High Concentration Of D-glucose Research Articles

Wnt/β‐catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes

Diabetic nephropathy is a major complication of diabetes and a frequent cause of end-stage renal disease and recent studies suggest that podocyte damage may play a role in the pathogenesis of this. At early onset of diabetic nephropathy there is podocyte drop-out, which is thought to provoke glomerular albuminuria and subsequent glomerular injury; however, the underlying molecular mechanisms of this remain poorly understood. Here we report that we tested the hypothesis that early diabetic podocyte injury is caused, at least in part, by up-regulation of transient receptor potential cation channel 6 (TRPC6), which is regulated by the canonical Wnt signalling pathway, in mouse podocytes. Mechanism of injury initiation in mouse podocytes, by high concentration of D-glucose (HG, 30 mM), was investigated by MTT, flow cytometry, real-time quantitative PCR, and western blot analysis. HG induced apoptosis and reduced viability of differentiated podocytes. It caused time-dependent up-regulation of TRPC6 and activation of the canonical Wnt signalling pathway, in mouse podocytes. In these cells, blockade of the Wnt signalling pathway by dickkopf related protein 1 (Dkk1) resulted in effective reduction of TRPC6 up-regulation and amelioration of podocyte apoptosis. Furthermore, reduction of cell viability induced by HG was attenuated by treatment with Dkk1. These findings indicate that the Wnt/β-catenin signalling pathway may potentially be active in pathogenesis of TRPC6-mediated diabetic podocyte injury.

The Abnormally High Level of Uric D-Ribose for Type-2 Diabetics

In 1815,the French chemist Michel Eugene Chevreul(1786-1889) discovered that the sweetness in the urine of diabetics comes from grape sugar or D-glucose.Diabetes mellitus(DM) is considered as a group of metabolic diseases characterized by hyperglycemia(high concentration of blood D-glucose) resulting from defects in insulin secretion,insulin action,or both.On the other hand,D-ribose as an energetic enhancer was found to decrease the concentration of blood D-glucose,and thus "Oral administration of D-ribose in diabetes mellitus" was ever described by Steinberg and colleagues(1970).As described previously in this laboratory,D-ribose rapidly glycates proteins,such as BSA,neuronal Tau and α-synuclein,producing advanced glycation end products(AGEs) with severe cyt o toxicity,leading to dysfunction and cell death,in vitro and in vivo.Intraperitoneal injection of D-ribose into mice significantly increases their glycated serum protein and blood AGEs though the concentration of D-glucose became slightly decreased,suggesting that D-ribose is much easier to produce AGEs than D-glucose in vivo.Here,using 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid(MOPBA) coupled with HPLC,we determined the concentration of uric D-ribose of type 2 diabetic patients(n=30) and the age-matched healthy controls(n=30).The results show that the yield of the derivative of MOPBA-ribose is linearly correlated with the concentration of D-ribose(r2=0.999) with a recovery of 99%.The isolated fractions of D-ribose and D-glucose from urine of type 2 diabetic patients through HPLC were analyzed by mass spectrometry,and the results showed that the fractions contained 569.19 u compound(C27 H29 N4 O10,D-ribose),and 599.20 u compound(C28 H31 N4 O11,D-glucose) respectively.The concentration of uric D-ribose of Type 2 diabetics(male(134.28±35.09) μmol/L,female(97.33± 23.68) μmol/L) was significantly(P 0.001) higher than that of the age-matched healthy control(male(35.99 ± 5.64) μmol/L,female(33.72 ±6.27) μmol/L).Under the experimental conditions,the uric D-glucose level of the patients was also markedly(P 0.001) higher than the control.Further analyses showed a marked increase in the level of uric D-ribose from either male(P 0.001) or female(P 0.001),but a significant difference of the uric levels between male and female could not be observed(P 0.05).The high levels of uric D-ribose and D-glucose of the patients suggest that type 2 diabetic patients are not only suffered from D-glucose metabolism disorders,but also from D-ribose metabolism disorders.

Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81

Megasphaera elsdenii T81 grew on either DL-lactate or D-glucose at similar rates (0.85 h(-1)) but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able to grow at much higher concentrations of D-glucose (500 mM), but never removed more than 80 mM of glucose from the medium, and nearly 60 % the glucose removed was sequestered as intracellular glycogen, with low yields of even-carbon acids (acetate, butyrate, caproate). In the presence of both substrates, glucose was not used until lactate was nearly exhausted, even by cells pregrown on glucose. Glucose-grown cultures maintained only low extracellular concentrations of acetate, and addition of exogenous acetate increased yields of butyrate, but not caproate. By contrast, exogenous acetate had little effect on lactate fermentation. At pH 6.6, growth rate was halved by exogenous addition of 60 mM propionate, 69 mM butyrate, 44 mM valerate, or 33 mM caproate; at pH 5.9, these values were reduced to 49, 49, 18, and 22 mM, respectively. The results are consistent with this species' role as an effective ruminal lactate consumer and suggest that this organism may be useful for industrial production of volatile fatty acids from lactate if product tolerance could be improved. The poor fermentation of glucose and sensitivity to caproate suggests that this strain is not practical for industrial caproate production.

Response to Comment on: Gorboulev et al. Na+-d-glucose Cotransporter SGLT1 Is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion. Diabetes 2012;61:187–196

In response to the commentary of Professor Kellett (1), we would like to clarify the following points. The data reported in our article (2) confirm the observation that SGLT1-mediated D-glucose uptake in the presence of a high D-glucose concentration in the small intestinal lumen mediates translocation of GLUT2 to the luminal membrane of the enterocytes; however, they contradict the hypothesis that GLUT2 is the major pathway of D-glucose absorption when glucose is plentiful (3–6). Comparing SGLT1-mediated and GLUT2-mediated glucose uptake into isolated brush-border membrane (BBM) vesicles, we present evidence that SGLT1 transports about 90% of D-glucose into the enterocytes in the presence of high luminal glucose whereas only about 10% …

A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro.

Diabetic retinopathy is a common complication of diabetes mellitus (DM). The oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose closely approximates the molecular basis for the loss of vision associated with this disease. We investigate a novel algae-derived polysaccharide compound for its role in protecting ARPE-19 cells from high glucose-induced oxidative damage. ARPE-19 cells were cultured for 4 d with normal concentration of D-glucose, and exposed to either normal or high concentrations of D-glucose in the presence or absence of the polysaccharide compound at variety of concentrations for another 48 h. Taurine was used as a positive control. Activity of super oxide dismutase (SOD) and concentration of glutathione (GSH) were measured as well as cytotoxicity of high glucose and the polysaccharide compound. To analyse cellular damage by high glucose, activation of Annexin V and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) were examined. Our results showed that a significant cellular damage on ARPE-19 cells after 48 h treatment with high glucose, accompanied by a decrease in SOD activity and GSH concentration; high glucose also caused ARPE-19 cell apoptosis and activation of p38MAPK and ERK. As the non-toxic polysaccharide compound protected ARPE-19 cells from high glucose-induced cellular damage, the compound recovered SOD activity and concentration of GSH in the cells. The compound also abrogated the cell apoptosis and activation of p38MAPK and ERK. Therefore, the polysaccharide compound derived from algae extracts could be unique candidate for a new class of anti-DM and anti-oxidative damage.

NADPH Oxidase 4 Mediates Monocyte Priming and Accelerated Chemotaxis Induced by Metabolic Stress

Metabolic disorders increase monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis in mice. The goal of this study was to determine the molecular mechanisms responsible for the enhanced responsiveness of monocytes to chemoattractants induced by metabolic stress. Chronic exposure of monocytes to diabetic conditions induced by human LDL plus high D-glucose concentrations (LDL+HG) promoted NADPH Oxidase 4 (Nox4) expression, increased intracellular H(2)O(2) formation, stimulated protein S-glutathionylation, and increased chemotaxis in response to MCP-1, platelet-derived growth factor B, and RANTES. Both H(2)O(2) added exogenously and overexpression of Nox4 mimicked LDL+HG-induced monocyte priming, whereas Nox4 knockdown protected monocytes against metabolic stress-induced priming and accelerated chemotaxis. Exposure of monocytes to LDL+HG promoted the S-glutathionylation of actin, decreased the F-actin/G-actin ratio, and increased actin remodeling in response to MCP-1. Preventing LDL+HG-induced protein S-glutathionylation by overexpressing glutaredoxin 1 prevented monocyte priming and normalized monocyte chemotaxis in response to MCP-1. Induction of hypercholesterolemia and hyperglycemia in C57BL/6 mice promoted Nox4 expression and protein S-glutathionylation in macrophages, and increased macrophage recruitment into MCP-1-loaded Matrigel plugs implanted subcutaneous in these mice. By increasing actin-S-glutathionylation and remodeling, metabolic stress primes monocytes for chemoattractant-induced transmigration and recruitment to sites of vascular injury. This Nox4-dependent process provides a novel mechanism through which metabolic disorders promote atherogenesis.

A Fluorescent sp2‐Iminosugar With Pharmacological Chaperone Activity for Gaucher Disease: Synthesis and Intracellular Distribution Studies

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.

A Role for Insulin on L-Arginine Transport in Fetal Endothelial Dysfunction in Hyperglycaemia

Endothelial cells are key in the regulation of vascular tone through the release of vasoactive molecules, including nitric oxide (NO). NO is a gas synthesized from the cationic amino acid L-arginine via the endothelial NO synthase (eNOS). The semi-essential amino acid L-arginine is a taken up by endothelial cells via systems y(+) and y(+)L in primary cultures of human umbilical vein endothelial cells (HUVEC). System y(+) is a family of membrane transporters including at least five transport systems for cationic amino acids (CAT) of which HUVEC express human CAT-1 (hCAT-1) and hCAT-2B. Exposure of HUVEC to high extracellular concentrations of D-glucose increases L-arginine transport, hCAT-1 mRNA expression and eNOS activity. These phenomena are also related with increased production of reactive oxygen species (ROS), thus supporting the possibility that changes in L-arginine/NO signalling pathway result from elevated ROS. It has been shown that insulin blocks D-glucose-increased L-arginine transport and cGMP accumulation in HUVEC, whereas in this cell type insulin also modulates high D-glucose effects by activating the transcriptional factors Sp1 and NFkappaB. These transcription factors have response elements in SLC7A1 (for hCAT-1) gene promoter region, thus representing 2 possible targets for regulation of the expression of this transporter by D-glucose and/or insulin in this cell type. Recent evidences suggest that insulin blocks the stimulatory effect of D-glucose on L-arginine transport by reducing the transcriptional activity of SLC7A1 via Sp1-, NFkappaB- and ROS-dependent mechanisms. Thus, a role for these transcription factors in response to insulin is proposed in fetal endothelial cells exposed to hyperglycaemia.

Aldose reductase inhibitor counteracts the attenuated adhesion of human corneal epithelial cells induced by high glucose through modulation of MMP-10 expression

Aims We investigated the preventive effect of aldose reductase inhibitor (ARI) on the adhesion of SV40-transformed human corneal epithelial cells (HCECs) exposed to high glucose, and the underlying mechanism focusing on the role of matrix metalloproteinase (MMP)-10. Methods HCECs were cultured in medium containing a normal (5.5 mM) or high (31.2 mM) concentration of d-glucose in the presence or absence of ARI, fidarestat. Cell attachment ability was evaluated by short-term adhesion assay. The levels of intracellular polyol were measured by liquid-gas chromatography. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were used to determine the expression levels. Results Decreased attachment activity and increased accumulation of polyol induced by exposure to high glucose were abrogated by ARI. Supply of recombinant MMP-10 decreased integrin α3β1-expression and cell adhesion. The expression level of MMP-10 was enhanced at both protein and mRNA levels by exposure to high glucose, while that of integrin α3β1 was decreased at the protein level, but remained unchanged at the mRNA level. These alterations in the expression levels of MMP-10 and integrin α3β1 were normalized by ARI. Conclusions ARI counteracts the decreased adhesion of HCECs induced by high glucose exposure, through the modulation of the expression of MMP-10.

High glucose induces transactivation of the human paraoxonase 1 gene in hepatocytes

Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein and inhibits oxidative modification of low-density lipoprotein in vitro. Therefore, PON1 is expected to protect against atherosclerosis in vivo. We and other investigators have shown that PON1 enzymatic activity is decreased in diabetic patients; however, an alteration in hepatic PON1 synthesis under hyperglycemic conditions remains unclear. We previously demonstrated that Sp1 is a positive regulator of PON1 transcription and that an interaction between Sp1 and protein kinase C (PKC) is a crucial mechanism for the effect of Sp1 on PON1 transcription in cultured HepG2 cells. Because several PKC isoforms are activated under hyperglycemic conditions, we examined the effect of d-glucose, which can activate the diacylglycerol-PKC pathway, on the transcription and expression of PON1. For a reporter gene assay, Huh7 human hepatocyte cell line incorporated with PON1 (−1232/−6)–luciferase expression vector was established using a cationic lipid method. d-Glucose dose dependently enhanced PON1 promoter activity. d-Glucose also enhanced both messenger RNA and protein expression of PON1. Increased PON1 expression was also detected in primary human hepatocytes treated with high d-glucose concentrations. Bisindolylmaleimide, a PKC inhibitor, significantly inhibited d-glucose–induced transactivation of PON1; and mithramycin, an inhibitor of Sp1, completely abrogated the transactivation. Our data suggest that high glucose concentrations transactivate the PON1 gene through Sp1 activation by PKC in cultured hepatocytes. Up-regulated hepatic PON1 expression under high glucose conditions may be a compensatory mechanism in diabetes in which antioxidant capacity, including PON1 enzymatic activity, is attenuated.

Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial β-Adrenergic Signaling

Although beta-adrenergic stimuli are essential for myocardial contractility, beta-blockers have a proven beneficial effect on the treatment of heart failure, but the mechanism is not fully understood. The stimulatory G protein alpha-subunit (Gsalpha) couples the beta-adrenoreceptor to adenylyl cyclase and the intracellular cAMP response. In a mouse model of conditional Gsalpha deficiency in the cardiac muscle (Gsalpha-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I (cTnI-ND) from restricted removal of the cTnI-specific N-terminal extension. To investigate the functional significance of the increase of cTnI-ND in Gsalpha-DF cardiac muscle, we generated double transgenic mice to overexpress cTnI-ND in Gsalpha-DF hearts. The overexpression of cTnI-ND in Gsalpha-DF failing hearts increased relaxation velocity and left ventricular end diastolic volume to produce higher left ventricle maximum pressure and stroke volume. Supporting the hypothesis that up-regulation of cTnI-ND is a compensatory rather than a destructive myocardial response to impaired beta-adrenergic signaling, the aberrant expression of beta-myosin heavy chain in adult Gsalpha-DF but not control mouse hearts was reversed by cTnI overexpression. These data indicate that the up-regulation of cTnI-ND may partially compensate for the cardiac inefficiency in impaired beta-adrenergic signaling.

P38MAPK and ERK promote nitric oxide production in cultured human retinal pigmented epithelial cells induced by high concentration glucose

Increased nitric oxide (NO) has been correlated with diabetic retinopathy. In this study we investigated the cell injury, production of NO in retinal pigment epithelial (RPE) cells exposed to increased glucose concentration, and its molecular mechanism involved. Cultured human RPE cells (ARPE-19) were exposed for 4 days with normal blood glucose concentration (5.5 mM d-glucose), followed by exposure to either normal (5.5 mM) or high (33 mM) concentrations of d-glucose for 48 h. To determine the cytotoxicity of high glucose, cell viability, ROS production and SOD activity were measured, respectively. The end product of NO (nitrite and nitrate) was determined by a colorimetric assay and nitrotyrosine levels were quantified by a competitive ELISA. The expression of iNOS and the activation of p38MAPK, ERK and JNK were analyzed by Western blot. Treatment of RPE cells with high glucose-induced a significant increased of iNOS, accompanied by an increase in cell damage, NO and nitrotyrosine levels. High glucose caused activation of p38MAPK and ERK, inhibition for p38MAPK and ERK abrogated the high glucose-induced increase in iNOS, cell injury and levels of NO and nitrotyrosine. High glucose causes increased cell damage and NO generation in RPE cells by a process of iNOS expression that requires the activation of p38MAPK and ERK.

Effects of plasma membrane Ca2+-ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells

In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

Folic acid supplementation affects apoptosis and differentiation of embryonic neural stem cells exposed to high glucose

Folic acid (FA) supplementation has been shown to be extremely effective in reducing the occurrence of neural tube defects (NTDs), one of the most common birth defects associated with diabetic pregnancy. However, the antiteratogenic mechanism of FA in diabetes-induced NTDs is unclear. This study investigated the neuroprotective mechanism of FA in neural stem cells (NSCs) exposed to high glucose in vitro. The undifferentiated or differentiated NSCs were cultured in normal D-glucose concentration (NG) or high D-glucose concentration (HG) with or without FA. FA supplementation significantly decreased apoptosis induced by HG and lowered the expression of p53 in the nucleus of undifferentiated NSCs exposed to HG. Administration of FA in differentiated NSCs did not alter their precocious differentiation induced by HG. The increased mRNA expression levels of the basic helix-loop-helix factors including Neurog1, Neurog2, NeuroD2, Mash1, Id1, Id2, and Hes5 in the presence of HG were not significantly affected by FA. The present results provided a cellular mechanism by which FA supplementation may have a potential role in prevention of NTDs in diabetic pregnancies. On the other hand, FA increased the mRNA expression levels of the above transcription factors and accelerated the differentiation of NSCs in the NG medium, suggesting that it may adversely affect the normal differentiation of NSCs. Therefore, the timing and dose of FA would be critical factors in considering FA supplementation in normal maternal pregnancy.

High glucose promotes collagen synthesis by cultured cells from rat cervical posterior longitudinal ligament via transforming growth factor-β1

Non-insulin-dependent diabetes is known as a risk factor of ossification of posterior longitudinal ligament, but the mechanism has not been well understood. We hypothesized that hyperglycemia, as a typical characteristic of diabetes, is closely associated with ligament hypertrophy in ossification of posterior longitudinal ligament. In this in vitro study, we investigated the effect of high glucose on collagen synthesis and transforming growth factor-beta1 (TGF-beta1) production using cells isolated from rat cervical posterior longitudinal ligament. The cells were subjected to high D-glucose concentration (25 mM) media for 4 days. Notable increases were observed in gene expression and protein synthesis of collagen types I, III in the cells. The increase was inhibited in the presence of anti-TGF-beta1 antibodies. Production of TGF-beta1 by the cells was also increased significantly by high glucose concentration. Exogenous application of TGF-beta1 was confirmed to increase collagen synthesis of the cells. These data suggested that high glucose could promote collagen synthesis in the posterior longitudinal ligament mainly via endogenous TGF-beta1, resulting in hypertrophy of the ligament.

The deleterious effect of high concentrations of D-glucose requires pro-inflammatory preconditioning

The present study investigated whether high concentrations of D-glucose can trigger pro-inflammatory mechanisms in human aortic smooth muscle cells. The expression and/or the activity of inducible nitric oxide synthase (iNOS), the extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF)-kappaB were studied in cultured human aortic smooth muscle cells (HASMC) in response to increasing concentrations of D-glucose and/or the inflammatory cytokine interleukin (IL)-1beta. Increasing D-glucose in the medium from 5.5 to 22 mmol/l had no effect on any of these parameters. However, the high concentration of D-glucose did increase iNOS expression in response to low concentrations of IL-1beta (2.5 and 5 ng/ml), as well as the IL-1beta-induced activation of both ERK 1/2 and NF-kappaB. D-glucose also enhanced, concentration-dependently, the expression and activity of iNOS induced by co-incubation with IL-1beta (10 ng/ml). Pretreatment with IL-1beta sensitized the cells to the subsequent effects of high D-glucose. The results indicate that high concentrations of D-glucose exacerbate the pro-inflammatory effects of IL-1beta. We suggest that the observed association between inflammation and diabetes is the result of elevated D-glucose enhancing a pre-existing inflammatory condition, rather than a direct effect of D-glucose on the production of inflammatory mediators.

Characterization and Role of Polyphenol Oxidase and Peroxidase in Browning of Fresh-Cut Melon

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from two different varieties of melon ( Cucumis melo L. cantalupensis cv. Charentais and C. melo L. inodorus cv. Amarillo) and characterized using reliable spectrophotometric methods. In both cases the enzymes followed Michaelis-Menten kinetics, showing different values of kinetics parameters between the two cultivars: K m = 7.18 +/- 0.70 mM ('Charentais') and 6.66 +/- 0.20 mM ('Amarillo') mM; V max = 7.93 +/- 0.35 units/min ('Charentais') and 13.82 +/- 0.37 units/min ('Amarillo'), relative to PPO; K m = 24.0 +/- 2.10 mM ('Charentais') and 5.05 +/- 0.19 mM ('Amarillo') mM; V max = 344.83 +/- 10.32 units/min ('Charentais') and 80.64 +/- 2.01 units/min ('Amarillo'), relative to POD. Optimum pH for PPO was 7.0 for 'Charentais' and 7.5 for 'Amarillo, whereas it was 4.5 for both cultivars relative to POD. Melon PPO had maximum activity at 60 degrees C in both 'Charentais' and 'Amarillo' cultivars, whereas POD maximum activity was found at 45 degrees C in 'Charentais' and at 25 degrees C in 'Amarillo'. POD from both cultivars showed higher thermolability compared with PPO, losing >90% of relative activity after only 5 min of incubation at 70 degrees C. POD's activation energy was much higher than that of PPO (Delta E (#) = 86.3 and 160.6 kJ mol (-1) for 'Charentais' and 'Amarillo', respectively). PPO and POD activities from both cultivars showed a decreasing pattern as sugar concentration in the assay medium increased, except in POD extract from 'Charentais', which maintained its activity in the presence of high d-glucose concentration (up to 5 M). Changes in L*, a*, b*, chroma, and hue angle values were chosen to describe the browning development in the samples during storage at 5 degrees C. A slight decrease in L* value and a more marked reduction of a* value were noted in both cultivars above all at the end of storage period. POD activity during storage at 5 degrees C was highly correlated with changes of parameters a*, b*, chroma, and hue angle ( r (2) from 0.82 to 0.97) for cultivar 'Charentais'. According to these results, only POD activity seemed to be involved in browning of minimally processed melon.

Adenosine A2B receptor mediates an increase on VEGF-A production in rat kidney glomeruli

Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A1, A2A, A2B and A3 in isolated rat kidney glomeruli. We localized the expression of A2BAR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A2BAR selective antagonist MRS1754 supplementation. Furthermore, we showed that A2BAR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.

Astaxanthin protects mesangial cells from hyperglycemia‐induced oxidative signaling

Astaxanthin (ASX) is a carotenoid that has potent protective effects on diabetic nephropathy in mice model of type 2 diabetes. In this study, we investigated the protective mechanism of ASX on the progression of diabetic nephropathy using an in vitro model of hyperglycemia, focusing on mesangial cells. Normal human mesangial cells (NHMCs) were cultured in the medium containing normal (5 mM) or high (25 mM) concentrations of D-glucose. Reactive oxygen species (ROS) production, the activation of nuclear transcription factors such as nuclear factor kappa B (NFkappaB) and activator protein-1 (AP-1), and the expression/production of transforming growth factor-beta 1 (TGFbeta(1)) and monocyte chemoattractant protein-1 (MCP-1) were evaluated in the presence or absence of ASX. High glucose (HG) exposure induced significant ROS production in mitochondria of NHMCs, which resulted in the activation of transcription factors, and subsequent expression/production of cytokines that plays an important role in the mesangial expansion, an important event in the pathogenesis of diabetic nephropathy. ASX significantly suppressed HG-induced ROS production, the activation of transcription factors, and cytokine expression/production by NHMCs. In addition, ASX accumulated in the mitochondria of NHMCs and reduced the production of ROS-modified proteins in mitochondria. ASX may prevent the progression of diabetic nephropathy mainly through ROS scavenging effect in mitochondria of mesangial cells and thus is expected to be very useful for the prevention of diabetic nephropathy.

Aldose reductase is implicated in high glucose‐induced oxidative stress in mouse embryonic neural stem cells

Oxidative stress caused by hyperglycemia is one of the key factors responsible for maternal diabetes-induced congenital malformations, including neural tube defects in embryos. However, mechanisms by which maternal diabetes induces oxidative stress during neurulation are not clear. The present study was aimed to investigate whether high glucose induces oxidative stress in neural stem cells (NSCs), which compose the neural tube during development. We also investigated the mechanism by which high glucose disturbs the growth and survival of NSCs in vitro. NSCs were exposed to physiological d-glucose concentration (PG, 5 mmol/L), PG with l-glucose (25 mmol/L), or high d-glucose concentration (HG, 30 or 45 mmol/l). HG induced reactive oxygen species production and mRNA expression of aldose reductase (AR), which catalyzes the glucose reduction through polyol pathway, in NSCs. Expression of glucose transporter 1 (Glut1) mRNA and protein which regulates glucose uptake in NSCs was increased at early stage (24 h) and became down-regulated at late stage (72 h) of exposure to HG. Inhibition of AR by fidarestat, an AR inhibitor, decreased the oxidative stress, restored the cell viability and proliferation, and reduced apoptotic cell death in NSCs exposed to HG. Moreover, inhibition of AR attenuated the down-regulation of Glut1 expression in NSCs exposed to HG for 72 h. These results suggest that the activation of polyol pathway plays a role in the induction of oxidative stress which alters Glut1 expression and cell cycle in NSCs exposed to HG, thereby resulting in abnormal patterning of the neural tube in embryos of diabetic pregnancy.