The Mechanism Of Olanzapine-mediated Down-regulation Of FGF21 In Brown Fat Causes Energy Metabolism Disorders

Background:Schizophrenia is a chronic disease with abnormal mental activity,which easily causes the patient to lose cognitive and social functions,and causes a greater burden on the family and society.Olanzapine(Olan)is one of the first-line drugs for the treatment of schizophrenia,but it is prone to cause metabolic disorders,such as weight gain,dyslipidemia,diabetes,and other adverse reactions,which reduce the life expectancy of patients.Weight gain is caused by the body’s energy metabolism disorder,and thermogenesis is an important way of energy expenditure.Brown adipose tissue is one of the body’s main heat-producing organs,which can resist the cold and maintain the body’s energy metabolism balance.It is currently a hot spot in obesity research.Brown fat mainly exerts a thermogenic effect through uncoupling protein 1(UCP1)molecules.It has been shown that Olan can reduce the thermogenic activity by inhibiting the expression of UCP1 in brown adipose tissue.Fibroblast growth factor 21(FGF21)is a metabolic regulator.It has become a candidate target drug for lowering blood glucose and weight loss.It is mainly expressed in liver,brown fat,white fat and pancreas,and is involved in controlling glucose homeostasis,insulin sensitivity and brown fat thermogenesis.In brown adipose tissue,FGF21 is activated by norepinephrine released by sympathetic nerves and acts on brown adipose cells through autocrine and paracrine methods to promote the expression of UCP1.According to previous studies in our laboratory,the histone methylation level in the promoter region of Fgf21 in the brown fat of mice administered with olanzapine has changed,therefore,we first need to clarify the role of FGF21 in the brown fat energy metabolism disorder caused by olanzapine.Objective:Through olanzapine-administered mouse models and cell models,explore the energy metabolism side-reaction mechanism of olanzapine-mediated FGF21 affecting brown fat thermogenesis,and provide a new solution for clinical interventions in olanzapineinduced weight gain and other side-effects.Methods and results:(1)The effect of olanzapine on the body weight and thermogenesis of miceTwenty 6-week-old female C57BL/6J mice were selected and divided into two groups: control group(Control)and olanzapine administration group(Olan,3 mg/kg,oral).During the administration,the food intake,water consumption,and body weight of the mice were measured every 4 days.Thermal imaging of the mice was taken before and after the administration to detect the temperature of the scapula of the mice.After four weeks of treatment of C57BL/6J mice with olanzapine,the body weight of the mice increased significantly(P<0.001),while the amount of diet and water consumption did not change significantly.The blood lipids and blood glucose levels of mice were measured by the kit,and it was found that compared with the control group,the HDL content of the olanzapine group was significantly decreased(P<0.05),the LDL and glucose content were significantly increased(P<0.001),the level of TG and TC was upregulated but not significant.After olanzapine treatment,the weight of brown fat in mice was significantly reduced(P<0.001),and the weight of white fat,liver and kidney increased;It was found by oil red O staining that olanzapine enlarged the lipid droplets in the brown adipose tissue(P<0.05)and liver(P<0.05)of mice.In addition,the measurement of the scapular temperature of the mice showed that,compared with the control group,the scapular temperature of the mice in the olanzapine group decreased significantly after 28 days of administration.(P<0.05).The above results indicate that olanzapine can cause significant weight gain and decrease of thermogenesis in mice,and the mice have shown a state of energy metabolism disorder.(2)The effect of olanzapine on thermogenesis factor of brown fatIn order to find molecules related to energy metabolism affected by olanzapine,we selected brown fat from mice treated with olanzapine for 28 days for gene transcriptome sequencing.We successfully enriched Fgf21,which is closely related to brown fat differentiation and thermogenesis.Subsequently,the protein expression level of FGF21 was detected by Western Blot(WB)experiment,and the protein expression level of FGF21 target molecule UCP1 and its transcription factors were also detected.The results showed that olanzapine not only reduced the protein levels of FGF21 and its transcription factor ATF2 in brown fat(P<0.05),but also significantly reduced the expression of UCP1(P<0.001)and its transcription factor PGC1(P<0.05).Therefore,we chosed FGF21 as the target molecule for follow-up research.(3)FGF21 is involved in the down-regulation of UCP1 expression mediated by olanzapineIn order to further verify whether FGF21 is involved in the effect of olanzapine on brown fat thermogenesis-related factors,we selected brown adipocytes differentiated from C3H10 T1/2 mesenchymal stem cells as the cell model and carried out a series of in vitro experiments.We used 0,1,5 μM olanzapine to treat brown adipocytes,and found that olanzapine dose-dependently down-regulated the protein expression levels of FGF21 and its target molecule UCP1.5 μM olanzapine had a significant effect on downregulation of FGF21 and UCP1 protein expression(P<0.05).We constructed FGF21 receptor(FGFR1)silencing vector and FGF21 overexpression vector,respectively,combined with olanzapine(5 μM)to treat brown adipocytes.The WB results of the FGFR1 silenced experiment showed that,compared with the control group,the FGFR1 and UCP1 protein expressions in the FGFR1 silenced group were significantly down-regulated(P<0.01),and the UCP1 transcription factors PPARγ,PGC1,and PRDM16 were also significantly down-regulated(P<0.05).Compared with the olanzapine group,the FGFR1 silencing plasmid and olanzapine combined treatment group had no significant changes in FGFR1,UCP1 and other factors.The WB results of the overexpression experiment showed that the expression of FGF21 and its transcription factor ATF2 was significantly up-regulated(P<0.05)after FGF21 overexpression plasmid was transfected into cells for 48 hours(P<0.05),and the expression of thermogenesis factor UCP1 and its transcription factors PPARγ(P<0.01)and PGC1(P<0.05)were significantly up-regulated.Mitochondrial probe experiments showed that the mitochondrial activity of brown adipocytes also increased significantly after FGF21 overexpression(P<0.001).Compared with the olanzapine group,the FGF21 overexpression and olanzapine combined treatment group had significantly up-regulated FGF21(P<0.01)and UCP1(P<0.05)expression,and significantly increased mitochondrial activity(P<0.001),ATF2,PPARγ,and PGC1 showed an up-regulation trend,but not significant.(4)Olanzapine affects the expression of FGF21 through β3AR signaling pathwayWe used the β3 adrenergic receptor agonist CL316243,which can promote FGF21 expression,to treat mice in combination with olanzapine to explore the mechanism by which olanzapine affects FGF21 expression.We divided 24 6-week-old female C57BL/6J mice into four groups: control group(Control),olanzapine administration group(Olan,3 mg/kg Olan,oral),β3 adrenergic receptor agonist group(CL,0.1 mg/kg CL316243,ip),olanzapine and agonist co-treatment group(Olan+CL,3 mg/kg Olan,oral+ 0.1 mg/kg CL316243,ip).During the treatment period,the mice’s diet,water intake,body weight and body temperature were measured.After 28 days of administration,compared with the control group,the weight of the mice in the Olan group increased significantly(P<0.05),and the body temperature decreased significantly(P<0.01).Olan and CL combined treatment alleviated the trend of weight gain and body temperature drop in mice caused by olanzapine(P<0.05).WB experiment results showed that compared with the control group,olanzapine down-regulated the protein expression levels of β3AR(P<0.05),PKA(P<0.05),ATF2(P<0.05),FGF21(P<0.01),FGFR1(P<0.05),PGC1(P<0.05),UCP1(P<0.05)in the brown fat of mice.Compared with the Olan group,the protein expression of PKA(P<0.05),ATF2(P<0.05),FGF21(P<0.01),FGFR1(P<0.05),AMPK(P<0.01),PGC1(P<0.05),UCP1(P<0.05)was significantly up-regulated in the Olan+CL group.Conclusion:Olanzapine can increase the weight of mice,reduce thermogenesis,and disturbance of energy metabolism is one of the important reasons for olanzapine to cause weight gain.We found that olanzapine reduced the expression of UCP1 and mitochondrial activity in brown adipocytes by down-regulated FGF21 to cause energy metabolism disorders,and it may affect the expression of FGF21 by inhibiting the β3AR signaling pathway,thereby affecting thermogenesis,which provides a theoretical basis for intervention in metabolic side effects such as weight gain caused by olanzapine.