Influence Of Apelin On Glucose Toxicity And Islet Cells Expression Of PDX-1 Protein

Objective:1. Exploring whether Apelin leads to the reduction of insulin by participating in glucose toxicity mechanism.2. Study on the relationship between Apelin and PDX-1 protein synthesis.Method:NIT-1 cells of islet β cell strain after resuscitation and culture is inoculated in 25ml cell culture bottle (1×107cells/bottle), culturing for 3 days in RPMI 1640 culture media, which is divided into six groups randomly by different concentration of glucose:(1) Group Con 1, the concentration of 5.6 mmol/L Glu. (2) Group Con 2, the concentration of 5.6 mmol/L Glu+ Apelin-36 (1μmol/L). (3) Group HG 1, the concentration of 16.7 mmol/L Glu. (4) Group HG 2, the concentration of 16.7 mmol/L Glu+Apelin-36 (1μmol/L). (5) Group EHG 1, the concentration of 33.3mmol/L Glu. (6) Group EHG 2, the concentration of 33.3mmol/L Glu+Apelin-36 (1μmol/L).Then:1. Base release experiment of Ins:collect 1 ml centrifuge tube (1×106cells/tube) of islet cell of each group respectively, and then pre-incubate them in HBSS fluid at the degree of 37 for 60 minutes, and after PBS washing for 2 times, pre-incubate them in HBSS buffer containing Glu at 5.6 mmol/L at the degree of 37 for 1 hour, then discard the buffer and incubate each cell respectively in new 1 ml HBSS buffer containing Glu (base) at 5.6 mmol/L and glucose (glucose stimulation) at 16.7mmol/L, then collect the reaction buffer and measure the INS with the method of ELISA.2. INS in cell:collect 1 ml centrifuge tube (1×106cells/tube) of islet cell of each group respectively, after freezing and thawing repeatedly and extracting with the acidified alcohol, incubate at the degree of 4℃ for 16-24 hours, and then measure the INS with the method of ELISA.3. Expression of PDX-1 protein:fix the smears of the NIT cells from the four groups with 4% paraformaldehyde for 20 minutes and then PBS wash them for 3minutes one time for 3 times, then follow the instruction of the Histostain-Plus Kit. Count the number of positive cells with the microscope.4. Expression of PDX-1 mRNA:fix the smears of the NIT-1 cells from the four groups with 4% paraformaldehyde/0.1% DEPC/0.1mmol/L PBS(pH 7.2～7.6) for 20-30 minutes and then PBS wash them for 3 minutes one time for 3 times, then follow the instruction of the In situ hybridization kit. Count the number of positive cells with the microscope.Result:① Comparing with Group Con 1, after base and stimulation, secretion of INS in cells of Group HG 1 and Group EHG 1;②After base and stimulation, secretion of INS in cells of Group Con 2 with Apelin has no obvious difference with Group Con 1;③After base and stimulation, secretion of INS in cells of Group HG 2 and Group EHG 2 with Apelin is obviously decreased comparing with Group HG land Group EHG 1;④Comparing with Group Con 1, expression of PDX-1 protein of Group HG 1 and Group EHG 1 is decreased;⑤ Group Con 2 with Apelin has no obvious difference with Group Con 1;⑥expression of PDX-1 protein of Group HG 2 and Group EHG 2 with Apelin is obviously decreased comparing with Group HG land Group EHG 1; ⑦The level of insulin is positive correlated with the PDX-1 protein, but is irrespective of PDX-1 mRNA.⑧Dfference of PDX-1 mRNA among each group is not statistically significant.Conclusion:Apelin can influence the toxicity of glucose by restraining the expression of PDX-A protein, decrease the production of insulin, play an important role in the pathogenesis of diabetic.