High Concentration Of D-glucose Research Articles

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells.

The present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

Cytoprotective Roles of a Novel Compound, MHY-1684, against Hyperglycemia-Induced Oxidative Stress and Mitochondrial Dysfunction in Human Cardiac Progenitor Cells.

Diabetic cardiomyopathy (DCM) is tightly linked to heart disorders and dysfunction or death of the cardiomyocytes including resident cardiac progenitor cells (CPCs) in diabetic patients. In order to restore loss of function of resident or transplanted CPCs, much research has focused on novel therapeutic strategies including the discovery of novel function-modulating factors such as reactive oxygen species (ROS) scavengers. Here, we developed and defined a novel antioxidant, MHY-1684, for enhancing the angiogenic potential of CPCs against ROS-related DCM. Short-term treatment with MHY-1684 restored ROS-induced CPC cell death. Importantly, MHY-1684 decreased hyperglycemia-induced mitochondrial ROS generation and attenuated hyperglycemia-induced mitochondrial fragmentation. We observed that the activation process of both Drp1 (phosphorylation at the site of Ser616) and Fis-1 is drastically attenuated when exposed to high concentrations of D-glucose with MHY-1684. Interestingly, phosphorylation of Drp1 at the site of Ser637, which is an inhibitory signal for mitochondrial fusion, is restored by MHY-1684 treatment, suggesting that this antioxidant may affect the activation and inhibition of mitochondrial dynamics-related signaling and mitochondrial function in response to ROS stress. In conclusion, our finding of the novel compound, MHY-1684, as an ROS scavenger, might provide an effective therapeutic strategy for CPC-based therapy against diabetic cardiomyopathy.

The Benefits of the Citrus Flavonoid Diosmin on Human Retinal Pigment Epithelial Cells under High-Glucose Conditions.

We investigate diosmin for its effect on the ARPE-19 human retinal pigment epithelial cells exposed to high glucose, a model of diabetic retinopathy (DR). After incubation for 4 days with a normal (5 mmol/L) concentration of D-glucose, ARPE-19 cells were exposed separately to normal or high concentrations of D-glucose (30 mmol/L) with or without diosmin at different concentrations (0.1, 1, 10 μg/mL) for another 48 h. Next, we assessed cell viability, reactive oxygen species (ROS) generation and antioxidant enzyme activities. In order to examine the underlying molecular mechanisms, we meanwhile analyzed the expressions of Bax, Bcl-2, total and phosphorylated JNK and p38 mitogen-activated protein kinase (MAPK). Diosmin dose dependently enhanced cell viability following high glucose treatment in ARPE-19 cells. The activities of superoxide dismutase and glutathione peroxidase, as well as the levels of reduced glutathione were decreased, while it was observed that levels of ROS in high glucose cultured ARPE-19 cells increased. High glucose also disturbed Bax and Bcl-2 expression, interrupted Bcl-2/Bax balance, and triggered subsequent cytochrome c release into the cytosol and activation of caspase-3. These detrimental effects were ameliorated dose dependently by diosmin. Furthermore, diosmin could abrogate high glucose-induced apoptosis as well as JNK and P38 MAPK phosphorylation in ARPE-19 cells. Our results suggest that treatment ARPE-19 cells with diosmin halts hyperglycemia-mediated oxidative damage and thus this compound may be a candidate for preventing the visual impairment caused by DR.

Role of ROS-TRPM7-ERK1/2 axis in high concentration glucose-mediated proliferation and phenotype switching of rat aortic vascular smooth muscle cells

This study investigated the change of transient receptor potential cation channel subfamily M member 7 (TRPM7) expression in rat aortic vascular smooth muscle cells (RAoSMCs) treated with a high concentration of d-glucose (HG) and its role in promoting the proliferative phenotype of RAoSMCs. Chronic exposure to HG increased TRPM7 protein expression and TRPM7 whole-cell currents in RAoSMCs. By contrast, RAoSMC exposure to high concentration of l-glucose and mannital exhibited no such effect. Mechanistically, HG treatment elevated TRPM7 expression by increasing oxidative stress. Data also demonstrated that HG significantly promoted RAoSMC proliferation. In addition, as indicated by the changes of the expression of VSMC differentiation marker molecules, phenotype switching of RAoSMCs occurred during exposing to HG. TRPM7 knockdown partially blocked the HG effect on phenotype switching and RAoSMC proliferation. This phenomenon was achieved through inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling pathway. These observations suggest that reactive oxygen species-TRPM7-ERK1/2 axis plays an important role in hyperglycemia-induced development of the proliferative phenotype in RAoSMC.

Long noncoding RNA BDNF-AS inversely regulated BDNF and modulated high-glucose induced apoptosis in human retinal pigment epithelial cells.

In this study, we characterized the functional role of long noncoding RNA (lncRNA), brain derived neurotrophic factor anti-sense (BDNF-AS) in regulating D-glucose-induced (DGI) apoptosis in human retinal pigment epithelial (RPE) cells. Human RPE cell line, ARPE-19 cells were cultured in vitro and treated with various concentrations of D-glucose for 24 h. A TUNEL assay was applied with immunohistochemical and quantitative approaches to assess the apoptotic effect of D-glucose. Under the condition of 50 mM D-glucose, qPCR was used to assess gene expression of BDNF and BDNF-AS in ARPE-19 cells. Using siRNA transfection, BDNF-AS was endogenously knocked down in ARPE-19 cells. The effects of BDNF-AS downregulation on DGI apoptosis and BDNF expression were assessed by TUNEL assay, qPCR, and Western blot, respectively. Furthermore, in BDNF-AS-downregulated ARPE-19 cells, secondary siRNA transfection was conducted to knock down endogenous BDNF expression. Its effect on BDNF-AS-associated apoptotic regulation was further evaluated. High concentrations of D-glucose induced significant apoptosis in ARPE-19 cells in vitro. With treatment of 50 mM D-glucose, BDNF was markedly downregulated whereas BDNF-AS upregulated in ARPE-19 cells. SiRNA-mediated BDNF-AS downregulation ameliorated DGI apoptosis and upregulated BDNF in ARPE-19 cells. In addition, inhibiting BDNF reversed the protective effect of BDNF-AS downregulation on DGI apoptosis. Our results suggest that BDNF-AS, through inverse regulation of BDNF, might play a critical role in the process of DGI apoptosis in diabetic retinopathy.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

Exposure to High Glucose Concentration Decreases Cell Surface ABCA1 and HDL Biogenesis in Hepatocytes.

Aim: To study atherosclerosis risk in diabetes, we investigated ATP-binding cassette transporter A1 (ABCA1) expression and high-density lipoprotein (HDL) biogenesis in the liver and hepatocytes under hyperglycemic conditions.Methods and Results: In streptozotocin-induced diabetic mice, plasma HDL decreased while ABCA1 protein increased without changing its mRNA in the liver, only in the animals that responded to the treatment to show hypoinsulinemia and fasting hyperglycemia but not in the poor responders not showing those. To study the mechanism for this finding, hepatocytes were isolated from the control and diabetic mice, and they showed no difference in expression of ABCA1 protein, its mRNA, and HDL biogenesis in 1 g/l d-glucose but showed decreased HDL biogenesis in 4.5 g/l d-glucose although ABCA1 protein increased without change in its mRNA. Similar findings were confirmed in HepG2 cells with d-glucose but not with l-glucose. Thus, these cell models reproduced the in vivo findings in hyperglycemia. Labeling of cell surface protein revealed that surface ABCA1 decreased in high concentration of d-glucose in HepG2 cells despite the increase of cellular ABCA1 while not with l-glucose. Immunostaining of ABCA1 in HepG2 cells demonstrated the decrease of surface ABCA1 but increase of intracellular ABCA1 with high d-glucose. Clearance of ABCA1 was retarded both in primary hepatocytes and HepG2 cells exposed to high d-glucose but not to l-glucose, being consistent with the decrease of surface ABCA1.Conclusions: It is suggested that localization of ABCA1 to the cell surface is decreased in hepatocytes in hyperglycemic condition to cause decrease of HDL biogenesis.

Screening, identification, and characterization of a novel saccharide-stimulated β-glycosidase from a soil metagenomic library.

MeBglD2, a β-glycosidase that is highly activated in the presence of various monosaccharides and disaccharides, was isolated from a soil metagenomic library. MeBglD2 had not only β-glucosidase activity but also β-galactosidase and β-fucosidase activities. MeBglD2 β-glucosidase activity increased in a cellobiose concentration-dependent manner and was not inhibited by a high concentration of D-glucose or cellobiose. MeBglD2 β-glucosidase and β-fucosidase activities were activated by various monosaccharides and disaccharides including D-glucose, D-xylose, D-galactose, maltose, and cellobiose. The saccharification yield of rice straw using Trichoderma reesei cellulase was improved by the addition of MeBglD2. These results show that MeBglD2 can be used to improve plant biomass saccharification, because both substrates and products can activate its enzymatic activity.

High glucose levels affect spermatogenesis: an in vitro approach.

Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas-liquid interface with either 5, 25 or 50mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increased Sertoli cell number (P<0.05) and decreased tubular luminal area (P<0.01), suggesting an impairment of this somatic cell type. Moreover, higher proliferative activity in a TM4 Sertoli cell line exposed to high D-glucose was found (P<0.05) without compromising cell viability (P>0.05), further suggesting altered Sertoli cell maturation. Overall, high D-glucose concentrations may lead to impairment of Sertoli cell function, which, given their significant role in spermatogenic control, may compromise male fertility.

Docosahexaenoic Acid Protects against High Glucose-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells

Diabetic retinopathy is a leading cause of vision loss and has been correlated with increased oxidative stress. The aim of the present study was to evaluate the protective properties of docosahexaenoic acid (DHA), an omega-3 fatty acid, in human retinal pigment epithelial (RPE) cells exposed to high glucose. Human RPE cell line (ARPE-19) was cultured for 4 days followed by 5 days of exposure to either a normal (5.5 mM) or a high (45 mM) D-glucose concentration in the absence and presence of DHA (100 µM). Reduced form of glutathione (GSH), total antioxidant capacity (TAC), total nitrites, and malodialdehyde (MDA) were assessed. 4-Hydroxynonenal (HNE), another lipid peroxidation product, was determined by immunocytochemistry. Cell viability was assessed by the MTT assay. The results showed that both TAC and GSH content were significantly decreased after the high glucose challenge. The presence of DHA prevented the reduction and maintained the TAC and GSH at the levels found in cells exposed to the normal glucose concentration (5.5 mM). Moreover, the levels of total nitrites and MDA were significantly increased after high glucose exposure compared to cell exposed to 5.5 mM glucose. Again, the presence of DHA prevented the increase and maintained the nitrites and MDA at the levels found in control cells. Notably, the high glucose condition led to a significantly increased number of HNE aggresomes as compared to control cells, and DHA completely prevented this increase. In line with the reduced oxidative stress, DHA treatment also completely prevented the high glucose-induced decrease in RPE cell viability. Taken together, this study demonstrated that DHA protected human retinal pigment epithelial ARPE-19 cells from high glucose-induced oxidative damage and cytotoxicity. The results of this study support a role for oxidative stress in high glucose-induced RPE injury and the potential use of omega-3 fatty acids to protect against diabetic retinopathy.

Adenosine A2 regulate cell proliferation via nitric oxide synthesis in normal and gestational diabetes

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.

Influence of P-Glycoprotein Inhibition or Deficiency at the Blood-Brain Barrier on (18)F-2-Fluoro-2-Deoxy-D-glucose ( (18)F-FDG) Brain Kinetics.

The fluorinated D-glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein's (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact (18)F-FDG incorporation, suggesting that P-gp function at the blood-brain barrier may also modulate (18)F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5μM) on the uptake of (18)F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for (18)F-FDG brain kinetics were then assessed using (i) (18)F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15mgkg(-1)h(-1)) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled D-glucose exposure to the brain. In vitro, the time course of (18)F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of D-glucose but not in the presence of high D-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K 1, k 2, k 3, V T , and CMRGlc. The lack of P-gp effect on in vivo (18)F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates (18)F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. (18)F-FDG is not a P-gp substrate at the BBB and (18)F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.

A strategy for improving FDG accumulation for early detection of metastasis from primary pancreatic cancer: Stimulation of the Warburg effect in AsPC-1 cells

IntroductionEarly detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro-d-glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells. Methods14C-FDG accumulation was assessed under the following conditions: 1) characteristics of 14C-FDG transport were examined using phloridzin, a Na+-free buffer, and various hexoses, and 2) accumulation of 14C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed. Results14C-FDG transport into AsPC-1 cells was mediated primarily by a Na+-independent transport mechanism. Aldohexoses such as d-glucose, d-mannose, and d-galactose inhibited 14C-FDG transport. Cells pretreated with d-glucose, d-mannose, or d-fructose exhibited augmented 14C-FDG accumulation. Pretreatment with higher concentrations of d-glucose or d-fructose tended to increase PFK-1 activity. ConclusionsVery little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis.

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Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

BackgroundEngineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.ResultsWe have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.ConclusionsEngineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0168-9) contains supplementary material, which is available to authorized users.

Reciprocal Encoding of Signal Intensity and Duration in a Glucose-Sensing Circuit

Cells continuously adjust their behavior in response to changing environmental conditions. Both intensity and duration of external signals are critical factors indetermining what response is initiated. To understand how intracellular signaling networks processsuch multidimensional information, we studied theAtRGS1-mediated glucose response system of Arabidopsis. By combining experiments with mathematical modeling, we discovered a reciprocal dose and duration response relying on the orchestrated action of three kinases (AtWNK1, AtWNK8, and AtWNK10) acting on distinct timescales and activation thresholds. Specifically, we find that high concentrations of D-glucose rapidly signal through AtWNK8 and AtWNK10, whereas low, sustained sugar concentration slowly activate the pathway through AtWNK1, allowing the cells to respond similarly to transient, high-intensity signals and sustained, low-intensity signals. This "dose-duration reciprocity" allows encoding of both the intensity and persistence of glucose as an important energy resource and signaling molecule.

Role of TRPM7 Channels in Hyperglycemia-Mediated Injury of Vascular Endothelial Cells

This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway

Chronic hyperglycemia is an established risk factor for endothelial damage. It remains unclear, however, whether brief hyperglycemic exposure exacerbates the damage to vascular endothelial cells induced by endotoxin. We hypothesize that brief hyperglycemic exposure enhances the permeability of the endothelium following stimulation with lipopolysaccharide (LPS). Correlations between modulation of nitric oxide synthase (NOS) pathways and altered endothelial homeostasis have been studied and demonstrated in various pathophysiological conditions. NOS activities are regulated by endogenous inhibitors, including asymmetric dimethylarginine (ADMA), which is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since previous data demonstrated that endothelial dysfunction may be related to reduced expression and/or activity of DDAH, in this study, we aimed to determine the effect of increased glucose levels on pulmonary microvascular endothelial cell (PMVEC) permeability, including effects on the NOS pathways. Human PMVECs were incubated with normal (5.5 mM) and high (33 mM) concentrations of D-glucose for 5 days to create a monolayer of cells prior to LPS stimulation (10 μg/ml) for 12 h. When stimulated with LPS, cells incubated with a high glucose (HG) concentration had significant microfilament rearrangement compared with cells incubated with a normal glucose concentration, as determined by immunofluorescence. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the hyperglycemic PMVECs when stimulated with LPS, compared with PMVECs cultured with a normal glucose concentration. The results demonstrated that a high concentration of glucose increases the LPS-stimulated horseradish peroxidase (HRP) permeability compared with a normal concentration of glucose. Furthermore, a HG concentration upregulated LPS-stimulated inducible NOS (iNOS) production and down-regulated endothelial NOS (eNOS) and DDAH-2 expression. Hyperglycemia significantly increased LPS-stimulated nitrite/nitrate production (stable NO end-products). Our results, thus, demonstrate that in vitro HG concentrations exacerbate LPS-stimulated cytoskeletal rearrangement and hyperpermeability of an endothelial monolayer, and cause further imbalance of the NO pathway. These results suggest that it is important to manage even short-term increases in blood glucose, particularly following acute infection.

CCL2/CCR2 augments the production of transforming growth factor-beta1, type 1 collagen and CCL2 by human CD45-/collagen 1-positive cells under high glucose concentrations

The migration and activation of circulating profibrotic cells including fibrocytes by the action of the chemokine/chemokine receptor system has been implicated in pathological fibrogenesis. In the present study, the involvement of collagen 1 (Col1)-producing cells, CD45-positive/collagen-1-positive (CD45(+)/Col1(+)) cells originally named as fibrocytes via CC chemokine receptor 2 (CCR2), a cognate receptor of CCL2/monocyte chemoattractant protein, was examined in diabetic conditions. Human CD45(+)/Col1(+) cells originating from the peripheral blood of healthy volunteers were incubated with high concentrations of D-glucose or D-mannitol as an osmotic control for 12, 24 or 48h. In addition, these cells were preincubated with CCL2 under high glucose concentrations. We also examined the effects of the inhibitors of glucose transporters (GLUTs), reactive oxygen species or CCR2 on the expression of transforming growth factor beta1 (TGF-β1), pro-α1 chain of Col1 (COL1A1), and CCL2. Stimulation of CD45(+)/Col1(+) cells with high glucose concentrations increased the mRNA and protein levels of TGF-β1 and CCL2 and those of pro-COL1A1, and this effect was mediated in part by increased osmolality. Preincubation of the cells with cytochalasin B (a GLUT inhibitor) or N-acetylcysteine (an antioxidant) blocked the stimulatory effect of high glucose concentrations on these profibrotic molecules. In addition, preincubation of the cells with CCL2 enhanced the high glucose-induced upregulation of TGF-β1, pro-COL1A1 and CCL2 and migration of the cells, and this effect was partly inhibited by treatment with CCR2 inhibitors. These results suggest that CD45(+)/Col1(+) cells may be directly involved, in part through CCL2/CCR2 signaling, in the fibrotic process under diabetic conditions.

Activation of a GH43 β-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity

RS223-BX of glycoside hydrolase family 43 is a β-d-xylosidase that is strongly activated (kcat/Km as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+. Slow activation by Mg2+ was demonstrated (kon 0.013s−1mM−1, koff 0.008s−1) at pH 7.0 and 25°C. koff and kon values are independent of Mg2+ concentration, but koff and kon are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-d-xylopyranoside. The kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E M2+, followed by slow isomerization to the activated enzyme, E∗ M2+. Moderately high values of kcat (7–30s−1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25°C. Certain M2+-activated RS223-BX exhibit the highest reported values of kcat/Km of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn2+, 190s−1mM−1), xylotriose (Ca2+, 150s−1mM−1) and xylotetraose (Ca2+, 260s−1mM−1). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.