Effect Of Advanced Glycation End Products On Activation Of Islet Stellate Cell And Pancreatic Beta-cell Function

Part 1 Effect of advanced glycation end products on activation of islet stellate cell and pancreatic β cell functionBackground and objective:Advanced glycation end products(AGEs)are oxidative derivatives of chronic hyperglycemia,which are considered as potential risk factors for islet β cell injury,peripheral IR,and diabetes.Previous studies have demonstrated that AGEs have a direct effect on islet β cells.The aim of this study was to observe the effect of AGEs on islet stellate cell(ISC),islet β cell function,vitality,through in vitro experiments,and to determine whether AGEs can activate pancreatic mesenchymal cell—ISC,and injure islet β cell during the activation.Methods:1.Bovine serum albumin(BSA)and glucose were incubated at 37 °C,for three months.2.Islets were isolated from SD rats,cultured with 200 mg / L AGEs and BSA separately,and characterized by Oil O staining.The outgrowth rate was observed and compared by a normal microscope.CCK8 assay was used to detect cell viability.qPCR was performed to verify the different expression of genes.3.Two parts of the supernatant of qISCs treated with AGEs were collected and used to treat INS-1 cells,respectively.INS-1 cells function and viability were detected by KSIS、qPCR and CCK8 assay.Results: 1.After 48 h of culture,ISC began to grow out from the islets.In AGEs treated medium,the exogenous rate of ISC was significantly faster than that of ISC in BSA.Oil O staining showed that the disappearance rate of ISC intracellular lipid droplets in AGEs medium was significantly faster than that of BSA.Additionally,intracellular lipid droplets in the AGEs treated medium completely disappeared after 96 hours.The results of CCK8 assay showed that the vitality of ISC in medium with AGEs increased significantly in 48 hours compared with the control group.The Wound Healing experiment showed that the ISC migration ability in AGEs medium was faster than that in BSA.qPCR showed a significant increase in the expression of α-SMA、Col I and FN of ISC in AGEs treated group.2.After 48 hours of intervention,the insulin secretion in INS-1 cells decreased significantly compared to the control group.qPCR results showed that the gene expression of Insulin1 and Insulin2 in the experimental group was lower than that of the control group.CCK8 results showed that INS-1 cell vitality was inhibited.TUNEL experiments showed that apoptosis occurred in the experimental group.At the same time,the expression of CHOP mRNA in the experimental group was significantly higher than that in the control group.Conclusions: Our result confirms that: AGEs promotes the activation of ISC by promoting the outgrowth,vitality,migration and ECM production.This process significantly impaired INS-1 cell function and vitality,and increased cell apoptosis.The mechanism may be the triggering of endoplasmic reticulum stress in INS-1.Part 2 Correlation between serum glycation end products peptide and islet β cell functionBackground and objective:Cell experiments have confirmed that AGEs can directly or indirectly decrease islet β cell function.However,it’s still unclear whether high AGEs in the circulation is also a risk factor for the injury of islet β cells.Due to the limitations of detecting methods,literature reports are still inconclusive.Based on the previous confirmation of the high correlation between AGEs-P and serum AGEs by the flow injection assay assembled with HPLC,in this study we observe the association between the two by assessing serum AGEs-P and islet beta cell function in the population.Methods: In the second phase of the National key research and development program "Research on prevention and control of major chronic non-communicable diseases",the data of the epidemic survey in Yancheng,Jiangsu Province were randomly sampled.Fasting plasma glucose,2-hour postprandial glucose(2h-PG),Hb A1 c,total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL),low-density lipoprotein(DL),blood urea nitrogen(BUN),creatinine(Cre),and uric acid(UA)were measured.The serum AGEs-P was detected by the flow injection assay assembled with HPLC,and the islet β cell function(HOMA-β)and insulin resistance(HOMA-IR)were calculated by HOMA model.The relationship between AGES-P and HOMA-β was studied by using Pearson correlation analysis and multivariate linear regression to correct the influence of potential confounding factors.Results: 1.This study included 391 subjects,of which 126 had diabetes(DM),132 had impaired glucose regulation(IGR),and 133 had normal glucose regulation(NGR).2.SBP、FPG、2h-PG、Hb A1c、AGEs-P、HOMA-IR、TC、TG、waist-to-hip ratio(WHR)and Heart rate in the DM group were significantly higher than the NGR and IGR groups,while HOMA-β was significantly lower than the other two groups(P<0.05).3.AGEs-P and FPG、2h PG、Hb A1c、HOMA-IR exhibited significant positive correlation(P<0.05),and significant negative correlation with HOMA-β(P=0.025).4.Pearson correlation analysis showed that gender、FINS and HOMA-IR were significantly and positively correlated with HOMA-β,but age、AGEs-P、FPG、2h-PG、Hb A1c、heart rate、systolic pressure(SBP)、Body mass index(body mass index,BMI)、 WHR showed significant negative correlation(P<0.05).5.After adjusting for age、gender、SBP、BMI、TC、TG、HDL、BUN、Cre and UA,AGEs-P was an independent risk factor for HOMA-β(P=0.031);However,when the FPG was included in the model,the relationship between AGEs and HOMA-β was no longer significant(P=0.646).Conclusion: Our study confirmed that serum AGEs-P and FPG,2h-PG,Hb A1 c,HOMA-IR,HOMA-β have significant correlation;At the same time,AGEs is an independent risk factor for islet β cell function.However,the impact on the islet β cell function is not independent of glucose.