Intermittent High Glucose Research Articles

Glucose exposure pattern determines glucagon-like peptide 1 receptor expression and signaling through endoplasmic reticulum stress in rat insulinoma cells

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72h to either sustained high glucose (SHG) conditions (30mM glucose) or IHG conditions (11 and 30mM glucose, alternating every 12h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.

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.

Cardiovascular disease in recent onset diabetes mellitus

Diabetes is associated with a marked increased risk of atherosclerotic vascular disorders, including coronary, cerebrovascular, and peripheral artery disease. Cardiovascular disease (CVD) could account for disabilities and high mortality rates in patients with diabetes. Conventional risk factors, including hyperlipidemia, hypertension, smoking, obesity, lack of exercise, and a positive family history, contribute similarly to macrovascular complications in type 2 diabetic patients and non-diabetic subjects. The levels of these factors in diabetic patients are certainly increased, but not enough to explain the exaggerated risk for macrovascular complications in the diabetic population. Furthermore, recently, macrovascular complications of diabetes have been shown to start before the onset of diabetes. Indeed, several clinical studies have confirmed the increased risk of CVD in patients with impaired glucose tolerance (IGT). Since insulin resistance-related postprandial metabolic derangements are thought to play a central role in the development and progression of CVD in patients with IGT, amelioration of postprandial metabolic disturbance is a therapeutic target for the prevention of CVD in these high-risk patients. Therefore, in this paper, we review the molecular mechanisms for the increased risk of CVD in recent onset diabetes mellitus, especially focusing on postprandial dysmetabolism. We also discuss here the potential therapeutic strategies that specially target the mechanisms responsible for vascular alterations in diabetes.

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.

Damage to the cultured rat mesangial cells by constant and intermittent high glucose

Objective To study the impairment and the expression of receptor of advanced glycation endproduct (RAGE) in cultured rat glomerular mesangial cells ( GMC ) induced by constant and intermittent high glucose, and to investigate the pathogenesis of diabetic nephropathy. Methods After being cultured under constant and intermittent high glucose with different concentrations for 24 and 48 hours, the morphological changes of rat mesangial cells were observed, the proliferation of GMC was detected by MTT assay, the activity of superoxide dismutase (SOD)and the level of malondialdehyde (MDA)in supernatant were measured by spectrophotometer,and the expressions of RAGE mRNA were evaluated by RT-PCR. Results ( 1 ) Compared with the control group,the cellular morphology was changed in case of constant and intermittent high glucose. The damage of GMC with intermittent high glucose concentrations was more serious. (2)The activity of SOD was decreased and the level of MDA was raised in case of intermittent high glucose concentrations compared with the constant high glucose concentrations (P＜0.05). (3)The expression of RAGE mRNA with intermittent high glucose concentrations was significantly higher than that with constant high glucose concentrations ( P＜0. 01 ). Conclusions The damaging effects and increased expression of RAGE in cultured rat GMC induced by blood glucose fluctuation was much worse than that with constant high glucose. The blood glucose fluctuation may be one of the causes that induce diabetic nephropathy. Key words: Diabetic nephropathy; Blood glucose concentrations; Rat glomerular mesangial cells; Receptor of advanced glycation end-products

Intermittent high glucose promotes expression of proinflammatory cytokines in monocytes

The aim of this study was to examine expression of proinflammatory cytokines in monocytes under fluctuating glucose conditions. Monocytic cells (THP-1) were divided into four groups and cultured in the presence of 5 or 15 mmol/L glucose or in fluctuating conditions (12 h exposure to 15 mmol/L glucose or mannitol medium followed by 12 h exposure to 5 mmol/L glucose or mannitol medium) respectively. Levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants and surface expression of CD11b in monocytes were measured after 72 h culture. Paired Student's t tests were used to compare two groups and ANOVA for multiple comparisons. Activation of monocytes was most pronounced in the fluctuating glucose conditions, as measured by concentrations of IL-6 and TNF-α in cultured supernatants and surface expression of CD11b in monocytes (P < 0.05). Fluctuating mannitol also induced a proinflammatory profile, but to a lesser extent than fluctuating glucose. The results indicated that exposure to fluctuating glucose concentrations enhanced activation of monocytes compared with stable elevation of glucose concentrations. The effects were partly attributable to the inherent osmotic changes.

The effect of NF-κB pathway on proliferation and apoptosis of human umbilical vein endothelial cells induced by intermittent high glucose

Our previous study found that blocking nuclear factor (NF)-κB signaling could protect human umbilical vein endothelial cells (HUVECs) from apoptosis and proliferation inhibition due to high glucose (HG). Intermittent HG makes glucose toxicity more significant. In this study, we aimed to investigate the effect of NF-κB pathway on HUVECs induced by intermittent HG (a daily alternating 5.5 or 30.5 mmol/l glucose). A recombinant adenovirus containing a RNAi cassette targeting the NF-κB/p65 gene was produced, and its silencing effect on p65 gene was detected by Western blot analysis in HUVECs cultured with intermittent HG. The subsequent effect on proliferation of HUVECs in the indicated conditions was measured by the AlamarBlue assay. The Bcl-2 expression was also detected by Western blot. The results showed that the expression of p65 protein could be inhibited efficiently by the RNAi adenovirus. Intermittent HG also induced the translocation of NF-κB in HUVECs. Inhibition of NF-κB with the RNAi adenovirus could prevent the effects. At the 6th day after HUVECs were exposed to intermittent HG, the proliferation of HUVECs with Ad-1566 was significantly higher than that of HUVECs with Ad-DEST (P < 0.01). Knockdown of NF-κB/p65 up-regulated the Bcl-2 expression of HUVECs under intermittent HG conditions (P < 0.01). These findings concluded that the NF-κB/p65-targeting RNAi adenovirus is an important tool, which can efficiently inhibit the expression of p65 gene in HUVECs. Intermittent HG reduces HUVECs proliferation with a concomitant increase in apoptosis. Knockdown of NF-κB/p65 partly protected HUVECs from proliferation inhibition and may reduce apoptosis.

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.

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, beta3 integrin receptor and beta5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [(3)H]thymidine incorporation in a time-dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [(3)H]thymidine incorporation, cell number, [(3)H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor beta3 integrin but not neutralizing antibodies to beta5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis.

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.

Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin

Objective Hyperglycemia induces vascular smooth muscle cells (VSMCs) proliferation and may thus contribute to the formation of atherosclerotic lesions. Glucose fluctuations are strong predictor of diabetic vascular complications. We investigate the effects of exposure to constant and intermittent high glucose concentrations on the proliferation and matrix metalloproteinase (MMP)-2 activity of rat aortic VSMCs in culture, as well as the expression of osteopontin (OPN). Methods Rat aortic VSMCs were grown to confluence and then exposed to 5 mmol/L glucose, 25 mmol/L glucose, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor. The cell proliferation, MMP-2 activity and the expression of OPN were assessed. Results In cultured VSMCs, treatment with constant or intermittent high glucose significantly increased [ 3H]thymidine incorporation in a time-dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum expression at 48 h. However, [ 3H]thymidine incorporation was more pronounced in intermittent high glucose than in constant high glucose. Treatment with constant high glucose for 48 h significantly increase cell number, MMP-2 activation, OPN protein and mRNA expression compared with VSMCs treated with the cells normal glucose, and these effects were further enhanced when VSMCs were treated with intermittent high glucose. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin significantly suppressed increase in [ 3H]thymidine incorporation and MMP-2 activity induced by constant or intermittent high glucose. Conclusions In cultured VSMCs, constant high glucose concentrations enhanced MMP-2 activity, cell proliferation and OPN expression. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis, which is mediated by the stimulation of OPN expression and synthesis.

Adiponectin Reduces Glucotoxicity-Induced Apoptosis of INS-1 Rat Insulin-Secreting Cells on a Microfluidic Chip

Diabetes mellitus is a metabolic disorder characterized by elevated blood sugar and progressive failure of insulin-producing beta-cells. Persistent hyperglycemia and blood sugar fluctuation are two general phenomena in diabetic patients, and both of them can result in an increased frequency of beta-cell apoptosis. Therefore, rescuing pancreatic beta-cells from glucotoxicity-induced beta-cell apoptosis is increasing viewed as a promising means for curing. The aim of this study was to investigate whether adiponectin, an important cytokine expressed in adipose tissue, has a potential for the application as the antiapoptotic strategy. INS-1 rat insulin-secreting cell line was used in this study as a model of pancreatic beta-cells, because INS-1 cells show the susceptibility to glucotoxicity, as seen in beta-cells. INS-1 cells were cultured on a novel microfluidic chip with persistent perfusion and subsequently exposed to sustained high glucose (SHG) (25 mmol/l) or intermittent high glucose (IHG) (11.1 and 25 mmol/l glucose alternating every 12 h) in the absence or presence of adiponectin for 72 h. Using this device, we showed that IHG induced more serious impairment in INS-1 cells than did SHG, and adiponectin partially rescued INS-1 cells from glucotoxicity-induced apoptosis, dysfunction and reduction of insulin gene expression. Simultaneously, the mRNA expression of AMP-activated protein kinase (AMPK), which is a signaling protein that acts to modulate glucose uptake in skeletal muscle, was restored in the presence of adiponectin. Based on the above evidence, we suggest that adiponectin could reduce glucotoxicity-induced apoptosis of beta-cells, at least in part, by transiently activating AMPK signaling pathway.

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.

Intermittent high glucose concentrations reduce neuronal precursor survival by altering the IGF system: the involvement of the neuroprotective factor DHCR24 (Seladin-1)

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy, whereas members of the IGF system are neurotropic factors. Here, we investigated the effects of constant and intermittent high glucose concentrations on IGF1 and IGF-binding proteins (IGFBPs) in human neuroblast long-term cell cultures fetal neuroepithelial cells (FNC). These cells express the IGF1 receptor, and express and release in the culture medium IGFBP2, IGFBP4, and IGF1. The release of IGF1 was significantly increased by 17beta-estradiol (10 nM). IGF1 (100 nM) treatment determined a significant increase of IGFBP2 and a decrease of IGFBP4 release. In addition, IGF1 (1-100 nM) stimulated FNC cell proliferation in a dose-dependent manner. We hypothesized that this effect may be, at least partially, due to IGF1-induced up-regulation of the expression of the Alzheimer's disease related gene SELADIN-1 (now known as DHCR24 ), which acts as a pro-survival factor for neuronal cells. Conversely, the exposure to intermittent (20/10 mM), but not stable (20 mM), high glucose concentrations decreased the release of IGF1 and IGFBP2 in the culture medium and inhibited FNC growth by inducing apoptosis. The latter was prevented by the addition of IGF1 to the culture medium. Furthermore, high glucose concentrations reduced the expression of DHCR24. In conclusion, our results indicate for the first time that intermittent high glucose concentrations, similar to those observed in poorly controlled diabetic patients, may contribute to the development of diabetic neuropathy by interfering with the tropic effects exerted by the IGF system, and suggest the involvement of the neuroprotective factor DHCR24.

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.