Glucose Metabolites Research Articles

Non-Enzymatic Methylglyoxal Formation From glucose Metabolites and Generation of Superoxide Anion Radical During Methylglyoxal-Dependent Cross-Links Reaction

The paper explores the formation of a-oxoaldehydes during the interaction of glucose metabolites with hydroxyl or alkoxyl radicals. Hydroxyl radicals were generated under radiolysis of aqueous solutions, and alkoxyl radicals (t-BuO) were obtained in the model system tert-butyl hydroperoxide/Fe2+. High-performance liquid chromatography revealed that methylglyoxal was one of the organic products resulting from t-BuO-induced transformations of fructose-1,6-bisphosphate under hypoxic conditions. The interaction of lysine and methylglyoxal one of the main targets of a-oxoaldehydes in proteins was also studied. As chemiluminescence and EPR spectroscopy demonstrated, this reaction generates a methylglyoxal anion radical, a cation-radical of methylglyoxal dialkylamine and a superoxide anion radical. EPR signal of methylglyoxal-derived free radicals was observed in hypoxia, whereas only the trace amounts of these free radicals were recorded in the aerated reaction medium.

Similarities and differences in the biotransformation and transcriptomic responses of Caenorhabditis elegans and Haemonchus contortus to five different benzimidazole drugs

We have undertaken a detailed analysis of the biotransformation of five of the most therapeutically important benzimidazole anthelmintics - albendazole (ABZ), mebendazole (MBZ), thiabendazole (TBZ), oxfendazole (OxBZ) and fenbendazole (FBZ) - in Caenorhabditis elegans and the ruminant parasite Haemonchus contortus. Drug metabolites were detected by LC-MS/MS analysis in supernatants of C. elegans cultures with a hexose conjugate, most likely glucose, dominating for all five drugs. This work adds to a growing body of evidence that glucose conjugation is a major pathway of xenobiotic metabolism in nematodes and may be a target for enhancement of anthelmintic potency. Consistent with this, we found that biotransformation of albendazole by C. elegans reduced drug potency. Glucose metabolite production by C. elegans was reduced in the presence of the pharmacological inhibitor chrysin suggesting that UDP-glucuronosyl/glucosyl transferase (UGT) enzymes may catalyze benzimidazole glucosidation. Similar glucoside metabolites were detected following ex vivo culture of adult Haemonchus contortus. As a step towards identifying nematode enzymes potentially responsible for benzimidazole biotransformation, we characterised the transcriptomic response to each of the benzimidazole drugs using the C. elegans resistant strain CB3474 ben-1(e1880)III. In the case of albendazole, mebendazole, thiabendazole, and oxfendazole the shared transcriptomic response was dominated by the up-regulation of classical xenobiotic response genes including a shared group of UGT enzymes (ugt-14/25/33/34/37/41/8/9). In the case of fenbendazole, a much greater number of genes were up-regulated, as well as developmental and brood size effects suggesting the presence of secondary drug targets in addition to BEN-1. The transcriptional xenobiotic response of a multiply resistant H. contortus strain UGA/2004 was essentially undetectable in the adult stage but present in the L3 infective stage, albeit more muted than C. elegans. This suggests that xenobiotic responses may be less efficient in stages of parasitic nematodes that reside in the host compared with the free-living stages.

Has Resveratrol a Modulatory Effect on Renal Injury Associating Diabetic Nephropathy in Albino Rats?

AbstractBackground: The involvement of abnormally stimulated renin angiotensin system, excessive formation of reactive oxygen species, increased glucose metabolite flux and up-regulation of pro-inflammatory and profibrotic cytokines in the pathogenesis of diabetic nephropathy has become an emerging concern. Exploration of novel therapies that specif-ically target such factors is a major goal in diabetes treatment.Aim of Study: Our aim is to explore the potential protective and therapeutic effect of resveratrol on DN progression through its antioxidant, anti-inflammatory and antifibrotic role in comparison with losartan as one of the therapeutic regimens used in diabetic nephropathy.Material and Methods: Forty eight male albino rats were assigned into six groups; control, diabetic nephropathy, pro-phylactic losartan, prophylactic resveratrol, therapeutic losartan and therapeutic resveratrol. Systolіc blood pressure, fasting glucose, glomerular filtration rate, urea, urea albumin excretion, tumor necrosis factor a, tansforming growth factor-(3, inter-leukin-б and superoxide dismutase have been measured. Histopathological assessment of inflammation, glomeruloscle-rosis and fibrosis using hematoxylin and eosin, Masson's trichrome and Periodic acid shift staining and histomorpho-metric analysis for glomerular and tubular parameters have also been performed.Results: Prophylactic resveratrol showed significant regression in inflammation, fibrosis and oxidative stress compared with its therapeutic effect.Conclusion: Resveratrol showed better outcome compared with losartan with significant change in the biochemical parameters.

Blocked O-GlcNAc cycling disrupts mouse hematopoeitic stem cell maintenance and early T cell development

Small numbers of hematopoietic stem cells (HSCs) balance self-renewal and differentiation to produce the diversity and abundance of cell types that make up the blood system. How nutrients are recruited to support this massive differentiation and proliferation process remains largely unknown. The unique metabolism of adult HSCs, which rely on glycolysis and glutaminolysis, suggests a potential role for the post-translational modification O-GlcNAc as a critical nutrient signal in these cells. Glutamine, glucose, and other metabolites drive the hexosamine biosynthetic pathway (HBP) ultimately leading to the O-GlcNAc modification of critical intracellular targets. Here, we used a conditional targeted genetic deletion of the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), to determine the consequences of blocked O-GlcNAc cycling on HSCs. Oga deletion in mouse HSCs resulted in greatly diminished progenitor pools, impaired stem cell self-renewal and nearly complete loss of competitive repopulation capacity. Further, early T cell specification was particularly sensitive to Oga deletion. Loss of Oga resulted in a doubling of apoptotic cells within the bone marrow and transcriptional deregulation of key genes involved in adult stem cell maintenance and lineage specification. These findings suggest that O-GlcNAc cycling plays a critical role in supporting HSC homeostasis and early thymocyte development.

Effect of IDH3a on glucose uptake in lung adenocarcinoma: A pilot study based on [18 F]FDG.

Subunit of isocitrate dehydrogenase 3 (IDH3a) as upstream of the hypoxia‐inducible factor was reported highly expressed in malignant tumors, playing an important role in glucose metabolism reprogramming. As one of rate‐limiting enzyme in the Krebs cycle, whether high expression of IDH3a affects glucose uptake in tumors has not been elucidated. This study was aimed to investigate the relationship between IDH3a expression and tumor glucose uptake. Sixty‐five patients who underwent 2‐[18F]‐2‐deoxy‐D‐glucose ([18F]‐FDG) positron emission tomography/computed tomography (PET/CT) imaging before surgery and pathologically diagnosed as lung adenocarcinoma were included. All patients were divided into high (n = 31) and low (n = 34) groups according IDH3a expression by immunohistochemistry. Comparatively higher [18F]‐FDG uptake was found in high IDH3a expression group. Glucose transporter 1 (GLUT1) level was demonstrated to correlate with IDH3a expression, but not for hexokinase 2 (HK2). Furthermore, A549 and H1299 cells experiment showed, the expression of p‐AKT and GLUT1 were significantly downregulated after IDH3a interference. The cellular uptake of [18F]‐FDG and lactate production were significantly reduced in treatment group. In summary, high expression of IDH3a in lung adenocarcinoma patients is associated with higher glucose uptake. IDH3a targets AKT‐GLUT1 pathway to affect glucose uptake and metabolites in lung adenocarcinoma.

Glucose-6-Phosphate Regulates Hepatic Bile Acid Synthesis in Mice.

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient‐sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose‐6‐phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver‐specific glucose‐6‐phosphatase knockout (L‐G6pc−/−) mice, and mice treated with a pharmacological inhibitor of the G6P transporter. Hepatic G6P accumulation induces sterol 12α‐hydroxylase (Cyp8b1) expression, which is mediated by the major glucose‐sensitive transcription factor, carbohydrate response element‐binding protein (ChREBP). Activation of the G6P‐ChREBP‐CYP8B1 axis increases the relative abundance of cholic‐acid–derived bile acids and induces physiologically relevant shifts in bile composition. The G6P‐ChREBP–dependent change in bile acid hydrophobicity associates with elevated plasma campesterol/cholesterol ratio and reduced fecal neutral sterol loss, compatible with enhanced intestinal cholesterol absorption. Conclusion: We report that G6P, the primary intracellular metabolite of glucose, controls hepatic bile acid synthesis. Our work identifies hepatic G6P‐ChREBP‐CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

O-GlcNAcylation Enhances Double-Strand Break Repair, Promotes Cancer Cell Proliferation, and Prevents Therapy-Induced Senescence in Irradiated Tumors

The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. IMPLICATIONS: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Chrysin in Combination with Insulin Promotes Glucose Uptake in Skeletal Muscle Cell: Impact of Combination Therapy in Diabetes Myopathy (P01-031-19)

ObjectivesThe objective of the current study was examining the beneficial effects of DPP4 inhibitor chrysin alone and in combination with insulin benefits diabetes myopathy. MethodsSulforhodamine B (SRB) protein-dye was used to determine the acute toxicity, and 1H NMR spectroscopy was used to identify and quantify glucose levels to assess glucose uptake on treatments chrysin and combination of chrysin with Insulin on differentiated skeletal muscle cells. Pathway analysis was carried out using omics net web server. All experiments were conducted in triplicates, and all data in the graph represent Mean ± S.D. Graph pad software was used to calculate One-way analysis of variance (ANOVA), computed p-value among different groups and values with P < 0.05 is significant. ResultsResults showed that 250 μM chrysin and combination (10 nM Insulin with 250 μM chrysin) treatments are not acutely toxic to skeletal muscle cells and proliferates the cells significant to insulin-treated skeletal muscle cells (Figure 1A). Glucose metabolite levels are studied as skeletal muscle cells adopted cell proliferative and to demonstrate our hypothesis on glucose metabolism. Significant differences were observed in skeletal muscle cells treated with insulin, chrysin, and combination (Figure 1B). It is observed that chrysin alone, combination increases glucose uptake significantly in comparison to control cells. Pathway analysis revealed that GPCR signaling and immune-related signaling plays a role in proliferating skeletal muscle cells to regulate glucose metabolism. ConclusionsThe results of this study propose the use of natural compound chrysin in combination with insulin to promote skeletal muscle health in diabetes mellitus. The combination identified herein must be considered for future therapies to control diabetic myopathy in preclinical and clinical studies. Chrysin can also be applied as a supplement in the diet as well, to control diabetic myopathy. Funding SourcesAuthors wish to express their gratitude to Escuela Nacional de Ciencias Biológicas-Instituto Politécnico Nacional (ENCB-IPN) for providing financial support for the present study. Supporting Tables, Images and/or Graphs▪

Characterization of Glycolytic Enzymes and Pyruvate Kinase M2 in Type 1 and 2 Diabetic Nephropathy.

Elevated glycolytic enzymes in renal glomeruli correlated with preservation of renal function in the Medalist Study, individuals with ≥50 years of type 1 diabetes. Specifically, pyruvate kinase M2 (PKM2) activation protected insulin-deficient diabetic mice from hyperglycemia-induced glomerular pathology. This study aims to extend these findings in a separate cohort of individuals with type 1 and type 2 diabetes and discover new circulatory biomarkers for renal protection through proteomics and metabolomics of Medalists' plasma. We hypothesize that increased glycolytic flux and improved mitochondrial biogenesis will halt the progression of diabetic nephropathy. Immunoblots analyzed selected glycolytic and mitochondrial enzymes in postmortem glomeruli of non-Medalists with type 1 diabetes (n = 15), type 2 diabetes (n = 19), and no diabetes (n = 5). Plasma proteomic (SOMAscan) (n = 180) and metabolomic screens (n = 214) of Medalists with and without stage 3b chronic kidney disease (CKD) were conducted and significant markers validated by ELISA. Glycolytic (PKM1, PKM2, and ENO1) and mitochondrial (MTCO2) enzymes were significantly elevated in glomeruli of CKD- versus CKD+ individuals with type 2 diabetes. Medalists' plasma PKM2 correlated with estimated glomerular filtration rate (r 2 = 0.077; P = 0.0002). Several glucose and mitochondrial enzymes in circulation were upregulated with corresponding downregulation of toxic metabolites in CKD-protected Medalists. Amyloid precursor protein was also significantly upregulated, tumor necrosis factor receptors downregulated, and both confirmed by ELISA. Elevation of enzymes involved in the metabolism of intracellular free glucose and its metabolites in renal glomeruli is connected to preserving kidney function in both type 1 and type 2 diabetes. The renal profile of elevated glycolytic enzymes and reduced toxic glucose metabolites is reflected in the circulation, supporting their use as biomarkers for endogenous renal protective factors in people with diabetes.

Postprandial Metabolic Effects of Fiber Mixes Revealed by in vivo Stable Isotope Labeling in Humans.

Food supplementation with a fiber mix of guar gum and chickpea flour represents a promising approach to reduce the risk of type 2 diabetes mellitus (T2DM) by attenuating postprandial glycemia. To investigate the effects on postprandial metabolic fluxes of glucose-derived metabolites in response to this fiber mix, a randomized, cross-over study was designed. Twelve healthy, male subjects consumed three different flatbreads either supplemented with 2% guar gum or 4% guar gum and 15% chickpea flour or without supplementation (control). The flatbreads were enriched with ~2% of 13C-labeled wheat flour. Blood was collected at 16 intervals over a period of 360 min after bread intake and plasma samples were analyzed by GC-MS based metabolite profiling combined with stable isotope-assisted metabolomics. Although metabolite levels of the downstream metabolites of glucose, specifically lactate and alanine, were not altered in response to the fiber mix, supplementation of 4% guar gum was shown to significantly delay and reduce the exogenous formation of these metabolites. Metabolic modeling and computation of appearance rates revealed that the effects induced by the fiber mix were strongest for glucose and attenuated downstream of glucose. Further investigations to explore the potential of fiber mix supplementation to counteract the development of metabolic diseases are warranted.

Mourning Doves, Zenaida macroura, are Resistant to Metabolic Effects of a High Fat Diet

Birds have naturally high glycemia relative to mammals and can serve as a natural animal model of hyperglycemia without associated mammalian‐like complications. In mice, consumption of a high fat diet (60% kcals from fat) for six weeks results in a pathologic condition characterized by increased body weight and elevated plasma insulin, glucose, and triglycerides. Thus, we hypothesized that adult Mourning Doves consuming a high fat diet (60% kcal from fat; HF; n=4) would likewise exhibit diabetes‐like pathology including hyperglycemia and altered metabolic profile when compared to birds fed a control diet (CON; n=5). All doves were housed individually and fed a nutritional balanced dove seed diet during a week‐long cage acclimation period. Next, birds were acclimated to their study diets for a week where the study diets were mixed into the seed diet in increasing amount until only the study diet remained. At the end of the four‐week experimental dietary period, we euthanized birds with an overdose of sodium pentobarbital and collected cardiac blood, liver, kidney, and pectoralis muscle for metabolomic analyses and biochemical assays. Contrary to our hypothesis, doves fed a HF diet developed few metabolite changes compared to control doves (number of altered metabolite concentrations: 19 of 137 in plasma, 4 of 152 in liver, 1 of 142 in pectoralis muscle, 3 in 150 in kidney; p&lt;0.05). Specifically, glucose metabolite concentrations did not differ between groups. Further, we observed alterations in several metabolic pathways, but changes in only two pathways were significant (p&lt;0.05) and concurrently of high impact (scale: 0–1; &gt;0.60): (a) Ubiquinone and other terpenoid‐quinone biosynthesis (plasma and kidney), and (b) alanine, aspartate, and glutamate metabolism (plasma and kidney). In addition, consumption of the HF diet did not alter body mass, plasma uric acid, pectoralis glycogen, or liver glycogen and triglycerides. In conclusion, consumption of a HF diet for four weeks did not instigate symptoms of diabetes. Elucidating the uniqueness of avian nutritional physiology, specifically their resistance to dietinduced metabolic complications, may help develop novel therapeutic agents for mammalian diabetes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucose-6-phosphate dehydrogenase deficiency and metabolic profiling in adolescence from the Chinese birth cohort: “Children of 1997”

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency affects 6.0% of the global population. G6PD deficiency has been associated with lower risk of cardiovascular disease and higher risk of diabetes, which could be etiologically informative, but these relations are uncertain. To clarify, we assessed the associations of G6PD deficiency with serum metabolite profiles in late adolescence. MethodsIn a nested case-control study of 50 G6PD-deficient late adolescents (~17.5 years) and 150 sex-matched non-G6PD-deficient controls from a Chinese birth cohort: “Children of 1997”, we compared 80 serum metabolites analyzed by nuclear magnetic resonance spectrometry using adjusted linear regression with Bonferroni correction for testing 12 traits (p < 0.0042). ResultsG6PD-deficiency was inversely associated with serum levels of total cholesterol (−0.27 mmol, 95% confidence interval (CI) −0.46, −0.09, p = 0.004), free cholesterol (−0.08 mmol, 95% CI −0.13, −0.03, p = 0.003) and creatinine (−0.004 mmol, 95% CI −0.007, −0.001, p = 0.003), adjusted for sex and parental education. G6PD deficiency was not associated with fatty acids, amino acids, glucose or related metabolites, ketone bodies or glycoprotein. ConclusionsG6PD deficiency is associated with lower serum levels of cholesterol and creatinine, but not other serum metabolites. Whether such differences are transient or become more evident in adulthood warrant further investigations.

Metabolomics Coupled with Transcriptomics Approach Deciphering Age Relevance in Sepsis.

Sepsis is a severe disease frequently occurred in the Intenisive Care Unit (ICU), which has a very high morbidity and mortality, especially in patients aged over 65 years. Owing to the aging effect and the ensuing deterioration of body function, the elder patients may have atypical responses to sepsis. Diagnosis and pathogenesis of sepsis in this population are thus difficult, which hindered effective treatment and management in clinic. To investigated age effects on sepsis, 158 elderly septic patients and 71 non-septic elderly participants were enrolled, and their plasma samples were collected for transcriptomics (RNA-seq) and metabolomics (NMR and GC-MS) analyses, which are both increasingly being utilized to discover key molecular changes and potential biomarkers for various diseases. Protein-protein interaction (PPI) analysis was subsequently performed to assist cross-platform integration. Real time polymerase chain reaction (RT-PCR) was used for validation of RNA-seq results. For further understanding of the mechanisms, cecal ligation and puncture (CLP) experiment was performed both in young and middle-aged rats, which were subjected to NMR-based metabolomics study and validated for several key inflammation pathways by western blot. Comprehensive analysis of data from the two omics approaches provides a systematic perspective on dysregulated pathways that could facilitate the development of therapy and biomarkers for elderly sepsis. Additionally, the metabolites of lactate, arginine, histamine, tyrosine, glutamate and glucose were shown to be highly specific and sensitive in distinguishing septic patients from healthy controls. Significant increases of arginine, trimethylamine N-oxide and allantoin characterized elderly patient incurred sepsis. Further analytical and biological validations in different subpopulations of septic patients should be carried out, allowing accurate diagnostics and precise treatment of sepsis in clinic.

Milk production in Saanen goats treated with repeated low doses of intermediate-release insulin during early lactation

ABSTRACT: The effect of insulin administration on the productive responses of Saanen goats during early lactation was investigated. Ten of 20 adult females were subjected to subcutaneous administration of intermediate-acting insulin (0.14UI/kg body weight) at 2, 9, and 14 days postpartum. Milk yield was measured twice daily for 13 weeks and milk samples were collected to measure protein and fat contents. Plasma levels of progesterone, insulin, non-esterifies fatty acids, glucose and other metabolites were measured. Results showed a significantly increased effect of insulin treatment on the content of milk fat and protein; moreover, milk production in the first and second postpartum weeks were higher than control group. The peak of lactation in the insulin group was achieved one week earlier in comparison to the control group. In addition, the milk production rate showed lower persistency (milk yield 13 week/milk yield at peak) in the same group. During the first four weeks of postpartum, treated animals showed greater weight loss and higher non-esterified fatty acid concentration, whereas no effect was observed on the concentration of progesterone and other metabolites. The above results indicated that repeated administration of insulin in dairy goats during early lactation increase yield and qualitative components of milk, but has substantial consequences on animal productive rate and metabolic response.

Mathematical study of feedback inhibition effects on the dynamics of metabolites on the central metabolism of a yeast cell: a combination of kinetic model and metabolic control analysis

The fermentation system is a metabolic system that can be considered as a complex system, since it involves metabolites linked by different reactions. Some outputs of the reaction act as input in the other reactions and they control the behaviour of the system. Glucose as the main carbon source of the yeast cells plays an important role in the cell’s reproduction cycle and product synthesis. Glucose promotes negative feedback to some reactions in the central metabolism of the yeast cell. In this paper we mathematically study the effects of the negative feedback (inhibition) by glucose and other metabolites on the dynamics of the fermentation system. We also study the sensitivity of the flux in the presence of inhibition. We found that high inhibition by glucose affects the concentration of acetyl-CoA, which will lead to the respiration pathway of the yeast cells. High inhibition by acetaldehyde affects the concentration of all metabolites, including the maximal concentration of ethanol. Based on the metabolic control analysis results, we found that glucose can be considered as the external regulating point in increasing the flux of ethanol. Although glucose acts as a negative feedback, it can also be used to promote certain processes in the metabolic pathway since it gives the highest positive control in increasing the flux of ethanol as the desired product. For the internal regulation point, the maximal concentration of ethanol can be increased significantly by regulating the maximal activity of alcohol dehydrogenase and acetaldehyde dehydrogenase simultaneously.

Methylglyoxal stress, the glyoxalase system, and diabetic chronic kidney disease.

Chronic kidney disease (CKD) remains a serious diabetic complication despite the use of widely employed interventions such as angiotensin-converting enzyme inhibitors and glucose-lowering treatments. Accumulation of methylglyoxal, a highly reactive glucose metabolite and a major precursor in the formation of advanced glycation end products, may link the hemodynamic, inflammatory, metabolic, and structural changes that drive diabetic CKD. Therefore, methylglyoxal may serve as a potential therapeutic target to prevent diabetic CKD. Higher plasma methylglyoxal levels were shown to be associated with a decline in the estimated glomerular filtration rate. Furthermore, interventions that lower methylglyoxal levels reduced albuminuria in rodent models of diabetes. In addition, the glyoxalase system, which detoxifies methylglyoxal into D-lactate, has been identified as a key protective enzymatic system against diabetic CKD in both human and rodent studies. Recently, several promising treatments to lower methylglyoxal directly or to boost the glyoxalase system have been identified. The review highlights the mechanisms through which methylglyoxal is formed in diabetes, and how methylglyoxal contributes to the mechanisms that drive CKD in diabetes. Furthermore, we discuss the role of glyoxalase-1 in diabetic CKD. Finally, we discuss recent data about treatments that lower methylglyoxal stress.

Grape pomace polyphenols improve insulin response to a standard meal in healthy individuals: A pilot study

Dietary polyphenols have beneficial effects on glucose/lipid metabolism in subjects at high risk to develop type 2 diabetes; however, the underlying mechanisms are not clear. We aimed to evaluate: 1) the acute effects of the consumption of a drink rich in polyphenols from red grape pomace (RGPD) on glucose/insulin and triglyceride responses to a standard meal in healthy individuals, and, 2) the relationship between plasma levels of phenolic metabolites and metabolic parameters. Twelve healthy men, aged 20-40 years participated in a randomized, controlled study according to a cross-over design. After a 3-day low-polyphenol diet, all participants consumed, on two different days and separated by a one week interval, after an overnight fast, a drink rich in polyphenols (1.562g gallic acid equivalents (GAE)) or a control drink (CD, no polyphenols), followed after 3h by a standard meal (960kcal, 18% protein, 30% fat, 52% CHO). Blood samples were taken at fasting, 3h after the drink, over 5h after the standard meal and at fasting on the next day to measure plasma concentrations of glucose, insulin, triglyceride and phenolic metabolites. Glycemic and triglyceride post-meal responses were similar after both the RGPD and the control drink. In contrast, postprandial insulin incremental area (iAUC0-5h) was 31% lower (p<0.05), insulin secretion index was 18% lower (p<0.016) and insulin sensitivity (SI) index was 36% higher (p=0.037) after the RGPD compared to CD. Among phenolic metabolites, gallic acid correlated inversely with the insulin response (r=-0.604; p=0.032) and positively with the SI index (r=0.588, p=0.037). RGPD consumption acutely reduced postprandial insulin levels and improved insulin sensitivity. This effect could be likely related to the increase in gallic acid levels. This drink, added to usual diet, could contribute to increase the daily intake of polyphenols, with potential health benefits. CLINICALTRIALS. NCT02865278.

Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression.

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr96/101. We found that fructose (5 mM) increased transport-related O2 consumption in TALs by 11.1 ± 3.2% ( P < 0.05). The effect of fructose on O2 consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% ( P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5, 49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness.We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples.We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A).The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus.Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation.In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. [Display omitted] DisclosuresSchimmer:Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.

Extracellular glucose levels in cultures of undifferentiated mouse trophoblast stem cells affect gene expression during subsequent differentiation with replicable cell line-dependent variation.

Mouse trophoblast stem cells (TSCs) have been established and maintained using hyperglycemic conditions (11 mM glucose) for no apparent good reason. Because glucose metabolites are used as resources for cellular energy production, biosynthesis, and epigenetic modifications, differences in extracellular glucose levels may widely affect cellular function. Since the hyperglycemic culture conditions used for TSC culture have not been fully validated, the effect of extracellular glucose levels on the properties of TSCs remains unclear. To address this issue, we investigated the gene expression of stemness-related transcription factors in TSCs cultured in the undifferentiated state under various glucose concentrations. We also examined the expression of trophoblast subtype markers during differentiation, after returning the glucose concentration to the conventional culture concentration (11 mM). As a result, it appeared that the extracellular glucose conditions in the stem state not only affected the gene expression of stemness-related transcription factors before differentiation but also affected the expression of marker genes after differentiation, with some line-to-line variation. In the TS4 cell line, which showed the largest glucose concentration-dependent fluctuations in gene expression among all the lines examined, low glucose (1 mM glucose, LG) augmented H3K27me3 levels. An Ezh2 inhibitor prevented these LG-induced changes in gene expression, suggesting the possible involvement of H3K27me3 in the changes in gene expression seen in LG. These results collectively indicate that the response of the TSCs to the change in the extracellular glucose concentration is cell line-dependent and a part of which may be epigenetically memorized.