4 frequency has been associated with greater cognitive function in Amazonian forager-horticulturalists with a high parasite burden (41). Similarly, Vila-Rodriguez et al. (42) reported that ApoE4 was associated with higher fertility rates in rural Ghanaian women exposed to high pathogen levels. Evidence that ApoE4 is associated with higher levels of progesterone supports the hypothesis that ApoE polymorphism is associated with variation in reproductive performance (43). Evidence of higher ApoE4 allele frequency in people who 1) inhabit northern latitudes (areas with diminished sunlight exposure), or 2) have darker skin (which allows less ultraviolet B (UVB) radiation absorption) suggests that ApoE4 is beneficial in populations at greater risk for vitamin D deficiency (35). This so-called UVB hypothesis is based in part on studies reporting higher serum vitamin D levels in people with the ApoE4 allele. A mechanistic link to vitamin D metabolism is suggested by the fact that ApoE transports cholesterol, a precursor in vitamin D synthesis. Multiple studies by Llewellyn and colleagues showed that low serum vitamin D in older adults was associated with increased odds of cognitive impairment in 1) the Health Survey for England 2000 (44), 2) the InCHIANTI study in Italy (45), and 3) the U.S. National Health and Nutrition Examination Survey (NHANES) (46). The fact that ApoE4 has been associated with enhanced fat storage, higher metabolic rates, protection against nephropathy and pathogens, greater fertility, UV exposure and vitamin D metabolism suggests that the ApoE4 allele was historically and perhaps remains beneficial for human species adaptation and survival. D5. Diabetes exacerbates Alzheimer’s disease pathology Home Diabetes is a widely recognized risk factor for Alzheimer’s disease (23). Ott et al. (47) reported that diabetes almost doubled the risk for dementia in elderly people, and that diabetes patients treated with insulin had higher dementia risk than those treated with other drugs. Ravona-Springer et al. (48) reported that among type 2 diabetes subjects, higher mean HbA1c levels were associated with lower cognitive performance in ApoE4 carriers but not in non-carriers. The authors suggested that ApoE4 carriers may be more vulnerable to the insults of poor glycemic control. Variation in blood glucose dynamics has also been associated with the severity of AD pathology. Long- term glycemic variability, as measured by the standard deviation of individual HbA1c over time, was associated with significantly higher white matter hyperintensity (a measure of ischemic injury and cortical/subcortical atrophy) in ApoE4 genotype carriers with type 2 diabetes (49). Li et al. (50) reported that the coefficients of variation of both FPG and HbA1c were significant predictors of AD even after adjustment for sociodemographic factors, lifestyle behaviors, diabetes-related variables, FPG, HbA1c, drug-related variables, and comorbidities. In contrast, Pruzin et al. (51) found no relationship between diabetes or HbA1c and global or regional AD pathology and suggested that the diabetes-dementia link is based on subcortical vascular pathology and not on regional AD pathology.

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D6. Insulin resistance and hyperglycemia are associated with Alzheimer’s disease in non-diabetic subjects Home Fava et al. (52) conducted annual metabolic and neuropsychological assessments in 335 diabetic patients and 142 non-diabetic subjects over a 6.8 y period. Subjects were subdivided into groups with low, moderate or high insulin resistance as measured by HOMA-IR. Compared to baseline, significant cognitive decline was observed at the end of the study in both diabetic and non-diabetic subjects with high baseline HOMA-IR. Pavlik et al. (53) reported that higher serum insulin was associated with significantly worse cognitive performance in both AD patients and non-demented older individuals, even after statistically controlling for variation in HbA1c or ApoE4 polymorphism. Carantoni et al. (54) reported that nondiabetic patients with vascular dementia or senile dementia characteristic of AD had higher fasting glucose and insulin levels than healthy controls. Razay et al. (55) also reported significantly higher mean plasma glucose in patients with AD compared to cognitively normal controls. Wagner et al. (56) reported lower BMI, lower blood pressure and higher fasting plasma glucose levels in dementia cases vs. controls over a 14 year follow-up period. Although Fujisawa and colleagues (57) reported no differences in fasting plasma insulin or glucose between AD patients and normal controls, the authors did observe a significantly greater increase in plasma insulin in the AD group during an oral (OGTT). Insulin resistance and hyperinsulinemia rarely exist in isolation in nondiabetic populations (58). Although a preponderance of reports support a link between higher insulin and greater AD risk, low insulin has also been associated with increased risk of dementia. Peila et al. (59) reported a U-shaped relationship where both low and high insulin levels were associated with AD in a population of Japanese-American men that included both diabetic and non-diabetic subjects. In a non-diabetic, mixed gender population of cognitively normal older Korean adults, Byun et al. (60) reported that lower insulin levels, but not higher HbA1c, was associated with increased beta amyloid positivity and decreased cerebral glucose metabolism. In contrast, higher HbA1c, but not higher insulin level, was positively associated with neurodegeneration in the AD-signature region. The authors concluded that whereas low insulin levels appear to contribute to increased cerebral beta amyloid deposition and neurodegeneration in non-diabetic subjects, impaired glycemic control as assessed by HbA1c appears to aggravate neurodegeneration through a non-amyloid mechanism. One non-amyloid mechanism that might explain the relationship between higher insulin and greater AD risk is insulin activation of glycogen synthase kinase 3beta (GSK-3) which not only phosphorylates glycogen synthase but also tau proteins that promote the formation of NFT (61). To date, however, studies of associations between GSK-3 polymorphism and AD risk have produced equivocal results (62; 63). D7. Insulin degrading enzyme (IDE) hypofunction adversely affects beta amyloid metabolism Home Besides insulin stimulation of tau phosphorylation by GSK-3, another possible mechanistic explanation for the relationship between higher insulin and greater AD risk is insulin-degrading enzyme (IDE) hypofunction. IDE is a zinc-binding metalloprotease that competitively cleaves insulin, amylin (which inhibits glucagon secretion) and beta amyloid (64-66). Qiu and Folstein (67) suggested that because IDE enzymatically degrades both insulin and beta amyloid, high insulin levels increase beta amyloid levels by outcompeting beta amyloid for proteolytic degradation by IDE. IDE is expressed in all tissues and its levels can be modulated by many signals, including cellular stress, glucagon, and free fatty acids (68). Farris et al. (69) reported that IDE knockout mice exhibited characteristics similar to those observed in humans with AD, including 1) decreased beta amyloid degradation in brain membrane fractions, 2) increased cerebral accumulation of endogenous beta amyloid, 3) decreased insulin degradation in liver, 4) hyperinsulinemia, and 5) glucose intolerance. The authors concluded that IDE hypofunction may contribute to some forms of AD and type 2 diabetes.

8 Version 2018-08-29 The GOP Research Project A role for IDE hypofunction in AD pathology is also supported by Cook et al. (70) who reported that hippocampal IDE mRNA levels averaged 50% lower in ApoE4 subjects. Given that ApoE4 is strongly associated with AD, the authors suggested that genetic risk associated with AD may be mediated in part by ApoE4 downregulation of IDE expression. Du et al. (71) reported that ApoE4 down-regulates IDE expression in by stimulating the N- methyl-d-aspartic acid (NMDA) receptor, an ion channel protein in nerve cells that allows positively charged ions to flow through a membrane when activated by glutamate and glycine. Polymorphisms in the IDE gene have been associated with susceptibility for LOAD (72) although a meta-analysis of published studies found only weak associations between AD and IDE polymorphism (73). A review by Qiu et al. (67) suggests that hyperinsulinemia and IDE gene variations were only related to the risk of AD when the ApoE4 allele was absent. The IDE hypofunction hypothesis for explaining the relationship between hyperinsulinemia and AD was questioned on the grounds that IDE was not secreted by cultured embryonic kidney cells or immortalized microglial cells (74). D8. GLP-1 analogs and DPP4 inhibitors promote insulin activity and improve cognitive function Home Glucagon-like peptide-1 (GLP-1) is an insulinotropic secreted by intestinal cells in response to food intake (75). GLP-1 receptor agonists and GLP-1 mimetics are used clinically in people with type 2 diabetes to upregulate GLP-1 activity. This in turn 1) stimulates pancreatic insulin release, 2) inhibits pancreatic glucagon release, and 3) slows glucose uptake from the intestine and helps control postprandial glucose levels. Perry and Greig (75) reported that GLP-1 has a role in neuronal development and the regulation of food intake. Growing evidence suggests that GLP-1 provides neuroprotection across a range of experimental models of AD (76). Treatment with the GLP-1 analog exendin-4 decreased the severity of diabetes and brain levels of beta amyloid in a streptozotocin-treated transgenic AD mouse model (77). Holscher (78) noted that GLP-1 mimetics cross the blood–brain barrier and showed impressive neuroprotective effects in numerous preclinical studies of AD, Parkinson’s disease, stroke and other neurodegenerative disorders. GLP-1 mimetics like exendin-4, liraglutide and lixisenatide have been shown to reduce beta amyloid plaques, prevent loss of synapses and impairments, and reduce oxidative stress and chronic inflammation in the of animal models of AD. Exogenous GLP-1 mimetics clearly show promise as a novel treatment for neurodegenerative conditions. So does bariatric surgery, which reportedly can delay or prevent the onset of Alzheimer's disease by upregulating GLP-1 activity (79). Ghanim et al. (80) reported that Roux-en-Y reversed the proinflammatory state of and was associated with a concomitant decrease in the expression of amyloid precursor protein (APP) and other AD-related genes in peripheral blood mononuclear cells (PBMC). The authors hypothesized that obesity and caloric intake could modulate the expression of APP in the brain which could have important implications for the pathogenesis and treatment of AD. Dipeptidyl peptidase-4 (DPP4) is a serine exopeptidase that cleaves X-proline dipeptides from the N-termi of GLP-1 and a host of other , chemokines and neuropeptides (81). DPP4 is mainly secreted by adipocytes and endothelial cells and has a central role as a regulatory protease for cytokines, chemokines, and neuropeptides involved in inflammation, immunity, and vascular function (82). DPP4 inhibits insulin-stimulated Akt phosphorylation in muscle and adipocytes thus impeding insulin signaling and Glut4 translocation. Because DPP4 inactivates GLP-1, DPP4 inhibitors are used clinically to extend naturally produced GLP-1 and insulin activity in diabetes patients. Isik et al. (83) assessed the effects of the DPP4 inhibitor sitagliptin on cognitive function in elderly diabetic patients with and without cognitive impairment. After 6 months there was no difference in body weight, BMI or HbA1c between the two groups. However, insulin use declined and sitagliptin improved cognitive assessment scores in elderly diabetic patients with or without AD. Zheng et al. (81) reported that high plasma DPP4 levels were associated with mild cognitive impairment in a cross-sectional sample of 1,160 elderly Chinese type 2 diabetes patients, none of whom were being treated with drugs known to affect cognitive function or DDP4 9 Version 2018-08-29 The GOP Research Project activity. Patients with higher DPP4 activity tended to be older with longer diabetes duration, higher BMI, more , more NSAID use, higher insulin requirement and higher levels of triglycerides, LDL- cholesterol, HbA1c, IL-6, CRP, nitrotyrosine and 8-iso-PGF2a. Patients in the highest quartile of DPP4 had significantly lower scores compared to those in the lowest quartile. The authors suggested that their observations could be mechanistically explained by a pro-inflammatory effect of DPP4 and an inadequate systemic response to oxidative stress. D9. Is Alzheimer’s disease is a condition of oxidative or reductive stress? Home Redox homeostasis is best defined as the continuously challenged balance between oxidative and reductive forces that maintains a healthy physiological state (84). Oxidative stress is a consequence of the imbalance between systemically produced reactive oxygen species and an organism’s capacity to detoxify reactive intermediates and repair oxidative damage. Reductive stress is an overexpression of antioxidant forces that produces excess reducing equivalents that can deplete physiologically important reactive oxidative species and disrupt normal redox homeostasis (85). Reductive stress has been implicated in the development of Alzheimer’s disease and other inflammation-associated including cardiomyopathy, muscular dystrophy, pulmonary , Parkinson’s disease, rheumatoid arthritis and metabolic syndrome (85). Vergallo et al. (86) reported low levels of biomarkers of oxidative stress and high levels of in individuals with AD compared to patients with mild cognitive impairment. Lloret et al. (87) reported that individuals with the ApoE4 allele suffer reductive stress long before the onset of the disease and even before the occurrence of mild cognitive impairment. Badia et al. (88) reported that young, healthy ApoE4 carriers exhibited characteristics of reductive stress based on observations of lower blood levels of oxidized glutathione (GSSG), lower lymphocyte phospho-p38 (P-p38, biomarker of oxidative stress), greater lymphocyte expression of glutamylcysteinyl ligase (GCL, first step in glutathione synthesis) and greater lymphocyte expression of glutathione peroxidase (GPx, which uses GSH to detoxify lipid peroxides and hydrogen peroxide). It is important to note that while younger ApoE4 carriers exhibited characteristics of reductive stress, the investigators found that compared to age matched controls, older ApoE4 carriers with diagnosed Alzheimer’s disease exhibited characteristics of oxidative stress based on the same set of biomarkers. One possible source of inflammation with AD onset is glycation of macrophage migration inhibitory factor (MIF) which downregulates MIF participation in the immune response of the brain (89). Another possible source of inflammation is senile plaques which are increasingly deposited in brain tissue over time. The plaques become surrounded by activated microglia which are resident innate brain immune cells that secrete a diverse array of proinflammatory molecules in response to contact with fibrillar beta-amyloid (90). The source of fibrillar beta amyloid induced reactive oxygen species is primarily microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multicomponent enzyme complex that produces the highly reactive free superoxide. Perez-Torres et al. (85) suggested that chronic reductive stress triggers back-and-forth episodes where systems responsible for maintaining redox homeostasis first overproduce more reducing agents, then produce more oxidizing agents to counterbalance, then more reducing agents in a back and forth feedback loop where reductive stress stimulates oxidative stress which stimulates reductive stress and so on, very slowly producing biomolecules that accumulate with pathological consequences over long modern day lifespans. Collectively, these observations suggest that AD can be a condition of both oxidative and reductive stress, which perhaps explains why therapies intended to augment antioxidant defenses have been generally shown to be ineffective in the extension of life span as reported by Sohal and Orr (91). The authors suggest that chronic consumption of antioxidant supplements, such as vitamins or flavonoids, may actually have pro-oxidant effects that unnaturally alter cellular redox equilibrium and contribute to reductive stress in ways that diminish life

10 Version 2018-08-29 The GOP Research Project expectancy. The authors further proposed the “redox stress hypothesis" of aging in which aging-associated functional losses are primarily caused by a progressive pro-oxidizing shift in the redox state of cells, which leads to the over-oxidation of redox-sensitive protein thiols (possibly including ApoE4 Cys112?) and the consequent disruption of redox-regulated signaling mechanisms. D10. RBC Glut1 transporter levels are elevated in both early and late onset Alzheimer’s disease Home Varady et al. (92) used flow cytometry to show that red blood cell (RBC) Glut1 transporter levels and RBC insulin receptor (IR) expression were elevated in both early and late onset AD patients compared to age-matched controls. Glut1 bi-directionally transports glucose by insulin-independent facilitated diffusion and is the primary glucose transporter of RBC, blood-brain barrier endothelial cells and astrocytes (93). Glut1 gene expression is tissue- dependently regulated by glucose, hypoxia, insulin and growth hormones. In contrast to the higher RBC Glut1 levels observed by Varady et al. (92), a review of brain glucose transporters by Szablweski (94) concluded that brain tissue levels of Glut1 and ATP production were lower in AD, especially in the , resulting in glucose hypometabolism and deficits in energy. Willette et al. (95) reported that insulin resistance measured by HOMA-IR was associated with significantly lower regional cerebral glucose metabolism measured by FDG-PET in a cross-sectional study of cognitively normal late middle-aged adults. Winkler et al. (96) reported that congenital Glut1 deficiency syndrome worsens Alzheimer's disease cerebrovascular degeneration, neuropathology and cognitive function in mice overexpressing beta amyloid precursor protein. Diabetes has also been associated with variation in RBC Glut1 levels. Garg et al. (97) reported 50% lower RBC Glut1 protein levels in pediatric patients and that HbA1c levels were negatively correlated with glucose influx. In contrast, Harik et al. (98) used cytochalasin B binding to report 22% higher RBC Glut1 levels in diabetes patients compared to controls. The authors concluded that chronic hyperglycemia upregulates RBC Glut1 transporter density. Kinetic studies by Bistritzer et al. (99) showed that RBC 3-O-methyl glucose uptake was significantly greater in diabetes patients and that Vmax (maximum velocity) was over 60% higher in RBC from diabetic patients while Km (glucose concentration required to achieve half Vmax) was not different between diabetic and control subjects. Porter-Turner et al. (100) compared glucose transporter function in RBC in a study population of 30 non-diabetic and 30 type 2 diabetic subjects. Although no differences were observed in phosphorylation rates or transporter density, RBC from diabetes patients had higher binding affinity and decreased glucose transport. D11. Low glutathione and vitamin C are characteristic of Alzheimer’s disease Home The endogenous antioxidant glutathione (GSH) declines with ageing and in several age-related degenerative diseases, including AD (101; 102). Liu et al. (103) reported lower RBC glutathione levels in males with AD compared to normal controls, but no difference in RBC glutathione in women with or without AD. Although some studies have also implicated glutathione S-transferase (GST) in AD pathology, a meta-analysis by Wang (104) found no association between GST polymorphisms and risk or morbidity in subjects with AD. Glyoxalase I uses GSH in reactions that detoxify dicarbonyls and suppress AGE formation. Chen et al. (105) reported that glyoxalase I was upregulated in brain tissues from mutant tau transgenic mice that are used as a model for AD. The authors also identified a single nucleotide polymorphism in the glyoxalase I gene as a focal point of selective forces in ethnically diverse human populations. Kharrazi et al. (106) compared biomarkers of redox status in 91 AD patients and 91 healthy age- and gender-matched controls. The results showed that compared to controls, AD patients had 1) lower serum total antioxidant status (TAS), 2) lower erythrocyte levels of the antioxidant enzymes catalase (CAT) and glutathione peroxidase (Gpx), but 3) higher levels of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD). Moreover, AD patients with the ApoE4 allele had even lower TAS, CAT and Gpx,

11 Version 2018-08-29 The GOP Research Project and higher SOD, than non-ApoE4 AD patients. Minghetti et al. (107) reported that serum total anti-oxidant capacity measured by copper reduction was lower in AD patients than in controls, and that higher anti-oxidant capacity was associated with longevity in AD patients. RBC and glutathione play crucial roles in vitamin C metabolism and systemic redox homeostasis (Figure 1). Reduced vitamin C (ascorbic acid, AA) enters most cells via high affinity, high specificity sodium and energy- dependent SLC2A transporters capable of generating a high intracellular concentration of vitamin C relative to plasma. In contrast, RBC lose SLC2A transporters during maturation, after which Glut1 transports glucose, oxidized vitamin C (dehydroascorbic acid, DHA) and glucosamine by facilitated diffusion. High glucose concentrations have been empirically shown to competitively inhibit cellular DHA uptake by Glut1 in a concentration-dependent manner (93; 108). Once inside RBC, DHA is reduced to AA both enzymatically and non-enzymatically in reactions involving reduced glutathione. Oxidized glutathione (GSSG) is then recycled by glutathione reductase which uses NADPH produced by the pentose phosphate pathway to regenerate GSH. Unlike most other species, humans, higher primates and guinea pigs are unable to synthesize vitamin C and thus have a dietary requirement (109). Vitamin C plays a role in neuronal differentiation, maturation, myelin formation and modulation of the cholinergic, catecholinergic, and glutaminergic systems in both cognitively intact and impaired individuals (110). Vitamin C has been regarded as the most important antioxidant in neural tissue because it decreases beta amyloid generation and acetylcholinesterase activity, and prevents endothelial dysfunction by regulating which has recently been implicated in the pathogenesis and progression of AD (111). Meta-analysis suggested that AD patients are at risk of low vitamin C and other nutritional insufficiencies because of both physiological and psychological factors (112). Accumulating evidence in a mouse model of accelerated senescence indicates a rescuing role of ascorbic acid in premature aging and supports a role for ascorbic acid in ameliorating factors linked to AD pathogenesis (113). However, clinical trials using antioxidants, including vitamin C, in patients with AD have yielded equivocal results (111). E. Alzheimer’s Disease from the GOP Perspective E1. Low HGI and Alzheimer’s disease are both associated with insulin resistance and reductive stress Home As reviewed in sections D6-8, AD has been associated with both hyperinsulinemia and hyperglycemia leading some to propose classifying AD as type 3 diabetes (61; 114). We discussed how higher plasma insulin levels have been consistently associated with greater cognitive dysfunction in both diabetic and non-diabetic populations. We reviewed the interrelated ways higher insulin levels can arise by insulin resistance at the Glut4 transporter level, IDE hypofunction, GLP-1 hyperfunction, DPP4 hypofunction, and exogenous insulin administration. We also discussed how higher insulin might be mechanistically linked to AD pathophysiology by 1) insulin stimulation of tau phosphorylation by GSK-3, and 2) competitive degradation of insulin and beta amyloid by IDE. Although IDE hypofunction and exogenous insulin in diabetes patients are associated with worse AD outcomes, enhancing endogenous insulin activity by administration of GLP-1 analogs or DPP4 inhibitors has beneficial effects on cognition in people with AD. This seeming inconsistency about the effects of insulin attests to the complexity of the highly coordinated metabolic processes involved, as do observations reviewed in section D9 indicating that AD has been associated with both reductive and oxidative stress. Observations of reductive stress and insulin resistance in AD are consistent with higher βVal1 glucosylamine reducing capacity and higher HOMA-IR that we have observed in people with a low HGI phenotype. We previously reported, for example, that low HGI was associated with higher levels of “labile A1c” as quantified by capillary isoelectric focusing (CIEF) in pediatric type 1 diabetes patients (115). This suggests that βVal1 glucosylamine reducing capacity was higher in the low HGI subjects because as we now know the CIEF labile 12 Version 2018-08-29 The GOP Research Project

A1c fraction contains only βVal1 glucosylamine (116). We also observed evidence of relative insulin resistance in low HGI people as part of an analysis of data collected by the National Health and Nutrition Examination Survey (NHANES). We published results of our NHANES study showing that high HGI was associated with higher levels of circulating biomarkers of inflammation (C-reactive protein, monocytes and polymorphonuclear cells) (117). What we did not publish, mainly because we didn’t know what to make of the results at the time, was that low HGI was associated with higher plasma insulin and insulin resistance as measured by HOMA-IR in non-diabetic, non-obese NHANES participants. Intriguingly, the association between HGI and HOMA-IR disappeared in obese subjects. From the GOP perspective, insulin resistance in AD appears to be a homeostatic response to oxidative stress that increases blood glucose and the constitutive rate of βVal1 glucosylamine synthesis. It is important to note that measuring βVal1 glucosylamine levels tells us little about the rate of βVal1 glucosylamine synthesis. That’s because any pro-glycation effect of higher blood glucose concentration on βVal1 glucosylamine supply (Figure 1) could be offset by an equal anti-glycation effect of higher DHA concentration on βVal1 glucosylamine demand thus producing no net effect on βVal1 glucosylamine levels despite spontaneously higher rates of synthesis at higher glucose concentrations. E2. Low HGI and ApoE4 are both associated with lower risk for nephropathy in type 2 diabetes Home As reviewed in sections D2-4, ApoE4 is harmfully associated with risk for AD. In contrast, ApoE4 has been beneficially associated with lower risk for kidney disease in diabetes patients. Jiang et al. (40) studied 845 Chinese type 2 diabetes patients who were divided into groups with or without diabetic nephropathy. Their results showed that ApoE4 allele frequency was significantly higher in the group without kidney disease (11.5% vs. 7.9%, p<0.05). ApoE3/E4 and E4/E4 genotypes were also significantly higher in the nephropathy-free control group vs. the diabetic nephropathy group (20.0% vs. 14.9%, p<0.05). The authors concluded that ApoE4 may play a protective role against diabetic nephropathy in type 2 diabetes patients. Lower risk for nephropathy is also a characteristic of a low HGI phenotype. We and others have reported that low HGI is associated with lower risk for microvascular disease, including retinopathy and nephropathy in non- diabetic subjects (118) or in type 1 (7) or type 2 (9) diabetes patients. The observation that low HGI and ApoE4 are both associated with lower risk for diabetic nephropathy suggests that HGI and ApoE metabolism or function could be mechanistically related. Evidence that ApoE polymorphism was not a strong risk factor for diabetic nephropathy and retinopathy in type 1 diabetes (119) suggests that the influence of ApoE polymorphism on nephropathy may be limited to people with type 2 diabetes. The observation that retinopathy was not associated with dementia risk in type 1 diabetes supports the hypothesis that the pathophysiological processes underlying dementia may be different in type 1 and type 2 diabetes (120). E3. Low HGI is associated with higher ApoE4 allele and genotype frequency Home As discussed in section D2, ApoE4 allele frequency is strongly associated with risk for AD. ApoE4 is the ancestral isoform which differs from the more recent ApoE3 isoform by a single Arg to Cys substitution. This small difference in amino acid composition is responsible for a variety of structural and charge differences between the two ApoE isoforms. From the GOP perspective, it seems possible that glutathione could play a role in maintaining the redox state of the ApoE3 cysteine sulfhydryl group. If so, and we accept the proposed competitive/cooperative relationship between GSH and βVal1 glucosylamine (Figure 1), the ApoE3 genotype could increase demand for GSH reducing power for sulfhydryl group reactions which could in turn increase demand for βVal1 glucosylamine for vitamin C recycling. In contrast, the guanidinium side chain of the ApoE4 arginine could play

13 Version 2018-08-29 The GOP Research Project a role in glycation reactions through its interactions with negatively charged molecules like phosphates, which regulate Glut1 transport (121) and catalyze glycation of βVal1 amino groups (122). Data from studies by Crane et al. (123; 124) hint at a relationship between low HGI and high ApoE4 allele frequency but the story is a little complicated. In the first of their two publications the authors reported that “higher glucose levels may be a risk factor for dementia, even among persons without diabetes”. This attracted our attention because higher blood glucose levels spontaneously produce higher βVal1 glucosylamine levels which could indicate that ApoE4 subjects have constitutively greater vitamin C recycling capacity. The authors’ conclusion about glucose levels is very misleading, however, because the investigators didn’t directly measure glucose levels. Instead, the authors reported their results in estimated average glucose (eAG) units, a metric derived from HbA1c based on a regression equation produced by the A1c Derived Average Glucose (ADAG) study (125). We previously demonstrated that eAG is a poor estimate of mean blood glucose because it systematically underestimates measured mean blood glucose in low HGI subjects and overestimates mean blood glucose in high HGI subjects (126). The upshot is that people with a high eAG in the Crane studies had higher HbA1c than predicted by blood glucose, which is the defining characteristic of a high HGI phenotype. Although the authors’ first report concluded that high eAG “may be a risk factor for dementia” their second report failed to duplicate the previously reported association between high eAG and greater cognitive risk (124). For the purposes of this discussion, however, what is most interesting about the second study by Crane et al. (124) is their observation that ApoE4 allele frequency in non-diabetic subjects was higher in low eAG subjects (32%, n=102) compared to moderate (27%, n=110) or high (22%, n=106) eAG subjects. Because low eAG and low HGI are essentially equivalent, this is the same as saying ApoE4 allele frequency was higher in low HGI subjects. E4. Can high RBC Glut1 transporter density mechanistically explain low HGI in Alzheimer’s disease? Home As discussed in section D10, reports by Varady et al. (92) and other investigators indicate that RBC from people with AD have higher RBC Glut1 transporter density. If we accept that 1) low HGI is a characteristic of AD, and 2) AD is associated with higher RBC Glut1 transporter levels, then by extension we can hypothesize that low HGI is mechanistically associated with higher RBC Glut1 transporter levels. We originally considered this hypothesis about 20 years ago when we first recognized the HGI phenomenon. At that time, glucose concentration was widely considered to be the only clinically relevant factor that affected population variation in glycated hemoglobin levels. Studies had shown that Glut1 transporter density affects Vmax (maximal transport velocity) but not Km (the substrate concentration that gives rise to 50% Vmax); which means Glut1 density doesn’t affect equilibrium distribution of glucose. It is important to note that RBC glucose transport is so fast that equilibrium is reached within a matter of seconds whenever there is a change in plasma glucose concentration. In contrast, hemoglobin glycation takes hours or days to reach equilibrium under steady state glucose conditions. We initially reasoned that if 1) intracellular glucose concentration was the only factor that affected hemoglobin glycation, and 2) RBC Glut1 transporter density doesn’t really affect hemoglobin exposure to glucose, then how could Glut1 have a role in the HGI phenomenon? If we now accept, however, that βVal1 fructosamine (HbA1c) accumulation in RBC is also a function of βVal1 glucosylamine oxidation, then population variation in RBC Glut1 density might mechanistically influence HGI if Glut1 transport is a rate limiting step in βVal1 glucosylamine oxidation. The supporting rationale is as follows. First, because βVal1 glycation and oxidation reactions are both non-enzymatic (Figure 1), we know that with all other conditions equal the rates for both reactions will be directly proportional to substrate concentrations. The substrates for the glycation reaction are deprotonated βVal1 amino groups and open chain glucose molecules. Under normal physiological conditions, over 99% of the glucose molecules in blood are in an unreactive closed 14 Version 2018-08-29 The GOP Research Project

chain glucopyranose or glucofuranose configuration. Moreover, because the pKa of the βVal1 amino group is close to intra-erythrocyte pH, only about half of the βVal1 amino groups will be unprotonated and reactive under normal conditions. Thus the concentrations of open chain glucose and unprotonated βVal1 amino groups are major determinants of how fast the non-enzymatic glycation phase spontaneously converts glucose and hemoglobin into βVal1 glucosylamine. Similarly, the concentrations of βVal1 glucosylamine and DHA are the major determinants of how fast the non-enzymatic oxidation phase spontaneously converts the glucose carbon chain into gluconic acid, while also producing ascorbic acid and the unprotonated βVal1 amino group. One way to look at the two phases of the GOP is that glucose and DHA have opposing pro-glycation (supply) and anti- glycation (demand) effects, respectively, on the accumulation of βVal1 fructosamine (HbA1c) in RBC. Predicting the effects of βVal1 glycation and oxidation reactions on HGI becomes more complicated, however, when variation in RBC Glut1 transporter activity is added to this model. In the following thought experiments, the biochemical differences between two hypothetical individuals are limited to three variables, 1) glucose concentration, 2) DHA concentration, and 3) RBC Glut1 density. To simplify the process, all other conditions are held constant, including factors like the concentrations of hemoglobin and organic and inorganic compounds like hydrogen ions, glutathione and phosphates that can influence transport, glycation and oxidation reactions. The analyses are further limited to the fasting state in order to model what happens inside RBC under steady state conditions. We will also mostly ignore competition between βVal1 glucosylamine and glutathione in vitamin C recycling, and the fact that gluconic acid produced by βVal1 glucosylamine oxidation could lower intracellular pH and increase the number of protonated and thus unreactive βVal1 amino groups. We will also mostly ignore the fact that DHA and glucose both enter RBC via the Glut1 transporter, except to note that higher glucose concentrations can significantly inhibit Glut1 uptake of DHA but not vice versa. That’s because although the Km for glucose and DHA transport by Glut1 are similar (127), the concentration of glucose is about a hundred times greater than that of DHA. Our calculations suggest that doubling the concentration of glucose will increase the rate of Glut1 glucose transport by only 10% while decreasing the rate of DHA transport by 40%. In contrast, doubling the concentration of DHA would nearly double the rate of DHA transport (90% increase) while only decreasing the rate of glucose transport by 1%. Although the six scenarios below discuss how HGI would be affected by characteristics that differ between two individuals (inter-individual variation), the conclusions also apply to changes within an individual over time (intra-individual variation). Other factorial combinations, like higher plasma glucose in someone with lower DHA, are also possible but are not necessary to review for this discussion. 1. High plasma glucose. Individuals A and B have similar plasma DHA concentrations and RBC Glut1 densities, but A has higher fasting plasma glucose (Figure 1). Higher plasma glucose spontaneously increases RBC Glut1 uptake and intra-erythrocyte glucose concentration. With all other conditions equal, higher intracellular glucose concentrations will increase the rates of βVal1 glucosylamine and βVal1 fructosamine (HbA1c) synthesis. As described in section B3, we propose that βVal1 fructosamine (HbA1c) levels reflect the balance between forces that affect βVal1 glucosylamine synthesis (supply) and oxidation (demand), while HGI is primarily a measure of βVal1 glucosylamine demand caused by factors other than blood glucose concentration. Our research has conclusively shown that HGI tends to be quantitatively and directionally consistent within individuals over time and across the physiological range of blood glucose concentrations (126). This evidence of intra-individual HGI consistency at different glucose levels is consistent with the hypothesis that βVal1 glucosylamine supply and demand both increase proportionally across the physiological range of blood glucose concentrations. In this single variable high glucose scenario, HGI would not differ between A and B. 15 Version 2018-08-29 The GOP Research Project

2. High plasma DHA. Individuals C and D have similar plasma glucose concentrations and RBC Glut1 density, but C has higher plasma DHA (Figure 1). This combination of factors would spontaneously and proportionally increase intracellular DHA concentration and the rate of βVal1 glucosylamine oxidation (demand). Under steady state conditions, the result would be greater production of ascorbic acid (AA) and gluconic acid, less accumulation of glucose carbon chains in the βVal1 fructosamine (HbA1c) compartment, and lower HGI. The magnitude of the decrease in HGI would be inversely proportional to the difference in DHA concentration. In this single variable high DHA scenario, HGI would be lower in C than D. 3. High RBC Glut1 density. Individuals E and F have similar plasma glucose and DHA concentrations, but E has higher RBC Glut1 density (Figure 1). Although higher Glut1 density can increase the maximal rate of transport (Vmax), intracellular glucose concentration reaches equilibrium so rapidly that higher RBC Glut1 density by itself would have little effect on the overall exposure of βVal1 amino groups to glucose or DHA, and thus have no effect on βVal1 glucosylamine supply or demand. In this single variable high Glut1 scenario, HGI would not differ between E and F. 4. High plasma glucose and high plasma DHA. Individuals G and H have similar RBC Glut1 density, but G has higher plasma glucose and higher DHA concentrations (Figure 1). Higher plasma glucose by itself proportionally increases both βVal1 glucosylamine supply and βVal1 glucosylamine demand and thus has no net effect on HGI as described in scenario 1. Higher DHA by itself spontaneously increases intracellular DHA concentration and the rate of non-enzymatic βVal1 glucosylamine oxidation (demand) resulting in less accumulation of glucose carbon chains in the βVal1 fructosamine (HbA1c) compartment and lower HGI as described in scenario 2. If we assume that the effects of high glucose and high DHA are additive, then HGI would be lower in G than H in this multivariable high glucose and high DHA scenario. 5. High plasma glucose and high RBC Glut1 density. Individuals I and J have similar plasma DHA concentrations, but I has higher plasma glucose and higher RBC Glut1 density (Figure 1). Higher plasma glucose by itself proportionally increases both βVal1 glucosylamine supply and βVal1 glucosylamine demand and thus has no net effect on HGI as described in scenario 1. Higher RBC Glut1 density by itself has no effect on the overall exposure of βVal1 amino groups to glucose or DHA, and thus no effect on βVal1 glucosylamine supply or demand as described in scenario 3. If we assume that the effects of high glucose and high RBC Glut1 density on HGI are additive, then HGI would not differ between I and J in this multivariable high glucose and high Glut1 scenario. 6. High DHA concentration and high RBC Glut1 density. Individuals K and L have similar fasting plasma glucose concentrations, but K has higher plasma DHA and higher RBC Glut1 density (Figure 1). Higher DHA by itself would spontaneously increase intracellular DHA concentration and the rate of non-enzymatic βVal1 glucosylamine oxidation (demand) resulting in less accumulation of glucose carbon chains in the βVal1 fructosamine (HbA1c) compartment and lower HGI as described in scenario 2. Higher RBC Glut1 density by itself would increase Vmax but have no effect on the overall exposure of βVal1 amino groups to glucose as described in scenario 3. If we assume that the effects of high DHA and high RBC Glut1 density on HGI are additive, then HGI would be lower in K than L for reasons described in scenario 2. It is possible, however, that the combined effects of high DHA and high Glut1 would be multiplicative, i.e. that RBC Glut1 density could amplify the effect of DHA on βVal1 glucosylamine oxidation (demand). This would only be true, however, if Glut1 transport is a rate limiting step for DHA transport and βVal1 glucosylamine oxidation. In this case the higher Vmax associated with higher Glut1 density could increase DHA uptake and amplify GOP throughput capacity. If we assume the effects of high DHA and high RBC Glut1 density on HGI are

16 Version 2018-08-29 The GOP Research Project multiplicative, then HGI would be even lower than expected with higher DHA alone in this multivariable high DHA and high Glut1 scenario. These limited mechanistic thought experiments consider how the non-enzymatic βVal1 glycation and oxidation reactions of the GOP might be affected by variation in glucose, DHA and RBC Glut1 density. While recognizing many potential caveats, we conclude that variation in blood glucose concentration likely has little effect on HGI by itself or in combination with variation in DHA concentration or RBC Glut1 density. In contrast, higher DHA concentrations should 1) increase βVal1 glucosylamine demand, 2) decrease βVal1 fructosamine (HbA1c) accumulation, and 3) lower HGI. Although higher RBC Glut1 density by itself would not be expected to affect βVal1 glucosylamine oxidation rates, variation in RBC Glut1 density might modulate GOP throughput capacity if Glut1 DHA transport is a rate limiting step in the GOP. Better understanding of the possible role of Glut1 in GOP biochemistry could have important clinical implications for conditions where RBC Glut1 activity varies from normal as reported for Alzheimer’s disease (92), diabetes (97) and Glut1 deficiency syndrome (128) or as might be caused by Glut1 polymorphisms (129; 130). E5. Alzheimer’s disease and salsalate therapy are both associated with higher pentosidine levels Home Meli et al. (131) reported that serum pentosidine levels were higher than normal in non-diabetic AD patients and concluded that pentosidine could be a useful biomarker of AD. But where did the pentosidine come from? Pentosidine is a crosslinked lysine-arginine advanced glycation end product (AGE) that can be non-enzymatically derived from either glucose or oxidized vitamin C (DHA) (132-134). Higher DHA concentration can drive the conversion of DHA to pentosidine (135-137). Because RBC have an important role in vitamin C recycling, one way to increase the intra-erythrocyte DHA concentration would be to decrease vitamin C recycling capacity (Figure 1). One known way that decreases vitamin C recycling capacity would be to lower glutathione reducing capacity either by 1) lowering glutathione synthesis, 2) increasing competition for GSH for use in other biochemical reactions, or 3) decreasing the availability of NADPH for glutathione recycling. Glutathione insufficiency and low antioxidant status are common in people with AD and other age-related degenerative diseases (101; 102). From the GOP perspective, however, a second way to decrease vitamin C recycling capacity and increase DHA conversion to pentosidine might be to lower βVal1 glucosylamine synthesis. Acetylsalicylate (aspirin) competitively inhibits the reaction between glucose and the βVal1 amino group (138). From the GOP perspective this would be expected to lower βVal1 glucosylamine synthesis and vitamin C recycling capacity. Salsalate is composed of two salicylates attached together to form a reactive ester. Barzilay et al. (132) reported that salsalate therapy reduced HbA1c and plasma levels of glucose and some types of early and advanced glycation products in type 2 diabetes patients, including furosine and carboxymethyllysine. These apparent beneficial effects of salsalate were surprisingly offset, however, by the presence of much higher plasma pentosidine levels. The authors noted that “Possible explanations for the rise in pentosidine include excess production of a pentosidine precursor, such as the vitamin C oxidation product dehydroascorbate.” If salsalate, like acetylsalicylate, inhibits βVal1 amino group glycation, then the higher pentosidine levels observed in diabetes patients receiving salsalate therapy might be due to excessive accumulation of intra-erythrocyte DHA caused by βVal1 glucosylamine insufficiency. Salsalate has other proposed effects, however, including uncoupling mitochondrial proton conductance, suppressing de novo lipogenesis and improving glycemia in overweight subjects with diabetes (139; 140). Salsalate also activates brown adipose tissue in mice via the PKA pathway (141) and reportedly protects mice from high fat diet-induced obesity (142). In the PS19 transgenic mouse model of frontotemporal dementia, administration of salsalate after disease onset inhibited acetyltransferase p300 activity, lowered levels of total tau 17 Version 2018-08-29 The GOP Research Project and acetylated tau, rescued tau-induced memory deficits and prevented hippocampal atrophy (19). Salsalate and other inhibitors of tau protein acetylation are actively being studied for use in the treatment of tau pathology and AD (18; 143). An ongoing clinical trial (https://www.nia.nih.gov/alzheimers/clinical-trials/salsalate-mild- moderate-alzheimers-disease) is studying the feasibility of salsalate for treatment of Alzheimer’s disease. F. Summary Higher GA/HbA1c ratios reported in AD subjects by Mukai et al. (2) suggest that people with AD have lower HGI and higher rates of βVal1 glucosylamine oxidation. Understanding why GOP activity might be greater than “normal” in Alzheimer’s disease could help mechanistically explain some of the pathophysiology observed in cognitively impaired people. F1. HGI is a comprehensive biomarker of systemic redox status Home Based on the GOP hypothesis, we propose that βVal1 glucosylamine is a physiologically important reducing agent with a role in vitamin C recycling and in the maintenance of systemic redox homeostasis (3). From the GOP perspective (Figure 1), βVal1 glucosylamine levels inside RBC are a function of factors that affect βVal1 glucosylamine synthesis (supply) and those that affect βVal1 glucosylamine oxidation (demand). Intra- erythrocyte glucose concentration is the main determinant of βVal1 glucosylamine supply and intra-erythrocyte DHA concentration is the main determinant of βVal1 glucosylamine demand. But RBC glucose and DHA concentrations are themselves determined by genetic and environmental variation in Glut1 transport, glutathione metabolism and a multitude of other biochemical factors that collectively produce characteristic person-to-person variation in βVal1 fructosamine (HbA1c) levels and HGI. We further propose that HGI is a biomarker of GOP activity. Look at it this way. Because the glycation (supply) and oxidation (demand) reactions are both non-enzymatic, anything that increases the βVal1 glucosylamine oxidation rate will decrease the rate at which βVal1 glucosylamine carbon chains rearrange to the βVal1 fructosamine (HbA1c) configuration. Because of how it is calculated, HGI mathematically factors out the effect of blood glucose concentration (supply) on βVal1 fructosamine (HbA1c) concentrations. Thus while βVal1 fructosamine (HbA1c) level is a function of βVal1 glucosylamine supply and demand, HGI factors out the main effector of supply (glucose concentration) and can thus be considered a function of person-to-person variation in βVal1 glucosylamine demand (oxidation), where low and high HGI reflect greater and lower βVal1 glucosylamine demand, respectively. In this scenario, an HGI of zero reflects average βVal1 glucosylamine demand in people with a “normal” metabolic phenotype. An HGI greater than zero indicates that an individual’s βVal1 glucosylamine demand is lower than normal resulting in more glucose carbon chains ending up as βVal1 fructosamine (HbA1c). An HGI less than zero indicates that an individual’s βVal1 glucosylamine demand is higher than normal resulting in more glucose carbon chains ending up as gluconic acid and fewer as βVal1 fructosamine (HbA1c). Low HGI in Alzheimer’s disease suggests that AD is a condition of greater than normal βVal1 glucosylamine demand. Low RBC glutathione, low plasma vitamin C, and higher blood glucose levels are common traits in both normal aging and AD. Given the proposed yin-yang relationship between GSH and βVal1 glucosylamine in vitamin C recycling (Figure 1), we propose that hyperinsulinemia and higher blood glucose in AD is a homeostatic adaptation that increases βVal1 glucosylamine supply in response to diminished antioxidant status. Why only some people with AD deploy hyperinsulinemia and hyperglycemia as a way to boost βVal1 glucosylamine and vitamin C recycling remains unclear. The situation is similar, however, to what is observed in obesity where only

18 Version 2018-08-29 The GOP Research Project 10% of obese people develop type 2 diabetes which, from the GOP perspective, means only 10% deploy hyperinsulinemia and hyperglycemia as a homeostatic response to the oxidative stress of obesity (3). It has been proposed that immunosenescence, defined as an age-related decrease in the ability to respond to foreign antigens and to tolerate self-antigens, leads to increased susceptibility to , cancer, and autoimmune diseases (113; 144). Given that insulin resistance increases with both age and AD we propose that: • Hypothesis 5: Hyperglycemia observed during normal aging and AD is a homeostatic adaptation that upregulates βVal1 glucosylamine reducing capacity in response to immunosenescence and diminished capacity to otherwise maintain systemic redox status within limits compatible with life functions This hypothesis is tempered by the fact that the complex array of interdependent metabolic processes that influence βVal1 glycation and oxidation reactions is far more complex than depicted in Figure 1. There are many known factors that influence blood glucose concentration and βVal1 glucosylamine supply, like insulin, glucagon, IDE, GLP-1, DPP4, Glut1 etc. We can only guess how many unknown factors might also impact blood glucose concentration. Moreover, plasma vitamin C and RBC glutathione are involved in many other essential biochemical redox reactions that can increase demand for both DHA and GSH recycling. And while this discussion focuses on the role of βVal1 glucosylamine in vitamin C recycling, it seems unlikely that oxidation- reduction reactions involving βVal1 glucosylamine are limited to DHA. It is also important to note that the glycation and oxidation reactions that occur with the βVal1 amino group are also thermodynamically favorable with the αVal1 amino group as well, they just do not occur as rapidly because the catalytic activity of βHis2 amplifies βVal1 amino group reactions. A comprehensive understanding of how different metabolic processes combine to mechanistically produce high and low HGI phenotypes, and how different HGI phenotypes produce different age-related pathologies, will require much more research. What we can more easily and usefully understand at this time, however, is that HGI is a biomarker of variation in redox status and that low HGI in AD may be indicative of greater demand for βVal1 glucosylamine reducing capacity. F2. HGI and the antagonistic pleiotropy theory of aging Home Pleiotropy is the well-established phenomenon where a single gene affects multiple traits (145). Antagonistic pleiotropy is the term proposed by Williams (146) to describe the condition where a gene variant is associated with at least one trait that is beneficial for an organism’s fitness and at least one that is harmful. The antagonistic pleiotropy theory of aging holds that natural selection favors alleles that have beneficial effects during reproductive years when selective forces are strong, even if the same alleles have harmful effects later in life when selection is weak. The persistence of low and high HGI phenotypes in modern human societies suggests that metabolic variation promotes extremes of HGI and GOP activity that were likely beneficial for species survival in shorter-lived ancestral humans. Our research suggests, however, that both low and high HGI are associated with different types of age-related pathologies in modern industrialized human societies. The term polygenic pleiotropy has been used to describe polygenic effects in complex disorders and traits (147). If we accept the hypothesis that low and high HGI phenotypes are of polygenic origin and that each has beneficial effects early in life but harmful effects later in life, then we can then propose that: • Hypothesis 6: Low and high HGI phenotypes are both examples of antagonistic polygenic pleiotropy where genetic and environmental variation produce characteristic metabolic phenotypes with early-life benefits but late-life harms Golubev et al. (148) proposed a theory that harmonizes the evolution-oriented antagonistic pleiotropy theory of aging with the mechanistic-oriented free radical theory of aging. As stated by the authors “Chemically reactive metabolites spontaneously modify slowly renewable macromolecules in a continuous way over time; the resulting 19 Version 2018-08-29 The GOP Research Project buildup of damage wrought by the genes coding for enzymes that generate such small molecules eventually masquerades as late-acting pleiotropic effects. In aerobic organisms, ROS are major agents of this damage but they are far from alone.” This merger of the antagonistic pleiotropy and free radical theories emphasizes the fact that normal physiological aging can mechanistically arise from the inherent chemical reactivity of many biomolecules, not just ROS. This view fits nicely with our conclusions that 1) high HGI is associated with chronic vascular disease, a late-acting pathology mechanistically caused by reactive metabolites like glucose and its derivatives that spontaneously modify slowly renewable macromolecules in vascular tissues, whereas 2) low HGI is associated with chronic neurological disease, a late-acting pathology mechanistically caused by reactive metabolites like hyperphosphorylated tau and beta amylin that spontaneously modify slowly renewable macromolecules in neurological tissues. The important question now is what can be done to improve long-term outcomes in people with low or high HGI? F3. Future directions Home Exactly how low and high HGI phenotypes produce different sets of spontaneously reactive metabolites with early- and late-acting pleiotropic effects remains unclear but there are some clues. That glucose metabolism is fundamentally different in low and high HGI phenotypes is supported by our observation that non-diabetic, non- obese NHANES participants with low HGI were mildly insulin resistant compared to participants with moderate or high HGI (3). Because of how HGI is calculated, we know that people with a low HGI phenotype have higher fasting plasma glucose levels compared to others with a moderate or high HGI phenotype and similar βVal1 fructosamine (HbA1c) levels. From the GOP perspective, higher fasting plasma glucose should produce higher rates of non-enzymatic βVal1 glucosylamine synthesis, greater fasting βVal1 glucosylamine reducing power, and constitutively greater vitamin C recycling capacity. Exactly how variation in βVal1 glucosylamine and vitamin C reducing capacity might have benefitted survival in ancestral humans is also unclear but there are some clues here too. For example, we reported that non-diabetic NHANES participants with a low HGI phenotype had lower levels of plasma C-reactive protein and other biomarkers of inflammation after statistically controlling for the effects of obesity (117). From the GOP perspective, one explanation could be that low HGI subjects rely more on insulin resistance and βVal1 glucosylamine for the maintenance of systemic redox homeostasis, while high HGI subjects rely more on GSH or other antioxidant systems. Insulin resistance and higher than normal fasting blood glucose concentrations are characteristic features of a variety of physiological states that are associated with transient or chronic oxidative stress, including aging, obesity, starvation, , immune activation, growth, cancer and . The innate immune system is an evolutionarily ancient collection of non-specific metabolic adaptations that provide immediate defense against infection and inflammation (149). From the GOP perspective: • Hypothesis 7: Hyperglycemia evolved to upregulate βVal1 glucosylamine reducing capacity as part of the innate immune response that helps maintain systemic redox homeostasis during periods of inflammation and oxidative stress Better understanding of how and why βVal1 glucosylamine synthesis and oxidation reactions differ in low and high HGI phenotypes could help explain why extremes of HGI promote such different pathological states. More research into the GOP and its role in systemic redox homeostasis could help mechanistically connect the dots between HGI, energy metabolism and immune function in ways that ultimately lead to new interventions that prevent or delay cardiovascular disease, dementia and other age-related chronic disorders.

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