Inhibition Of IGF-1 Signaling Pathway Is Involved In The Inhibition Of Islet β Cells Proliferation By TXNIP

IntroductionThe prevalence of diabetes mellitus(DM)is increasing year by year which has become one of the diseases that seriously threaten human health,islet β cells dysfunction and shortage are key factors in the pathogenesis of DM.Therefore,inhibiting the reduction of islet β cells and promoting their proliferation become critical for the treatment of diabetes.Studies have found that thioredoxin interacting protein(TXNIP)expression is significantly up-regulated in diabetic patients and diabetic rodent models.Further researches showed that TXNIP could promote the apoptosis of islet β cells and accelerate the development of DM.All the above studies suggest that TXNIP is of great significance in the development of DM.However,more and more studies confirmed that the expression of TXNIP was significantly decreased in tumor tissues.And TXNIP over-expression can inhibit the proliferation and migration of tumor cells,which indicated TXNIP could be regarded as a tumor suppressor in the body.Therefore,we speculated that TXNIP may affect the proliferation of islet β cells in DM,but its specific mechanism remains to be explored.It is well known that insulin-like growth factor-1(IGF-1)can stimulate the proliferation of islet β cells and maintain the number of β cells.And the activation of PI3K/AKT/m TOR which are the downstream signaling molecules of IGF-1 receptor(IGF-1R),participate in β cell proliferation induced by IGF-1.Studies have shown that TXNIP can significantly induce the expression of micro RNA-200 b and apoptosis of islet β cells,while micro RNA-200 b can inhibit the expression of IGF-R and the phosphorylation of AKT,indicating that IGF-1 signaling pathway may be involved in the role of TXNIP in inhibiting the proliferation of islet β cells.Therefore,the main purpose of the present study was to investigate whether TXNIP can inhibit the expression of IGF-1R and its downstream signaling pathways and then affect the proliferation inhibition of islet β cells by TXNIP.The study may provide the new directions for DM treatment.Part One Changes of TXNIP,IGF-1 and the proliferation of islet β cells in DM miceObjective: The purpose of this study was to observe the changes in TXNIP,IGF-1 and its receptors,and islet β cells proliferation in db/db mice.Method: 1.Detected the weights of db/m and db/db mice at 8,10,12,and 16 weeks 2.Detected fasting blood glucose of db/m and db/db mice at 8,10,12,and 16 weeks 3.Detected TXNIP,PCNA and IGF-1R expression in pancreatic tissues of db/m and db/db mice by western blot 4.Observed PCNA expression of db/m and db/db mice by IHC 5.Observed of the morphology and structure of islets in db/m and db/db mice by HE 6.Observed of the islets β cells numbers in db/m and db/db mice by ICC 7.Detected the expression of IGF-1 and insulin in serum of db/m and db/db mice by ELISAResult: 1.With the increasing of the mice age,the weights of db/db mice were significantly higher than that of db/m mice.The fasting blood glucoses of db/db mice were much higher than that of db/m mice.Fasting insulin levels in db/db mice were higher than that in db/m mice 2.The structure of islets was destroyed,the number of islet β cells reduced and the expression of PCNA in islets reduced in db/db mice3.Serum IGF-1 levels decreased,IGF-1 receptor levels increased in pancreatic tissues,and TXNIP expression in pancreatic tissues increased in db/db miceConclusion: The structure of islets was destroyed,the number of islet β cells decreased,and the expression of PCNA in the islets decreased in the diabetic mice.The serum IGF-1 levels decreased,the IGF-1 receptor levels in pancreatic tissues increased,and TXNIP expression in pancreatic tissues increased in diabetic mice.It is suggested that there may be a correlation among TXNIP and IGF-1 and the proliferation of islet β cells in diabetic patients.Part Two IGF-1 signaling pathway participates in TXNIP’s inhibition of MIN6 cell proliferationObjective: 1.Observe whether TXNIP can inhibit the proliferation of islet β cells in MIN6 cell line.2.Observe whether TXNIP can affect the expression of IGF-1R,and whether TXNIP can reduce the proliferation effect of IGF-1 on islet β cells by affecting IGF-1R and its downstream PI3K/AKT/m TOR signaling pathway.Method: 1.Constructed TXNIP overexpression(Ad-TXNIP-GFP)and silencing(TXNIP Sh-RNA)MIN6 islet β cell lines with lentivirus 2.Observed the transfection rate of cells by fluorescence microscope 3.Added 100 nmol/L IGF-1 to each group and incubated for 48 h.4.Detected cell proliferation by Ed U method 5.Detected Ki67 expression by flow cytometry 6.Detected cell proliferation rate by Cell Counting Kit-8 method 7.Detected the expressions of TXNIP,PCNA and IGF-1R,and detected protein expressions of p-PI3 K,p-AKT and p-m TOR by western blot 8.Observed IGF-1R and PCNA expressions in cells by IFResult: 1.TXNIP overexpression and silencing models successfully constructed 2.Proliferation of islet β cells reduced when TXNIP was overexpressed 3.When TXNIP was overexpressed,the proliferation effect of IGF-1 on islet β cells was reduced.TXNIP silencing increased IGF-1 proliferation effect on islet β cells 4.TXNIP overexpression decreased the expression of IGF-1R and its downstream PI3K/AKT/m TOR signaling pathway.TXNIP silencing increased IGF-1R expressionConclusion: TXNIP can inhibit the proliferation of islet β cells.This effect is achieved by suppressing the expression of IGF-1R and its downstream signaling pathways.Part Three IGF-1 signaling pathway is involved in TXNIP inhibition of islet β cells proliferation in miceObjective: 1.Observe whether TXNIP can affect the proliferation of islet β cells in TXNIP knockin mice and TXNIP knockout mice.2.Observe whether TXNIP affected IGF-1R expression in TXNIP knockin mice and TXNIP knockout mice,and whether TXNIP can reduce the proliferation effect of IGF-1 in islet β cells by affecting IGF-1R and its downstream PI3K/AKT/m TOR signaling pathway.Method: 1.Ordinary C57 mice,TXNIP knockin mice and TXNIP knockout mice,6 mice randomly selected from each group,intraperitoneally injected with IGF-1,50 μg/kg/day for 4 weeks.2.Observed shape changes of islets in TXNIP knockin mice and TXNIP knockout mice by HE 3.Observed the number of islet β cells in pancreas tissue of TXNIP knockin mice and TXNIP knockout mice by ICC 4.Observed PCNA expression in pancreas tissue of TXNIP knockin mice and TXNIP knockout mice by IHC 5.Detected TXNIP,PCNA and IGF-1R expression in TXNIP knockin mice and TXNIP knockout mice by western blot 6.Detected the expression of IGF-1 and serum insulin in TXNIP knockin mice by ELISAResult: 1.HE observed the changes of islet shape and structure in TXNIP knockin mice and TXNIP knockout mice.The number of islet β cells in the islets of TXNIP knockin mice decreased,and the number of islet β cells in the islets of TXNIP knockout mice increased.2.When TXNIP was overexpressed,the proliferation effect of IGF-1 on islet β cells was reduced.TXNIP silencing increased IGF-1 proliferation effect on islet β cells 3.TXNIP overexpression decreased the expression of IGF-1R and its downstream PI3K/AKT/m TOR signaling pathway.Silencing TXNIP increased IGF-1 receptor expression.Conclusion: TXNIP can inhibit the proliferation of islet β cells.This effect is achieved by suppressing the expression of IGF-1R and its downstream signaling pathways.