The Mechanism Of Oxidative Stress And JNK Pathway In The High Glucose Toxicity Of INS-1 Cells

Glucose is the most original and major nutrition to regulate the function of theβ-cells in the physiologica condition. Normalβ-cells can compensate for insulin resistance by increasing insulin secretion orβ-cell mass to maintain the glucose homeostasis. Type 2 diabetes arises when the endocrine pancreas fails to secrete sufficient insulin to cope with the metabolic demand, because of acquiredβ-cells secretory dysfunction and/or decreasedβ-cells mass. Once hyperglycemia becomes apparent, which can exert deleterious effects onβ-cell function gradually. This process is called"glucose toxicity".Oxidative stress can, therefore, be defined as the pathogenic outcome of the overproduction of oxidants that overwhelms the cellular antioxidant capacity. Experimental support for oxidative stress as a mediator of cell death and function impaired.Under diabetic conditions, reactive oxygen species(ROS) increase in various tissues due to the hyperglycemia and hyperlipemia which including pancteatic.Pancreaticβ-cells have recently emerged as a target of oxidative stress-mediated tissue damage.In our study we investigate the relationship of the ROS production and the glucose concentration in islet, how the ROS impaire the function ofβ-cells, whether it is involved in the glucose toxicity . Insulin receptor substrates signal are response for mediating intracellular signaling transduction to regulate the function ofβ-cells and the insulin sensitivity in liver, musle, adipose tissue. Abnormality of IRS signaling might explain the eventual loss of compensatory insulin secretory and contribute to failure ofβ-cells function. C-Jun N-terminal kinase (JNK), or stress-activated protein kinase, is an important member of the mitogen-activated protein kinase superfamily, the members of which are readily activated by many environmental stimuli, and mediating cell growth and survive. The mechanisms whereby chronic elevations of glucose might damageβ-cells are our study will to investigate. We will explore whether that chronic oxidative stress and JNK pathway is involved in the mechanism that glucose excess can damageβ-cells and provides a target for that lessen glucose toxicity .1. To investigate the effect of different concentration glucose on the function and apoptosis of INS-1 cells.(1) Culturing the INS-1 cells at three glucose concentration: 5.6mmol/l, 11.2 mmol/l, 33.3 mmol/l, within 48 hours and 72 hours respectively. MTT used to measure cell viability, RT-PCT used to measure the insulin, PDX-1, IAPP gene expression of the INS-1 cells, apoptosis were examined by immuno- fluorescence and flow-cytometry analysis. After treating with IGF-1 measuring these items again.(2) Expose INS-1 cells to elevated glucose concentrations lead to impaired cell function by time-dependent method.We cultured the cells in 5.6mmol/l and 11.2 mmol/l glucose for 48h, the glucose dose not inhibit the cell viability and induce apoptosis.There were remarkably increasing of the apoptise when INS-1 cells expose to 33.3mmol/l glucose, with a 2.49-fold increase to cells in the basal 5.6mmol/l glucose.In 11.2G a nd 33.3G group caused a 11.2% and 42.4% decrease of the insulin gene expression (P <0.001),also reduced the PDX-1 gene expression by 50% and 63%(p<0.001)After IGF-1 plus, insulin and PDX-1 mRNA level are increased, wherase the effctor were lessen with the elevated glucose.2. Reactive oxygen species (ROS) were measured by enzyme linked immunosorbent assay.NAC added can improve the cells function in high glucose with decrease the ROS in the cells.(1).The ROS production were increased with the elevated glucose concentration, longer the cells exposed to high concentration glucose, more the ROS production were. NAC decreased the ROS in any concentration of glucose.(2).NAC treatment remarkably improve the function of INS-1 cells exposed to high glucose. NAC treatment led to cell viability increased and apoptosis cell decreased to 13% in 33.3G group. The mRNA level of insulin and PDX-1 were also increased after the INS-1 cell treated with NAC for 48h and 72h,for insulin were 41% and 77% increased(P<0.01) respectively,and for PDX-1 were 56% and 113%(P<0.01)increased respectively in 33.3G group.NAC treatment led to insulin and PDX-1 gene expression restoration nearly to the base level.3. Western-blot methods to test the serine270 phosphorylation of IRS and phosphorylation of JNK inβcells, and investigate the mechanisms of glucose toxitity.(1)SP600125 improves INS-1 cells function by inhibiting the JNK pathway. After SP600125 treatment caused a 45% decreased of the apoptosis cells in 33.3G group (P<0.01), and improveing the cytoactive. The mRNA level of insulin and PDX-1 were partly restored by inhibit the JNK signal pathway.(2)High glucose lead to the the serine 270 phosphorylation of IRS correlates with JNK phosphorylation in INS-1 cells. Using Western-blot analysis, the levels of serine 270 phosphorylation of IRS were 1.17 fold increased in 33.3G JNK compares to 11.2G group(P<0.01). SP600125 treatment completely blocked the JNK phosphorylation, but partly blocked serine 270 phosphorylation of IRS.Conclusion Chronic high glucose toxicity impair the INS-1 function and induce the cells developed apoptosis might be related to elevated glucose concentrations increase levels of reactive oxygen species in INS-1 cells. The antioxidant agent NAC protect INS-1 cells from glucose toxicity with the effect to improve the oxidative stress response in INS-1 cells.JNK pathway contribute to the glucose toxicity by inhibiting the IRS signal througe phosphorylate Ser270 of IRS in INS-1 cells.