Mingzhan Xue

Activation of NF-E2-related factor-2 reverses biochemical dysfunction of endothelial cells induced by hyperglycemia linked to vascular disease

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.

Glyoxalase in ageing.

The glyoxalase system has been studied since 1913. The biochemical function of this enzymatic system is the metabolism of reactive dicarbonyl metabolites, glyoxal and methylglyoxal, to less reactive products. In the last decade research has shown that methylglyoxal is the precursor of quantitatively important damage to the proteome and genome, forming mainly hydroimidazolone and imidazopurinone adducts in protein and DNA respectively. The aim of this article is to review the evidence of the involvement of the glyoxalase system in ageing and role of glyoxalase in future research into healthy ageing-mainly in mammalian systems for insights into consequences and interventions in human health. Protein and DNA damage by glyoxalase system substrates is linked to dysfunction of proteins susceptible to dicarbonyl modification-the dicarbonyl proteome, and DNA instability and mutation. A component of the glyoxalase system, glyoxalase 1, is a gene with expression influential on lifespan-increasing longevity being associated with increased expression of glyoxalase 1. The glyoxalase 1 gene is also a site of copy number variation in both transcribed and non-transcribed regions giving rise to population variation of expression. The glyoxalase system and Glo1 expression particularly is therefore likely linked to healthy ageing.

Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity: A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes

OBJECTIVE To study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity. RESEARCH DESIGN AND METHODS Human LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping. RESULTS MGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding. CONCLUSIONS MG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

Improved Glycemic Control and Vascular Function in Overweight and Obese Subjects by Glyoxalase 1 Inducer Formulation

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/m2), tRES-HESP coformulation increased expression and activity of Glo1 (27%, P < 0.05) and decreased plasma methylglyoxal (−37%, P < 0.05) and total body methylglyoxal-protein glycation (−14%, P < 0.01). It decreased fasting and postprandial plasma glucose (−5%, P < 0.01, and −8%, P < 0.03, respectively), increased oral glucose insulin sensitivity index (42 mL ⋅ min−1 ⋅ m−2, P < 0.02), and improved arterial dilatation Δbrachial artery flow-mediated dilatation/Δdilation response to glyceryl nitrate (95% CI 0.13–2.11). In all subjects, it decreased vascular inflammation marker soluble intercellular adhesion molecule-1 (−10%, 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.

Activity, regulation, copy number and function in the glyoxalase system

Molecular, catalytic and structural properties of glyoxalase pathway enzymes of many species are now known. Current research has focused on the regulation of activity and expression of Glo1 (glyoxalase I) and Glo2 (glyoxalase II) and their role in health and disease. Human GLO1 has MRE (metal-response element), IRE (insulin-response element), E2F4 (early gene 2 factor isoform 4), AP-2α (activating enhancer-binding protein 2α) and ARE (antioxidant response-element) regulatory elements and is a hotspot for copy number variation. The human Glo2 gene, HAGH (hydroxyacylglutathione hydrolase), has a regulatory p53-response element. Glo1 is linked to healthy aging, obesity, diabetes and diabetic complications, chronic renal disease, cardiovascular disease, other disorders and multidrug resistance in cancer chemotherapy. Mathematical modelling of the glyoxalase pathway predicts that pharmacological levels of increased Glo1 activity markedly decrease cellular methylglyoxal and related glycation, and pharmacological Glo1 inhibition markedly increases cellular methylglyoxal and related glycation. Glo1 inducers are in development to sustain healthy aging and for treatment of vascular complications of diabetes and other disorders, and cell-permeant Glo1 inhibitors are in development for treatment of multidrug-resistant tumours, malaria and potentially pathogenic bacteria and fungi.

Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics

The reactive dicarbonyl metabolite methylglyoxal (MG) is the precursor of the major quantitative advanced glycation endproducts (AGEs) in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. The glyoxalase system in the cytoplasm of cells provides the primary defence against dicarbonyl glycation by catalysing the metabolism of MG and related reactive dicarbonyls. Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased protein and DNA modification contributing to cell and tissue dysfunction in ageing and disease. It is produced endogenously by increased formation and/or decreased metabolism of dicarbonyl metabolites. Dicarbonyl stress contributes to ageing, disease and activity of cytotoxic chemotherapeutic agents. It contributes to ageing through age-related decline in glyoxalase 1 (Glo-1) activity. Glo-1 has a dual role in cancer as a tumour suppressor protein prior to tumour development and mediator of multi-drug resistance in cancer treatment, implicating dicarbonyl glycation of DNA in carcinogenesis and dicarbonyl-driven cytotoxicity in mechanism of action of anticancer drugs. Glo-1 is a driver of cardiovascular disease, likely through dicarbonyl stress-driven dyslipidemia and vascular cell dysfunction. Dicarbonyl stress is also a contributing mediator of obesity and vascular complications of diabetes. There are also emerging roles in neurological disorders. Glo-1 responds to dicarbonyl stress to enhance cytoprotection at the transcriptional level through stress-responsive increase of Glo-1 expression. Small molecule Glo-1 inducers are in clinical development for improved metabolic, vascular and renal health and Glo-1 inhibitors in preclinical development for multidrug resistant cancer chemotherapy.

Methylglyoxal-induced dicarbonyl stress in aging and disease: first steps towards glyoxalase 1-based treatments

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.

Frequency Modulated Translocational Oscillations of Nrf2 Mediate the Antioxidant Response Element Cytoprotective Transcriptional Response

Abstract Aims: Stress responsive signaling coordinated by nuclear factor erythroid 2-related factor 2 (Nrf2) provides an adaptive response for protection of cells against toxic insults, oxidative stress and metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). The aim of this study was to examine how Nrf2 signals cell stress status and regulates transcription to maintain homeostasis. Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component. Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2. Conclusion: Frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response. Antioxid. Redox Signal. 23, 613–629.

Frequency modulated translocational oscillations of Nrf2, a transcription factor functioning like a wireless sensor

The discovery that nuclear factor erythroid 2-related factor 2 (Nrf2) undergoes translocational oscillations from cytoplasm to nucleus in human cells with frequency modulation linked to activation of a stress-stimulated cytoprotective response raises the prospect that the Nrf2 works mechanistically analogous to a wireless sensor. Herein, we consider how this new model of Nrf2 oscillation resolves previous inexplicable experimental findings on Nrf2 regulation and why it is fit-for-purpose. Further investigation is required to assess how generally applicable the oscillatory mechanism is and if characteristics of this regulatory control can be found in vivo. It suggests there are multiple, potentially re-enforcing receptors for Nrf2 activation, indicating that potent Nrf2 activation for improved health and treatment of disease may be achieved through combination of Nrf2 system stimulants.

Copy number variation of glyoxalase I

The glyoxalase I gene GLO1 is a hotspot for copy number variation in the human and mouse genomes. The additional copies are often functional, giving rise to 2-4-fold increased glyoxalase I expression and activity. The prevalence of GLO1 copy number increase in the human population appears to be approximately 2% and may be linked to a risk of obesity, diabetes and aging. Increased GLO1 copy number has been found in human tumour cell lines and primary human tumours. The minimum common copy number increase region was approximately 1 Mb and it contained GLO1 and seven other genes. The increased copy number was generally functional, being associated with increased glyoxalase I protein and multidrug resistance in cancer chemotherapy. Glo1 duplication in the mouse genome is found within approximately 0.5 Mb of duplicated DNA. It was claimed to be linked to anxiety phenotypes, but other related discordant findings have doubted the association with glyoxalase I and further investigation is required.