Research On The Mechanism Of Insulin Resistance Caused By Olanzapine Based On FABP4 Protein

OBJECTIVE:In the clinic,the long-term administration of olanzapine is associated with increased risk of severe metabolic abnormalities,such as insulin resistance(IR).Insulin resistance is an important factor leading to diabetes and metabolic abnormalities in patients,however,the mechanism of olanzapine-induced IR is unclear.Previous studies have shown that inflammatory TNF-α,fatty acid-binding protein,adipocyte(FABP4),insulin receptor substrate-1(IRS-1),protein kinase B(AKT),and palmitoylated protein DHHC7 of glucose transporter 4(GLUT4)may be closely related to IR.This study aims to investigate the potential mechanisms and proteins in pathways of long-term olanzapine-induced IR based on FABP4 protein through in vivo and in vitro experiments.METHODS:(1)Twelve female Sprague-Dawley rats(weighing 200±20g)were randomly assigned to two groups:the blank group(BL group),the olanzapine group(OLZ group).The OLZ group received olanzapine orally(1.5mg/kg/day)for 8 weeks,while the BL group received drug vehicle.The rats were weighed and took blood from the orbit in the third,sixth and eighth week to measure the fasting blood glucose,fasting insulin,and calculate the insulin resistance index(HOMA-IR)of each group of rats.At the end of the study,the rats in each group performed the oral glucose tolerance test(OGTT).And the level of TNF-αin adipose tissue of rats were determined by Elisa kit.Then 3T3-L1 adipocytes were treated with different concentrations of TNF-α,and the expressions of FABP4 protein,total GLUT4 and plasma membrane GLUT4 were measured by western blot.(2)3T3-L1 adipocytes were transfected with DHHC7 si RNA.The expressions of total GLUT4 and plasma membrane GLUT4 was measured by western blot.Then3T3-L1 adipocytes were administered with FABP4 inhibitor,and DHHC7 m RNA levels was detected by PCR.The expressions of total GLUT4,plasma membrane GLUT4,p-IRS-1(Ser307)and p-AKT(Ser473)were measured by western blot.(3)First,an adenovirus containing FABP4 si RNA was constructed.Then twenty-four female C57bl/6 mice were randomly assigned to three groups:the blank group(Control group),the olanzapine group(OLZ group)and the FABP4 gene silencing+olanzapine group(FABP4 si RNA group).The mice in the olanzapine group and the FABP4 gene silencing+olanzapine group received olanzapine intraperitoneally(10mg/kg/day)for 8 weeks,and the blank group mice received drug vehicle.Meanwhile,the FABP4 si RNA group received Adenovirus intraperitoneally every three days(10~7pfu/time).The mice were weighed and took blood from the orbit in the second,forth,sixth,and eighth week to determine the fasting insulin,fasting blood glucose,and HOMA-IR of each group of mice.At the end of the study,the mice in each group performed the oral glucose tolerance test(OGTT).Protein expressions of FABP4,total GLUT4,plasma membrane GLUT4,p-IRS-1(Ser307)and p-AKT(Ser473)were measured by western blot,and DHHC7 m RNA levels was detected by PCR.RESULTS:(1)The results of studies in rats are as follows:compared with the BL group,the HOMA-IR,weight,fasting insulin and fasting blood glucose were further significantly increased in OLZ group.And olanzapine can up-regulate the expressions of TNF-αand FABP4 in OLZ group.Cell experiments found that TNF-αcan up-regulate the expression of FABP4.(2)A 48h exposure of 3T3-L1 adipocytes to olanzapine(5u M)significantly inhibited the expression of DHHC7 and AKT(Ser473),significantly increased the expression of IRS-1(Ser307);3T3-L1 adipocytes transfected with DHHC7 si RNA significantly reduced the expression of plasma membrane GLUT4.Then 3T3-L1adipocytes were administered FABP4 inhibitors and olanzapine.The FABP4 inhibitor efficiently blocked the effects of olanzapine on DHHC7,GLUT4,IRS-1(Ser307)and AKT(Ser 473).(3)The results of studies in mice are as follows:compared with the Control group and FABP4 si RNA group,the HOMA-IR,fasting insulin and fasting blood glucose were significantly increased in OLZ group,and the expressions of FABP4and IRS-1(Ser307)in white adipose tissue were significantly increased in OLZ group,there was no significant difference between the FABP4 si RNA group and the Control group.Moreover,compared with the Control group and FABP4 si RNA group,the expressions of DHHC7,AKT(Ser473),and plasma membrane GLUT4 were significantly decreased in adipose tissue in the OLZ group,there was no significant difference between the si RNA group and the Control group.CONCLUSION:Chronic olanzapine treatment may up-regulate TNF-α,thereby promoting the expression of FABP4.On the one hand,FABP4 could inhibit the expression of DHHC7,the upper membrane of GLUT4 and glucose transport;on the other hand FABP4 could induce serine phosphorylation of IRS-1 and inhibit AKT phosphorylation,and inhibition of glucose transport ultimately lead to insulin resistance.