Dicarbonyl Stress Research Articles

An integrated proteomics and metabolomics analysis of methylglyoxal-induced neurotoxicity in a human neuroblastoma cell line

This study aimed to highlight the molecular and biochemical changes induced by methylglyoxal (MGO) exposure in SH-SY5Y human neuroblastoma cells, and to explore how these changes contribute to its neurotoxicity, utilizing an integrated proteomics and metabolomics approach. Using label-free quantitative nanoLC-MS/MS proteomics and targeted LC-TQ-MS/MS-based metabolomics, the results revealed that MGO exposure, particularly at cytotoxic levels, significantly altered the proteome and metabolome of SH-SY5Y cells. Analysis of proteomics data showed significant alterations in cellular functions including protein synthesis, cellular structural integrity, mitochondrial function, and oxidative stress responses. Analysis of metabolomics and integration of metabolomics and proteomics data highlighted significant changes in key metabolic pathways including arginine biosynthesis, glutathione metabolism, cysteine and methionine metabolism, and the tricarboxylic acid cycle. These results suggest that MGO exposure induced both toxic effects and adaptive responses in cells. MGO exposure led to increased endoplasmic reticulum stress, disruptions in cellular adhesion and extracellular matrix integrity, mitochondrial dysfunction, and amino acid metabolism disruption, contributing to cellular toxicity. Conversely, cells exhibited adaptive responses by upregulating protein synthesis, activating the Nrf2 pathway, and reprogramming metabolism to counteract dicarbonyl stress and maintain energy levels. Furthermore, a set of key proteins and metabolites associated with these changes were shown to exhibit a significant concentration-dependent decrease or increase in their expression levels with increasing MGO concentrations, suggesting their potential as biomarkers for MGO exposure. Taken together, these findings provide insight into the molecular mechanisms underlying MGO-induced neurotoxicity and potential targets for therapeutic intervention.

Molecular mechanism of ectopic lipid accumulation induced by methylglyoxal via activation of the NRF2/PI3K/AKT pathway implicates renal lipotoxicity caused by diabetes mellitus.

Patients with chronic kidney disease (CKD) have a high incidence of dyslipidemia comprising high triglyceride (TG) and low high-density lipoprotein (HDL)-cholesterol levels. An abnormal increase of TGs within cells can lead to intracellular lipid accumulation. In addition to dyslipidemia, hyperglycemia in diabetes may elicit ectopic lipid deposition in non-adipose tissues. Hyperglycemia increases intracellular levels of methylglyoxal (MG) leading to cellular dysfunction. A deficit of glyoxalase I (GLO1) contributes to dicarbonyl stress. Whether dicarbonyl stress induced by MG causes renal lipotoxicity through alteration of lipid metabolism signaling is still unknown. In this study, mice with high fat diet-induced diabetes were used to investigate the renal pathology induced by MG. NRK52E cells treated with MG were further used in vitro to delineate the involvement of lipogenic signaling. After treatment with MG for 12 weeks, plasma TG levels, renal fatty changes, and tubular injuries were aggravated in diabetic mice. In NRK52E cells, MG activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and sterol regulatory element-binding protein 1 (SREBP1), resulting in stimulation of fatty acid synthase. The intracellular accumulation of lipid droplets was mainly contributed by TGs, which increased the oxidative stress accompanied by high Nrf2 expression. In addition, MG time-dependently activated cyclin D, cyclin-dependent kinase 4 (CDK4), and cleaved caspase-3, evidencing that G0/G1 arrest was associated with apoptosis of NRK52E cells. In conclusion, our studies revealed the mechanism of lipotoxicity caused by MG. The target of such dicarbonyl stress may become a promising therapy for diabetic CKD.

Long-Term Casein-Bound Lactulosyllysine Consumption Induced Nonobese Nonalcoholic Fatty Liver Disease by Promoting Carbonyl Glycation in the Liver of C57BL/6 Mice.

Lactulosyllysine (LL) is abundant in thermally processed dairy products, with its concentration increasing in response to more intense heat treatment. However, there are limited studies on the potential harmful effects of LL on human health. This study investigated the negative impact of casein-bound LL on liver health by feeding healthy C57BL/6 mice diets containing varying levels of casein-bound LL. After 16 weeks of LL diet administration, mice exhibited a nonobese nonalcoholic fatty liver disease (NONAFLD) phenotype, characterized by reduced body weight gain, hypolipidemia, and intrahepatic lipid accumulation. Nontarget metabolomic analysis showed that casein-bound LL induced alterations in plasma levels of compounds associated with lipid degradation. Mechanistically, casein-bound LL may impair the function of 5'-adenosine monophosphate-activated protein kinase and apolipoprotein B100 by inducing dicarbonyl stress, thereby promoting carbonyl glycation in the liver. Consequently, the long-term consumption of LL-rich dairy products may be a contributing factor to the risk of developing NONAFLD.

Taurine, alpha lipoic acid and vitamin B6 ameliorate the reduced developmental competence of immature mouse oocytes exposed to methylglyoxal

Advanced glycation end products (AGEs) are the final products of the Maillard reaction, formed through the interaction of carbohydrates and proteins. Reactive dicarbonyl compounds such as methylglyoxal (MGO) serve as precursors for AGEs formation. Elevated levels of MGO/AGEs are observed in conditions like obesity, polycystic ovarian syndrome (PCOS), and diabetes, negatively impacting oocyte development. Previous studies have shown that hydrogen sulfide, a gasotransmitter with anti-AGEs effects, is produced in a process influenced by vitamin B6. R-α-lipoic acid (ALA) inhibits protein glycation and AGEs formation while stimulating glutathione (GSH) production. Taurine mitigates oxidative stress and acts as an anti-glycation compound, preventing in vitro glycation and AGEs accumulation. This study aimed to explore the ameliorative effects of a micronutrient support (Taurine, ALA and B6: TAB) on mouse oocytes challenged with MGO. Our results indicate that MGO reduces oocyte developmental competence, while TAB supplementation improves maturation, fertilization, and blastocyst formation rates. TAB also restores cell lineage allocation, redox balance and mitigates mitochondrial dysfunction in MGO-challenged oocytes. Furthermore, cumulus cells express key enzymes in the transsulfuration pathway, and TAB enhances their mRNA expression. However, TAB does not rescue MGO-induced damage in denuded oocytes, emphasizing the supportive role of cumulus cells. Overall, these findings suggest that TAB interventions may have significant implications for addressing reproductive dysfunctions associated with elevated MGO/AGEs levels. This study highlights the potential of TAB supplementation in preserving the developmental competence of COCs exposed to MGO stress, providing insights into mitigating the impact of dicarbonyl stress on oocyte quality and reproductive outcomes.

Genistein Prevents Apoptosis and Oxidative Stress Induced by Methylglyoxal in Endothelial Cells.

Glycolytic overload promotes accumulation of the highly reactive dicarbonyl compounds, resulting in harmful conditions called dicarbonyl stress. Methylglyoxal (MG) is a highly reactive dicarbonyl species and its accumulation plays a crucial pathophysiological role in diabetes and its vascular complications. MG cytotoxicity is mediated by reactive oxygen species (ROS) generation, a key event underlying the intracellular signaling pathways leading to inflammation and apoptosis. The identification of compounds able to inhibit ROS signaling pathways and counteract the MG-induced toxicity is a crucial step for developing new therapeutic strategies in the treatment of diabetic vascular complications. In this study, the effect of genistein, a natural soybean isoflavone, has been evaluated on MG-induced cytotoxicity in human endothelial cells. Our results show that genistein is able to counteract the MG-induced apoptosis by restraining ROS production, thus inhibiting the MAPK signaling pathways and caspase-3 activation. These findings identify a beneficial role for genistein, providing new insights for its potential clinical applications in preserving endothelial function in diabetic vascular complications.

Accumulation of Non-Pathological Liver Fat Is Associated with the Loss of Glyoxalase I Activity in Humans.

The underlying molecular mechanisms for the development of non-alcoholic fatty liver (NAFL) and its progression to advanced liver diseases remain elusive. Glyoxalase 1 (Glo1) loss, leading to elevated methylglyoxal (MG) and dicarbonyl stress, has been implicated in various diseases, including obesity-related conditions. This study aimed to investigate changes in the glyoxalase system in individuals with non-pathological liver fat. Liver biopsies were obtained from 30 individuals with a narrow range of BMI (24.6-29.8 kg/m2). Whole-body insulin sensitivity was assessed using HOMA-IR. Liver biopsies were analyzed for total triglyceride content, Glo1 and Glo2 mRNA, protein expression, and activity. Liquid chromatography-tandem mass spectrometry determined liver dicarbonyl content and oxidation and glycation biomarkers. Liver Glo1 activity showed an inverse correlation with HOMA-IR and liver triglyceride content, but not BMI. Despite reduced Glo1 activity, no associations were found with elevated liver dicarbonyls or glycation markers. A sex dimorphism was observed in Glo1, with females exhibiting significantly lower liver Glo1 protein expression and activity, and higher liver MG-H1 content compared to males. This study demonstrates that increasing liver fat, even within a non-pathological range, is associated with reduced Glo1 activity.

Effects of contamination and warming on ragworms Hediste diversicolor: A laboratory experiment with Oder estuary sediments

Knowledge about the effects of sediment contamination on estuarine benthos has come mainly from observational studies and spiking experiments. There is a lack of experimental studies with field-contaminated sediments, especially those assessing the combined effects of contamination and other environmental stressors. Here we investigated the biomarker responses of ragworms Hediste diversicolor to contaminated sediments and warming. Ragworms were exposed to sediments with different contamination levels from the Oder estuary (German-Polish border) at 10 and 20 °C for three weeks. The most contaminated sediments induced dicarbonyl stress, but did not result in enhanced detoxification, significant oxidative stress, or impaired energy balance. Warming altered antioxidant defense in ragworms and increased their energy reserves. The effects of contamination and warming were generally mild, and the interactive effects were absent. This study suggests the relatively high resilience of H. diversicolor to multiple stressors.

The metabolism and transformation of casein-bound lactulosyllysine in vivo: Promoting dicarbonyl stress and the formation of advanced glycation end products accompanied by systemic inflammation

Lactulosyllysine (LL) widely exists in thermally processed dairy products, while the metabolism and transformation of LL remain poorly understood. We aimed to elucidate the metabolic pathways of LL and its impact on body health by subjecting C57BL/6 mice to a short-term ll-fortified casein diet. Our findings indicated that casein-bound LL might be metabolized and transformed into 3-deoxyglucosone through fructosamine-3-kinase (FN3K) in vivo, which promoted α-dicarbonyl stress, ultimately leading to the formation of advanced glycation end products (AGEs) in various tissues/organs, accompanied by systemic inflammation. The levels of AGEs formation in tissues/organs at various stages of casein-bound LL intake exhibited dynamic changes, correlating with alterations in the expression of FN3K and α-dicarbonyl compounds metabolic detoxification enzymes. The negative effects induced by casein-bound LL cannot be fully reversed by switching to a standard diet for equal periods. Consumption of dairy products rich in LL raises concerns as a potential risk factor for healthy individuals.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

In vitro study of antioxidant, antigylycation, sugar hydrolysis enzyme inhibitory effect and molecular in silico docking study of angularly condensed diquinothiazines

BackgroundAngularly condensed diquinothiazines (MY1, MY2, and MY3) a phenothiazine derivative was evaluated for antioxidant, antiglycation, sugar hydrolysis enzyme inhibitory effect and molecular in silico docking. MethodAntioxidants were evaluated using DPPH, chelating ion, FRAP and lipid peroxidation. Alpha glucosidase and alpha amylase inhibition was used for sugar hydrolysis enzyme inhibitory activity and antiglycation with BSA-Glucose and BSA-MGO. In silco docking was done with AutoDock Vina and ADMET properties with pkCSM software. ResultAngularly condensed diquinothiazines (MY1, MY2, and MY3) showed free radical scavenging, antiglycation and alpha-glucosidase and alpha-amylase inhibition. Insilco docking and pkCSM ADME shows interaction with aldose reductase, glyoxalase 1, receptor AGE, alpha-glucosidase, and alpha-amylase. ConclusionAntiglycation, alpha-glucosidase, and alpha-amylase inhibition activity of Angularly condensed diquinothiazines maybe through deactivation of aldose reductase enzyme activity, activation of glyoxalase 1 pathway, reduction of dicarbonyl stress and oxidative stress.

Molecular Link between Glo-1 Expression and Markers of Hyperglycemia and Oxidative Stress in Vascular Complications of Type 2 Diabetes Mellitus.

Chronic hyperglycemia and oxidative stress in Type 2 Diabetes Mellitus trigger cellular dysfunction via the formation of Advanced Glycation End Products (AGEs), resulting in dicarbonyl stress. Glyoxalase-1 (Glo-1) is the main defense against dicarbonyl stress. The aim of this study was to explore any cross-talk between Glo-1 and markers of hyperglycemia and oxidative stress. The siRNA-mediated downregulation of Glo-1 was performed in human microvascular endothelial cell line (HMEC-1). A Glo-1 transgenic rat model was developed. Glo-1 activity, as determined spectrophotometrically, and methylglyoxal were quantified using UPLC-MS/MS and the expression of representative markers of hyperglycemia and oxidative stress was performed using quantitative real-time PCR. A significant increase in the expression of Vascular Cell Adhesion Molecule-1 (VCAM-1) was observed in the case of the siRNA-mediated downregulation of Glo-1 in the microvasculature model under hyperglycemic conditions (p-value < 0.001), as well the as overexpression of Glo-1 in the macrovasculature (p-value = 0.0125). The expression of thioredoxin interacting protein (TXNIP) was found to be significantly upregulated in wildtype diabetic conditions vs. Glo-1 transgenic control conditions (p-value = 0.008), whereas the downregulation of Glo-1 had no impact on TXNIP expression. These findings substantiate the role of VCAM as an important marker of dicarbonyl stress (represented by Glo-1 downregulation), as well as of hyperglycemia, in diabetic vascular complications. Our findings also suggest a potential feedback loop that may exist between Glo-1 and TXNIP, as the highest expression of TXNIP is observed in cases of wildtype diabetic conditions, and the lowest expression of TXNIP is observed when Glo-1 transgene is being expressed in absence of dicarbonyl stress.

Pathophysiology of Red Blood Cell Dysfunction in Diabetes and Its Complications.

Diabetes Mellitus (DM) is a complex metabolic disorder associated with multiple microvascular complications leading to nephropathy, retinopathy, and neuropathy. Mounting evidence suggests that red blood cell (RBC) alterations are both a cause and consequence of disturbances related to DM-associated complications. Importantly, a significant proportion of DM patients develop varying degrees of anemia of confounding etiology, leading to increased morbidity. In chronic hyperglycemia, RBCs display morphological, enzymatic, and biophysical changes, which in turn prime them for swift phagocytic clearance from circulation. A multitude of endogenous factors, such as oxidative and dicarbonyl stress, uremic toxins, extracellular hypertonicity, sorbitol accumulation, and deranged nitric oxide metabolism, have been implicated in pathological RBC changes in DM. This review collates clinical laboratory findings of changes in hematology indices in DM patients and discusses recent reports on the putative mechanisms underpinning shortened RBC survival and disturbed cell membrane architecture within the diabetic milieu. Specifically, RBC cell death signaling, RBC metabolism, procoagulant RBC phenotype, RBC-triggered endothelial cell dysfunction, and changes in RBC deformability and aggregation in the context of DM are discussed. Understanding the mechanisms of RBC alterations in DM provides valuable insights into the clinical significance of the crosstalk between RBCs and microangiopathy in DM.

419-P: Glucose-Induced Cellular Resistance to Methylglyoxal Is Lost in Patients with Diabetes Complications and Further Deteriorated after Caloric Restriction

Aim: Glucose stress can be protective against dicarbonyl-mediated toxicity. This study aimed to investigate cellular resistance against methylglyoxal (MG) after glucose stress in healthy humans (CON), patients with (T2D+) and without diabetes complications (T2D-) and whether this is affected by caloric restriction. Fibroblast growth factor 21 (FGF21) and β- hydroxybutyrate (β-HB) were analyzed as marker for metabolic adaptation to stress response and ketogenesis. Methods: Peripheral blood mononuclear cells were isolated before and 2h after oGTT and incubated at each time-point with increasing MG-levels for 24hrs. Cell viability was measured with CellTiter-Glo to determine changes of MG-resistance at 2h after oGTT (ΔEC50). Experiments were repeated after a 5-day fasting-mimicking diet (FMD). FGF21 and β-HB were measured in venous blood. Results: Thirty participants (18m/12f, age 64.6 [62.2, 67.0] y, BMI 28.6 [27.2, 30.0] kg/m2) were included. HbA1c was higher in T2D+ (7.4 [6.9, 7.9] %) and T2D- (7.0 [6.2, 7.7] %) compared to CON (5.5 [5.1, 5.8] %, p&amp;lt;0.001), age and BMI were comparable. ΔEC50 of MG increased in CON before and after FMD (12.9% vs. 12.6%), was unaffected in T2D- (1.8% vs. -1.7%) and decreased in T2D+ after FMD (-3.9% vs. -19.0%, p&amp;lt;0.01) and when compared to T2D- and CON (p&amp;lt;0.05). ΔFGF21 increased in CON after FMD (7.7% vs. 115.0%, p&amp;lt;0.05) and tended to be higher compared to T2D- and T2D+ (115.0% vs. 69.3% vs. 47.5%, p=0.5). ΔFGF21 correlated with β-HB (r = 0.48, p&amp;lt;0.01), with strongest correlation for T2D+. Conclusion: Acute glucose stress induces cellular resistance to MG in healthy humans, a response which is lost in T2D with diabetic complications and aggravates after caloric restriction. Our data suggest for an association between loss of capacity for handling dicarbonyl stress and the presence of diabetic complications. FGF21 might play an important role in the metabolic adaptation to dicarbonyl stress and ketosis. Disclosure E.V.Rauchhaupt: None. C.Rodemer: None. E.Kliemank: None. S.Kopf: Advisory Panel; Novo Nordisk, Bayer Inc., Recordati Rare Disease, Speaker's Bureau; Novo Nordisk, Boehringer-Ingelheim. S.Herzig: Consultant; Novo Nordisk, Actimed, Research Support; Novo Nordisk, Boehringer-Ingelheim, Almac. P.Nawroth: None. J.Szendroedi: None. J.Zemva: None. A.Sulaj: None. Funding DFG; German Research Foundation (236360313-SFB, 1118DZD); German Center for Diabetes Research (82DZD07C2G)

Hot and heavy: Responses of ragworms (Hediste diversicolor) to copper-spiked sediments and elevated temperature

Sediment contamination and seawater warming are two major stressors to macrobenthos in estuaries. However, little is known about their combined effects on infaunal organisms. Here we investigated the responses of an estuarine polychaete Hediste diversicolor to metal-contaminated sediment and increased temperature. Ragworms were exposed to sediments spiked with 10 and 20 mg kg−1 of copper at 12 and 20 °C for three weeks. No considerable changes were observed in the expression of genes related to copper homeostasis and in the accumulation of oxidative stress damage. Dicarbonyl stress was attenuated by warming exposure. Whole-body energy reserves in the form of carbohydrates, lipids and proteins were little affected, but the energy consumption rate increased with copper exposure and elevated temperature, indicating higher basal maintenance costs of ragworms. The combined effects of copper and warming exposures were mostly additive, with copper being a weak stressor and warming a more potent stressor. These results were replicable, as confirmed by two independent experiments of similar settings conducted at two different months of the year. This study suggests the higher sensitivity of energy-related biomarkers and the need to search for more conserved molecular markers of metal exposure in H. diversicolor.

Advanced glycation endproducts and dicarbonyl compounds are not associated with coronary atherosclerosis characteristics in middle-aged and older male athletes

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation. Background Cardiovascular disease is the leading cause of mortality worldwide and is dominated by coronary atherosclerosis. Moreover, coronary atherosclerosis is the most common cause of sudden cardiac death among athletes and current screening methods lack sufficient accuracy for identifying individuals at high risk. Advanced glycemic end products (AGEs) and dicarbonyl stress markers are generated during normal metabolism and aging, but increase in the presence of hyperglycemia, oxidative stress and inflammation. Several studies have demonstrated that higher concentrations of AGEs in plasma predict both the presence and severity of coronary atherosclerosis, as well as mortality. AGEs and its dicarbonyl precursors in atherosclerotic plaques have also been demonstrated to predict rupture-prone phenotypes. Plasma AGEs and dicarbonyl precursors might be a novel way to screen for high-risk coronary atherosclerosis in middle-aged and older athletes. Purpose To assess the association between plasma AGEs and dicarbonyl compounds with the presence of coronary plaques, plaque characteristics and coronary artery calcification scores in a population of middle-aged and older amateur athletes. Methods We measured concentrations of AGEs and dicarbonyl stress markers in plasma with dilution ultra-performance liquid chromatography tandem mass spectrometry in middle-aged and older athletes from the Measuring Athletes’ Risk of Cardiovascular Events (MARC) 2 study cohort. Coronary plaques, predominating plaque characteristics (&amp;gt;50% calcified, non-calcified, or mixed plaque composition), and coronary artery calcification scores were assessed using coronary computed tomography. We explored associations with linear and logistic regression analyses and compared groups with the Mann-Whitney U and Kruskal-Wallis tests, while correcting for multiple testing. Results 284 men were included with a median age of 60 [56-66] years, body mass index of 24.5 [22.9-26.7] kg/m2, and an average weekly exercise volume of 41 [26-57] Metabolic Equivalent of Task-hours. Coronary plaques were detected in 241 participants (83%), with calcified, non-calcified, and mixed plaques constituting &amp;gt;50% of plaques in 42%, 12%, and 21%, respectively. Median CAC score was 31 [0-132]. None of the AGEs or dicarbonyl stress markers were associated with presence of plaques, any of the plaque characteristics (Figure 1), or coronary artery calcification score categories (Figure 2) in adjusted analyses (P&amp;gt;0.05). Conclusion Concentrations of AGEs and dicarbonyl stress markers could not be used to identify coronary plaques, different plaque characteristics or coronary calcifications in this cohort of middle-aged and older athletes. These findings suggest that AGEs and dicarbonyl stress markers cannot be used as a screening method for high-risk coronary atherosclerosis.

Hypolipidemic and insulin sensitizing effects of salsalate beyond suppressing inflammation in a prediabetic rat model.

Background and aims: Low-grade chronic inflammation plays an important role in the pathogenesis of metabolic syndrome, type 2 diabetes and their complications. In this study, we investigated the effects of salsalate, a non-steroidal anti-inflammatory drug, on metabolic disturbances in an animal model of prediabetes-a strain of non-obese hereditary hypertriglyceridemic (HHTg) rats. Materials and Methods: Adult male HHTg and Wistar control rats were fed a standard diet without or with salsalate delivering a daily dose of 200mg/kg of body weight for 6weeks. Tissue sensitivity to insulin action was measured ex vivo according to basal and insulin-stimulated 14C-U-glucose incorporation into muscle glycogen or adipose tissue lipids. The concentration of methylglyoxal and glutathione was determined using the HPLC-method. Gene expression was measured by quantitative RT-PCR. Results: Salsalate treatment of HHTg rats when compared to their untreated controls was associated with significant amelioration of inflammation, dyslipidemia and insulin resistance. Specificaly, salsalate treatment was associated with reduced inflammation, oxidative and dicarbonyl stress when inflammatory markers, lipoperoxidation products and methylglyoxal levels were significantly decreased in serum and tissues. In addition, salsalate ameliorated glycaemia and reduced serum lipid concentrations. Insulin sensitivity in visceral adipose tissue and skeletal muscle was significantly increased after salsalate administration. Further, salsalate markedly reduced hepatic lipid accumulation (triglycerides -29% and cholesterol -14%). Hypolipidemic effects of salsalate were associated with differential expression of genes coding for enzymes and transcription factors involved in lipid synthesis (Fas, Hmgcr), oxidation (Pparα) and transport (Ldlr, Abc transporters), as well as changes in gene expression of cytochrome P450 proteins, in particular decreased Cyp7a and increased Cyp4a isoforms. Conclusion: These results demonstrate important anti-inflammatory and anti-oxidative effects of salsalate that were associated with reduced dyslipidemia and insulin resistance in HHTg rats. Hypolipidemic effects of salsalate were associated with differential expression of genes regulating lipid metabolism in the liver. These results suggest potential beneficial use of salsalate in prediabetic patients with NAFLD symptoms.

Reader Response: Association of Amount of Weight Lost After Bariatric Surgery With Intracranial Pressure in Women With Idiopathic Intracranial Hypertension.

I read the article by Mollan et al.1 regarding weight loss and intracranial hypertension (IH) with interest. Gastric bypass results in decreased dicarbonyl stress (DS).2 DS-related compounds, such as methylgloxal, are associated with the breakdown of the blood-brain barrier,3 as seen in IH through the glycation of biomolecules and the production of advanced glycation end products (AGEs).3,4 Inhibiting semicarbazide-sensitive amine oxidase, which causes DS, with caffeine may lead to decreased weight gain and AGEs.5

Glyoxalase I Assay as a Possible Tool for Evaluation of Biological Activity of Antioxidant-Rich Plant Extracts

The health-promoting properties of natural plant bioactive compounds are mainly attributable to their ability to counteract oxidative stress. This is considered a major causative factor in aging and aging-related human diseases, in which a causal role is also ascribed to dicarbonyl stress. This is due to accumulation of methylglyoxal (MG) and other reactive dicarbonyl species, leading to macromolecule glycation and cell/tissue dysfunction. The glyoxalase (GLYI) enzyme, catalyzing the rate-limiting step of the GSH-dependent MG detoxification pathway, plays a key role in cell defense against dicarbonyl stress. Therefore, the study of GLYI regulation is of relevant interest. In particular, GLYI inducers are important for pharmacological interventions to sustain healthy aging and to improve dicarbonyl-related diseases; GLYI inhibitors, allowing increased MG levels to act as proapoptotic agents in tumor cells, are of special interest in cancer treatment. In this study, we performed a new in vitro exploration of biological activity of plant bioactive compounds by associating the measurement of their antioxidant capacity (AC) with the evaluation of their potential impact on dicarbonyl stress measured as capability to modulate GLYI activity. AC was evaluated using TEAC, ORAC, and LOX-FL methods. The GLYI assay was performed using a human recombinant isoform, in comparison with the recently characterized GLYI activity of durum wheat mitochondria. Different plant extracts were tested, obtained from plant sources with very high phytochemical content ('Sun Black' and wildtype tomatoes, black and 'Polignano' carrots, and durum wheat grain). Results showed high antioxidant properties of the tested extracts, associated with different modes (no effect, activation, and inhibition) and effectiveness in modulating both GLYI activity sources. Overall, results indicate the GLYI assay as an advisable and promising tool for researching plant foods as a source of natural antioxidant compounds acting as GLYI enzymatic regulators to be used for dietary management associated the treatment of oxidative/dicarbonyl-promoted diseases.

Molecular dynamics simulations and bioinformatics’ analysis of deleterious missense single nucleotide polymorphisms in Glyoxalase-1 gene

Glyoxalase-1 (Glo-1) is a key member of the Glyoxalase system, the primary line of defense against dicarbonyl stress which, in tandem, with reduced levels of expression or activity of Glyoxalase-1 enzyme, has been implicated in various human diseases like type 2 diabetes mellitus (T2DM) and its vascular complications. The association of Glo-1 single nucleotide polymorphisms with genetic susceptibility to T2DM and its vascular complications is yet to be explored. Therefore, in this study, we have employed a computational approach to identify the most damaging missense or nonsynonymous SNPs (nsSNPs) in Glo-1 gene. Initially, we characterized missense SNPs that are damaging to the structural and functional integrity of Glo-1 using various bioinformatic tools. These tools included SIFT, PolyPhen-2, SNAP, PANTHER, PROVEAN, PhD-SNP, SNPs&GO, I-Mutant, MUpro and MutPred2. One of these missense SNPs (rs1038747749; corresponding to amino acid change Arginine to Glutamine at position 38) was found to be highly conserved in evolution and is an important part of the enzyme’s active site, glutathione binding site, as well as the dimeric interface based on the results obtained from ConSurf and NCBI Conserved Domain Search tools. Project HOPE reported that this mutation replaces a positively charged polar amino acid (Arginine) with a small, neutrally charged amino acid (Glutamine). Comparative modelling of wildtype and mutant (R38Q) Glo-1 proteins was performed in the run up to molecular dynamics simulation analysis which showed that rs1038747749 adversely impacts Glo-1 protein’s stability, rigidity, compactness, hydrogen bonds/interactions as demonstrated by the results of various parameters computed during the analysis.Communicated by Ramaswamy H. Sarma

The antioxidant glutathione.

Reduced glutathione (GSH) is an essential non-enzymatic antioxidant in mammalian cells. GSH can act directly as an antioxidant to protect cells against free radicals and pro-oxidants, and as a cofactor for antioxidant and detoxification enzymes such as glutathione peroxidases, glutathione S-transferases, and glyoxalases. Glutathione peroxidases detoxify peroxides by a reaction that is coupled to GSH oxidation to glutathione disulfide (GSSG). GSSG is converted back to GSH by glutathione reductase and cofactor NADPH. GSH can regenerate vitamin E following detoxification reactions of vitamin E with lipid peroxyl radicals (LOO). GSH is a cofactor for GST during detoxification of electrophilic substances and xenobiotics. Dicarbonyl stress induced by methylglyoxal and glyoxal is alleviated by glyoxalase enzymes and GSH. GSH regulates redox signaling through reversible oxidation of critical protein cysteine residues by S-glutathionylation. GSH is involved in other cellular processes such as protein folding, protecting protein thiols from oxidation and crosslinking, degradation of proteins with disulfide bonds, cell cycle regulation and proliferation, ascorbate metabolism, apoptosis and ferroptosis.