There is increasing evidence that genome-wide association (GWA) studies represent a powerful approach to the identification of genes involved in common human diseases. We describe a joint GWA study (using the Affymetrix GeneChip 500K Mapping Array Set) undertaken in the British population, which has examined approximately 2,000 individuals for each of 7 major diseases and a shared set of approximately 3,000 controls. Case-control comparisons identified 24 independent association signals at

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5m genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N≤71,225 European ancestry, N=12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N=29,136). We identified association between systolic or diastolic blood pressure and common variants in 8 regions near the CYP17A1 (P=7×10−24), CYP1A2 (P=1×10−23), FGF5 (P=1×10−21), SH2B3 (P=3×10−18), MTHFR (P=2×10−13), c10orf107 (P=1×10−9), ZNF652 (P=5×10−9) and PLCD3 (P=1×10−8) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

Word count: 5056. 2 SYNOPSISAbnormal cellular accumulation of the dicarbonyl metabolite methylglyoxal occurs on exposure to high glucose concentration, inflammation, cell ageing and senescence. It is associated with increased methylglyoxal-adduct content of protein and DNA linked to increased DNA strand breaks and mutagenesis, mitochondrial dysfunction and reactive oxygen species formation and cell detachment from the extracellular matrix. Methylglyoxalmediated damage is countered by glutathione-dependent metabolism by glyoxalase-1. It is not known, however, if glyoxalase-1 has stress responsive up-regulation to counter periods of high methylglyoxal concentration or dicarbonyl stress. We identified a functional antioxidant response element in the 5'-untranslated region of exon-1 of the mammalian glyoxalase-1 gene. Transcription factor Nrf2 binds to this antioxidant response element increasing basal and inducible expression of glyoxalase 1. Activators of Nrf2 induced increased glyoxalase-1 mRNA, protein and activity. Increased expression of glyoxalase-1 decreased cellular and extracellular concentrations of methylglyoxal, methylglyoxal -derived protein adducts, mutagenesis and cell detachment. Hepatic, brain, heart, kidney and lung glyoxalase-1 mRNA and protein were decreased in Nrf2 (-/-) mice and urinary excretion of methylglyoxal protein and nucleotide adducts were increased ca. 2-fold. We conclude that dicarbonyl stress is countered by up-regulation of glyoxalase-1 in the Nrf2 stress responsive system, protecting protein and DNA from increased damage and preserving cell function.Key words: Nrf2, glyoxalase, methylglyoxal, DNA damage, protein damage, glycation.Abbreviations used: AITC, allyl isothiocyanate; ARE, antioxidant response element; CDDOMe, methyl 2-cyano-3,12-dioxo-oleana-1,9(11)dien-28-oate; ChIP, chromatin immunoprecipitation; CNC, cap 'n' collar; ECL, enhanced chemiluminescence; Glo1, glyoxalase 1; keap1, kelch (β-propeller tertiary structure)-like erythroid cell-derived protein with CNC homology-associated protein 1; IRE, insulin response element; MG, methylglyoxal; MGdG, 3-(2'-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purin-9(8)one; MG-H1, N  -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine; Nrf2, nuclear erythroid factor E2 related factor-2; ROS, reactive oxygen species; SFN, sulforaphane; TBST, tris-buffered saline with Tween-20. 3 INTRODUCTIONModification of proteins and DNA by the dicarbonyl metabolite methylglyoxal (MG) has emerged as an important endogenous threat to the functional integrity of the proteome and genome. MG is formed by the spontaneous degradation of triosephosphate intermediates and is an unavoidable by-product of anaerobic glycolysis [1]. MG reacts with proteins and DNA forming quantitatively major adducts of endogenous damage, similar to and in some cases exceeding the steady-state levels of adducts produced by oxidative damage. Modification of proteins by MG is directed to arginine residues forming the hydroimidazolone, N  -(5-hydro-5-methyl-4-imi...

Risk of insulin resistance, impaired glycemic control, and cardiovascular disease is excessive in overweight and obese populations. We hypothesized that increasing expression of glyoxalase 1 (Glo1)-an enzyme that catalyzes the metabolism of reactive metabolite and glycating agent methylglyoxal-may improve metabolic and vascular health. Dietary bioactive compounds were screened for Glo1 inducer activity in a functional reporter assay, hits were confirmed in cell culture, and an optimized Glo1 inducer formulation was evaluated in a randomized, placebo-controlled crossover clinical trial in 29 overweight and obese subjects. We found trans-resveratrol (tRES) and hesperetin (HESP), at concentrations achieved clinically, synergized to increase Glo1 expression. In highly overweight subjects (BMI >27.5 kg/m 2 ), tRES-HESP coformulation increased expression and activity of Glo1 (27%, P < 0.05) and decreased plasma methylglyoxal (237%, P < 0.05) and total body methylglyoxal-protein glycation (214%, P < 0.01). It decreased fasting and postprandial plasma glucose (25%, P < 0.01, and 28%, P < 0.03, respectively), increased oral glucose insulin sensitivity index (42 mL $ min 21 $ m 22, P < 0.02), and improved arterial dilatation Dbrachial artery flowmediated dilatation/Ddilation response to glyceryl nitrate (95% CI 0.13-2.11). In all subjects, it decreased vascular inflammation marker soluble intercellular adhesion molecule-1 (210%, P < 0.01). In previous clinical evaluations, tRES and HESP individually were ineffective. tRES-HESP coformulation could be a suitable treatment for improved metabolic and vascular health in overweight and obese populations.

OBJECTIVE-Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia.RESEARCH DESIGN AND METHODS-Human microvascular HMEC-1 endothelial cells were incubated in low and high glucose concentrations (5 and 30 mmol/l, respectively), and activation of nrf2 was assessed by nuclear translocation. The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed.RESULTS-Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three-to fivefold increased expression of transketolase and glutathione reductase. Hyperglycemia increased the formation of ROS-an effect linked to mitochondrial dysfunction and prevented by sulforaphane. ROS formation was increased further by knockdown of nrf2 and transketolase expression. This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression. Sulforaphane also prevented hyperglycemia-induced activation of the hexosamine and PKC pathways and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal.CONCLUSIONS-We conclude that activation of nrf2 may prevent biochemical dysfunction and related functional responses of endothelial cells induced by hyperglycemia in which increased expression of transketolase has a pivotal role.

Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased protein and DNA modification contributing to cell and tissue dysfunction in aging and disease. It is produced by increased formation and/or decreased metabolism of dicarbonyl metabolites. MG (methylglyoxal) is a dicarbonyl metabolite of relatively high flux of formation and precursor of the most quantitatively and functionally important spontaneous modifications of protein and DNA clinically. Major MG-derived adducts are arginine-derived hydroimidazolones of protein and deoxyguanosine-derived imidazopurinones of DNA. These are formed non-oxidatively. The glyoxalase system provides an efficient and essential basal and stress-response-inducible enzymatic defence against dicarbonyl stress by the reduced glutathione-dependent metabolism of methylglyoxal by glyoxalase 1. The GLO1 gene encoding glyoxalase 1 has low prevalence duplication and high prevalence amplification in some tumours. Dicarbonyl stress contributes to aging, disease and activity of cytotoxic chemotherapeutic agents. It is found at a low, moderate and severe level in obesity, diabetes and renal failure respectively, where it contributes to the development of metabolic and vascular complications. Increased glyoxalase 1 expression confers multidrug resistance to cancer chemotherapy and has relatively high prevalence in liver, lung and breast cancers. Studies of dicarbonyl stress are providing improved understanding of aging and disease and the basis for rational design of novel pharmaceuticals: glyoxalase 1 inducers for obesity, diabetes and cardiovascular disease and glyoxalase 1 inhibitors for multidrug-resistant tumours. The first clinical trial of a glyoxalase 1 inducer in overweight and obese subjects showed improved glycaemic control, insulin resistance and vascular function.

Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20 mM versus 5 mM control) in vitro for 3–6 days. Flux of glucose metabolism, MG formation and glycation and changes in cytosolic protein abundances, MG modification and proteotoxic responses were assessed. Similar studies were performed with human microvascular endothelial HMEC-1 cells where similar outcomes were observed. HAECs exposed to high glucose concentration showed increased cellular concentration of MG (2.27 ± 0.21 versus 1.28 ± 0.03 pmol/10 6 cells, P < 0.01) and formation of MG-modified proteins (24.0 ± 3.7 versus 14.1 ± 3.2 pmol/10 6 cells/day; P < 0.001). In proteomics analysis, high glucose concentration increased proteins of the heat shock response – indicating activation of the unfolded protein response (UPR) with downstream inflammatory and pro-thrombotic responses. Proteins susceptible to MG modification were enriched in protein folding, protein synthesis, serine/threonine kinase signalling, glycolysis and gluconeogenesis. MG was increased in high glucose by increased flux of MG formation linked to increased glucose metabolism mediated by proteolytic stabilisation and increase of hexokinase-2 (HK-2); later potentiated by proteolytic down regulation of glyoxalase 1 (Glo1) - the major enzyme of MG metabolism. Silencing of Glo1, selectively increasing MG, activated the UPR similarly. Silencing of HK-2 prevented increased glucose metabolism and MG formation. trans -Resveratrol and hesperetin combination (tRES-HESP) corrected increased MG and glucose metabolism by increasing expression of Glo1 and decreasing expression of HK-2. Increased MG glycation activates the UPR in endothelial cells and thereby may contribute to endothelial cell dysfunction in diabetic vascular disease where tRES-HESP may provide effective therapy.