Effects Of Atypical Antipsychotics Clozapine On The Blood Glucose In The Male C57BL/6 Mice And Its Mechanism Research

OBJECTIVETo study the effects of coadministration of clozapine and fluvoxamine versus clozapine monotherapy on the metabolism and investigate the mechanism of clozapine' hyperglycemia in the male C57BL/6 mice.METHODS1.The effects of coadministration of clozapine and fluvoxamine versus clozapine monotherapy on the metabolism in the male C57BL/6 mice120 male C57BL/6 mice were divided into 6 groups,(1)vehicle,(2) clozapine 2 mg/kg,(3)clozapine 10 mg/kg,(4)fluvoxamine 2 mg/kg, (5)clozapine 2mg/kg + fluvoxamine 2 mg/kg,(6)clozapine 10 mg/kg + fluvoxamine 2 mg/kg.The mice were treated with corresponding drug intraperitoneally(IP)QD at 8:30 AM for 28 consecutive days.Body weight was recorded on days 0,7,14,21,and 28.To assess glucose tolerance,mice underwent ovemight fasting on day 29,and their blood glucose responses to IP administration of 25%glucose(2 g/kg)were determined.Blood glucose concentration were assessed in the whole blood collected from the tail vein before glucose administration(ie,time 0,FBG)and 30,60,and 120 minutes after glucose administration.The mice were fasted for 6 hours after glucose administration and were obtained 0.6 mL blood by removing the mice' eyeball after anesthetized using sodium pentobarbital 130 mg/kg.The blood samples were immediately placed in heparin-coated tubes for plasma lipid(including triglyceride,total cholesterol,high density lipoprotein cholesterol and low density lipoprotein cholesterol),insulin determination and plasma drug concentration.2.The effects of coadministration fluvoxamine on the plasma clozapine and its metabolites concentrationThe plasma clozapine and its main metabolites(desmethylclozapine and clozapine N-oxide)concentration were determined in the following experiment group:(2)clozapine 2 mg/kg,(3)clozapine 10 mg/kg,(5) clozapine 2 mg/kg + fluvoxamine 5 mg/kg and(6)clozapine 10 mg/kg + fluvoxamine 2 mg/kg.The number of plasma determined in every group was ten.The correlation between the plasma clozpine and its metabolites (desmethylclozapine and clozapine N-oxide)concentration,plasma lipid and fasted blood glucose was analyzed.A high performance liquid chromatography electrospray mass spectrometry(HPLC-MS/ESI)methods to simultaneously determine clozapine and its metabolites desmethyl-clozapine and clozapine-N-oxide in mice plasma.The extracted sample was performed on liquid chromatography using a Waters Xtera^TMMS C₁₈column(150 mm×3.9 mm,5μm)with column temperature 40℃.Mobile phase consisted of acetonitrile and water(36:64,including 10mmol/L ammonium acetate and 0.01%formic acid,pH=5.96)with a flow rate 1.0mL/min.The compounds were ionized in the electrospray ionization(ESI)ion source, fragmented by in-source collisions and the ionized molecular and fragment ions detected in the selected ion recording(SIR)mode.The samples were evaporated by N₂ and dissolved mobile phase and sample size were 20μL.3.The mechanism of clozapine on the blood glucose in the male C57BL/6 miceMale C57BL/6 mice were sacrificed by ceruical uertebra luxation after obtaining plasma by moving mice' eyeball.Then pancreatic salivary gland and musculus quadriceps fexoris of C57BL/6 mice were taken out.Pancreatic gland were placed in percent 10 neutral formaldehyde,fixed,imbedded by paraffin and cut paraffin section(two portion,thickness 4μm).Musculus quadriceps fexoris of C57BL/6 mice were deepfreezed by placing liquid nitrogen and transferred into -70℃refrigerator until determination.(1)GLUT4 mRNA expression in the mice musculus quadriceps fexoris were determined by reverse transcription pclymerase(RT-PCR).(2)A paraffin section of mice pancreatic gland were routinely deparaffinaged,stained by hematine-eosine(HE),and observed whether they have the insulitis.(3)A paraffin section of mice pancreatic gland were routinely deparaffinaged.Apoptosisβcell were marked by in situ nick end-labeling(TUNEL),βcell were labeled by streptavidin-biotin-peroxidase complex(SABC)and the count ofβcell apoptosis were counted.(4)The correlation were analysed between the expression of GLUT mRNA,the count of pancreasβcell apoptosis,the degree of pancreatic island lymphocyte infiltrate and fasted blood glucose,plasma lipid, plasma clozapine and its metabolites concentration.RESULTS1.Results of effects of coadministration of clozapine and fluvoxamine versus clozapine monotherapy on the metabolism in the male C57BL/6 miceThere were no siginificance in body weight and fasted blood glucose among trial groups before experiment(P＞0.05).The body weight significantly decreased in the 10 mg/kg clozapine-treated groups at days 14,21,and 28(all,P＜0.05)compared with the vehicle-treated group.In the(2 mg/kg clozapine + 10 mg/kg fluvoxamine)-treated group, body weight significantly increased compared with the vehicle-treated group(P＜0.05).Compared with clozapine monotherapy group,there were no significance in the body weight of clozapine and fluvoxamine coadministration(P＞0.05).In the clozapine 10 mg/kg group,blood glucose concentration significantly increased 0,30,60,and 120 minutes after glucose administration(all,P＜0.05).The clozapine 10 mg/kg group also had significant increase in plasma insulin concentration and IRI compared with the vehicle(12.70±5.27 vs 7.62±4.54μIU/mL;P＜0.05;3.01±1.26 vs 1.51±0.96;P＜0.05).Plasma HDL-C concentration also significantly decreased in the clozapine 10 mg/kg group compared to the vehicle(1.47±0.16 vs 1.23±0.25;P＜0.05).2.Results of the effects of coadministation fluvoxamine on the plasma clozapine and its metabolites concentration in the male C57BL/6 miceFluvoxamine could inhibite the clozapine metabolism.There were significant difference between clozapine monotherapy group and clozapine -fluvoxamine coadministration group in the plasma drug concentration(p＜0.05).The plasma drug concentration between 2 mg/kg clozapine treatment group and 2 mg/kg clozapine- 2 mg/kg fluvoxamine treatment group were 97.61±10.62 ng/mL and 176.97±13.61 ng/mL (clozapine),72.17±10.11 ng/mL and 20.35±2.71 ng/mL (desmethylclozapine),8.42±1.88 ng/mL and 14.89±3.21 ng/mL (clozapine N-oxide),respectively.The plasma drug concentration between 10 mg/kg clozapine treatment group and 10 mg/kg clozapine- 2 mg/kg fluvoxamine treatment group were 325.07±43.49 ng/mL and 727.30±73.34 ng/mL(clozapine),112.77±14.00 ng/mL and 23.99±6.25 ng/mL(desmethylclozapine),31.74±6.37 ng/mL and 39.32±7.16 ng/mL(clozapine N-oxide),respectively.Correlation analysis which were done between fasted blood glucose,plasma lipid and plasma drug (clozapine,desmethylclozapine and clozapine N-oxide)concentration showed there were no significan correlation(all,P＞0.05).However, mice fasted blood glucose had the elevation tendency along with the increase of plasma desmethylclozapine concentration.3.Results of the mechanism of elozapine on the blood glucose in the male C57BL/6 mice(1)The expression ratio between GLUT4 mRNA in the mice musculus quadriceps fexoris andβ-actin mRNA in the internal reference in every group were following,vehicle treatment group were 1.02±0.21,2 mg/kg clozapine treatment group were 0.81±0.20,10 mg/kg clozapine treatment group were 0.79±0.18,fluvoxamine treatment group were 0.95±0.09,(2 mg/kg clozapine - 2 mg/kg fluvoxamine)treatment group were 0.98±0.06, (10 mg/kg clozapine- 2 mg/kg fluvoxamine)treatment group were 1.01±0.18.Concering the expression ratio between GLUT4 mRNA in the mice musculus quadriceps fexoris andβ-actin mRNA in the internal reference,there were significant decrease in the 2 mg/kg and 10 mg/kg cloapine treatment group compared with vehicle treatment group(P＜0.05);There were no significant difference between vehicle and fluvoxamine treatment group(P＞0.05);The expression ratio of GLUT mRNA in the clozapine-fluvoxamine coadministration were more than clozapine monotherapy,however,there were significan difference between the 2 mg/kg clozapine treatment group and(2 mg/kg clozapine-2 mg/kg fluvoxamine)treatment group(P＜0.05).(2)HE slices were observed in the high power light microscope.The results showed that pancreatic island located in the pars ecocrina pancreatic.They were well-rounded,edge sharpness,uniform distribution and unseen lumphocyte among the vehicle group,clozapine 2 mg/kg group,clozapine 10 mg/kg group,fluvoxamine 2 mg/kg group, clozapine 2 mg/kg + fluvoxamine 2 mg/kg group,clozpaine 10 mg/kg + fluvoxamine 2 mg/kg group.Through two HE slices in the clozapine 10 mg/kg group were lymphocyte around pancreatic island,there were not lymphocyte in the pancreatic island.(3)Islet cell were well-distributed andβcell kytoplasm were buffy insulin positive expression.βcell apoptosis were unseen. (4)There were negative correlation between the expression of GLUT 4 mRNA and fasted blood glucose,plasma desmethylclozapine concentration(P＜0.05,r=-0.814,r=-0.940).There were no correlation between the expression of GLUT4 mRNA and plasma lipid,plasma clozapine,clozapine N-oxide concentration(P＞0.05,r=0.463～0.555).CONCLUSIONThe body weight and high density lipoprotein of the male C57BL/6 mice were decreased,fasted blood glucose,glucose tolerance and insulin level were increased in the clozapine 10 mg/kg treatment group for 28 consecutive days intraperitoneal injection.The coadministration(2 mg/kg)could attenuate the clozapine-induced hyperglycemia.Fluvoxamine could inhibit the clozapine metabolism which resulted in the decrease of plasma desmethyclozapine concentration.Correlation analysis which were done between fasted blood glucose and plasma drug(clozapine,desmethylclozapine and clozapine N-oxide) concentration showed there were no significan correlation.However, mice fasted blood glucose had the elevation tendency along with the increase of plasma desmethylclozapine concentration.Fluvoxamine could attenuate the clozapine-induced hyperglycemia which may be by inhibiting the clozapine metabolism.Clozapine 2 mg/kg and clozapine 10 mg/kg could decrease the GLUT mRNA expression in the male C57BL/6 mice musculus quadriceps fexoris which could be attenuated by coadministration 2 mg/kg fluvoxamine.There were negative correlation between the expression of GLUT mRNA and fasted blood glucose,plasma desmethylclozapine concentration.There were no correlation between the expression of GLUT mRNA and plasma clozapine,clozapine N-oxide concentration.Fluvoxamine and different dose clozapine had no effect on theβcell apoptosis.