RESUMO
Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder in children. It is currently diagnosed by behaviour-based assessments made by observation and interview. In 2018 we reported a discovery study of a blood biomarker diagnostic test for ASD based on a combination of four plasma protein glycation and oxidation adducts. The test had 88% accuracy in children 5-12 years old. Herein, we present an international multicenter clinical validation study (N = 478) with application of similar biomarkers to a wider age range of 1.5-12 years old children. Three hundred and eleven children with ASD (247 male, 64 female; age 5.2 ± 3.0 years) and 167 children with typical development (94 male, 73 female; 4.9 ± 2.4 years) were recruited for this study at Sidra Medicine and Hamad Medical Corporation hospitals, Qatar, and Hospital Regional Universitario de Málaga, Spain. For subjects 5-12 years old, the diagnostic algorithm with features, advanced glycation endproducts (AGEs)-Nε-carboxymethyl-lysine (CML), Nω-carboxymethylarginine (CMA) and 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and oxidative damage marker, o,o'-dityrosine (DT), age and gender had accuracy 83% (CI 79 - 89%), sensitivity 94% (CI 90-98%), specificity 67% (CI 57-76%) and area-under-the-curve of receiver operating characteristic plot (AUROC) 0.87 (CI 0.84-0.90). Inclusion of additional plasma protein glycation and oxidation adducts increased the specificity to 74%. An algorithm with 12 plasma protein glycation and oxidation adduct features was optimum for children of 1.5-12 years old: accuracy 74% (CI 70-79%), sensitivity 75% (CI 63-87%), specificity 74% (CI 58-90%) and AUROC 0.79 (CI 0.74-0.84). We conclude that ASD diagnosis may be supported using an algorithm with features of plasma protein CML, CMA, 3DG-H and DT in 5-12 years-old children, and an algorithm with additional features applicable for ASD screening in younger children. ASD severity, as assessed by ADOS-2 score, correlated positively with plasma protein glycation adducts derived from methylglyoxal, hydroimidazolone MG-H1 and Nε(1-carboxyethyl)lysine (CEL). The successful validation herein may indicate that the algorithm modifiable features are mechanistic risk markers linking ASD to increased lipid peroxidation, neuronal plasticity and proteotoxic stress.
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Transtorno do Espectro Autista , Biomarcadores , Produtos Finais de Glicação Avançada , Oxirredução , Humanos , Masculino , Feminino , Biomarcadores/sangue , Criança , Pré-Escolar , Produtos Finais de Glicação Avançada/sangue , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/sangue , Glicosilação , Lisina/análogos & derivados , Lisina/sangue , Transtorno Autístico/sangue , Transtorno Autístico/diagnóstico , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/análise , Lactente , Sensibilidade e EspecificidadeRESUMO
The study of the glyoxalase system by Thornalley and co-workers in clinical diabetes mellitus and correlation with diabetic complications revealed increased exposure of patients with diabetes to the reactive, dicarbonyl metabolite methylglyoxal (MG). Twenty-eight years later, extended and built on by Thornalley and co-workers and others, the glyoxalase system is an important pathway contributing to the development of insulin resistance and vascular complications of diabetes. Other related advances have been: characterization of a new kind of metabolic stress-'dicarbonyl stress'; identification of the major physiological advanced glycation endproduct (AGE), MG-H1; physiological substrates of the unfolded protein response (UPR); new therapeutic agents-'glyoxalase 1 (Glo1) inducers'; and a refined mechanism underlying the link of dysglycemia to the development of insulin resistance and vascular complications of diabetes.
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Doenças Cardiovasculares , Complicações do Diabetes , Diabetes Mellitus , Hiperglicemia , Resistência à Insulina , Lactoilglutationa Liase , Doenças Cardiovasculares/metabolismo , Humanos , Hiperglicemia/complicações , Lactoilglutationa Liase/genética , Aldeído Pirúvico , Estresse Fisiológico , Resposta a Proteínas não DobradasRESUMO
Protein damage by glycation, oxidation and nitration is a continuous process in the physiological system caused by reactive metabolites associated with dicarbonyl stress, oxidative stress and nitrative stress, respectively. The term AGEomics is defined as multiplexed quantitation of spontaneous modification of proteins damage and other usually low-level modifications associated with a change of structure and function-for example, citrullination and transglutamination. The method of quantitation is stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS). This provides robust quantitation of normal and damaged or modified amino acids concurrently. AGEomics biomarkers have been used in diagnostic algorithms using machine learning methods. In this review, I describe the utility of AGEomics biomarkers and provide evidence why these are close to the phenotype of a condition or disease compared to other metabolites and metabolomic approaches and how to train and test algorithms for clinical diagnostic and screening applications with high accuracy, sensitivity and specificity using machine learning approaches.
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Produtos Finais de Glicação Avançada , Espectrometria de Massas em Tandem , Biomarcadores , Cromatografia Líquida , Produtos Finais de Glicação Avançada/química , Aprendizado de Máquina , Proteínas/químicaRESUMO
On 20-24 September 2021, leading researchers in the field of glycation met online at the 14th International Symposium on the Maillard Reaction (IMARS-14), hosted by the authors of this introductory editorial, who are from Doha, Qatar [...].
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Alimentos , Reação de Maillard , Glicosilação , Alérgenos , Produtos Finais de Glicação Avançada/metabolismoRESUMO
The abnormal accumulation of methylglyoxal (MG) leading to increased glycation of protein and DNA has emerged as an important metabolic stress, dicarbonyl stress, linked to aging, and disease. Increased MG glycation produces inactivation and misfolding of proteins, cell dysfunction, activation of the unfolded protein response, and related low-grade inflammation. Glycation of DNA and the spliceosome contribute to an antiproliferative and apoptotic response of high, cytotoxic levels of MG. Glyoxalase 1 (Glo1) of the glyoxalase system has a major role in the metabolism of MG. Small molecule inducers of Glo1, Glo1 inducers, have been developed to alleviate dicarbonyl stress as a prospective treatment for the prevention and early-stage reversal of type 2 diabetes and prevention of vascular complications of diabetes. The first clinical trial with the Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP)-a randomized, double-blind, placebo-controlled crossover phase 2A study for correction of insulin resistance in overweight and obese subjects, was completed successfully. tRES-HESP corrected insulin resistance, improved dysglycemia, and low-grade inflammation. Cell permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a prospective treatment for cancer-particularly for high Glo1 expressing-related multidrug-resistant tumors. The prototype Glo1 inhibitor is prodrug S-p-bromobenzylglutathione cyclopentyl diester (BBGD). It has antitumor activity in vitro and in tumor-bearing mice in vivo. In the National Cancer Institute human tumor cell line screen, BBGD was most active against the glioblastoma SNB-19 cell line. Recently, potent antitumor activity was found in glioblastoma multiforme tumor-bearing mice. High Glo1 expression is a negative survival factor in chemotherapy of breast cancer where adjunct therapy with a Glo1 inhibitor may improve treatment outcomes. BBGD has not yet been evaluated clinically. Glycation by MG now appears to be a pathogenic process that may be pharmacologically manipulated for therapeutic outcomes of potentially important clinical impact.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glutationa/análogos & derivados , Hesperidina/uso terapêutico , Lactoilglutationa Liase/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Resveratrol/uso terapêutico , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Quimioterapia Combinada , Indução Enzimática/efeitos dos fármacos , Glutationa/química , Glutationa/uso terapêutico , Glicosilação/efeitos dos fármacos , Hesperidina/química , Humanos , Resistência à Insulina/fisiologia , Lactoilglutationa Liase/antagonistas & inibidores , Camundongos , Estrutura Molecular , Neoplasias Experimentais/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/fisiopatologia , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Resveratrol/químicaRESUMO
The recent discovery of the glucose-induced stabilization of hexokinase-2 (HK2) to proteolysis in cell dysfunction in model hyperglycemia has revealed a likely key initiating factor contributing to the development of insulin resistance and vascular complications in diabetes. Consequently, the increased flux of glucose metabolism without a change in the expression and activity of glycolytic enzymes produces a wave of increased glycolytic intermediates driving mitochondrial dysfunction and increased reactive oxygen species (ROS) formation, the activation of hexosamine and protein kinase C pathways, the increased formation of methylglyoxal-producing dicarbonyl stress, and the activation of the unfolded protein response. This is called HK2-linked glycolytic overload and unscheduled glycolysis. The conditions required to sustain this are GLUT1 and/or GLUT3 glucose uptake and the expression of HK2. A metabolic biomarker of its occurrence is the abnormally increased deposition of glycogen, which is produced by metabolic channeling when HK2 becomes detached from mitochondria. These conditions and metabolic consequences are found in the vasculature, kidneys, retina, peripheral nerves, and early-stage embryo development in diabetes and likely sustain the development of diabetic vascular complications and embryopathy. In insulin resistance, HK2-linked unscheduled glycolysis may also be established in skeletal muscle and adipose tissue. This may explain the increased glucose disposal by skeletal uptake in the fasting phase in patients with type 2 diabetes mellitus, compared to healthy controls, and the presence of insulin resistance in patients with type 1 diabetes mellitus. Importantly, glyoxalase 1 inducer-trans-resveratrol and hesperetin in combination (tRES-HESP)-corrected HK2-linked glycolytic overload and unscheduled glycolysis and reversed insulin resistance and improved vascular inflammation in overweight and obese subjects in clinical trial. Further studies are now required to evaluate tRES-HESP for the prevention and reversal of early-stage type 2 diabetes and for the treatment of the vascular complications of diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Angiopatias Diabéticas , Resistência à Insulina , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glicólise , Hexoquinase/metabolismo , HumanosRESUMO
Accumulation of advanced glycation endproducts (AGEs) is linked to decline in renal function, particularly in patients with diabetes. Major forms of AGEs in serum are protein-bound AGEs and AGE free adducts. In this study, we assessed levels of AGEs in subjects with and without diabetes, with normal renal function and stages 2 to 4 chronic kidney disease (CKD), to identify which AGE has the greatest progressive change with decline in renal function and change in diabetes. We performed a cross-sectional study of patients with stages 2-4 CKD, with and without diabetes, and healthy controls (n = 135). Nine protein-bound and free adduct AGEs were quantified in serum. Most protein-bound AGEs increased moderately through stages 2-4 CKD whereas AGE free adducts increased markedly. Methylglyoxal-derived hydroimidazolone MG-H1 free adduct was the AGE most responsive to CKD status, increasing 8-fold and 30-fold in stage 4 CKD in patients without and with diabetes, respectively. MG-H1 Glomerular filtration flux was increased 5-fold in diabetes, likely reflecting increased methylglyoxal glycation status. We conclude that serum MG-H1 free adduct concentration was strongly related to stage of CKD and increased in diabetes status. Serum MG-H1 free adduct is a candidate AGE risk marker of non-diabetic and diabetic CKD.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Insuficiência Renal Crônica , Humanos , Reação de Maillard , Aldeído Pirúvico , Produtos Finais de Glicação Avançada , Estudos TransversaisRESUMO
The reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in obesity and diabetes and is implicated in the development of insulin resistance, type 2 diabetes mellitus and vascular complications of diabetes. Dicarbonyl stress is the metabolic state of abnormal high MG concentration. MG is an arginine-directed glycating agent and precursor of the major advanced glycation endproduct, arginine-derived hydroimidazolone MG-H1. MG-H1 is often formed on protein surfaces and an uncharged hydrophobic residue, inducing protein structural distortion and misfolding. Recent studies indicate that dicarbonyl stress in human endothelial cells and fibroblasts in vitro induced a proteomic response consistent with activation of the unfolded protein response (UPR). The response included: increased abundance of heat shock proteins and ubiquitin ligases catalysing the removal of proteins with unshielded surface hydrophobic patches and formation of polyubiquitinated chains to encapsulate misfolded proteins; and increased low grade inflammation. Activation of the UPR is implicated in insulin resistance. An effective strategy to counter increased MG is inducing increased expression of glyoxalase-1 (Glo1). An optimized inducer of Glo1 expression, trans-resveratrol and hesperetin combination, normalized increased MG concentration, corrected insulin resistance and decreased low grade inflammation in overweight and obese subjects. We propose that dicarbonyl stress, through increased formation of MG-glycated proteins, may be an important physiological stimulus of the UPR and Glo1 inducers may provide a route to effective suppression and therapy. With further investigation and validation, this may provide key new insight into physiological activators of the UPR and association with dicarbonyl stress.
Assuntos
Glucose/metabolismo , Proteínas/química , Glicosilação , Humanos , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.
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Glicemia/metabolismo , Microbioma Gastrointestinal/fisiologia , Índice Glicêmico/fisiologia , Degeneração Macular/metabolismo , Retina/metabolismo , Animais , Produtos Finais de Glicação Avançada/metabolismo , Metaboloma/fisiologia , Metabolômica , CamundongosRESUMO
Recent research has identified glycation as a non-enzymatic post-translational modification of proteins in plants with a potential contributory role to the functional impairment of the plant proteome. Reducing sugars with a free aldehyde or ketone group such as glucose, fructose and galactose react with the N-terminal and lysine side chain amino groups of proteins. A common early-stage glycation adduct formed from glucose is Nε-fructosyl-lysine (FL). Saccharide-derived reactive dicarbonyls are arginine residue-directed glycating agents, forming advanced glycation endproducts (AGEs). A dominant dicarbonyl is methylglyoxal-formed mainly by the trace-level degradation of triosephosphates, including through the Calvin cycle of photosynthesis. Methylglyoxal forms the major quantitative AGE, hydroimidazolone MG-H1. Glucose and methylglyoxal concentrations in plants change with the developmental stage, senescence, light and dark cycles and also likely biotic and abiotic stresses. Proteomics analysis indicates that there is an enrichment of the amino acid residue targets of glycation, arginine and lysine residues, in predicted functional sites of the plant proteome, suggesting the susceptibility of proteins to functional inactivation by glycation. In this review, we give a brief introduction to glycation, glycating agents and glycation adducts in plants. We consider dicarbonyl stress, the functional vulnerability of the plant proteome to arginine-directed glycation and the likely role of methylglyoxal-mediated glycation in the activation of the unfolded protein response in plants. The latter is linked to the recent suggestion of protein glycation in sugar signaling in plant metabolism. The overexpression of glyoxalase 1, which suppresses glycation by methylglyoxal and glyoxal, produced plants resistant to high salinity, drought, extreme temperature and other stresses. Further research to decrease protein glycation in plants may lead to improved plant growth and assist the breeding of plant varieties resistant to environmental stress and senescence-including plants of commercial ornamental and crop cultivation value.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Plantas/química , Produtos Agrícolas , Secas , Técnicas Genéticas , Produtos Finais de Glicação Avançada , Glicosilação , Glioxal/metabolismo , Lactoilglutationa Liase/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Aldeído Pirúvico/química , Proteínas Recombinantes/biossíntese , Tolerância ao Sal , TemperaturaRESUMO
Glyoxalase 1 (Glo1) is part of the glyoxalase system in the cytoplasm of all human cells. It catalyses the glutathione-dependent removal of the endogenous reactive dicarbonyl metabolite, methylglyoxal (MG). MG is formed mainly as a side product of anaerobic glycolysis. It modifies protein and DNA to form mainly hydroimidazolone MG-H1 and imidazopurinone MGdG adducts, respectively. Abnormal accumulation of MG, dicarbonyl stress, increases adduct levels which may induce apoptosis and replication catastrophe. In the non-malignant state, Glo1 is a tumour suppressor protein and small molecule inducers of Glo1 expression may find use in cancer prevention. Increased Glo1 expression is permissive for growth of tumours with high glycolytic activity and is thereby a biomarker of tumour growth. High Glo1 expression is a cause of multi-drug resistance. It is produced by over-activation of the Nrf2 pathway and GLO1 amplification. Glo1 inhibitors are antitumour agents, inducing apoptosis and necrosis, and anoikis. Tumour stem cells and tumours with high flux of MG formation and Glo1 expression are sensitive to Glo1 inhibitor therapy. It is likely that MG-induced cell death contributes to the mechanism of action of current antitumour agents. Common refractory tumours have high prevalence of Glo1 overexpression for which Glo1 inhibitors may improve therapy.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactoilglutationa Liase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Aldeído Pirúvico/metabolismo , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Glicosilação/efeitos dos fármacos , Humanos , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Aldeído Pirúvico/toxicidadeRESUMO
Advanced glycation end products (AGEs) are stable posttranslational modifications of proteins formed by the spontaneous reaction with glucose and related metabolites. Important AGEs quantitatively are methylglyoxal (MG)-derived hydroimidazolone MG-H1, Nε-carboxymethyl-lysine (CML), and glucosepane. They contribute to the development of chronic kidney disease (CKD). Cellular proteolysis of AGE-modified proteins forms AGE free adducts, glycated amino acids, which are cleared by the kidneys and excreted in urine. Dietary AGEs mainly supplement the endogenous flux of AGE free adduct formation. AGE free adducts accumulate markedly in plasma with decline in glomerular filtration rate. A key precursor of AGEs is the dicarbonyl metabolite MG, which is metabolized by glyoxalase 1 (Glo1) of the cytoplasmic glyoxalase system. Proteins susceptible to MG modification are collectively called the dicarbonyl proteome. Abnormal increase of MG dicarbonyl stress is a characteristic of CKD, driven by down-regulation of renal Glo1, increasing flux of MG-H1 formation. Protein inactivation and dysfunction linked to the dicarbonyl proteome contributes to CKD development. The receptor for AGEs, RAGE, is important in development of CKD, but its interaction with AGEs in vivo remains enigmatic; other ligands and ternary complexation may be influential. Prevention of diabetic kidney disease (DKD) by overexpression of Glo1 in transgenic animal models has stimulated the development of small-molecule inducers of Glo1 expression, Glo1 inducers, to prevent AGE formation. trans-Resveratrol-hesperetin combination therapy is a Glo1 inducer. In clinical trial it demonstrated a profound improvement in insulin resistance and vascular inflammation. It may find future therapeutic application for treatment of DKD.
Assuntos
Nefropatias Diabéticas/sangue , Produtos Finais de Glicação Avançada/sangue , Rim/metabolismo , Insuficiência Renal Crônica/sangue , Animais , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/terapia , Progressão da Doença , Humanos , Rim/fisiopatologia , Lactoilglutationa Liase/metabolismo , Prognóstico , Carbonilação Proteica , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Eliminação Renal , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/terapia , Fatores de RiscoRESUMO
[This corrects the article DOI: 10.1186/s12953-018-0131-y.].
RESUMO
Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE-/-) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.
RESUMO
Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress - the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease.
Assuntos
Células-Tronco Embrionárias/metabolismo , Lactoilglutationa Liase/deficiência , Lactoilglutationa Liase/metabolismo , Animais , Células Cultivadas , Hibridização Genômica Comparativa , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Dosagem de Genes/efeitos dos fármacos , Dosagem de Genes/genética , Genótipo , Humanos , Lactoilglutationa Liase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição/genética , Aldeído Pirúvico/farmacologiaRESUMO
Hyperglycemia results in accumulation of the reactive dicarbonyl methylglyoxal (MG). Methylglyoxal is detoxified by the glyoxalase system (glyoxalase 1 and 2). The influence of glyoxalase 1 knockdown on expression of collagens 1, 3, 4, and 5 in L6 myoblasts under hyperglycemic conditions was investigated. Increased biosynthesis of collagens 1, 3, 4, and 5 was detected at mRNA-level following knockdown of glyoxalase 1 (GLO1). At the protein level a significant elevation of the concentration of collagen 1 and 4 was shown, whereas no increase of collagen 5 and a non-significant increase in collagen 3 were detectable. These results could partially explain MG-induced changes in the extracellular matrix (ECM) which account for increased fibrosis and impaired function in myocytes. The mechanisms by which reactive glucose metabolites influence ECM composition deserve further investigation.
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Colágeno/metabolismo , Homeostase , Lactoilglutationa Liase/metabolismo , Mioblastos/metabolismo , Aldeído Pirúvico/metabolismo , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Glucose/metabolismo , Lactoilglutationa Liase/genética , RatosRESUMO
Decreased transketolase activity is an unexplained characteristic of patients with end-stage renal disease and is linked to impaired metabolic and immune function. Here we describe the discovery of a link to impaired functional activity of thiamine pyrophosphate cofactor through the presence, accumulation, and pyrophosphorylation of the thiamine antimetabolite oxythiamine in renal failure. Plasma oxythiamine was significantly increased by 4-fold in patients receiving continuous ambulatory peritoneal dialysis and 15-fold in patients receiving hemodialysis immediately before the dialysis session (healthy individuals, 0.18 [0.11-0.22] nM); continuous ambulatory peritoneal dialysis patients, 0.64 [0.48-0.94] nM; and hemodialysis patients (2.73 [1.52-5.76] nM). Oxythiamine was converted to the transketolase inhibitor oxythiamine pyrophosphate. The red blood cell oxythiamine pyrophosphate concentration was significantly increased by 4-fold in hemodialysis (healthy individuals, 15.9 nM and hemodialysis patients, 66.1 nM). This accounted for the significant concomitant 41% loss of transketolase activity (mU/mg hemoglobin) from 0.410 in healthy individuals to 0.240 in hemodialysis patients. This may be corrected by displacement with excess thiamine pyrophosphate and explain lifting of decreased transketolase activity by high-dose thiamine supplementation in previous studies. Oxythiamine is likely of dietary origin through cooking of acidic thiamine-containing foods. Experimentally, trace levels of oxythiamine were not formed from thiamine degradation under physiologic conditions but rather under acidic conditions at 100(°)C. Thus, monitoring of the plasma oxythiamine concentration in renal failure and implementation of high-dose thiamine supplements to counter it may help improve the clinical outcome of patients with renal failure.
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Antimetabólitos/toxicidade , Falência Renal Crônica/metabolismo , Oxitiamina/toxicidade , Deficiência de Tiamina/induzido quimicamente , Tiamina Pirofosfato/metabolismo , Transcetolase/antagonistas & inibidores , Adulto , Dieta/efeitos adversos , Feminino , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Oxitiamina/sangue , Oxitiamina/metabolismo , Processamento de Proteína Pós-Traducional , Diálise Renal , Eliminação Renal , Tiamina Pirofosfoquinase/metabolismo , Tiamina/uso terapêutico , Deficiência de Tiamina/tratamento farmacológico , Complexo Vitamínico B/uso terapêuticoRESUMO
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.
Assuntos
Envelhecimento/fisiologia , Tratamento Farmacológico , Lactoilglutationa Liase/uso terapêutico , Aldeído Pirúvico/toxicidade , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , Doença/genética , Humanos , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
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.
Assuntos
Envelhecimento/metabolismo , Dislipidemias/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Lactoilglutationa Liase/metabolismo , Neoplasias/metabolismo , Aldeído Pirúvico/metabolismo , Doenças Vasculares/metabolismo , Animais , Dislipidemias/tratamento farmacológico , Humanos , Neoplasias/tratamento farmacológico , Doenças Vasculares/tratamento farmacológicoRESUMO
Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1-5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N(ε)-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them - amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine - particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.