TXNIP Reduces Hepatic Insulin Sensitivity By Impairing Insulin Signaling Pathway

Background:Insulin resistance is one of the main pathological features of type 2 diabetes.In the liver tissue,it is mainly manifested as decreased glycogen synthesis,and increased output of glucose to the outside from the liver.Studies have shown that thioredoxin-interacting protein（TXNIP）expression is significantly increased in diabetic patients and diabetic mice.TXNIP plays an important role in the body’s glucose metabolism and the development of diabetes.However,the role and significance of TXNIP in hepatic insulin resistance are still unclear.Objectives:1.Establish a TXNIP overexpression（TXNIP-KI）and knockout（TXNIP-KO）homozygous mouse model,observe the general characteristics of mouse body weight and fasting blood glucose,and observe its oral glucose tolerance,insulin tolerance and hepatic insulin resistance.2.Confirm that TXNIP regulates hepatic insulin sensitivity by affecting the insulin signaling pathway IRS-1/AKT/GSK3β.Methods:1.Establish a TXNIP-KI and TXNIP-KO homozygous mouse model.2.Detect mouse body weight and fasting blood glucose every 4 weeks,and detect serum biochemical indicators at the 24^thweek.3.Perform glucose tolerance and insulin tolerance tests on mice at the 8^thweek and the 24^thweek.4.Use RT-PCR method to detect liver TXNIP gene expression.5.Apply PAS staining to observe liver glycogen accumulation,and use glycogen detecting kit to determine liver tissue glycogen content.6.Employ western blot method to detect the liver TXNIP,insulin receptor（IR）,insulin signal pathway-related indicators:IRS-1,AKT,GSK3βprotein,and GLUT2protein expressions.Results:1.TXNIP homozygous mouse model was successfully established.The mouse tail DNA was detected by ordinary PCR,and homozygous mice were identified.TXNIP-KI mice had both 329bp and 335bp fragments,and there was no 412bp fragment.TXNIP-KO mice had 262 bp but no 407 bp fragment.Real-time fluorescence quantitative PCR was used to detect the expression of TXNIP gene in mouse liver and perform reverse amplification.The results showed that TXNIP of overexpression mice was greatly amplified,and knockout mice did not amplify TXNIP.The expression of TXNIP protein in mouse liver was detected by Western blot experiment,and it was found a significant increase in TXNIP-KI mice,but almost no expression in TXNIP-KO mice.The above results showed that the TXNIP-KI and TXNIP-KO homozygous mouse models were successfully constructed.2.General characteristics of TXNIP homozygous mice.Compared with WT mice,the body weight of TXNIP-KI mice decreased,and the body weight of TXNIP-KO mice increased before but decreased after 12 weeks of normal feeding.Compared with the WT mice,the fasting blood glucose of TXNIP-KI mice was high,and it continued to increase with time,while the fasting blood glucose of TXNIP-KO mice was reduced,and it kept at a low level over time.The results of serum lipid metabolism-related indicators at the 24^thweek showed that compared with WT mice,TXNIP-KI mice had increased LDL,and TXNIP-KO mice had increased TC and HDL content.Changes in body weight,blood glucose,and lipid metabolism related indicators suggest that TXNIP homozygous mice have abnormal glucose and lipid metabolism.3.TXNIP overexpression impaired glucose tolerance and insulin tolerance.The OGTT test was used to detect changes in oral glucose tolerance in mice.The results showed that compared with WT mice,the blood glucose of TXNIP-KI mice at the 8^thweek and the 24^thweek increased rapidly,but decreased slowly,while the blood glucose of TXNIP-KO mice rose slowly and quickly fell back to the normal range.The ITT test was used to detect the insulin tolerance of mice.Compared with WT mice,the blood glucose of TXNIP-KI mice at the 8^thweek and the 24^thweek decreased slowly and with a lower amplitude.Compared with WT mice,there was no difference in blood glucose changes in TXNIP-KO mice at the 8^thweek,but blood glucose dropped rapidly and rose rapidly at the 24^thweek.The above results suggest that TXNIP-KI mice have abnormal glucose tolerance and decreased insulin sensitivity,while TXNIP-KO mice can improve such manifestations.4.TXNIP-KI mice developed insulin resistance.The insulin resistance index（HOMA-IR）was calculated by combining fasting blood glucose and fasting insulin values.HOMA-IR results showed that compared with WT mice,TXNIP-KI mice developed insulin resistance at the 8^thweek and the24^thweek,but TXNIP-KO mice did not develop insulin resistance.Compared with WT mice,TXNIP-KI mice had no difference in first-phase insulin secretion,suggesting that the function of pancreaticβ-cells in TXNIP-KI mice was temporarily not defective.5.The hepatic glycogen content was decreased in TXNIP-KI mice.PAS staining was used to observe the accumulation of glycogen in liver tissue.The results showed that compared with WT mice,TXNIP-KI mouse liver had less rose-red substance and a lighter color,indicating that glycogen accumulation was reduced,while in TXNIP-KO mouse liver tissue,rose-red substance was darker and more abundant,and glycogen accumulated.The test kit was used to detect glycogen content in liver tissue,and the results were consistent with PAS staining.The above results suggest that TXNIP-KI mice do have insulin resistance.6.The GLUT2 protein expression in TXNIP-KI mice was increased on the cell membrane.Western blot experiment was used to detect the proteins in the liver either on the cell membrane or within the cells by extracting them respectively first.The results showed that,compared with WT mice,the expression of GLUT2 protein in TXNIP-KI mice increased on the cell membrane and decreased in the cell.Compared with WT mice,TXNIP-KO mouse GLUT2 protein expression decreased on the cell membrane and increased in the cell.The above results strongly suggest that TXNIP-KI mice have insulin resistance.7.TXNIP overexpression inhibited IR protein expression in mouse liver and downstream insulin signal transduction.Western blot was used to detect the expression of IR in mouse liver and the expression of P-IRS-1,P-AKT,and P-GSK-3βprotein in the insulin signaling pathway.The results showed that compared with WT mice,TXNIP-KI mouse IR protein and P-IRS-1,P-AKT,P-GSK-3βprotein expressions were all reduced,while TXNIP-KO mouse expression of these proteins was increased.The above results suggest that the insulin signaling pathway in the liver of TXNIP-KI mice is impaired.Conclusion:TXNIP reduces the sensitivity of liver insulin by down-regulating the expression of insulin receptor and downstream IRS-1/AKT/GSK3βinsulin signaling pathway,thereby participates in the regulation of the occurrence and development of diabetes.