Atorvastatin impaired glucose metabolism in C2C12 cells partly via inhibiting cholesterol-dependent glucose transporter 4 translocation.

Skeletal muscle accounts for approximately 75% of glucose disposal in body and statins impair glucose metabolism. We aimed to investigate the effect of atorvastatin on glucose metabolism in C2C12 cells. Glucose metabolism and expression of glucose transporter 4 (GLUT4) and hexokinase II (HXKII) were measured following incubation with atorvastatin or pravastatin. Roles of cholesterol in atorvastatin-induced glucose metabolism impairment were investigated via adding cholesterol or mevalonic acid and confirmed by cholesterol depletion with methyl-ß-cyclodextrin. Hypercholesterolemia mice induced by high fat diet (HFD) feeding, orally received atorvastatin (6 and 12 mg/kg) or pravastatin (12 mg/kg) for 22 days. Results showed that atorvastatin not pravastatin concentration-dependently impaired glucose consumption, glucose uptake and GLUT4 membrane translocation in C2C12 cells without affecting expression of HXKII or total GLUT4 protein. The atorvastatin-induced alterations were reversed by cholesterol or mevalonic acid. Cholesterol depletion exerted similar impact to atorvastatin, which could be alleviated by cholesterol supplement. Glucose consumption or GLUT4 translocation was positively associated with cellular cholesterol levels. In HFD mice, atorvastatin not pravastatin significantly increased blood glucose levels following glucose or insulin dose and decreased expression of membrane not total GLUT4 protein in muscle. Glucose exposure following glucose or insulin dose was negatively correlated to muscular free cholesterol concentration. Expression of membrane GLUT4 protein was positively related to free cholesterol in muscle. In conclusion, atorvastatin impaired glucose utilization in muscle cells partly via inhibiting GLUT4 membrane translocation due to inhibition of cholesterol synthesis by atorvastatin, at least, partly contributing to glucose intolerance in HFD mice.

Differential effects of pravastatin on the pharmacokinetics of paroxetine in normal and diabetic rats.

1. Diabetes is often accompanied with depression and hypercholesterolemia. It is possible that paroxetine and pravastatin are co-administered to diabetic patients. The aim of this study was to research the differential effect of pravastatin on plasma exposure of paroxetine in normal and diabetic rats. 2. Pharmacokinetics of paroxetine was investigated following oral administration of paroxetine with and without pravastatin in normal and diabetic rats. Effects of pravastatin on metabolism, intestinal absorption and hepatic uptake of paroxetine were investigated. Activity and expression of hepatic Oatp1 and Oatp2 were also assessed. 3. Pravastatin decreased plasma exposure of paroxetine in normal rats, but increased exposure of paroxetine in diabetic rats. Pravastatin neither affected metabolism nor intestinal absorption of paroxetine. Data from hepatocytes demonstrated that hepatic uptake of paroxetine were involved in Oatp1 and Oatp2. Diabetes suppressed Oatp1 activity and expression, but enhanced Oatp2 activity and expression. Pravastatin stimulated Oatp1 but inhibited Oatp2 activity. 4. We concluded that differential effects of pravastatin on plasma exposure of paroxetine in normal and diabetic rats was partly due to the fact that diabetes suppressed Oatp1 activity and expression but enhanced Oatp2 activity and expression as well as that pravastatin stimulated Oatp1 activity but inhibited Oatp2 activity.

The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1.

Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation.

Interspecies differences in metabolism of deoxypodophyllotoxin in hepatic microsomes from human, monkey, rat, mouse and dog.

Deoxypodophyllotoxin (DPT) is a natural lignan product which has drawn much attention due to its pharmacological properties including antitumor effect. The purpose of this study was to investigate interspecies differences in metabolism of DPT in hepatic microsomes from human (HLM), cynomolgus monkey (CyLM), rat (RLM), mouse (MLM) and dog (DLM). Incubation of DPT with hepatic microsomes from five species in the presence of NADPH resulted in formation of seven metabolites, five of which were compared with the synthetic standards. M2 was the most abundant metabolite in microsomes from all species. Rank order of intrinsic clearance for M2 formation was RLM > CyLM > MLM > HLM > DLM. In HLM, sulfaphenazole showed the strongest inhibition effect on M2 formation, but neither ticlopidine nor ketoconazole inhibited M2 formation in HLM. Results from cDNA-expressed human CYP450s experiments showed that clearance of M2 formation was much higher in CYP2C9 and CYP2C19 than that in CYP3A4. Contributions of the three CYP450 isoforms to M2 formation in HLM were estimated using relative activity factor (RAF) method or correction by amount of CYP450 isoforms in HLM. M2 formation in HLM was mainly attributed to CYP2C9, followed by CYP2C19. Involvement of CYP3A4 was minor.

Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly attribute to hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia suppressed BCRP expression and function. Impaired BCRP at BBB might enhanced pharmacological/toxic effects of corresponding substrates on CNS.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

AIM: Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. METHODS: Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). RESULTS: BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 µmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. CONCLUSION: UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Ciprofloxacin blocked enterohepatic circulation of diclofenac and alleviated NSAID-induced enteropathy in rats partly by inhibiting intestinal ß-glucuronidase activity.

AIM: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), which may cause serious intestinal adverse reactions (enteropathy). In this study we investigated whether co-administration of ciprofloxacin affected the pharmacokinetics of diclofenac and diclofenac-induced enteropathy in rats. METHODS: The pharmacokinetics of diclofenac was assessed in rats after receiving diclofenac (10 mg/kg, ig, or 5 mg/kg, iv), with or without ciprofloxacin (20 mg/kg, ig) co-administered. After receiving 6 oral doses or 15 intravenous doses of diclofenac, the rats were sacrificed, and small intestine was removed to examine diclofenac-induced enteropathy. ß-Glucuronidase activity in intestinal content, bovine liver and E coli was evaluated. RESULTS: Following oral or intravenous administration, the pharmacokinetic profile of diclofenac displayed typical enterohepatic circulation, and co-administration of ciprofloxacin abolished the enterohepatic circulation, resulted in significant reduction in the plasma content of diclofenac. In control rats, ß-glucuronidase activity in small intestinal content was region-dependent: proximal intestine

In vitro metabolism of l-corydalmine, a potent analgesic drug, in human, cynomolgus monkey, beagle dog, rat and mouse liver microsomes.

l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1-M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1-3) and hydroxylation (M4-6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations.

Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2.

1. Atorvastatin is frequently prescribed for lowering blood cholesterol and for prevention of events associated with cardiovascular disease. The aim of this study was to investigate the pharmacokinetics of atorvastatin in diabetic rats. 2. Diabetes was induced in rats by combination of high-fat diet and low-dose streptozotocin (35 mg/kg). Plasma concentrations of atorvastatin following oral (10 mg/kg) and intravenous (2 mg/kg) administrations to rats were measured by LC-MS. Metabolism and uptake of atorvastatin in primary hepatocytes of experimental rats were assessed. Protein expressions and activities of hepatic Cyp3a and Oatp2 were further investigated. 3. Clearances of atorvastatin in diabetic rats following oral and intravenous administrations were remarkably increased, leading to marked decreases in area-under-the-plasma concentration-time curve (AUC). The estimated oral and systematic clearances of atorvastatin in diabetic rats were 4.5-fold and 2.0-fold of control rats, respectively. Metabolism and uptake of atorvastatin in primary hepatocytes isolated from diabetic rats were significantly increased, which were consistent with the up-regulated protein expressions and activities of hepatic Cyp3a and Oatp2. 4. All these results demonstrated that the plasma exposure of atorvastatin was significantly decreased in diabetic rats, which was partly due to the up-regulated activities and expressions of both hepatic Cyp3a and Oatp2.

Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes.

Accumulating evidences demonstrated that statins impaired glucose utilization. This study was aimed to investigate whether PXR was involved in the atorvastatin-impaired glucose utilization. Rifampicin/PCN served as PXR activator control. Glucose utilization, glucose uptake, protein levels of GLUT2, GCK, PDK2, PEPCK1 and G6Pase in HepG2 cells were measured. PXR inhibitors, PXR overexpression and PXR siRNA were applied to verify the role of PXR in atorvastatin-impaired glucose utilization in cells. Hypercholesterolemia rats induced by high fat diet feeding, orally received atorvastatin (5 and 10 mg/kg), pravastatin (10 mg/kg) for 14 days, or intraperitoneally received PCN (35 mg/kg) for 4 days. Results showed that glucose utilization was markedly inhibited by atorvastatin, simvastatin, pitavastatin, lovastatin and rifampicin. Neither rosuvastatin nor pravastatin showed the similar effect. Atorvastatin and pravastatin were selected for the following study. Atorvastatin and rifampicin significantly inhibited glucose uptake and down-regulated GLUT2 and GCK expressions. Similarly, overexpressed PXR significantly down-regulated GLUT2 and GCK expressions and impaired glucose utilization. Ketoconazole and resveratrol attenuated the impaired glucose utilization by atorvastatin and rifampicin in both parental and overexpressed PXR cells. PXR knockdown significantly up-regulated GLUT2 and GCK proteins and abolished the decreased glucose consumption and uptake by atorvastatin and rifampicin. Animal experiments showed that atorvastatin and PCN significantly elicited postprandial hyperglycemia, leading to increase in glucose AUC. Expressions of GLUT2 and GCK in rat livers were markedly down-regulated by atorvastatin and PCN. In conclusion, atorvastatin impaired glucose utilization in hepatocytes via repressing GLUT2 and GCK expressions, which may be partly due to PXR activation.

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high-performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone derivatization: application to gluconeogenesis studies.

A new pre-column derivative high-performance liquid chromatography (HPLC) method for determination of d-glucose with 3-O-methyl-d-glucose (3-OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1-phenyl-3-methy-5-pyrazolone at 70°C for 50 min. Glucose and 3-OMG were extracted by liquid-liquid extraction and separated on a YMC-Triart C18 column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri-ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39-25 µm (R(2) = 0.9997, n = 5) and the lower limit of quantitation was 0.39 µm (0.070 mg/mL). Intra- and inter-day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1-2.5 µm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits.

Co-administration of paroxetine increased the systemic exposure of pravastatin in diabetic rats due to the decrease in liver distribution.

1. Liver distribution and systemic exposure of pravastatin were the determinant factors of efficacy and toxicity of pravastatin. Aim of the present study was to investigate the effect of paroxetine on the liver distribution and systemic exposure of pravastatin in diabetic rats induced by combining high fat diet (HFD) and low-dose streptozotocin (STZ). 2. Plasma concentrations and liver distribution of pravastatin were measured in the presence of paroxetine. Effect of paroxetine on pravastatin excretion via bile, intestine, feces and urine, as well as pravastatin absorption via intestine was documented. Freshly isolated hepatocytes and Caco-2 cells were used to investigate the effect of paroxetine on pravastatin transport. 3. Paroxetine increased the systemic exposure of pravastatin and decreased hepatic distribution of pravastatin in diabetic rats. In vitro, paroxetine inhibited the hepatic uptake of pravastatin and promoted the efflux of pravastatin in freshly isolated hepatocytes, which may partly explain the decreased hepatic distribution of pravastatin by paroxetine. It was also observed that paroxetine promoted the absorption of pravastatin via jejunum and the uptake of pravastatin in Caco-2 cells. 4. We concluded that paroxetine increased the systemic exposure of pravastatin partly via promoting absorption via jejunum and inhibiting hepatic uptake of pravastatin.

Aggravation of clozapine-induced hepatotoxicity by glycyrrhetinic acid in rats.

Clozapine (CLZ) was reported to be associated with hepatotoxicity. Glycyrrhetinic acid (GA) has a liver protective effect. Our preliminary experiments showed that GA aggravated rather than attenuated CLZ-induced hepatotoxicity in primary cultured rat hepatocytes. The study aimed to describe the enhancing effect of GA on CLZ-induced hepatotoxicity in vivo and in vitro. Data from primary cultured rat hepatocytes showed the decreased formation of metabolites demethylclozapine (nor-CLZ) and clozapine N-oxide (CLZ N-oxide). The results in vivo showed that 7-day CLZ treatment led to marked accumulation of triglyceride (TG) and increase in γ-glutamyl transpeptidase (γ-GT) activity, liver weight, and serum AST in rats. Co-administration of GA enhanced the increases in hepatic TG, γ-GT, liver weight, and serum total cholesterol induced by CLZ. GA decreased plasma concentrations of nor-CLZ and CLZ N-oxide. Compared with control rats, hepatic microsomes of GA rats exhibited the decreased formations of nor-CLZ and CLZ N-oxide, accompanied by decreases in activities of CYP2C11 and CYP2C19 and increased activity of CYP1A2. QT-PCR analysis demonstrated that GA enhanced expression of CYP1A2, but suppressed expression of CYP2C11 and CYP2C13. All these results support the conclusion that GA aggravated CLZ-induced hepatotoxicity, which was partly via inhibiting CYP2C11 and CYP2C13 or inducing CYP1A2.