Effect Of Intermittent High Glucose Research Articles

Intermittent high glucose induces pyroptosis of rat H9C2 cardiomyocytes via sodium–glucose cotransporter 1

Cardiomyocyte death is an important pathogenic process in cardiac complications of diabetes. Diabetic patients often suffer glycemic variability. Pyroptosis is a form of programmed cell death triggered by inflammasomes and related with caspase-1 and gasdermin D activation. The present study was designed to examine the effects of intermittent high glucose simulating glycemic variability on the pyroptosis of cardiomyocytes in vitro. Rat H9C2 cardiomyocytes were incubated with normal glucose (NG), constant high glucose (CHG) and intermittent high glucose (IHG). Results showed that compared to CHG treatment, IHG further inhibited cell proliferation and promoted cell death of H9C2 cardiomyocytes. In addition, IHG upregulated higher levels of the expressions of inflammasome NLR family pyrin domain containing 3 (NLRP3) and adaptor protein apoptosis-associated speck-like protein containing CARD domain (ASC) and increased higher levels of activated caspase-1 and gasdermin D than CHG treatment. Moreover, the production of reactive oxygen species (ROS) and activation of NF-κB that is induced by IHG were significantly higher than that induced by CHG. Knockdown of sodium-glucose cotransporters 1 (SGLT1) in H9C2 cardiomyocytes was performed and the effects of SGLT1 on IHG-induced pyroptosis was evaluated. The results demonstrated that knockdown of SGLT1 partially reduced IHG-induced pyroptosis, ROS generation and NF-κB activation. Our results indicated that IHG is harmful to cardiomyocytes and it might bepartially because of the SGLT1-depedent pyroptosis in cardiomyocytes.

Effect of intermittent high glucose on oxygen-glucose deprivation/refurnish neuronal survival

To investigate the effect of intermittent high glucose on oxygen-glucose deprivation/refurnish (OGD/R) neuronal survival. The primary cultured hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80%. Cells in logarithmic growth phase were placed in a hypoxic incubator (37 centigrade, 5% CO2, 95% N2) to simulate cell hypoxia. The culture medium was replaced by glucose-free Hank equilibrium salt solution (HBSS) to simulate cell hypoglycemia. The normal glucose and oxygen control group was set up. Cell morphology was observed under inverted phase contrast microscope after 6 hours of hypoxia and hypoglycemia treatment, and cell viability was detected by CCK-8 cell proliferation assay kit, and then grouping experiment was carried out. The cells were randomly divided into four groups. The cells were cultured in different concentration glucose medium under normal oxygen, 5% CO2 and 37 centigrade for 72 hours to prepare OGD/R model of cell ischemia/reperfusion. The low-glucose control group was cultured in medium containing 5.5 mmol/L glucose. The constant high-glucose group was cultured in medium containing 33.0 mmol/L glucose. The intermittent high-glucose group was cultured in medium containing 33.0 mmol/L glucose for 3 hours then in medium containing 5.5 mmol/L glucose for 2 hours alternately for 3 times during the day, and overnight in medium containing 33.0 mmol/L glucose at night. The hyperosmotic control group was made up of 5.5 mmol/L glucose medium and mannitol. The osmotic pressure was the same as that of the constant high-glucose group, and the effective glucose concentration was the same as that of the normal glucose and oxygen group, so as to eliminate the effect of osmotic pressure changes caused by the high-glucose medium on the results. Cell morphology was observed under inverted phase contrast microscope after 72 hours of cell culture in each group. Cell viability was measured by CCK-8 kit, and apoptotic rate was measured by flow cytometry. The inverted phase contrast microscope showed that the cells in the normal glucose and oxygen control group were plump and refractive, and had obvious nucleus, clear processes and high cell activity. After 6 hours of hypoxia and hypoglycemia treatment, the cells were shrunk, refractive index was poor, the nucleus was unclear, the processes were not clear, and the cell activity was significantly lower than that of normal glucose and oxygen control group (A value: 0.34±0.06 vs. 1.09±0.06, P < 0.01), which indicated that the model of oxygen-glucose deprivation (OGD) was successfully prepared. After 72 hours of culture with different concentrations of glucose, the cells in the low-glucose control group were shrunk, the cell membrane was incomplete, the nucleus was unclear, and number of necrotic cells were more. In the constant high-glucose group, the refractive index of cells was poor, a large number of cells floated, and the nucleus was not obvious. In the intermittent high-glucose group, the cell morphology was normal, the refractive rate of cells was decreased slightly, and the necrotic cells were less. In the hypertonic control group, the cell status was close to that in the constant high-glucose group. Compared with the low-glucose control group or constant high-glucose group,the cell viability in the intermittent high-glucose group was significantly increased (A value: 2.04±0.15 vs. 0.64±0.18, 1.16±0.16, both P < 0.01), the apoptotic rate was significantly decreased [(59.60±2.55)% vs. (78.15±15.77)%, (95.60±0.14)%, both P < 0.05]. There was no significant difference in cell activity or apoptotic rate between the hypertonic control group and the constant high-glucose group [cell activity (A value): 1.07±0.07 vs. 1.16±0.16, apoptotic rate: (87.80±4.53)% vs. (95.60±0.14)%, both P > 0.05]. Intermittent high glucose within a certain range had protective effect on OGD/R neuronal survival.

Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1

BackgroundIn this study, we evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein.MethodsCultured human RPE cell line ARPE-19 cells were exposed to intermittent high glucose-induced oxidative stress. Reactive oxygen species (ROS) was determined by 2′, 7′-dichlorofluorescin diacetate (DCFH-DA); and malonyldialdehyde (MDA), superoxide dismutase (SOD) by commercial kits. Transmission electron microscopy was used to observe the generation of autophagosome. And MTT assay was used to examine the effect of autophagy on cell viability. For the inhibition experiments, cells were pre-incubated with lysosomal inhibitors NH4Cl or N-acetyl cysteine (NAC).Western blot was used to measure the expression patterns of autophagic markers, including LC3 and p62. The expression of HMGB1 was detected by immunohistochemistry.Cells were pre-incubated with HMGB1 inhibitor ethyl pyruvate (EP) ,then detected the expression pattern of autophagic markers and level of cellular ROS.ResultsWe found that intermittent high glucose significantly increased oxidative stress levels (as indicated by ROS, MDA, SOD), increased in the generation of autophagosome, decreased the level of p62, induced conversion of LC3 I to LC3 II. We further demonstrated that the NH4Cl/NAC inhibited intermittent high glucose-induced autophage by altered level of LC3 and p62. Intermittent high glucose-induced autophagy is independent of HMGB1 signaling, inhibition of HMGB1 release by EP decreased expression pattern of autophagic markers and level of cellular viability.ConclusionsUnder intermittent high glucose condition, autophagy may be required for preventing oxidative stress-induced injury in RPE. HMGB1 plays important roles in signaling for both autophagy and oxidative stress.

Effect of intermittent high glucose on INS-1 cell proliferation and the expression of skp2-p27

Objective To investigate the effect of intermittent high glucose on proliferation, apoptosis, and cell cycle progression of INS-1 cells, and the possible intracellular pathways activated by intermittent high glucose. Methods Cell viability was evaluated by cell counting kit, the cell cycle was determined by flow cytometry,Annexin-V/PI double-labeled cell apoptosis detection kit was used to monitor cell apoptosis. Cell cycle related protein Skp2 and p27 expressions were detected by Western blot. Results ( 1 ) Both intermittent and constant high glucose significantly inhibited the growth of INS-1 cells, and the former effect was more significant. ( 2 ) Intermittent and constant high glucose levels significantly increased apoptosis in INS-1 cells, and the former effect was more significant. (3) Intermittent and constant high glucose levels significantly inhibited the cell process, the G0/G1 cell cycle arrest also was induced by intermittent high glucose, resulting in lowered proportion of the G2/M phase and S phase of INS-1 cells. (4) Intermittent and constant high glucose significantly decreased the level of protein Skp2 and increased the level of cell cycle related protein p27. Conclusion Intermittent high glucose levels affect INS-1 cell growth and proliferation, as well as induce cell apoptosis, probably by decreasing the level of protein Skp2 and increasing the level of p27 in the cells, resulting in arrest of progression through the G1 phase to the S phase of INS1 cells, and thus impairment of cell proliferation. Key words: Islet β-cells; Intermittent high glucose; Skp2; p27; Proliferation

Effects of intermittent high glucose on BeWo choriocarcinoma cells in culture

The aim of this study was to investigate the cellular effects of intermittent high glucose on the human BeWo placental choriocarcinoma cell line, used as a model of the effects of glucose fluctuation in diabetic pregnancies. BeWo cells were subjected to three different glucose conditions for 48 h: 7 mmol/L (control), 42 mmol/L (high glucose), or 7 and 42 mmol/L glucose (intermittent, alternated every 6 h). Cell viability was assessed using cell counts, a cell proliferation assay, and a cell viability assay. Apoptosis was also studied using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and by immunocytochemistry of fractin, the N-terminal fragment of actin, which can distinguish apoptotic from necrotic cells. Furthermore, the expression of the well-known survival factors of trophoblast cells, heparin-binding epidermal growth factor-like growth factor and leptin, was evaluated by real-time polymerase chain reaction and Western blot analyses. Intermittent high-glucose conditions significantly decreased cell viability and enhanced apoptosis compared with control or continuous high-glucose conditions. Furthermore, the expression of heparin-binding epidermal growth factor-like growth factor, but not that of leptin, was significantly increased under intermittent high-glucose conditions compared to its expression under either control or continuous high-glucose conditions. These data indicate that intermittent high glucose is more deleterious to BeWo cells than continuous high-glucose conditions. Although further in vitro and in vivo study is necessary, excess fluctuation of glucose levels in the placental circulation might be involved in the impairment of placental development leading to the placental dysfunction.

Intermittent high glucose enhances apoptosis in INS-1 cells.

To investigate the effect of intermittent high glucose (IHG) and sustained high glucose (SHG) on inducing β-cell apoptosis and the potential involved mechanisms, INS-1 beta cells were incubated for 72 h in the medium containing different glucose concentrations: control (5.5 mmol/L), SHG (33.3 mmol/L), and IHG (5.5 mmol/L and 33.3 mmol/L glucose alternating every 12 h). Cell viability, apoptosis rate, and oxidative-stress markers were determined. The results showed that the apoptosis induced by IHG was more obvious than that by SHG. Simultaneously, the intracellular level of oxidative stress was more significantly increased in INS-1 cells exposed to IHG. These findings suggest that intermittent high glucose could be more deleterious to β-cell than a constant high concentration of glucose, this may be due to the aggravation of oxidative stress triggered by intermittent high glucose.

Intermittent high glucose enhances cell proliferation and VEGF expression in retinal endothelial cells: the role of mitochondrial reactive oxygen species

Proliferation of human retinal endothelial cells (HRECs) is an important event in the development of diabetic retinopathy. Glucose fluctuations are strong predictor of diabetic vascular complications. In this study we have investigated the effect of intermittent high glucose on proliferation and expression of vascular endothelial growth factor (VEGF) in HRECs. The possible involvement of mitochondrial reactive oxygen species (ROS) was assessed. HRECs were incubated for 72 h in media containing different glucose concentrations: 5, 25, 5 mmol/l alternating with 25 mmol/l glucose, with or without Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) and thenoyltri-fluoroacetone (TTFA). The cell proliferation, VEGF expression, mitochondrial ROS, nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) were measured. In cultured HRECs, treatment with constant or intermittent high glucose significantly increased [(3)H]thymidine incorporation in a time-dependent manner. Treatment with constant high glucose for 48 h resulted in significant increases in [(3)H]thymidine incorporation, mRNA and protein levels of VEGF compared with HRECs treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose. The levels of mitochondrial ROS, nitrotyrosine and 8-OhdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA can effectively prevent cell proliferation and overexpression of VEGF, as well as overproduction of mitochondrial ROS, nitrotyrosine and 8-OhdG in HRECs induced by constant or intermittent high glucose. Intermittent high glucose enhances cell proliferation and overexpression of VEGF through reactive oxygen species (ROS) overproduction at the mitochondrial transport chain level in HRECs, indicating that glycemic variability have important pathological effects on the development of diabetic retinopathy dependent of mitochondrial ROS.

Involvement of JNK signal transduction pathway in endothelial cell apoptosis induced by intermittent high glucose

To evaluate the effect of intermittent high glucose on the apoptosis of human umbilical vein endothelial cells (HUVECs) and its mechanism. Intermittent high glucose and constant high glucose were applied to HUVEC-12 for 7 days. Flow cytometer and fluorescent staining with Hoechst 33258 were used to detect apoptosis of HUVEC-12. The superoxide dismutase (SOD) activity and the content of malonaldehyde (MDA) in culture solution were detected with colorimetry, and the changes of p-JNK level were examined by Western blot. The apoptosis rate was obviously higher in the intermittent high glucose group than that in the constant high glucose group (P < 0.05). The SOD activity was significantly lower in the intermittent high glucose group (P < 0.05), but MDA level was higher than those of constant high glucose(P < 0.05). SP600125, the inhibitor of JNK, decreased the apoptosis rate induced by intermittent high glucose (P < 0.05). Antioxidant (Vitamin C) inhibited the p-JNK, decreased the apoptosis rate (P < 0.05). Intermittent high glucose is easier to worsen the proapoptotic effects on HUVECs than that of constant high glucose, which may account for the increased oxidative stress, and then activates JNK signal transduction pathway.

Effect of intermittent high glucose on synthesis of nitric oxide in human umbilical vein endothelial cells and its mechanism.

To compare the effect of intermittent and constant high glucose media on the synthesis of vascular relaxing factor nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVECs), and to investigate its mechanism. HUVECs were exposed to 5.5 mmol/L glucose (normal control), 20 mmol/L glucose (constant high glucose), and 5.5 mmol/L alternating with 20 mmol/L glucose (intermittent high glucose) for 7 days. The NO level was measured using Griess Reaction in cell culture supernatants; the expressions of PI3K, PKB and eNOS mRNA and protein were measured with RT-PCR or Western blot. NO level in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05). The expression of PI3K, PKB, and eNOS in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05). Intermittent high glucose could be more deleterious to endothelial cells than constant high glucose, and may lead to decrease synthesis of NO by inhibiting PI3K/PKB/eNOS pathway activation.

Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through mitochondrial superoxide overproduction in adipocytes

Hypoadiponectinemia and hyperresistinemia may be important in mediating signals from adipocytes to insulin-sensitive tissue and vasculature. However, the mechanism that mediates the aberrant production of adipokines remains poorly understood. In this study, we have investigated the effect of intermittent high glucose on the expression of adiponectin and resistin, and the production of 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine in the adipocytes, either in the presence or in the absence of Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) or thenoyltrifluoroacetone (TTFA). 3T3-L1 adipocytes were incubated for 72 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, 5 mmol/l alternating with 20 mmol/l glucose, with or without MnTBAP and TTFA. We measured the expression of resistin and adiponectin. The production of nitrotyrosine and 8-OHdG as oxidative stress parameter was measured. Both constant and intermittent high glucose significantly suppressed the expression and secretion of adiponectin, and increased expression and secretion of resistin in mature adipocytes compared to normal glucose conditions. However, these effects were significantly greater under intermittent high glucose conditions compared to constant high glucose. The levels of nitrotyrosine and 8-OHdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA reversed the aberrant production of adiponectin and resistin, as well as overproduction of nitrotyrosine and 8-OHdG in adipocytes induced by constant or intermittent high glucose. Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through reactive oxygen species overproduction at the mitochondrial transport chain level in adipocytes, indicating that glycemic variability has important pathological effects on the secretion of adipokines.

Intermittent High Glucose Stimulate MCP-l, IL-18, and PAI-1, but Inhibit Adiponectin Expression and Secretion in Adipocytes Dependent of ROS

Elevated circulating concentrations of interleukin-18 (IL-18), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and decrease of adiponectin are associated with obesity-related diseases. The mechanism that mediates the aberrant production of the adipokines remains poorly understood. The aim of this study was to investigate the effect of intermittent high glucose on the expression of IL-18, MCP-1, and PAI-1 and adiponectin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated for 24 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l and a daily alternating 5 or 20 mmol/l glucose, with or without the addition of1.0 mmol/l N-acetylcysteine (NAC). The expression and secretion of IL-18, MCP-1, PAI-1, and adiponectin were determined by real-time RT-PCR and ELISA, respectively.The production of reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine(8-OHdG) were measured. Stable high glucose significantly increased expression and secretion of IL-18, MCP-1, and PAI-1, and reduced adiponectin expression and secretion compared to normal glucose conditions.These effects were significantly greater under intermittent high glucose conditions compared to stable high glucose. The level of ROS and 8-OHdG were significantly elevated under both intermittent and stable high glucose conditions, the effect being greater under intermittent high glucose. The intermittent glucose was more effective in triggering the generation of ROS than stable high glucose. The adding of the NAC, aspecific pharmacological inhibitor of ROS, normalized the expression of these adipokines and the levels of ROS and 8-OHdG under both stable and intermittent glucose conditions.Intermittent high glucose induces a greater aberrant production of key adipokines than stable high glucose, and this effect seems to be related to over-production of ROS.

Effects of intermittent high glucose on oxidative stress in endothelial cells

The objective of this study is to explore the mechanism of oxidative stress induced by intermittent high glucose in porcine iliac endothelial cells (PIECs). The PIECs were exposed to intermittent or constant high glucose for 3 or 6 days, and the mean fluorescent intensity (MFI) was measured via intracellular reactive oxygen species (ROS) captured by flow cytometry. The NADPH oxidase activity was measured by chemiluminescence with lucigenin. Intermittent high glucose induced a greater over-production of ROS than constant high glucose in PIECs; the NADPH oxidase activity was increased under both constant and intermittent high glucose conditions, being more marked in the latter (P < 0.05). In conclusion, intermittent high glucose induced more ROS in PIECs than constant high glucose, this effect seemed to be, at least in part related to the enhanced activation of NADPH oxidase. Glucose fluctuation may be involved in the development of vascular complications.

Impairment of pancreatic islet beta cell function induced by intermittent high glucose through oxidative and endoplasmic reticulum stress: experiment with rat pancreatic islet beta cells

To investigate the effect of intermittent high glucose (IHG) on the pancreatic islet beta-cell function and mechanism thereof. Rat pancreatic islet p-cells of the line INS-1 were cultured and randomly divided into 3 groups: IHG group exposed to fluctuating concentrations of glucose, stable high glucose (SHG) group exposed to 16. 7 mmol/L glucose, and control group exposed to normal concentration (5.5 mmol/L) glucose. 24, 48, and 72 hours later radioimmunoassay was used to detect the insulin secretion index (ISI). 72 h later, the concentration of insulin in the cells was detected with radioimmunoassay. The contents of oxidative stress markers, nitrotyrosine (NT) and 8-hydroxy-2-deoxyguanosine (8-OHdG) were detected. Real-time PCR was used to detect the mRNA expression of peroxiredoxin 1 (PDX-1), ATF-4, one of the transcription factors of the family bZIP, and insulin. Western blotting was used to detect the protein expression of ATF-4. The ISI of the IHG and SHG groups decreased time-dependently, The ISI of IHG and SHG groups were 0.64 +/- 0.11 and 1.31 +/- 0. 04 respectively, both significantly lower than that of the control group (1.67 +/- 0.23, both P < 0.05). The intracellular insulin contents of the IHG and SHG groups were (10.91 +/- 0.14) and (11.08 +/- 0.03) +/- U/microg respectively, both significantly lower than that of the control group [(12.37 +/- 0.37) microU/microg, both P < 0.05]. The intracellular concentrations of 8-OHdG and NT of the SHG and IHG groups, were significantly higher than those of the control group (all P < 0.01), and those of the IHG group were significantly higher than those of the SHG group (both P < 0.05). The mRNA and protein expression levels of ATF-4 of the IHG group were all significantly higher than those of the control group (all P < 0.05) and those of the IHG group were significantly higher than those of the SHG group (both P < 0.05). IHG and SHG induce severe impairment in pancreatic islet beta cell functions, especially IHG, which is closely associated with the aggravation of oxidative stress and endoplasmic reticulum stress triggered by intermittent high glucose.

Involvement of chronic stresses in rat islet and INS-1 cell glucotoxicity induced by intermittent high glucose

In order to investigate the toxic effect of intermittent high glucose (IHG) and sustained high glucose (SHG) on rat pancreatic beta cell functions and the potential involved mechanisms, isolated rat islets and INS-1 beta cells were exposed to SHG (25 mmol/l) or IHG (11.1 and 25 mmol/l glucose alternating every 12 h) for 72 h. The results showed that IHG induced a more significant impairment of insulin release response in rat islets and INS-1 cell than SHG. Simultaneously, the intracellular levels of endoplasmic reticulum and oxidative stress were more markedly increased in islets and INS-1 cells exposed to IHG. However, there was no significant difference between reducing cell viability, insulin content and gene expression induced by SHG and IHG. Taken together, this study suggested the more serious toxic effect on rat pancreatic beta cell function induced by IHG treatment may be due to excessive activation of cellular stress.

Effect of constant and intermittent high glucose on apoptosis of cultured bovine retinal capillary pericytes

目的 探讨恒定性高糖及不同频率的波动性高糖对原代培养的牛视网膜周细胞增生、细胞周期及凋亡的影响.方法 以体外培养3代近融合的牛视网膜毛细血管周细胞为对象,分别在对照组(5.5 mmol/L)、恒定性高糖组(25 mmol/L)及不同波动频率的高糖组(5.5 mmol/L与25 mmol/L交替,频率分别为6 h和24 h)中孵育6 d,采用MTT比色法观察周细胞增生情况;流式细胞术观察周细胞周期情况;TUNEL法检测培养后周细胞的凋亡率.结果 周细胞增生受抑制,周期阻滞,出现典型的细胞凋亡特征.恒定性高糖组的作用高于波动性高糖组(P＜0.05);波动性高糖组内波动频率越高作用越明显(P＜0.05).结论 恒定性高糖较波动性高糖可能具有更强的抑制周细胞增生、周期阻滞及诱导凋亡作用,波动组间波动频率高者比频率低者作用更明显。