Chronic High Glucose Exposure Research Articles

Canagliflozin alleviates high glucose-induced peritoneal fibrosis via HIF-1α inhibition.

The cardioprotective effects of sodium-glucose cotransporter type 2 (SGLT2) inhibitors have been demonstrated in many studies. However, their benefits for end-stage kidney disease patients, particularly those on peritoneal dialysis, remain unclear. SGLT2 inhibition has shown peritoneal protective effects in some studies, but the mechanisms are still unknown. Herein, we investigated the peritoneal protective mechanisms of Canagliflozin in vitro by simulating hypoxia with CoCl2 in human peritoneal mesothelial cells (HPMCs) and rats by intraperitoneal injection of 4.25% peritoneal dialysate simulating chronic high glucose exposure. CoCl2 hypoxic intervention significantly increased HIF-1α abundance in HPMCs, activated TGF-β/p-Smad3 signaling, and promoted the production of fibrotic proteins (Fibronectin, COL1A2, and α-SMA). Meanwhile, Canagliflozin significantly improved the hypoxia of HPMCs, decreased HIF-1α abundance, inhibited TGF-β/p-Smad3 signaling, and decreased the expression of fibrotic proteins. Five-week intraperitoneal injection of 4.25% peritoneal dialysate remarkably increased peritoneal HIF-1α/TGF-β/p-Smad3 signaling and promoted peritoneal fibrosis and peritoneal thickening. At the same time, Canagliflozin significantly inhibited the HIF-1α/TGF-β/p-Smad3 signaling, prevented peritoneal fibrosis and peritoneal thickening, and improved peritoneal transportation and ultrafiltration. High glucose peritoneal dialysate increased the expression of peritoneal GLUT1, GLUT3 and SGLT2, all of which were inhibited by Canagliflozin. In conclusion, we showed that Canagliflozin could improve peritoneal fibrosis and function by ameliorating peritoneal hypoxia and inhibiting the HIF-1α/TGF-β/p-Smad3 signaling pathway, providing theoretical support for the clinical use of SGLT2 inhibitors in patients on peritoneal dialysis.

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKCδ activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKCδ activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKCδ mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKCδ-wild type (WT). Inhibition of PKCδ by PKCδ-kinase-deficient (KD) or rottlerin not only inhibited cardiac PKCδ activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKCδ-WT plasmids reversed the inhibitory effects of DATS on PKCδ activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKCδ activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKCδ signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

Propofol Alleviates Apoptosis Induced by Chronic High Glucose Exposure via Regulation of HIF-1α in H9c2 Cells.

Background The sedative anesthetic, propofol, is a cardioprotective agent for hyperglycemia-induced myocardial hypertrophy and dysfunction in rats. However, the specific protective mechanism has not been clarified. Methods and Results In this experiment, we used H9c2 cells subjected to 22 mM glucose lasting for 72 hours as an in vitro model of cardiomyocyte injury by hyperglycemia and investigated the potential mechanism of propofol against hyperglycemic stress in cells. Propofol (5, 10, or 20 μM) was added to the cell cultures before and during the high glucose culture phases. Cell viability and levels of ROS were measured. The levels of proinflammatory cytokines were tested by ELISA. The levels of SIRT3, SOD2, PHD2, HIF-1α, Bcl-2, P53, and cleaved caspase-3 proteins were detected by western blotting. Our data showed that propofol attenuated high glucose-induced cell apoptosis accompanied by a decrease in the level of reactive oxygen species (ROS) and proinflammatory cytokines. Meanwhile, propofol decreased the apoptosis of H9c2 cells via increasing the expression of Bcl-2, SIRT3, SOD2, and PHD2 proteins and decreasing the expression of cleaved caspase-3, P53, and HIF-1α. Real-time PCR analysis showed that propofol did not significantly change the HIF-1α but increase PHD2 at mRNA level. HIF-1α silence significantly decreased apoptosis and inflammation in H9c2 cell during high glucose stress. Pretreatment of IOX2 (the inhibitor of PHD2) inhibited cell viability until the concentration reached 200 μM during high glucose stress. However, 50 μM TYP (the inhibitor of SIRT3) significantly inhibited cell viability during high glucose stress. Delayed IOX2 treatment for 6 hours significantly inhibited cell viability during high glucose stress. Conclusions Propofol might alleviate cell apoptosis via SIRT3-HIF-1α axis during high glucose stress.

Upregulation of UCP2 in beta-cells confers partial protection against both oxidative stress and glucotoxicity

Deterioration of pancreatic beta-cells plays a critical role in the development of type 2 diabetes. Among the various stressors contributing to these deleterious effects, glucotoxicity and superoxides have been proposed as major players. In this context, the mitochondrial uncoupling protein UCP2 is regularly associated with the stress response. In the present study, we tested the effects of UCP2 upregulation in mouse islets with beta-cell specific overexpression of UCP2 (RIP-UCP2). Islets were subjected to both chronic glucotoxicity (7 days at 30mM glucose) and acute oxidative stress (200µM H2O2 for 10min). Increased UCP2 expression did not alter mitochondrial potential and ATP generation but protected against glucotoxic effects. Glucose-stimulated insulin secretion was altered by both glucotoxicity and oxidative stress, in particular through higher basal insulin release at non-stimulatory glucose concentrations. The secretory response to glucose stimulation was partially preserved in beta-cells overexpressing UCP2. The higher rate of cell death induced by chronic high glucose exposure was lower in RIP-UCP2 islets. Finally, superoxide production was reduced by high glucose, both under acute and chronic conditions, and not modified by UCP2 overexpression. In conclusion, upregulation of UCP2 conferred protective effects to the stressed beta-cell through mechanisms not directly associated with superoxide production.

An excessive increase in glutamate contributes to glucose-toxicity in \u03b2-cells via activation of pancreatic NMDA receptors in rodent diabetes

In the nervous system, excessive activation of NMDA receptors causes neuronal injury. Although activation of NMDARs has been proposed to contribute to the progress of diabetes, little is known about the effect of excessive long-term activation of NMDARs on β-cells, especially under the challenge of hyperglycemia. Here we thoroughly investigated whether endogenous glutamate aggravated β-cell dysfunction under chronic exposure to high-glucose via activation of NMDARs. The glutamate level was increased in plasma of diabetic mice or patients and in the supernatant of β-cell lines after treatment with high-glucose for 72 h. Decomposing the released glutamate improved GSIS of β-cells under chronic high-glucose exposure. Long-term treatment of β-cells with NMDA inhibited cell viability and decreased GSIS. These effects were eliminated by GluN1 knockout. The NMDAR antagonist MK-801 or GluN1 knockout prevented high-glucose-induced dysfunction in β-cells. MK-801 also decreased the expression of pro-inflammatory cytokines, and inhibited I-κB degradation, ROS generation and NLRP3 inflammasome expression in β-cells exposed to high-glucose. Furthermore, another NMDAR antagonist, Memantine, improved β-cells function in diabetic mice. Taken together, these findings indicate that an increase of glutamate may contribute to the development of diabetes through excessive activation of NMDARs in β-cells, accelerating β-cells dysfunction and apoptosis induced by hyperglycemia.

Altered expression of uncoupling protein 2 in GLP-1-producing cells after chronic high glucose exposure: implications for the pathogenesis of diabetes mellitus.

Glucagon-like peptide-1 (GLP-1) is a gut L-cell hormone that enhances glucose-stimulated insulin secretion. Several approaches that prevent GLP-1 degradation or activate the GLP-1 receptor are being used to treat type 2 diabetes mellitus (T2DM) patients. In T2DM, GLP-1 secretion has been suggested to be impaired, and this defect appears to be a consequence rather than a cause of impaired glucose homeostasis. However, although defective GLP-1 secretion has been correlated with insulin resistance, little is known about the direct effects of chronic high glucose concentrations, which are typical in diabetes patients, on GLP-1-secreting cell function. In the present study, we demonstrate that glucotoxicity directly affects GLP-1 secretion in GLUTag cells chronically exposed to high glucose. Our results indicate that this abnormality is associated with a decrease in ATP production due to the elevated expression of mitochondrial uncoupling protein 2 (UCP2). Furthermore, UCP2 inhibition using small interfering RNA (siRNA) and the application of glibenclamide, an ATP-sensitive potassium (KATP(+)) channel blocker, reverse the GLP-1 secretion defect induced by chronic high-glucose treatment. These results show that glucotoxicity diminishes the secretory responsiveness of GLP-1-secreting cells to acute glucose stimulation. We conclude that the loss of the incretin effect, as observed in T2DM patients, could at least partially depend on hyperglycemia, which is typical in diabetes patients. Such an understanding may not only provide new insight into diabetes complications but also ultimately contribute to the identification of novel molecular targets within intestinal L-cells for controlling and improving endogenous GLP-1 secretion.

Advances in molecular mechanisms of pancreatic beta-cell glucotoxicity

The deterioration of beta-cell function and survival caused by chronic high glucose exposure is termed glucotoxicity.The mechanisms of pancreatic beta-cell glucotoxicity involve in oxidative stress,endoplasmic reticulum stress,mitochrondrial dysfunction,inflammation,advanced glycation end products,hypoxia and activation of HIF-1 α,and loss of differentiation in the alteration of beta-cell phenotype.The role of oxidative stress,endoplasmic reticulum stress,and loss of differentiation in the alteration of beta-cell phenotype is well ascertained.Oxidative stress is the central mechanism for glucotoxicity in pancreatic beta cells in diabetes. Key words: Pancreatic beta-cell; Glucotoxicity; Molecular mechanism

The influence of high glucose on the aerobic metabolism of endothelial EA.hy926 cells

The endothelium is considered to be relatively independent of the mitochondrial energy supply. The goals of this study were to examine mitochondrial respiratory functions in endothelial cells and isolated mitochondria and to assess the influence of chronic high glucose exposure on the aerobic metabolism of these cells. A procedure to isolate of bioenergetically active endothelial mitochondria was elaborated. Human umbilical vein endothelial cells (EA.hy926 line) were grown in medium containing either 5.5 or 25 mM glucose. The respiratory response to elevated glucose was observed in cells grown in 25 mM glucose for at least 6 days or longer. In EA.hy926 cells, growth in high glucose induced considerably lower mitochondrial respiration with glycolytic fuels, less pronounced with glutamine, and higher respiration with palmitate. The Crabtree effect was observed in both types of cells. High glucose conditions produced elevated levels of cellular Q10, increased ROS generation, increased hexokinase I, lactate dehydrogenase, acyl-CoA dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression, and decreased E3-binding protein of pyruvate dehydrogenase expression. In isolated mitochondria, hyperglycaemia induced an increase in the oxidation of palmitoylcarnitine and glycerol-3-phosphate (lipid-derived fuels) and a decrease in the oxidation of pyruvate (a mitochondrial fuel); in addition, increased UCP2 activity was observed. Our results demonstrate that primarily glycolytic endothelial cells possess highly active mitochondria with a functioning energy-dissipating pathway (UCP2). High-glucose exposure induces a shift of the endothelial aerobic metabolism towards the oxidation of lipids and amino acids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1156-1) contains supplementary material, which is available to authorized users.

High Glucose Predisposes Gene Expression and ERK Phosphorylation to Apoptosis and Impaired Glucose-Stimulated Insulin Secretion via the Cytoskeleton

Chronic high glucose (HG) inflicts glucotoxicity on vulnerable cell types such as pancreatic β cells and contributes to insulin resistance and impaired insulin secretion in diabetic patients. To identify HG-induced cellular aberrations that are candidate mediators of glucotoxicity in pancreatic β cells, we analyzed gene expression in ERoSHK6, a mouse insulin-secreting cell line after chronic HG exposure (six-day exposure to 33.3 mM glucose). Chronic HG exposure which reduced glucose-stimulated insulin secretion (GSIS) increased transcript levels of 185 genes that clustered primarily in 5 processes namely cellular growth and proliferation; cell death; cellular assembly and organization; cell morphology; and cell-to-cell signaling and interaction. The former two were validated by increased apoptosis of ERoSHK6 cells after chronic HG exposure and reaffirmed the vulnerability of β cells to glucotoxicity. The three remaining processes were partially substantiated by changes in cellular morphology and structure, and instigated an investigation of the cytoskeleton and cell-cell adhesion. These studies revealed a depolymerized actin cytoskeleton that lacked actin stress fibers anchored at vinculin-containing focal adhesion sites as well as loss of E-cadherin-mediated cell-cell adherence after exposure to chronic HG, and were concomitant with constitutive ERK1/2 phosphorylation that was refractory to serum and glucose deprivation. Although inhibition of ERK phosphorylation by PD98059 promoted actin polymerization, it increased apoptosis and GSIS impairment. These findings suggest that ERK phosphorylation is a proximate regulator of cellular processes targeted by chronic HG-induced gene expression and that dynamic actin polymerization and depolymerization is important in β cell survival and function. Therefore, chronic HG alters gene expression and signal transduction to predispose the cytoskeleton towards apoptosis and GSIS impairment.

Early activation of the fatty acid metabolism pathway by chronic high glucose exposure in rat insulin secretory β-cells

Pancreatic beta-cells are responsible for insulin secretion that regulates blood glucose homeostasis. In the development of type II diabetes, a progressive impairment of insulin secretion by the pancreatic beta-cells occurs called beta-cell dysfunction or beta-cell failure. Chronic hyperglycemia has been shown being involved in beta-cell dysfunction, a phenomenon known as glucotoxicity. The molecular mechanisms underlying the impairment of insulin secretion by beta-cells induced by glucotoxicity are still not fully understood. In this work, quantitative proteomics was employed to identify early key players involved in beta-cell dysfunction induced by glucotoxicity. For this, the stable isotope labeling by amino acids in cell culture strategy was used on the slowly-growing rat beta-cell line INS-1E. We showed that the stable isotope labeling by amino acids in cell culture approach did not induce any detectable biological effects on these beta-cells, as measured at both the transcriptomic and proteomic levels. Proteins differentially expressed between control cells and cells submitted to chronic high glucose concentrations were identified and verified. The results obtained reinforce the link between glucotoxicity and lipogenesis and suggest that the fatty acid metabolism pathway may rapidly be stimulated in beta-cells submitted to chronic high glucose concentrations.

A Compensatory Mechanism for Acute Hyperglycemia Induced Apoptosis

Studies have shown that prolonged exposure of hyperglycemia may injure cells and contribute in the development of renal diseases in diabetes. In a recent study, we have demonstrated that acute (10–20 min) exposure of human proximal tubule epithelial cells (hPTECs) to 25 mM high glucose (HG) induces a time‐dependent dual effect, involving an early proliferation and a late apoptosis. Acute exposure of HG initiated intrinsic apoptotic pathway activated by reactive oxygen species (ROS). Furthermore, we observed increased protein expression of p53 that was inhibited by ROS inhibitor NAC. Under similar conditions we observed changes in Bcl‐2 and Bax proteins expression. Chronic HG exposure characteristic of diabetes induces a high Bax to Bcl‐2 ratio indicative of cellular apoptosis. However, acute HG exposure reversed the Bax to Bcl‐2 ratio indicating a possible physiological regulatory and recovery phase in which the hPTECs attempt to overcome damage caused by the reactive oxygen species produced by HG exposure. Consistent with these observations, we identified time‐dependent increase in caspase‐3 protein expression. Our study demonstrates for the first time that a single HG exposure for 10–20 min alone is sufficient to elicit cellular apoptosis inducing epithelial cell injury. However hPTECs may initiate intrinsic physiological machinery towards a recovery phase. Supported by NIH Grant DK072140

Identification of glucose response proteins in two biological models of beta-cell adaptation to chronic high glucose exposure.

High resolution 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with computerized analysis of gel images was used to search for proteins whose biosynthesis was induced or repressed in pancreatic islet cells chronically exposed to high glucose in an in situ and a tissue culture model of islet cell adaptation to excessive fuel load. The in situ model involved a 4-day intravenous infusion of either 50% glucose or 0.45% saline solution, followed by islet isolation, [35S]methionine labeling at 3 and 18 mM glucose for both groups, and protein analysis by 2-dimensional SDS-PAGE. The tissue culture model involved a 7-day culture of isolated rat islets in RPMI 1640 with 10% fetal calf serum containing either 3 or 30 mM glucose, followed by radiolabeling and 2-dimensional PAGE of proteins as in the in situ model. A small fraction of about 1.5% of the approximately 2000 identifiable proteins can be characterized as adaptive proteins. Of these altogether 58 proteins in the two models, 5 proteins were demonstrable in both models and two of these (proteins 1526 and 7622) are particularly noteworthy. Protein 1526 (Mr 57,000; pI 5.09) showed the same response pattern in both models and its expression was most enhanced when islets from chronically glucose-infused animals or those cultured for 7 days at 30 mM were radiolabeled at 18 mM glucose. Protein 7622 (Mr 68,000; pI 6.50) (also known as GSP-65; Collins, H.W., Buettger, C., and Matschinsky, F.M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5494-5498) showed a different labeling pattern in the two models: stimulation of [35S]methionine incorporation by 18 mM glucose both in control and experimental islets from the infusion study, but lack of such stimulation of radiolabeling in islets cultured for 7 days at 30 mM glucose in contrast to islets cultured at 3 mM. The experimental strategy and the methodology are evaluated and the significance of the results is discussed. Potentials of the approach and plans for future experiments are considered.