Mechanism Of Olanzapine Regulates The Expression Of GLP1R In The Hypothalamus To Mediate Increased Food Intake

Background:Schizophrenia is a serious chronic mental illness that affects behavior and cognition,and patients with schizophrenia have a life expectancy that is 15-20 years shorter than normal.Olanzapine(Olan)is the most widely used first-line drug for the clinical treatment of schizophrenia,but long-term use can lead to serious metabolic side effects,such as weight gain and obesity,which not only seriously affect the patient’s medication compliance,but also lead to increased morbidity and mortality of cardiovascular and cerebrovascular diseases.Increased food intake is one of the important manifestations of olanzapine-induced obesity.The hypothalamus is the regulatory center of food intake and energy metabolism.Glucagon like peptide 1 receptor(GLP1R)is widely distributed in the hypothalamus.Activation of GLP1 R will affect the downstream energy and food intake signaling pathway related factors AMP-activated protein kinase(AMPK)and neuropeptide like Proopiomelanocortin(POMC)in the hypothalamus to reduce food intake.Previous laboratory results found that olanzapine can significantly increase food intake in rats and significantly down-regulate the secretion of GLP1 and the expression of GLP1 R in the small intestine.GLP1 R in the hypothalamus is also affected by olanzapine,but the specific mechanism is still unclear.Therefore,understanding the role of hypothalamic GLP1 R in olanzapine-induced increased food intake and the mechanism are crucial for improving the effect of olanzapine-induced increased food intake.This study explored the mechanism of olanzapine regulates the expression of GLP1 R in the hypothalamus to mediate the increase in food intake through in vivo and in vitro experiments in rats,and provided a new reference for elucidating the mechanism of olanzapine-induced metabolic side effects.Methods and results:1.The effect of different doses of olanzapine on body weight,food intake and body fat of SD rats40 adult SD rats weighing 180-220 g were fed by active diet containing 0,0.25,1.0and 4.0 mg/kg olanzapine(t.i.d.)respectively for 14 days.Body weight and food intake were monitored during administration,and biochemical indicators such as TG,TC,HDL-C and LDL-C in serum were detected after administration.After 14 days of olanzapine treatment,the body weight,cumulative food intake,TG and TC concentration of the 1.0 mg/kg olanzapine group were significantly higher than the control group(p<0.01),while there was no significant difference in the low-dose group.Compared with the control group,the cumulative food intake and TC concentration of the rats in the 4.0 mg/kg group increased significantly,but the body weight and TG concentration did not increase significantly.2.Olanzapine down-regulated the expression of GLP1 R in the PVN of the hypothalamusWestern Blot experiments found that olanzapine dose-dependently down-regulated the protein expression of GLP1 R in the hypothalamus.Olanzapine mainly down-regulated the protein expression of GLP1 R and phosphorylation levels of AMPK/ACC in the PVN,and significantly down-regulated the gene expression of the anorexigenic peptide Cart,significantly up-regulated the gene expression of the orexigenic peptides Npy and Agrp.3.GLP1 R was involved in olanzapine-mediated down-regulation of AMPK/ACC expressionTo further verify whether GLP1 R is involved in the effect of olanzapine in hypothalamic feeding-related factors like AMPK/ACC,MHYPOE-N43/5 hypothalamic cells were selected for in vitro experiments.Olanzapine concentrations of 0,5,10,and20 μM were used to act on hypothalamic cells,and immunofluorescence experiments showed that olanzapine dose-dependently down-regulated the expression level of GLP1 R,and the expression of GLP1 R was decreased extremely significantly when the concentration of olanzapine was 20 μM(p<0.0001).Glp1r overexpression and silencing plasmids were constructed and transfected into MHYPOE-N43/5 hypothalamic cells respectively.Western Blot results showed that GLP1 R overexpression could significantly reverse the inhibitory effect of olanzapine on GLP1R(p<0.05),and activate AMPK/ACC phosphorylation level.While silencing GLP1 R achieves low expression of GLP1 R in hypothalamic cells,further inhibiting AMPK/ACC phosphorylation level.4.Olanzapine affected the expression of GLP1 R through the 5-HTR2A-PKA pathwayThe results of fluorescence quantitative RT-PCR showed that olanzapine significantly inhibited the transcription level of 5-htr2 a gene in the PVN of the hypothalamus(p<0.05).In in vitro experiments,serotonin was administered to stimulate the 5-HTR2 A in hypothalamic cells.Western Blot results showed that the expression level of 5-HTR2 A and the phosphorylation of PKA were significantly down-regulated after olanzapine treatment(p<0.01),the expression of GLP1 R was also significantly down-regulated(p<0.05).Compared with the olanzapine group,the expression of these factors were significantly up-regulated after simultaneous administration of 5-HT receptor agonists and olanzapine,and the 5-HTR2 A expression was positively correlated with GLP1 R expression(r=0.80,p=0.01).The phosphorylation of PKA and the expression of GLP1 R were significantly down-regulated after olanzapine treatment(p<0.05).Compared with the olanzapine group,the GLP1 R expression and the phosphorylation of PKA were significantly down-regulated in the PKA inhibitor H89 and olanzapine group(p<0.05).The above results indicated that olanzapine down-regulated the expression of 5-HT2 A receptor,inhibited the phosphorylation of PKA,and then down-regulated the expression of GLP1 R.5.Olanzapine down-regulated the expression of GLP1 R transcription factor PPARαTo further explore the regulation of olanzapine on the expression of GLP1 R,the GLP1R-related transcription factors like Pparα,Ebf1,and Stat4 were detected by fluorescence quantitative RT-PCR.The results showed that olanzapine significantly down-regulated the transcription level of Pparα gene(p<0.05).Immunofluorescence experiments showed that olanzapine dose-dependently down-regulated the expression of PPARα,which was consistent with the fluorescence results of GLP1 R gradient administration of olanzapine.The results of immunofluorescence and Western Blot experiments showed that PPARα and GLP1 R in the olanzapine group were significantly down-regulated(p<0.05).Compared with the olanzapine group,the expression of PPARα and GLP1 R were significantly up-regulated after Pparα overexpression plasmid transfection and administered with olanzapine(p<0.01).The above results suggest that olanzapine may interfere with the positive activation of GLP1 R by PPARα.6.Combination of liraglutide alleviated the effect of olanzapine-induced increased food intakeThe GLP1 R agonist liraglutide and olanzapine were treated rats to explore whether liraglutide could alleviate the effect of olanzapine-induced increased food intake.Thirty-two SD rats were randomly divided into four groups: control group(Control),olanzapine group(Olan,1 mg/kg,b.i.d.),and liraglutide group(Lir,0.125 mg/kg,b.i.d.),olanzapine combined with liraglutide group(Olan+Lir,1 mg/kg Olan;0.125 mg/kg Lir,b.i.d.),the body weight and remaining food of the rats were weighed and record every 4days during the period.After administration,biochemical indexes such as TG,TC,HDL-C and LDL-C in serum were detected.After continuous administration for 42 days,compared with the control group,the food intake,body weight and blood lipid concentration of the rats in the Olan group were significantly increased;Olan+Lir could alleviate the food intake,body weight gain and blood lipid elevation of the rats caused by olanzapine.Western Blot showed that compared with the control group,the phosphorylation of GLP1 R and its downstream AMPK/ACC in the PVN of the hypothalamus were significantly down-regulated after Olan treatment(p<0.05).The phosphorylation levels of GLP1 R and its downstream AMPK/ACC were significantly up-regulated in the group(p<0.05).The combined treatment of Olan+Lir effectively reduced the high expression of the orexigenic peptides Npy/Agrp gene caused by olanzapine,significantly increased the expression of the anorexigenic peptide Cart gene,and significantly improved the effect of olanzapine-induced food-intake increasing.Conclusion:Olanzapine inhibits the 5-HTR2A-PKA signaling pathway and the protein expression of the GLP1 R transcription factor PPARα,down-regulates the expression of GLP1 R in the hypothalamus,reduces the phosphorylation of AMPK/ACC and the expression of the food inhibitory peptide CART,thereby affecting the ingestion.Olan Combines with the GLP1 R agonist liraglutide can effectively attenuate the down-regulation of olanzapine on GLP1 R and alleviate the effect of olanzapine-induced increased food intake.This experiment provids new clues for elucidating the mechanism of olanzapine-induced metabolic side effects.