Study On The Effect And Possible Mechanism Of Olanzapine On Thermogenic Activity Of Brown Fat Based On Poly I:C Model

Background:Olanzapine(OLZ)is one of the most frequently used antipsychotic drugs in clinical practice,but it has a high risk of metabolic syndrome.Epidemiological and animal experiments have shown that olanzapine can cause metabolic abnormalities,especially weight gain and glucose and lipid metabolic disorders.Although olanzapine caused metabolic abnormalities with high clinical relevance,there is lack of evaluation of the relationship between metabolic abnormalities and schizophrenia phenotype.The body's metabolic abnormalities associated about schizophrenia disease itself and the antipsychotic drug olanzapine affect worth to study and the specific mechanism of olanzapine cause weight gain need to further clarifyWeight gain is due to the imbalance between energy intake and energy expenditure,and body heat production is a key factor affecting energy expenditure.Brown adipose tissue,a maj or source of non-shivering thermogenesis in mammals and humans,plays an important role in maintaining an animal's body temperature and energy balance.The substance in the mitochondria of brown fat cells is called uncoupled protein 1(UCP1),which causes glucose and fatty acids decomposition and converts chemical energy into heat.As a key factor of brown adipose tissue thermogenesis,UCP1 expression is mainly regulated by regulatory factors such as PPAR-?,PGC-1?,PRDM16 and AMPK,etc However,the relationship between weight gain caused by olanzapine and brown fat thermogenesis is rarely reported,and the relevant mechanism also needs further studyObjective:The effects of olanzapine on the thermogenesis of brown fat and the differentiation of brown fat cells were investigated by poly I:C-induced schizophrenia model combined with the brown fat cell model of directed differentiation of C3H10T1/2 mesenchymal stem cells.The molecular mechanism of the effect of olanzapine on energy metabolism was analyzed under different physiological conditions,which can provide a theoretical basis for the side effects caused by clinical antipsychoticsMethods:1.Eight adult female rats and two male rats were selected and divided into 2 cages(4 females+1 male).The first day after conception was recorded as gestational day 1(GD1)Pregnant rats were intraperitoneally injected with poly I:C(10 mg/kg)or equivalent saline at GD13 days.Blood samples(total blood samples<1.5ml)were collected from the eyes at GD15 days,and the expressions of IL-6 and CXCL1 inflammatory cytokines in serum were detected by elisa.Behavioral tests(1%sucrose preference test and forced swimming test)were performed to assess neurodevelopmental effects in the offspring of pregnant rats on the 56th days of feeding2.The offspring rats whose behavioral results met the requirements were randomly divided into 4 groups(12 in each group,half male and half female):(i)Veh(Saline);(ii)OLZ(Saline);(iii)Veh(Poly I:C)and OLZ(Poly I:C).The rats were trained to take blank sugar pills orally for 7 days.And then OLZ(Saline)and OLZ(Poly I:C)groups were given olanzapine(1.0 mg/kg/day,tid),Veh(Saline)and Veh(Poly I:C)groups were given equivalent of blank sugar pills for 28 days.Body weight and food intake were measured every 4 days during administration,and scapular temperature was measured at the 2nd and the 4th weeks.3.After the administration,the blood was collected from the abdominal aorta and the contents of TG,TC,HDL-C,LDL-C and NEFA in the serum were detected with the kit.Brown fat and white fat were obtained as well as weighed,and their sections were stained with HE and oil red4.Adipogenic differentiation solution was used to differentiate C3H10T1/2 cells into brown adipocytes.Oil red staining,immunofluorescence and mitochondrial probe were used to detect lipid droplet formation and UCP1 expression after differentiation of C3H10T1/2 cells to evaluate the brown adipocyte model5.The appropriate olanzapine concentration for the adipogenic differentiation of C3H10T1/2 cells was screened by MTT method and ?3-adrenergic receptor agonist(CL316243)/cAMP agonist(Forskolin)was used to to explore the possible mechanism of olanzapine's effect on brown fat cells6.Western Blot and RT-PCR were used to detect the changes of thermogenesis-related proteins and genes in brown adipose cells and rats'brown adipose tissue.Results:1.Poly I:C-induced schizophrenia modelThe serum levels of the inflammatory factor IL-6(860.3 ± 18.91 mmol/L vs.144.7± 10.26 mmol/L,p<0.001)and the chemokine CXCL1(4349 ± 167.1 mmol/L vs.599.3± 18.6 mmol/L,p<0.001)were significantly higher in pregnant rats with poly I:C than in pregnant rats with saline injection.The results of behavior test in the adult offspring of saline/poly I:C pregnant rats showed that compared with the offspring of saline pregnant rats,the sugar water preference rate in the offspring of poly I:C pregnant rats was significantly reduced(62.52± 0.5754 vs.80.73 ± 0.6316,p<0.001)and the time of immobility in the water increased significantly(167.4 ± 4.343 vs.99.22 ± 3.407,p<0.001).These results indicated that the progeny of poly I:C pregnant rats had significantly more anhedonia and despair behaviors than the progeny of saline pregnant rats,demonstrating successful establishment of schizophrenia model.2.Effects of olanzapine on offspring weight,feeding intake and lipid profile of poly I:C pregnant ratsIn the absence of olanzapine intervention,compared with the Veh(Saline)group with normal neurodevelopment,schizophrenia model in Veh(Poly I:C)group body weight increased,while the cumulative food intake of the latter group was significantly lower than that of the former group(504.25±37.79 g vs.534.51±16.50 g,p<0.001);combined with the physiological status of rats and the olanzapine intervention,the body weight of the OLZ(Saline)group increased significantly,compared with the Veh(Saline)group(295.44±14.25 g vs.276.22±21.13 g,p<0.01),there was no significant change in the food intake of the OLZ(Saline)group compared with the Veh(Saline)group;the OLZ(Poly I:C)group had significantly higher weight gain(333.11±11.42 g vs.284.56±19.66 g,p<0.001)and food intake(576.58±26.67 g vs.504.25±37.79 g,p<0.05)than the Veh(Poly I:C)group.Compared with the Veh(Saline)group,the TG level(0.827±0.294 mmol/L vs.0.343±0.058 mmol/L,p<0.001)and the pWAT weight in the Veh(Poly I:C)group significantly increased(10.4±0.6 g vs.7.3±0.4 g,p<0.001).While TC,LDL-C and NEFA all increased,and HDL-C decreased,but it did not show any significance;compared with the Veh(Saline)group,the levels of TC(1.653 ± 0.334 mmol/L vs.1.233± 0.062 mmol/L,p<0.01)and NEFA(471.883±39.160 mmol/L vs.318.943 ± 50.569 mmol/L,p<0.05)in the OLZ(Saline)group significantly increased.TG,LDL-C,HDL-C were no significant difference;compared with the Veh(Poly I:C)group,the TG level(1.105 ± 0.356 mmol/L vs 0.827 ± 0.294 mmol/L,p<0.05)and NEFA level(467.26 ±36.383 mmol/L vs 368.043 ± 61.557 mmol/L,p<0.05)of the OLZ(Poly I:C)group significantly increased,but the TC,LDL-C and HDL-C levels were no significant difference.3.Olanzapine effect on lipid accumulation and brown adipose function in offspring of poly I:C pregnant ratsThe results of scapular temperature detection and cold tolerance test showed that the scapular temperature and adaptive heat production in the Veh(Poly I:C)group decreased compared with the Veh(Saline)group,but there was no significant difference.The staining of histologic section showed that the volume of WAT increased and the density of brown fat decreased in the Veh(Poly I:C)group;compared with the Veh(Saline)group,the scapular temperature of OLZ(Saline)decreased significantly(29.1±0.4? vs.30.9±0.3?,p<0.001),the temperature of rats was lower under cold stimulation,but there was no significant difference;the scapular temperature in OLZ(Poly I:C)group was significantly lower than that in Veh(Poly I:C)group(29.0±0.5? vs.30.5±0.4?,p<0.05)and the temperature of rats was lower under cold stimulation;moreover,olanzapine treated groups showed significantly bigger lipid droplet volume and triglyceride accumulation in white fat,and decreased density and increased lipid droplets in brown fat.4.Olanzapine effect on the thermogenic protein production and gene transcription level of the offspring brown adipose of poly I:C pregnant ratsWestern Blot results showed no significant changes in UCP1 expression in model rats and normal rats without olanzapine treatment;compared with the Veh(Saline)group,UCP1 and PPAR-? in the OLZ(Saline)group were significantly reduced by?54.9%and?63.2%,respectively;compared with the Veh(Poly I:C)group,UCP1,PPAR-? and PRDM16 in the OLZ(Poly I:C)group were significantly reduced,with the reduction levels of?56.5%,?25.3%and?73.8%,respectively.RT-PCR results showed that compared with the Veh(Saline)group,Ucpl transcription level in the Veh(Poly I:C)group was down-regulated by?12.8%,and the transcription levels of upstream regulators and genes related to fatty acid oxidation,such as Ppar-?,Prdm16,Pgc-1?,Accl and Hsl were also significantly down-regulated;compared with the Veh(Saline)group,the transcription levels of Ucpl,Ppar-?,Prdm16,Pgc-1?,Acc1,Hsl and Cpt-1? in the OLZ(Saline)group were significantly down-regulated,with Ucpl down-regulated by?70.1%;compared with the Veh(Poly I:C)group,Ucpl transcription level in the OLZ(Poly I:C)group was significantly down-regulated by?67.0%,with significant downregulation of transcription levels in Ppar-y,Prdm16,Pgc-1?,Accl,Hsl and Cpt-1?.5.Effects of olanzapine on the differentiation of C3H10T1/2 cellsOlanzapine(1 ?M and 10 ?M)inhibited the directed differentiation of C3H10T1/2 cells into brown adipocytes,significantly down-regulated the protein levels of UCP1(?5%and-27%,p<0.05),PGC-1?,PPAR-?,PRDM16,AMPK and p-AMPK,as well as the transcription levels of Ucpl(?25.6%and?80.2%,p<0.05)and the genes related to mitochondrial biosynsynthesis and fatty acid oxidation in brown fat cells,such as Cox4,Cpt1?,Pdk4,Dio2 and NE-R.In addition,olanzapine(1 ?M and 10 ?M)significantly reduced the mitochondrial activity of brown fat cells(?13.9%and?22.9%,p<0.05)and mitochondrial membrane potential(?11.6%and?19.6%,p<0.001)with dose-dependent 6.Effects of CL316243/Forskolin and olanzapine co-treatment on the brown fat cellsWhen C3H10T1/2 cells differentiated into mature brown fat cells,the transcription level of Ucpl treated with olanzapine(10 ?M)for 2 h significantly decreased by?66.4%(p<0.001),and the levels of Pka,Ampk and Ppar-y transcription also decreased significantly,compared with the control group.After 45 min of CL316243 treatment,the transcription level of in the CL316243+olanzapine group increased by?55.5%(p<0.05),and the transcription level of Ampk also significantly increased by a negative feedback regulation versus the CL316243 alone group;the transcriptional level of Ucpl in the CL316243+olanzapine group increased by?85.05%(p<0.01)and the transcriptional levels of Pka,Ampk,and Ppar-y were also significantly increased versus the olanzapine alone group.In addition,after Forskolin treatment for 45 min,the transcription levels of Ucpl,Pka,Ampk,and Ppar-? in the Forskolin+olanzapine group were significantly lower than that in the Forskolin group alone with a?12.4%reduce of Ucpl(p<0.05),and the transcription levels of Ucpl,Pka,Ampk,and Ppar-? treatment in the co-treatment group were higher than that in the olanzapine alone groupConclusion:In the animal model of schizophrenia induced by poly I:C,the pWAT,weight gain,TC,LDL-C,NEFA and TG levels of offspring were increased with significances of pWAT and TG,indicating that there was a high risk of abnormal lipid metabolism in the disease state.But olanzapine had a more severe effect onthe metabolic abnormalities than schizophrenia itself.Olanzapine's reduction of the thermogenic activity of brown fat in the poly I:C model laid the foundation for weight gain and energy metabolic imbalance Moreover,olanzapine's effect on brown fat thermogenesis in poly I:C model rats may exert through inhibiting AMPK activation.In vitro,olanzapine inhibited the directed differentiation of C3H10T1/2 mesenchymal stem cells into brown adipocytes.Meanwhile,the expression of middle heat protein and mitochondrial activity in brown fat cells were down-regulated in a concentration-dependent manner.The ?3-AR-cAMP-PKA-AMPK-PPAR-?-UCP1 signaling pathway may involve in the regulation of olanzapine on the thermogenic activity of brown fat,which provides experimental evidence and theoretical basis for elucidating the mechanism of metabolic side effects of antischizophrenic drugs.