Metabolomics And Proteomics Analyses Reveal Large Effects Of Hypercholesterolemia On Expressions Of Hepatic Drug Transporters And Its Related Clinical Significances

Drug transporters are located in cell membrane and play a key role in transmembrane transport of xenobiotic or endogenous substrates. Uptake transporters such as OATP1B1、OATP1B3、OATP2B14、OAT2、OAT7、 OCT1、NTCP, and efflux transporters such as MRP3、MRP4、MRP6 are expressed in the sinusoidal (basolateral) membrane of hepatocyte, but efflux transporters BCRP (ABCG2)、BSEP (ABCB11)、MRP2 (ABCC2)、P-GP、MATE1 are localized to the canalicular membrane of hepatocyte. Uptake transporters in the sinusoidal membrane help drugs to enter the hepatocytes to gain access to the drug metabolizing enzymes (DMEs). Efflux transporters in the canalicular membrane remove drugs and their metabolites to bile. Therefore, hepatic transporters play a critical role in hepato-biliary excretion of drugs. Dysfunction of hepatic transporters leads to the alteration of plasma concentrations of drugs, therefore contributing to the changed pharmacodynamics of drugs such as ineffectiveness or toxicity.Expressions and activities of hepatic transporters are regulated by various drugs and diseases. Although many diseases including diabetes and obesity regulated expressions and activities of hepatic transporters, little is about how hypercholesterolemia influences hepatic transporters. To explore possible drug transporters-mediated disease-drug interactions in hypercholesterolemic condition, the effect and molecular mechanism of hypercholesterolemia on hepatic transporters were investigated in this study.Chapter 1. Metabolomics analysis using LC-MS/MS method for anotating the serum metabolic alterations in hypercholesterolemiaAims:Metabolomic approach was employed to examine the alterations of serum metabolites and the related metabolite pathways in hypercholesterolemia, and to analyze the latent transporters which are responsible for the transport of serum metabolites.Methods:1. To establish hypercholesterolemic model, male Sprague-Dawley rats were fed high cholesterol diet for 8 weeks.2. Serum biochemical indices were determined.3. High-throughput metabolomics analysis using LC-MS/MS was employed to investigate the alterations of serum metabolites.4. Multivariate statistics was performed to identify the biomarker metabolites related to hypercholesterolemia.Results:1. Male Sprague-Dawley rats were fed high cholesterol diets for 8 weeks, we observed the significantly elevated total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TC/HDL-C and LDL-C/HDL-C (P<0.05), but the unchanged triglyceride (TG) in rats receiving high cholesterol diets, compared to rats receiving standard diets. The results suggested that hypercholesterolemia was successfully established.2. Compared to those of the control, total bile acid, total and direct bilirubin, creatinine, urea and uric acid were markedly increased (P<0.05), but blood glucose, AST, ALT were not affected in the hypercholesterolemia. The results indicated that bile acid, bilirubin and creatinine metabolisms were perturbed in hypercholesterolemia.3. Differentiated metabolites were identified using metabolomics analysis coupled with multivariate statistics, including creatine, creatinine, glycocholic acid, animo acids such as methionine. The results indicated that creatine, bile acid and animo acid metabolisms were perturbed in hypercholesterolemia.Conclusions:Bile acid, bilirubin, animo acid and creatine metabolism were perturbed in hypercholesterolemic condition. Elevation of metabolites such as bile acid and direct bilirubin suggested that function of hepatic transporters which mediated hepato-biliary excretion of bile acid and direct bilirubin was inhibited in hypercholesterolemic condition.Chapter 2. iTRAQ-based proteomics analysis for the alterations of expressions of hepatic drug transporters in hypercholesterolemiaAims:Proteomic analysis using iTRAQ labeling coupled with LC TRIPLE-TOF was performed to explore the expression alterations of drug transporters in the livers of hypercholesterolemic rats.Methods:1. Proteomic analysis using iTRAQ labeling coupled with LC TRIPLE-TOF was performed to examine the differentially expressed proteins in the livers of hypercholesterolemic rats.2. Bioinformatics analysis including gene ontology, pathway analysis and upstream analysis was explored to identify the candidate drug transporters and the rellated molecular mechanisms in hypercholesterolemia.3. Candidate transporters were validated by RT-qPCR and western blot, respectively.Results:1. Total 2245 unique proteins were identified. Among these,239 (188 upregulated but 51 down-regulated) were significantly changed in hypercholesterolemia, compared with control (P＜0.05).2. GO analysis showed that differentially expressed proteins exerted the following functions, such as binding, catalytic activity and transporter activity. Drug transporters MRP2 and OAT2 were identified and were down-regulated in hypercholesterolemia.3. Pathway and upstream regulator analysis showed that expressions and activities of MRP2 and OAT2 were modulated by nuclear receptors including FXR, PXR, CAR, and pro-inflammatory cytokines TNF-a and IL-1β.4. Two of the differentially expressed protein candidates, MRP2 and OAT2, detected by iTRAQ, were confirmed by RT-qPCR and western blot respectively.Conclusions:Proteomic analysis in combination with bioinformatics analysis, RT-qPCR and western blot were performed to screen out two important hepatic drug transporters MRP2 and OAT2, which were down-regulated in hypercholesterolemia. The elevated pro-inflammatory cytokines in hypercholesterolemia as an inflammatory disease suppressed the expressions of MRP2 and OAT2 via the down-regulated function of nuclear receptors.Chapter 3. Molecular mechanisms for the reduced expressions of MRP2 and OAT2 in hypercholesterolemiaAims:Molecular mechanisms for the reduced expressions of MRP2 and OAT2 in hypercholesterolemia were investigated in this study.Methods:1. RT-qPCR and western blot were performed to examine the alterations of nuclear receptors in the livers of hypercholesterolemic rats, respectively.2. Western blot was utilized to investigate that the influences of LXRa agonist T0901317 on the reduced expressions of MRP2 and OAT2 in hypercholesterolemic serum or TNF-a treatment.3. Flow cytometry was performed to measure the influences of LXRa agonist T0901317 on the reduced transport activities of MRP2 in hypercholesterolemic serum or TNF-a treatment respectively.Results:1. Compared with those of the control, mRNA level of LXRa was markedly decreased (P＜0.05), but mRNA level of CAR was markedly increased (P<0.05). mRNA levels of FXR、PXR、PPARy were not changed in hypercholesterolemic livers. LXRa protein level was also markedly reduced in hypercholesterolemic livers (P<0.05). The results suggested that LXRa and/or CAR mediated the reduced expressions of MRP2 and OAT2 in hypercholesterolemia.2. Compared with those of normal serum treatment, protein levels of MRP2 and OAT2 in HepG2 cells were significantly reduced in hypercholesterolemic serum treatment (P＜0.05), but LXRa agonist T0901317 rescued the reduced expressions in hypercholesterolemic serum treatment (P＜0.05). Similarly, compared with those of control, protein levels of MRP2 and OAT2 in HepG2 cells were significantly reduced in TNF-a treatment (P<0.05), but LXRa agonist T0901317 counteracted it (P<0.05). The results indicated further that inflammatory disease hypercholesterolemia repressed the expressions of hepatic MRP2 and OAT2 via the suppression of nuclear receptor LXRa signal.3. Compared with that of normal serum treatment, efflux of MRP2 substrate CDCFDA in HepG2 cells was significantly inhibited in hypercholesterolemic serum treatment (P＜0.05), but LXRa agonist T0901317 rescued the reduced transport in hypercholesterolemic serum treatment (P＜0.05). Similarly, compared with that of control, efflux of MRP2 substrate CDCFDA in HepG2 cells was significantly reduced in TNF-a treatment (P＜0.05), but LXRa agonist T0901317 counteracted it (P＜0.05). The results indicated further that inflammatory disease hypercholesterolemia reduced the transport activity of hepatic MRP2 via the suppression of nuclear receptor LXRa signal.Conclusions:Suppression of nuclear receptor LXRa signal is at least partly responsible for the reduced expressions and activities of MRP2 and OAT2 in hypercholesterolemia, in which pro-inflammatory cytokine TNF-a plays a partial role.Chapter 4. Clinical significances for the reduced expressions of MRP2 and OAT2 in hypercholesterolemiaAims:Clinical significances for the reduced expressions of MRP2 and OAT2 in hypercholesterolemia were investigated in this study.Methods:1. MRP2 substrate direct bilirubin and OAT2 substrates uric acid and creatinine were analyzed between hypercholesterolemia and normal control patients.2. Correlations for serum lipid levels and MRP2 as well as OAT2 substrates were examined.Results:1. Compared to those of normal control, body mass index (BMI), systolic and diastolic blood pressure, ALT, AST, TC, LDL-C, HDL-C, TG, total bilirubin, indirect bilirubin and urea were markedly increased in hypercholesterolemia patients (P<0.05). However, sex, age, blood glucose, total protein, albumin, globulin remained similar between hypercholesterolemia and normal control patients.2. Compared to that of normal control, MRP2 substrate direct bilirubin was markedly increased in hypercholesterolemia patients (P<0.05), suggesting the activity of hepatic MRP2 was reduced in hypercholesterolemia. Additionally,OAT2 substrates uric acid and creatinine were markedly increased in hypercholesterolemia patients (P<0.05), suggesting the activity of OAT2 was reduced in hypercholesterolemia.3. TC and LDL-C were positively related with direct bilirubin, uric acid and creatinine respectively (P<0.05), and TG were only positively related with uric acid and creatinine (P<0.05), but HDL-C was slightly inversely related with uric acid and creatinine. Results suggested that TC and LDL-C levels control the activities of MRP2 and OAT2.Conclusions:The activities of MRP2 and OAT2 were reduced in hypercholesterolemic condition, which were inversely related with TC and LDL-C levels.