Mechanism Of Olanzapine Regulating Appetite-Related Neurons In Hypothalamic Arcuate Nucleus

Objective:Atypical antipsychotics(AAPs)are the most commonly prescribed drugs for the clinical treatment of schizophrenia.However,most AAPs,especially olanzapine,have been associated with serious metabolic disorders,and the mechanism underlying olanzapine-induced metabolic abnormalities remains unclear.In our previous studies,we identified that increased appetite induced by olanzapine plays a critical role in olanzapineinduced metabolic disorders.Agouti-related protein(Ag RP)and proopiomelanocortin(POMC)neurons in the arcuate nucleus of the hypothalamus are dominant in appetite regulation.Based on this,we hypothesize that olanzapine may increase appetite by affecting the appetite-regulating centers.Our study aims to investigate changes in the activity of appetite-regulating central neurons in mice treated with olanzapine using techniques such as calcium imaging,patch clamp,and immunofluorescence.Further verification will be carried out through functional correction experiments and pharmacological simulation experiments.Our ultimate goal is to explore the key targets of the appetite regulating center affected by olanzapine,and gain insight into the underlying mechanisms of olanzapine-induced appetite increase.The results of our study may provide a theoretical basis for the research and development of more effective methods to control appetite increase,weight gain and metabolic disorders induced by antipsychotics.Methods:(1)In order to investigate the metabolic phenotype induced by olanzapine,we established three cohorts of C57 mice.The first two cohorts of 6-week-old C57 mice were randomly assigned to control and olanzapine groups(n=6-8)and fed control and olanzapine diets for 8 weeks,respectively.Weight and food intake were regularly recorded,and an oral glucose tolerance test(OGTT)was conducted along with assessment of changes in appetite-related peptide expression at the end of the eight-week period.In the second cohort of mice,a metabolic cage was used to record energy intake and expenditure at baseline and after 8 weeks on the olanzapine diet.The third cohort of C57 mice was also separated into two groups and underwent OGTT and insulin tolerance test(ITT)after a single intraperitoneal injection of 6 mg/kg olanzapine.(2)Ag RP-Cre and POMC-Cre mice,aged 8 weeks,were divided into5 groups(n=4-6 mice),designated as control group and olanzapine groups with 4 different concentrations.After injection of the calcium-signaling virus into the arcuate nucleus of the hypothalamus via stereotactic surgery,calcium imaging was used to detect changes in the calcium signals of Ag RP/POMC neurons following intraperitoneal injection of olanzapine at different concentrations.Similarly,Ag RP-AI3-Cre and POMC-AI3-Cre mice were also divided into 5 groups,respectively.Olanzapine was administered intraperitoneally at different concentrations for 30 minutes,and then brain sections were obtained through perfusion.The immunofluorescence method was employed to detect changes in c-fos expression in arcuate nucleus Ag RP/POMC neurons.Finally,the membrane electrophysiology method was used to re-confirm the results at the cellular level.(3)Ag RP neurons can be eliminated by diphtheria toxin.Ag RP-Cre mice were separated into an ablation group and a control group(n=6-8),and were respectively injected with diphtheria toxin and control virus.The mice were fed an olanzapine diet for 1 month.Ag RP neurons were further activated by chemogenetics and separated into an activation group and a control group(n=6-8).The activated and control viruses were injected respectively,and both groups were fed an olanzapine diet for 7 weeks.Changes in appetite and metabolism of mice in both groups were observed throughout the experiment.Similarly,POMC neurons can also be eliminated with diphtheria toxin.POMC-Cre mice were separated into an ablation group and a control group,and were respectively injected with diphtheria toxin virus and control virus.These mice underwent an olanzapine diet for 5 weeks,and changes in appetite and metabolism were observed.Another cohort of POMC ablation mice were divided into a control group and an olanzapine group,which were fed a control diet and an olanzapine diet for 5 weeks,respectively.Changes in appetite and metabolism were also observed.(4)In order to explore the receptor mechanism,after a 30-minute pretreatment with lorcaserin,a 5-HT2 C receptor agonist,calcium signals and c-fos expression of POMC neurons were recorded again following intraperitoneal injection of 6mg/kg olanzapine.Then,sh RNA was used to downregulate the 5-HT2 C receptor in POMC neurons,and changes in calcium signaling were recorded.Finally,lorcaserin was used as an intervention to observe whether it could reverse olanzapine-induced appetite and weight gain.Results:(1)After 8 weeks of continuous olanzapine gavage,there was no significant difference in body weight between the two groups.However,the cumulative intake of the olanzapine group was significantly higher than that of the control group(F=7.25,P=0.02).Further metabolic cage tests showed that the increase in the olanzapine group was attributable to increased daytime intake(F=2.95,P=0.017),and there was no significant difference in energy expenditure and activity levels between the two groups.In olanzapine-fed mice,the expression of the appetite-promoting peptide,Ag RP,increased(F=-3.70,P=0.003),while the expression of the appetitesuppressing peptide,POMC,decreased(F=2.36,P=0.035).Both long-term feeding and acute administration of olanzapine can cause abnormal glucose metabolism in C57 mice.(2)Calcium signals and immunofluorescence indicated that acute intraperitoneal injection of olanzapine induced decreased activity of Ag RP and POMC neurons.The two neurons were sensitive to different concentrations of olanzapine.High concentrations(6-9mg/kg)of olanzapine caused a significant decrease in the activity of Ag RP neurons,while different concentrations of olanzapine led to a decrease in the activity of POMC neurons,with the most significant decrease occurring at 3-6mg/kg.Patch clamp experiments also indicated that olanzapine could induce a decrease in the electrical activity of Ag RP neurons.(3)After the ablation of Ag RP neurons,the cumulative food intake and body weight of olanzapine-fed mice significantly increased compared to the non-ablation group(P<0.05).After activating Ag RP neurons,there was a trend of increased cumulative food intake and body weight in olanzapine-fed mice compared with the non-activated group,but there was no significant difference between the two groups.After the ablation of POMC neurons,olanzapine treatment significantly reduced the cumulative food intake and body weight of mice(P<0.05).However,there was no significant difference in the expression of appetite-related regulatory peptides among all groups.(4)Both lorcaserin pretreatment and downregulation of the 5-HT2 C receptor in POMC neurons reversed olanzapine-induced decreased activity of POMC neurons.Lorcaserin significantly decreased the cumulative food intake(P<0.05),but did not improve dyslipidemia,and there was no significant difference in appetite-related regulatory peptides between the two groups.Conclusions:(1)The appetite of olanzapine-fed mice increased significantly,and weight gain was attributed to increased food intake,independent of energy expenditure and levels of physical activity.Both acute and chronic olanzapine exposure can cause abnormal glucose metabolism in mice.(2)Olanzapine can directly regulate the activity of the arcuate appetite-regulating neurons,Ag RP,and POMC,and the sensitivity ranges of the two neurons to olanzapine concentration are heterogeneous.(3)The appetite and weight gain induced by olanzapine could be reversed by regulating the activity of Ag RP and POMC neurons.(4)The 5-HT2 C receptor on POMC neurons is the main target of olanzapine.Activation of the 5-HT2 C receptor in POMC neurons could reverse the increased appetite and weight gain induced by olanzapine.