Naila Rabbani

Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome.

Methylglyoxal (MG) is a potent protein glycating agent. Glycation is directed to guanidino groups of arginine residues forming mainly hydroimidazolone Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) residues. MG-H1 formation is damaging to the proteome as modification is often directed to functionally important arginine residues. MG-H1 content of proteins is quantified by stable isotopic dilution analysis tandem mass spectrometry and also by immunoblotting with specific monoclonal antibodies. MG-glycated proteins undergo cellular proteolysis and release MG-H1 free adduct for excretion. MG-H1 residues have been found in proteins of animals, plants, bacteria, fungi and protoctista. MG-H1 is often the major advanced glycation endproduct in proteins of tissues and body fluids, increasing in diabetes and associated vascular complications, renal failure, cirrhosis, Alzheimer’s disease, arthritis, Parkinson’s disease and ageing. Glyoxalase 1 and aldo–keto reductase 1B1 metabolise >99% MG to innocuous products and thereby protect the proteome, providing an enzymatic defence against MG-mediated glycation. Proteins susceptible to MG modification with related functional impairment are called the “dicarbonyl proteome” (DCP). DCP includes albumin, haemoglobin, transcription factors, mitochondrial proteins, extracellular matrix proteins, lens crystallins and other proteins. DCP component proteins are linked to mitochondrial dysfunction in diabetes and ageing, oxidative stress, dyslipidemia, cell detachment and anoikis and apoptosis. Biochemical and physiological susceptibility of a protein to modification by MG and sensitivity of biochemical pathways and physiological systems to related functional impairment under challenge of physiologically relevant increases in MG exposure are key concepts. Improved understanding of the DCP will likely have profound importance for human health, longevity and treatment of disease.

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.

High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor α. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Glyoxalase in diabetes, obesity and related disorders

Diabetes was the first disease state where evidence emerged for increased formation of methylglyoxal. Metabolism of methylglyoxal by the glyoxalase system has been linked to the development of vascular complications of diabetes - nephropathy, retinopathy, neuropathy and cardiovascular disease. Increased formation of methylglyoxal in hyperglycaemia associated with diabetes and down regulation of glyoxalase 1 by inflammatory signalling in vascular cells leads to a marked increased modification of proteins by methylglyoxal to form advanced glycation endproducts at the sites of vascular complications. Hotspot protein targets of methylglyoxal that suffer functional impairment - the dicarbonyl proteome - likely play a key role in the mechanisms underlying the development of vascular complications in diabetes: particularly modification of integrin binding sites in extracellular matrix proteins leading to endothelial cell shedding and anoikis, modification of mitochondrial proteins and increased formation of reaction oxygen species, and modification of apolipoprotein B100 of low density lipoprotein leading to its increased atherogenicity. Some current therapeutic agents counter partially dysfunctional metabolism of methylglyoxal by the glyoxalase system in diabetes - including the recent development of high dose thiamine therapy for early stage diabetic nephropathy. Further pharmacologic strategies are required to overcome the down regulation of glyoxalase1 in diabetes. The glyoxalase system is likely to be a continuing and future focus for research on clinical biomarkers and therapeutic development for respectively assessment of metabolic control and prevention of vascular complications in diabetes and obesity.

C. elegans as Model for the Study of High Glucose– Mediated Life Span Reduction

OBJECTIVE Establishing Caenorhabditis elegans as a model for glucose toxicity–mediated life span reduction. RESEARCH DESIGN AND METHODS C. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied. RESULTS High glucose conditions reduced mean life span from 18.5 ± 0.4 to 16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal–detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days) and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner. CONCLUSIONS C. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1–dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.

Advanced Glycation End Products in Extracellular Matrix Proteins Contribute to the Failure of Sensory Nerve Regeneration in Diabetes

OBJECTIVE The goal of this study was to characterize glycation adducts formed in both in vivo extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats and in vitro by glycation of laminin and fibronectin with methylglyoxal and glucose. We also investigated the impact of advanced glycation end product (AGE) residue content of ECM on neurite outgrowth from sensory neurons. RESEARCH DESIGN AND METHODS Glycation, oxidation, and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-induced diabetic rat sciatic nerve (3–24 weeks post-STZ) and of laminin and fibronectin that had been glycated using glucose or methylglyoxal were examined by liquid chromatography with tandem mass spectrometry. Methylglyoxal-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration. RESULTS STZ-induced diabetes produced a significant increase in early glycation Nε-fructosyl-lysine and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by methylglyoxal and glucose increased glycation adduct residue contents with methylglyoxal-derived hydroimidazolone and Nε-fructosyl-lysine, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite outgrowth, which was prevented by aminoguanidine and nerve growth factor. CONCLUSIONS Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.

Dicarbonyl stress in cell and tissue dysfunction contributing to ageing and disease

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. Enzymes metabolising dicarbonyls, glyoxalase 1 and aldoketo reductases, provide an efficient and stress-response enzyme defence against dicarbonyl stress. Dicarbonyl stress is produced by increased formation and/or decreased metabolism of dicarbonyl metabolites, and by exposure to exogenous dicarbonyls. It contributes to ageing, disease and activity of cytototoxic chemotherapeutic agents.

Glyoxalase in tumourigenesis and multidrug resistance

Since the discovery by Warburg of high aerobic glycolysis in most tumours in the 1920s, it has remained unclear how to exploit this in chemotherapy. The aim of this review is to assess the evidence for the involvement of the glyoxalase system in tumour growth and multidrug resistance and the importance of the glyoxalase system as a target for anticancer drug development and a source of biomarkers for tumour diagnosis. Increased expression of glyoxalase 1 appears to support the viability of tumour cells with high glycolytic rates. Multidrug resistance conferred by overexpression of glyoxalase 1 suggests mechanisms of toxicity of most current antitumour agents involve, in some part, accumulation of methylglyoxal to cytotoxic levels. The recent finding of glyoxalase 1 gene amplification in tumours and induction of increased glyoxalase 1 expression by malignant transformation and conventional antitumour drug treatment implies a critical role of glyoxalase 1 in innate and acquired multidrug resistance in cancer treatment. Improved understanding of glyoxalase 1 in cancer chemotherapy multidrug resistance is likely vital to achieve improvement of cancer patient survival rates. Advances made to counter glyoxalase 1-linked multidrug resistance with glyoxalase 1 inhibitors and related prodrugs has been translated from in vitro to pre-clinical in vivo studies. Further research is required urgently for next stage clinical translation. Finally, overexpression of glyoxalase 1 may be linked to multidrug resistance in chemotherapy of other disease - such as microbial infections.

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.