The Mechanism Of Olanzanine-induced Liver Steatosis Based On The Regulation Of Transporters Involved In Fatty Acid Uptake And Oxidation

Antipsychotic drugs are classified into typical antipsychotic drugs and atypical antipsychotic drugs(AAPDs).AAPDs have become the first-line drugs for treatment of schizophrenia because of less vertebral body reaction compared to typical antipsychotics.However,AAPDs,including olanzapine,clozapine and aripiprazole,are well known for their propensity to induce metabolic disturbances.Olanzapine is extensively applied for the treatment of various mental illnesses with a high rate of inducing metabolic disturbances.Liver is the essential organ for lipid metabolic homeostasis.The metabolic side effects of olanzapine on liver,liver steatosis,have been widely reported.Liver steatosis is characterized by accumulation of triglyceride(TG)in hepatocytes.Hepatic TG is derived from non-esterified fatty acids.Therefore,an imbalance between fatty acid uptake and mitochondrial fatty acid oxidation will induce liver steatosis.Fatty acid transport proteins(FATPs),fatty acid binding proteins(FABPs)and CD36(also known as fatty acid translocase,FAT)are key contributors to the transmembrane process of fatty acid.FATP2 and FATP5 are two isoforms of FATPs highly expressed in liver.FABP1 is an isoform of FABPs and is abundant in the cytosol of hepatocytes.FATP2,FATP5 and FABP1 promote the uptake of free fatty acids into the liver.If drugs affect the expression of these proteins,it will disturb the uptake of fatty acid and lead to fatty liver or other related diseases.Mitochondrial(3-oxidation of fatty acids contributes to the steady balance of hepatic TG in the liver.L-Carnitine(L-Car)is an important physiological substance in the body that plays an essential role in fatty acid β-oxidation.The main function of L-Car is the transfer of long-chain fatty acids to mitochondria for subsequent β-oxidation.Camitine/organic cation transporter 2(OCTN2)is a high-affinity transporter of L-Car and is expressed in various tissues including liver.OCTN2 is responsible for high-affinity L-Car uptake in liver and L-Car is essential for normal liver function.As a result,inhibition or decreasing expression of OCTN2 will reduce L-Car levels in livers,and subsenquently attenuate β-oxidation of fatty acid and finally induce accumulation of TG in hepatocytes.In the present study,we investigated whether olanzapine up-regulated FATP2,FATP5,FABP1 and CD36 in mouse liver.In addition,we explored whether olanzapine reduced L-Car concentration in the liver by inhibiting hepatic OCTN2 function or/and down-regulating its expression.The results may account for the mechanism of olanzapine-induced liver steatosis and provide more information for improvement of fatty liver.1.Effects of olanzapine on lipid homeostasis in liverWe proved that chronic treatment of olanzapine induced simple steatosis in mouse liver.6-week-old C57BL/6 mice were daily intragastric administration with olanzapine(6,12,1 8 mg/kg)for 12 weeks.Hepatic TG content was increased after olanzapine treatment in a dose dependent manner,for instance,in 18 mg/kg olanzapine treated group,the TG was 1.78 folds of the control group in male and female mice.Livers of mice fed with 18 mg/kg olanzapine daily exhibited typical morphological changes of steatosis with excessive lipid deposition as assessed by way of Oil Red O staining.Serum ALT and AST levels were unchanged.H&E staining also showed no symptom of nonalcoholic steatohepatitis(NASH).The above data suggested that chronic exposure of olanzapine induced simple steatosis.2.Effects of olanzapine on hepatic FABP1,FATP2 and FATP5 expressionTo explore the mechanism of olanzapine-induced liver steatosis,we examined theexpression of genes involved in fatty acid uptake in the liver.Our results revealed that the mRNA levels of FABP1,FATP2 and FATP5 were up-regulated in primary mouse hepatocytes after a 24-h treatment with different concentrations(25,50,100 μM)of olanzapine,while CD36 was unchanged.Moreover,mRNA and protein levels of FABP1 and FATP2 were also increased in livers of female mice but not male mice after a 12-week treatment of olanzapine(6,12,18 mg/kg).mRNA levels of CD36 were unchanged in livers of female and male mice.Therefore,olanzapine increased hepatic fatty acid uptake by up-regulation of FABP1 and FATP2 in female mice3.Olanzapine attenuated fatty acid β-oxidation via inhibition/down-regulation of hepatic OCTN2To extensively explore the mechanism of olanzapine-induced liver steatosis,we investigated the effect of olanzapine on hepatic fatty acid(3-oxidation.Our data demonstrated that the levels of hepatic β-hydroxybutyrate,a product of hepatic β-oxidation,were decreased in mice treated with olanzapine(6,12,18 mg/kg)for 12 weeks However,expression of carnitine palmitoyltransferase 1A(CPT1A),carnitine palmitoyltransferase2(CPT2)and mediumchainacyl-CoAdehydrogenase(MCAD)was increased or unchanged in livers of olanzapine treated mice.Based on the important role of L-Car in mitochondrial β-oxidation,we examined the L-Car levels in livers of mice The concentrations of L-Car in livers were significantly reduced,which is a potential mechanism of decreasing β-oxidation after a 12-week treatment of olanzapine(6,12,18 mg/kg).Furthermore,our results showed that olanzapine had a strong inhibitory effect on L-Car accumulation in MDCK-hOCTN2 cells with the IC50 value of 1.06 μM.The inhibitory effect was also confirmed in primary mouse hepatocytes and HepG2 cells.On the other hand,the mRNA and protein expression of OCTN2 in livers of male mice was down-regulated after chronic exposure of olanzapine.The mRNA expression of OCTN2 in livers of female mice was increased slightly while protein expression of OCTN2 was unchanged after olanzapine treatment.Additionally,our results also revealed that olanzapine down-regulated OCTN2 mRNA in primary mouse hepatocytes after treatment with olanzapine(25,50,100 μM)for 24 hours.The above data implied that olanzapine may reduce hepatic L-Car levels by inhibiting the function or/and down-regulating the expression of OCTN2,which subsequently contributed to decreasing β-oxidation and increasing TG content in liver.To explore whether exogenous supplementation of L-Car would improve olanzapine-induced liver steatosis,mice were orally administered with L-Car(50～800 mg/kg)concomitantly with olanzapine(18 mg/kg)for 12 weeks.Our results revealed that L-Car deficiency and fatty acid β-oxidation in livers of L-Car co-dosed mice were improved,and the hepatic TG levels were significantly lower than that of the olanzapine alone group.The oil red O staining results of mouse liver slices also showed that L-Car supplementation significantly improved olanzapine-induced liver dyslipidemia.Taken together,olanzapine reduced hepatic L-Car levels via inhibition and down-regulation of hepatic OCTN2 in male mice and inhibition of hepatic OCTN2 in female mice.This effect of olanzapine attenuated mitochondrial β-oxidation of fatty acid in liver,which is a potential mechanism of olanzapine-induced liver steatosis.Supplementation of L-Car can improve olanzapine-induced liver steatosis.