Study Of Clozapine-induced Lipid Metabolism Abnormality Based On Regulation Of Key Proteins In Fatty Acid Oxidation And Transport

Mental illness is a refractory illness caused by a variety of factors.Currently,drug treatment is an effective way to control the disease progression.There are two classes of antipsychotic drugs,including typical antipsychotic drugs,such as chlorpromazine,sulpiride,and atypical antipsychotic drugs,such as clozapine,olanzapine,risperidone.Because typical antipsychotic drug treatment associated with more serious vertebral body reaction,it has been gradually replaced by atypical antipsychotics.Atypical antipsychotics have a superior effect on the negative and positive symptoms of mental illness,but there is a risk of developing metabolic syndrome during long-term treatment.Many studies on the mechanism of atypical antipsychotics-induced lipid metabolism abnormalities have been reported.However,because the mechanism of lipid metabolism is too complicated,the crucial mechanism has not been confirmed yetL-carnitine(L-Car)is an important physiological substance in the body,and it is a crucial substance in the metabolism of fatty acid.About 75%of L-Car in the human body comes from exogenous uptake,while endogenous synthetic L-Car only accounts for about 25%.In human,the plasma L-Car is maintained at about 50μM.L-Car is filtrated by glomeruli and reabsorbed in kidney,and un-reabsorded L-Car is excreted from urine Because L-Car is a highly polar ionic compound,generally transporters are required to mediate L-Car into cells.OCTN2 has high affinity to L-Car and with high expression level in kidney,liver,muscle and other tissues,it is the main transporter mediates L-Car reabsorption.When OCTN2is inhibited by drugs or primary OCTN2 expression lost,it will lead to a decrease in L-Car levels in vivo,and subsenquently causes metabolic disorders such as abnormal lipid oxidation and hypoglycemia.On the other,liver is one of the major organs for fatty acid oxidation.The long-chain fatty acids entering the liver require a series of proteins involved in the oxidative function.CD36 and FATP2/5,a fatty acid transporter expressed in cell membrane,can transport long chain fatty acids into cells CPT1/2 expressed in mitochondrial membrane assists fatty acids into the mitochondria.If drugs affect the expression of these proteins,it will broke the balance of fatty acid oxidation and lead to fatty liver or other related diseases.Therefore,in the present study we investigated whether clozapine reduced L-Car reabsorption by inhibiting renal OCTN2 function or/and down regulating its expression,and subsequently decreased fatty acid oxidation.On the other hand,we also explored whether clozapine regulated the main proteins involved in liver lipid oxidation.1.Clozapine inhibited OCTN2 function and down regulated OCTN2 expressionWe proved that clozapine has a direct inhibitory effect on L-Car transport mediated by OCTN2 through a variety of cell models and down-regulated the expression of OCTN2 in kidney.The inhibitory effect of several atypical antipsychotics(clozapine,olanzapine,risperidone,quetiapine,ziprasidone,haloperidol,aripiprazole)on OCTN2 in the uptake were studied using substrates of meldonium hydrochloride and deuterated L-Car(d3-L-Car).The results showed that the above drugs have different extent of inhibition on OCTN2 mediated uptake of d3-L-Car,clozapine and olanzapine showed a strong inhibitory effect with the IC50 values of 1.78 and 1.06μM.The inhibitory effect was also confirmed in d3-L-Car uptake study using mouse primary tubular cells and HK-2 cells Moreover,we found that clozapine down regulated OCTN2 mRNA expression in HK-2 cells probably through PPARa pathway.Furthermore,our in vivo study also demonstrated that clozapine down regulated Octn2 expression in mice after single ip dose or successively oral administration of clozapine for 21 days.2.Effects of clozapine on L-Car homeostasis and fatty acid oxidation in miceIn this chapter,we studied the effects of clozapine on L-Car homeostasis and fatty acid oxidation in vivo.We studied the change of urinary L-Car excretion of mice after single intraperitoneal injection of clozapine and successively oral administration of clozapine for 21 days.The results showed that after single ip dose and successively oral administration,urinary L-Car excretion increased significantly.Successively oral administration of clozapine at 50mg/kg for 21 days,the L-Car excretion was approximate 3 times higher than that of the vehicle control group.Plasma L-Car levels were reduced about 30%and 40%at 50mg/kg dose after single intraperitoneal injection or successively oral administration of clozapine for 21 days.L-Car levels were reduced in the liver,kidney and skeletal muscle of mice when successively oral administration of clozapine for 21 days.Which indicating that clozapine reduced carnitine levels in the body,broke the normal carnitine homeostasis.Successively oral administration of clozapine for 21 days,6 h after the last administration of 50mg/kg,liver and kidney drug concentrations reached 10 μmol/g and 30 μmol/g.Meanwhile,the liver triglyceride(TG)and otal cholesterol(TCHO)level were incereased about 1.5 and 2 times compared with the vehicle control.To explore whether exogenous supplementation of L-Car can improve clozapine-induced abnormal lipid metabolism,mice were oral administered 3 doses of L-Car(40 mg/kg～1 g/kg)concomitantly oral administration of clozapine(25 mg/kg)for 28 days.As we expected,our results revealed that L-Car deficiency in mouse plasma and tissues was improved,and the liver TG and TCHO levels were significantly lower than that of the clozapine group.The oil red O staining results of mouse liver slices also showed that L-Car significantly improved clozapine-induced liver dyslipidemia3.Regulation of clozapine on crucial proteins in fatty acid transport and oxidationThe crucial proteins in fatty acid transport and oxidation were studied.Out data showed that after successively oral administration of clozapine(50mg/kg)for 21 days,the intestinal CD36(Cluster of Differentiation 36),FATP4(fatty acid transport protein 4)and NPC1L1(Niemann-Pick-C 1-Like 1)mRNA expression were up-regulated.FATP2 and FATP5 were also up-regulated after HepG2 cells were incubated with 20μM clozapine for 24 hours,or mice were single ip administered or successively orally administered with clozapine for 21 days.Moreover,CPT1A(Carnitine palmitoyltransferase 1a)mRNA expression was found to be down-regulatedin mice after successively oral administration of clozapine for 21 days.Taken together,clozapine increased intestinal and liver lipid uptake,which probably reduced the efficiency of fatty acid oxidation in liver.