Effects Of Elevated Glucose Concentrations Research Articles

Elevated Glucose Levels Increase Oxidative Stress in Human Cerebral Microvascular Endothelial Cells

Although glucose is the primary fuel source for cerebral metabolism and function, chronic exposure to high glucose levels impairs cerebral endothelial cell function and increases the risk of cerebrovascular accidents (CVA). Indeed, hyperglycemia is linked to an increased risk and prevalence of ischemic stroke, primarily attributed to the detrimental impact of glucose on cerebral endothelial cell function. The direct and indirect effects of glucose on brain endothelial cells are complex and not well understood. It is known that hyperglycemia can induce a profound pro-oxidative state with deleterious effects on peripheral vascular function resulting in pathologic outcome. The experimental aim of this study was to determine, in vitro, the effect of elevated glucose concentrations on cerebral endothelial cell oxidative stress. Human cerebral microvascular endothelial cells (hCMECs) were cultured (3rd passage) and incubated with culture media containing 5 mM D-glucose (normal glucose condition [NG]), 6.5 mM glucose (concentration representative of impaired fasting glucose [IFG]), and 8 mM (concentration representative of type 2 diabetes [T2D]) for 3 hours. Cells were harvested and intracellular reactive oxygen species (ROS) production was determined using CellROX Deep Red Reagent and expression of key anti-oxidant defense proteins, superoxide dismutase 1 (SOD-1) and catalase, were determined by capillary electrophoresis immunoassay. In separate experiments, the effect of each glucose condition was determined in the presence of the ROS scavenger, vitamin C (200 μmol). Cells treated with glucose concentration representing IFG (137+8 %) and T2D (162+12%) significantly increased intracellular ROS compared with NG (98+3 %). Concordantly, expression of SOD-1 and catalase were significantly higher in hCMECs treated with glucose concentration representing IFG (190.2+17.8 AU; 134.7+15.4 AU, respectively) and T2D (186.7+19.6 AU; 137.2+17.5 AU, respectively) compared with NG (24.4+1.8 AU; 65.6+4.3 AU, respectively). Pretreatment of cells with vitamin C prevented the increase in ROS in both the IFG and T2D conditions. Of note, there were no significant differences in ROS or the expression of SOD-1 and catalase in hCMECs between the IFG and T2D glucose conditions. In conclusion, glucose concentrations associated with IFG and T2D markedly increased ROS in hCMECs. In addition, there was a compensatory increase in SOD-1 and catalase to counteract the increase in cellular oxidative burden. Similar effects between the IFG and T2D glucose conditions on the oxidative milieu of hCMECs is consistent with the similar clinical cerebrovascular risk burden between these pathologic conditions. Glucose-induced increased oxidative stress in cerebral endothelial cells may contribute to the increased risk of CVA, such as ischemic stroke, associated with IFG and T2D. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results.

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

High Glucose Enhances Neurotoxicity and Inflammatory Cytokine Secretion by Stimulated Human Astrocytes.

Chronic neuroinflammation caused by activation of microglia and astrocytes in the brain contributes to neuronal loss and disease progression in Alzheimer's disease (AD). Recent research has identified type 2 diabetes mellitus (T2DM) as a risk factor for AD. High blood glucose (hyperglycemia) and the phenomenon of insulin resistance are being considered as the major factors contributing to an increased risk of AD. However, the mechanisms involved in this interaction remain unclear. High glucose has been shown to increase release of pro-inflammatory mediators from various immune cells, including microglia. Since astrocytes are the most abundant glial cell type in the brain, we investigated the effects of elevated glucose concentrations (5.5-30.5 mM) on selected functions of cultured human astrocytes in the presence of inflammatory stimuli. Experiments were conducted using primary human astrocytes and U-118 MG astrocytoma cells. High glucose (30.5 mM) increased mRNA expression of interleukin (IL)-6 and secretion of both IL-6 and IL-8 by astrocytes. This astrocytic inflammatory response to high glucose did not appear to be mediated by augmented p38 or p44/42 mitogen activated protein kinase (MAPK) signaling pathways. In addition, high glucose increased the susceptibility of undifferentiated human SH-SY5Y neuronal cells and retinoic-acid differentiated SH-SY5Y cells to injury by hydrogen peroxide (H2O2) and fibrillar amyloid beta-42 protein (Aβ42), respectively. Our data indicate that hyperglycemia in T2DM may be one of the factors contributing to the observed increased risk of AD by exacerbating astrocyte-mediated neuroinflammation and neuronal injury caused by disease-associated agents.

A quick, simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation

ObjectiveAdvanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control. MethodsfAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis. ResultsIn in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p≤0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p≤0.005) and 12 (p≤0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs. ConclusionsApplying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.

Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

441 Cystathionine pathway-dependent cytotoxicity of diethyl maleate and diamide in Fa32 and Hep G2 cells

In this study neutrophil (PMN) oxidative burst activity was investigated ex vivo and in vitro in comparison to the PMN-phagocytic functions ingestion and bacterial killing in poorly-controlled type 1 diabetic patients. Luminol enhanced chemiluminescence in response to phorbolesters as a measure of oxidative burst was assessed in a parallel detecting microtiterplate luminometer in 40 poorly-controlled type 1 diabetic subjects. PMN ingestion was measured with [3H]thymidine-labelled Staphylococcus aureus in a kinetic radiometric assay. Microbicidal activity was determined by pure plate counting of surviving bacteria (colony forming units, cfu) after defined pmn challenge. PMNs of type 1 diabetic subjects showed a highly significant reduction of peak CL response in response to PMA compared to nondiabetic controls (P < 0.001) and PMN ingestion (51.8 ± 4.6%) and bacterial killing (28.6 ± 3.2%) were reduced as well (78.2 ± 5.2% (IN) and 18.4 ± 4.1% (BK), P < 0.01, respectively). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations ≥ 13.8. mmol/l whereas PMN IN and BK were significantly reduced at glucose levels ≥ 27.75 mmol/l. In the control group there was a positive correlation of peak CL response and IN as well as BK (P < 0.05); in type 1 diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN oxidative burst activity and markedly reduced ingestion and bacterial killing in type 1 diabetic patients ex vivo and in vitro. These findings suggest inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning impaired host defense.

Proximal Tubular Epithelial Cell Integrins Respond to High Glucose by Altered Cell-Matrix Interactions and Differentially Regulate Matrixin Expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mm glucose underwent a reduction of the expression of α3, β1, αvβ3, and α5 integrin subunits, with a concomitant increase of the α2 subunit, compared with cells grown in 5 mm glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mm glucose used mainly αvβ3 and α5β1 integrins to adhere to colIV, whereas in 25 mm glucose they additionally used α2β1. In the case of laminin, a β1-mediated adhesion was observed when HK-2 cells were in 5 mm glucose, whereas in 25 mm glucose, α2β1 and αvβ3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of α3β1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of αvβ3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.

Hyperglycemia-induced protein kinase C activation inhibits phagocytosis of C3b- and immunoglobulin g-opsonized yeast particles in normal human neutrophils.

The aim of this study was to investigate the effects of elevated glucose concentrations on complement receptor– and Fcγ receptor–mediated phagocytosis in normal human neutrophils. D-Glucose at 15 or 25 mM dose-dependently inhibited both complement receptor– and Fcγ receptor– mediated phagocytosis, as compared to that at a normal physiological glucose concentration. The protein kinase C (PKC) inhibitors GF109203X and Go6976 both dose dependently and completely reversed the inhibitory effect of 25 mM D-glucose on phagocytosis. Complement receptor– mediated phagocytosis was dose-dependently inhibited by the cell permeable diacylglycerol analogue 1,2-dioctanoylsn- glycerol (DAG), an effect that was abolished by PKC inhibitors. Furthermore, suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect on complement receptor–mediated phagocytosis. The authors conclude that elevated glucose concentrations can inhibit complement receptor and Fcγ receptor–mediated phagocytosis in normal human neutrophils by activating PKCα and/or PKCβ, an effect possibly mediated by DAG.

Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction.

Nitric oxide release from the endothelium plays an important role in regulation of vascular tone, inhibition of both platelet and leukocyte aggregation and adhesion, and inhibition of cell proliferation. These properties suggest that the level of NO production by the endothelium may play a pivotal role in the regulation of vascular disease. Analysis using mass spectrometry has revealed that NO is produced by NOS from the terminal guanidino nitrogen of the precursor amino acid L-arginine. Thus, utilization of L-arginine and conversion to NO may establish a regulatory site in the development of endothelial dysfunction. Endothelial dysfunction is characterized by defective endothelium-dependent relaxation, and some reviews regarding endothelial dysfunction in diabetes have been published. These reviews have focused on factors that might contribute to defective relaxation, some of which will not be addressed in detail in this review. The purpose of the present review is to summarize evidence that specifically supports either decreased NO production by diabetic vascular endothelium and/or impaired NO-mediated endothelium-dependent relaxation. Second, this review provides reasonable alternatives to explain some of the controversies in this research area. Third, since there is growing evidence that arginine appears to have some benefits for diabetes-associated abnormalities, this review summarizes the current state of knowledge of effects of acute and chronic administration of L-arginine on diabetesinduced endothelial dysfunction and discusses potential NOdependent and -independent mechanisms whereby therapeutic intervention with L-arginine might benefit the diabetic endothelium.

Glucose elevations alter bradykinin-stimulated intracellular calcium accumulation in cultured endothelial cells.

Diabetes selectively injures receptor-mediated endothelium-dependent relaxation. In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells. [Ca2+i] was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production. After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell [Ca2+i] in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered. When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in [Ca2+i] signal was observed after exposure to 35 mM glucose for a similar time period. Defective [Ca2+i] signaling was also seen in cells using MEM medium. [Ca2+i] signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure. The defect in [Ca2+i] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake. Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca2+i] in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure. Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.

Effect of elevated glucose concentrations on cellular lipid peroxidation and growth of cultured human kidney proximal tubule cells.

This study has examined whether elevated glucose can induce lipid peroxidation and contribute to the inhibition of cell growth in human kidney proximal tubule(HPT) cells. HPT cells were cultured in media containing glucose concentrations of 8 mM (control), 25 mM, and 50 mM. Lipid peroxidation was assessed by the thiobarbituric acid reactivity and cell growth was assessed by 3H-thymidine uptake. Results show decreased (59%, p < 0.01) growth of HPT cells cultured in 50 mM glucose. Cells cultured in 50 mM mannitol did not show any growth inhibition, suggesting that the decreased cell growth associated with glucose is not due to osmolarity changes. There was an increase (108%, p < 0.02) in lipid peroxidation in cells cultured with high levels of glucose (50 mM) compared with controls and cells cultured with 50 mM mannitol. To examine if membrane lipid peroxidation or malondialdehyde (MDA, an end product of lipid peroxidation) has any role in the inhibition of cell growth, we examined the effect of tertiary butylhydroperoxide (TBH, known to cause lipid peroxidation and generate MDA) on the growth of HPT cells. TBH decreased cell growth (49, 17 and 3% of controls at 0.1, 0.25, and 0.5 mumole TBH/ml medium). Similarly, a marked reduction in the growth was observed with exogenous MDA (72, 69 and 34% of controls at 0.1, 0.25, and 0.5 mumole MDA/ml medium). This suggests that elevated glucose can induce membrane lipid peroxidation and accumulation of MDA, which in turn can inhibit cellular growth and contribute to the altered structure and function of HPT cells in diabetes.

Glucose-enriched medium enhances cell-mediated low density lipoprotein peroxidation

A 1 week preculture of endothelial or smooth muscle cells in glucose-enriched (11.2 to 44.8 mM) media resulted in a marked enhancement of the subsequent ability of cells to oxidize low density lipoprotein, as assessed by the lipid peroxidation end product and conjugated diene content of the particle, its relative electrophoretic mobility and its degradation by macrophages. This phenomenon is correlated to a marked stimulation of superoxide anion secretion by cells. Such an effect of elevated glucose concentration on cell-induced LDL oxidative modification could be involved in the increased occurrence of atherosclerotic lesions in diabetes mellitus.

Plasminogen activator inhibitor-1 expression by brain microvessel endothelial cells is inhibited by elevated glucose.

Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 mM glucose compared with BBEC cultured in media with 5.5 mM glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.

Glucose-induced protein kinase C activity regulates arachidonic acid release and eicosanoid production by cultured glomerular mesangial cells.

Changes in glomerular eicosanoid production have been implicated in the development of diabetes-induced glomerular hyperfiltration and glomerular mesangial cells (GMC) are major eicosanoid-producing cells within the glomerulus. However, the mechanism for the effect of diabetes mellitus on glomerular mesangial eicosanoid production is unknown. The present study therefore examined whether elevated glucose concentrations activate protein kinase C (PKC) in GMC and whether this PKC activation mediates an effect of elevated glucose concentrations to increase the release of arachidonic acid and eicosanoid production by GMC. The percentage of [3H]arachidonic acid release per 30 min by preloaded GMC monolayers was significantly increased after 3-h exposure to high glucose (20 mM) medium (177% vs control medium) and this increase was sustained after 24-h exposure to high glucose concentrations. 3-h and 24-h exposure to high glucose medium also increased PGE2, 6-keto-PGF1 alpha, and thromboxane (TXB2) production by GMC. High glucose medium (20 mM) increased PKC activity in GMC at 3 and 24 h (168% vs control). In contrast, osmotic control media containing either L-glucose or mannitol did not increase arachidonic acid release, eicosanoid production, or PKC activity in GMC. Inhibiting glucose-induced PKC activation with either H-7 (50 microM) or staurosporine (1 microM) prevented glucose-induced increases in arachidonic acid release and eicosanoid production by GMC. These data demonstrate that elevated extracellular glucose concentrations directly increase the release of endogenous arachidonic acid and eicosanoids by GMC via mechanisms dependent on glucose-induced PKC activation.

Effect of Elevated Glucose Concentration on Membrane Voltage Regulation in Retinal Capillary Pericytes

The influence of elevated glucose concentration on resting membrane voltage, electrogenic Na(+)-K(+)-ATPase, and ATP-sensitive potassium channels (KATP channels) was studied in cultured bovine retinal capillary pericytes using conventional microelectrodes. The resting membrane voltage in cells grown in medium containing 5 mM glucose (control) averaged -27 +/- 1.2 mV (mean +/- SE, n = 26) and was not different from cells grown in medium containing 22.5 mM glucose (-26 1.2 mV, n = 26). Addition of ouabain (10(-4) M), a specific inhibitor of the Na(+)-K(+)-ATPase, depolarized the membrane potential by 3.6 +/- 0.4 mV (n = 10) in cells grown under control conditions and 0.7 +/- 0.2 mV (n = 6) in cells grown under elevated glucose conditions. Thus, electrogenic activity of the Na(+)-K(+)-ATPase was significantly (P < 0.0001) reduced to 19% compared with control conditions. Electrogenic Na(+)-K(+)-ATPase activity could be partially restored (ouabain-induced depolarization delta V = 2.0 +/- 0.2 mV, n = 6) in cells grown with high glucose in the presence of the aldose reductase inhibitor tolrestat (10(-5) M). The potassium channel opener Hoe 234 (10(-6) M) induced membrane potential hyperpolarization in control cells (delta V = 7.3 +/- 1.2 mV, n = 13), which could be completely inhibited by the KATP channel blocker glibenclamide (10(-7) M, n = 5). This indicates that pericytes possess KATP channels. The effect of KATP channels on membrane voltage was not significantly changed (P = 0.16) in cells cultured under high-glucose conditions (delta V = 9.6 +/- 2.0 mV, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of elevated glucose concentration on membrane voltage regulation in retinal capillary pericytes

Effect of elevated glucose concentration on membrane voltage regulation in retinal capillary pericytes

Evidence of ex vivo and in vitro impaired neutrophil oxidative burst and phagocytic capacity in type 1 diabetes mellitus

In this study neutrophil (PMN) oxidative burst activity was investigated ex vivo and in vitro in comparison to the PMN-phagocytic functions ingestion and bacterial killing in poorly-controlled type 1 diabetic patients. Luminol enhanced chemiluminescence in response to phorbolesters as a measure of oxidative burst was assessed in a parallel detecting microtiterplate luminometer in 40 poorly-controlled type 1 diabetic subjects. PMN ingestion was measured with [ 3H]thymidine-labelled Staphylococcus aureus in a kinetic radiometric assay. Microbicidal activity was determined by pure plate counting of surviving bacteria (colony forming units, cfu) after defined pmn challenge. PMNs of type 1 diabetic subjects showed a highly significant reduction of peak CL response in response to PMA compared to nondiabetic controls ( P < 0.001) and PMN ingestion (51.8 ± 4.6%) and bacterial killing (28.6 ± 3.2%) were reduced as well (78.2 ± 5.2% (IN) and 18.4 ± 4.1% (BK), P < 0.01, respectively). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations ≥ 13.8. mmol/l whereas PMN IN and BK were significantly reduced at glucose levels ≥ 27.75 mmol/l. In the control group there was a positive correlation of peak CL response and IN as well as BK ( P < 0.05); in type 1 diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN oxidative burst activity and markedly reduced ingestion and bacterial killing in type 1 diabetic patients ex vivo and in vitro. These findings suggest inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning impaired host defense.

Elevated glucose decreases the content of a basement membrane associated heparan sulphate proteoglycan in proliferating cultured porcine mesangial cells.

The effect of elevated glucose concentrations on the synthesis of basement membrane components was investigated in proliferating cultured porcine mesangial cells. Basement membrane associated heparan sulphate proteoglycan was determined by enzyme immunoassay with a specific antiserum recognizing the core protein of the heparan sulphate proteoglycan. When cells were exposed to increasing glucose concentrations up to 40 mmol/l, the heparan sulphate proteoglycan content was concomitantly decreased to 53% when compared to cells cultured under normal glucose concentrations or in the presence of 40 mmol/l sorbitol. The fibronectin content was essentially unchanged under these conditions. No significant effect of insulin on these basement membrane components was found. The results indicate that hyperglycaemia may be responsible for a decrease of mesangial heparan sulphate proteoglycan content in diabetes mellitus. This supports the view that loss of heparan sulphate proteoglycan may be an important step or even an initial event of mesangial alterations in diabetic glomerulopathy.

Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes.

In this study, ingestion of Staphylococcus aureus and "bacteria killing" (BK) were measured to evaluate polymorphonuclear leukocyte (PMN) phagocytic functions and chemiluminescence response (CL) to phorbol-myristic acetate (PMA) as respiratory burst activity with regard to metabolic control parameters in diabetic patients. PMN phagocytic functions were assessed in 40 diabetic patients, all receiving insulin and in poor metabolic control, with 3H-thymidine-labeled Staphylococcus aureus in a modified radiometric assay. Bacteria killing was determined by pure-plate counting of surviving bacteria (colony-forming units [cfu]) and luminol-enhanced CL in response to PMA as a measure of respiratory burst. PMN function data were correlated to HbA1 as parameter of recent metabolic control. PMN of diabetic patients showed a significant reduction in Staphylococcus aureus (50.7 +/- 4.1%) and BK (29.4 +/- 4.2%) compared with healthy nondiabetic control subjects (76.6 +/- 4.6% and 16.3 +/- 3.1%, respectively, P less than 0.001), and PMN CL response was markedly reduced in diabetic patients also. Linear regression analysis showed a highly significant negative correlation of HbA1 versus Staphylococcus aureus (r = -0.67, P = 0.001) and a positive correlation for BK (r = 0.73, P less than 0.001). This was also true for CL, although this did not reach statistical significance (P = 0.06). The data obtained demonstrate impaired PMN phagocytic functions and CL response in diabetic patients. These findings suggest inhibitory effects of elevated glucose concentrations on PMNs, a possible role of protein glycosylation for impairing PMN function, thus contributing in part to altered host defense.

Glucose inhibition of human fibroblasts proliferation and response to growth factors is prevented by inhibitors of aldose reductase

Diabetes mellitus is associated with premature senescence of cultured dermal fibroblasts. The present study investigated the effect of elevated glucose concentrations on cultured human fibroblasts from normal donors. Mean population doubling times, population, doublings until senescence, saturation density at confluence (cells/cm 2), tritiated thymidine incorporation, and response to platelet-derived growth factor (PDGF) were inhibited with the increasing glucose concentrations (11.0, 22, 44, or 55 mM glucose) ( P s ̌ 0.05 ). Replicative life span was markedly diminished by multiple passages in high glucose medium ( 5.5 mM glucose: 62.4 ± 7.9 population doublings; 22 mM glucose: 22.8 ± 3.4 population doublings: P s ̌ 0.05 ). Aldose reductase activity was present in the cultured fibroblasts (3.9 ± 0.5 nmol/min per mg protein), and inhibitors of aldose reductase, includng sorbinil (10 −4 M−10 −6 M) and tolrestat (10 −6 M−10 −8 M), completely prevented glucose-mediated inhibition of fibroblast proliferation, restored the response to PDGE, and allowed a normal replicative life span. Myo-inositol (11 μM-5.5 mM) also reversed the adverse effects of glucose. These in vitro data demonstrate that elevated concentrations of glucose inhibit cell growth and promote premature senescence, effects which can be prevented with inhibitors of aldose-reductase or supplemental myo-inositol. These aldose reductase-related effects may explain the impaired growth and premature senescence of cultured connective tissue from diabetic patients.