Mice overexpressing hepatic Abcb11 rapidly develop cholesterol gallstones

Cholelithiasis is a polygenic disease, although the genes responsible for gallstone formation have not yet been clearly identified. QTL analysis has identified the Lith 1 loci on mouse Chromosome 2, and the hepatic bile salt transporter Abcb11 maps to the Lith 1 locus. We have used recently developed TTR-Abcb11 transgenic mice that overexpress Abcb11 to determine the effects of Abcb11 overexpression on cholesterol gallstone formation. TTR-Abcb11 and FVB/NJ strain control mice were fed a lithogenic or chow diet and cholesterol crystal and gallstone formation were measured. Biliary lipids in gallbladder bile and gene expression of canalicular lipid transporters were also analyzed. TTR-Abcb11 mice fed a lithogenic diet had an increased rate of cholesterol crystal and gallstone formation. This was associated with an increase in both the hydrophobic bile salt and cholesterol content of gallbladder bile. Expression of Abcb4, Abcg5, and Abcg8 did not change before gallstone formation. These data indicate that hepatic overexpression of Abcb11 increases the rate of cholesterol gallstone formation. This is likely because of increases in bile salt hydrophobicity but not because of alterations of other biliary lipid transporters. These findings strongly support Abcb11 as a Lith 1 gene.     

Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3−1 and Can 10

Sinusoidal and apical transporters are responsible for the uptake and biliary elimination of many compounds by hepatocytes. Few in vitro models are however available for analyzing such functions. The expression and bile-acid inducibility of 13 transporters and two nuclear receptors were investigated in the new rat polarized lines, Can 3−1 and Can 10, and in their unpolarized parent, Fao. The relative abundance of mRNA, the protein level, and their localization were examined by real-time quantitative PCR, Western blotting, immunofluorescence, and confocal microscopy. Compared with rat liver, mRNA levels of Fao cells were: negligible for Bsep/Abcb11; lower for the uptake transporters Ntcp and Oatps; similar for SHP, FXR, and Bcrp/Abcg2; and higher (four–fold to 160-fold) for the efflux pumps Mdr1b/Abcb1b, Mdr2/Abcb4, Mrp1/Abcc1, Mrp2/Abcc2, Mrp3/Abcc3, Abcg5, and Abcg8. This profile was mostly maintained (and improved for Bsep) in Can 10. Some transporters were less well expressed in Can 3−1. In both lines, sinusoidal (Ntcp, Mrp3) and canalicular transporters (Mdr-P-glycoproteins detected with C219 antibody, Mrp2) were localized at their correct poles. Bile-acid effects on polarity and mRNA levels of transporters were analyzed after a 6-day treatment with 50 μM taurocholic, chenodeoxycholic (CDCA), or ursodeoxycholic acid (UDCA). No polarization of Fao cells was induced; Can 10 and Can 3−1 polarity was maintained. CDCA and UDCA induced marked enhancement of the volume of Can 10 bile canaliculi. CDCA upregulated Bsep, Mdr2, SHP, Mdr1b, and Oatp2/1a4 in Can 10 (two- to seven-fold) and in Fao cells. Thus, Can 10 constitutes an attractive polarized model for studying vectorial hepatobiliary transport of endogenous and xenobiotic cholephilic compounds. This work was supported by a grant from Egide (PAI Picasso) and the Acción Integrada Hispano-Francesa (HF2003-0089). This research group is part of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF; grant BFU2005-24983-E/BFI) and belongs to the “Centro de Investigación Biomédica en Red” for Hepatology and Gastroenterology Research (CIBERehd), Instituto de Salud Carlos III, Spain.     

Abcg5/Abcg8-independent pathways contribute to hepatobiliary cholesterol secretion in mice

The ATP-binding cassette (ABC) half-transporters ABCG5 and ABCG8 heterodimerize into a functional complex that mediates the secretion of plant sterols and cholesterol by hepatocytes into bile and their apical efflux from enterocytes. We addressed the putative rate-controlling role of Abcg5/Abcg8 in hepatobiliary cholesterol excretion in mice during (maximal) stimulation of this process. Despite similar bile salt (BS) excretion rates, basal total sterol and phospholipid (PL) output rates were reduced by 82% and 35%, respectively, in chow-fed Abcg5(-/-) mice compared with wild-type mice. When mice were infused with the hydrophilic BS tauroursodeoxycholate, similar relative increases in bile flow, BS output, PL output, and total sterol output were observed in wild-type, Abcg5(+/-), and Abcg5(-/-) mice. Maximal cholesterol and PL output rates in Abcg5(-/-) mice were only 15% and 69%, respectively, of wild-type values. An infusion of increasing amounts of the hydrophobic BS taurodeoxycholate increased cholesterol excretion by 3.0- and 2.4-fold in wild-type and Abcg5(-/-) mice but rapidly induced cholestasis in Abcg5(-/-) mice. Treatment with the liver X receptor (LXR) agonist T0901317 increased the maximal sterol excretion capacity in wild-type mice (fourfold), concomitant with the induction of Abcg5/Abcg8 expression, but not in Abcg5(-/-) mice. In a separate study, mice were fed chow containing 1% (wt/wt) cholesterol. As expected, hepatic expression of Abcg5 and Abcg8 was strongly induced (fivefold and fourfold) in wild-type but not LXR-alpha-deficient (Lxra(-/-)) mice. Surprisingly, hepatobiliary cholesterol excretion was increased to the same extent, i.e., 2.2-fold in wild-type mice and 2.0-fold in Lxra(-/-) mice, upon cholesterol feeding. Our data confirm that Abcg5, as part of the Abcg5/Abcg8 heterodimer, strongly controls hepatobiliary cholesterol secretion in mice. However, our data demonstrate that Abcg5/Abcg8 heterodimer-independent, inducible routes exist that can significantly contribute to total hepatobiliary cholesterol output.     

Uric acid metabolism of kidney and intestine in a rat model of chronic kidney disease

ABSTRACT

Uric acid (UA) is a potential risk factor of the progression of chronic kidney disease (CKD). Recently, we reported that intestinal UA excretion might be enhanced via upregulation of the ATP-binding cassette transporter G2 (Abcg2) in a 5/6 nephrectomy (Nx) rat model. In the present study, we examined the mRNA and protein expressions of UA transporters, URAT1, GLUT9/URATv1, ABCG2 and NPT4 in the kidney and ileum in the same rat model. Additionally, we investigated the Abcg2 mRNA expression of ileum in hyperuricemic rat model by orally administering oxonic acid. Male Wistar rats were randomly assigned to three groups consisting of Nx group, oxonic acid-treated (Ox) group and sham-operated control group, and sacrificed at 8 weeks. Creatinine and UA were measured and the mRNA expressions of UA transporters in the kidney and intestine were evaluated by a real time PCR. UA transporters in the kidney sections were also examined by immunohistochemistry. Serum creatinine elevated in the Nx group whereas serum UA increased in the Ox group. Both the mRNA expression and the immunohistochemistry of the UA transporters were decreased in the Nx group, suggesting a marginal role in UA elevation in decreased kidney function. In contrast, the mRNA expression of Abcg2 in the ileum significantly increased in the Ox group. These results suggest that the upregulation of Abcg2 mRNA in the ileum triggered by an elevation of serum UA may play a compensatory role in increasing intestinal UA excretion.     

Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro

ATP-driven efflux transport proteins at the blood-brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF-α and IL-1β rapidly decrease Abcg2 ( BMDP/BCRP ) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter-regulated by the anti-inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p-glycoprotein) protein expression was transiently increased after TNF-α addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL-1β caused a continuous decrease in protein expression of both ABC-transporters. Long-term treatment with an assumed TNF-α-downstream agent, the vasoconstrictor endothelin-1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance-associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC-transporters at the blood-brain barrier during early inflammation stages suggesting that Abcb1 (p-glycoprotein) is an early target of TNF-α-signalling counterbalanced by Abcg2.     

Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts

Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1^−/−-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1^−/−-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1^−/−-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs.     

Prevention of estradiol 17beta-D-glucuronide-induced canalicular transporter internalization by hormonal modulation of cAMP in rat hepatocytes

In estradiol 17β-d-glucuronide (E17G)-induced cholestasis, the canalicular hepatocellular transporters bile salt export pump (Abcb11) and multidrug-resistance associated protein 2 (Abcc2) undergo endocytic internalization. cAMP stimulates the trafficking of transporter-containing vesicles to the apical membrane and is able to prevent internalization of these transporters in estrogen-induced cholestasis. Hepatocyte levels of cAMP are regulated by hormones such as glucagon and adrenaline (via the β2 receptor). We analyzed the effects of glucagon and salbutamol (a β2 adrenergic agonist) on function and localization of Abcb11 and Abcc2 in isolated rat hepatocyte couplets exposed to E17G and compared the mechanistic bases of their effects. Glucagon and salbutamol partially prevented the impairment in Abcb11 and Abcc2 transport capacity. E17G also induced endocytic internalization of Abcb11 and Abcc2, which partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained internalized and mainly colocalizing with Rab11a in the perinuclear region after incubation with glucagon. Glucagon prevention was dependent on cAMP-dependent protein kinase (PKA) and independent of exchange proteins activated directly by cAMP (Epac) and microtubules. In contrast, salbutamol prevention was PKA independent and Epac/MEK and microtubule dependent. Anticholestatic effects of glucagon and salbutamol were additive in nature. Our results show that increases in cAMP could activate different anticholestatic signaling pathways, depending on the hormonal mediator involved.     

Functional analysis of candidate ABC transporter proteins for sitosterol transport

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.     

Basolateral and canalicular transport of xenobiotics in the hepatocyte: A review

The molecular and functional characterization of severalproteins involved in the uptake and excretion of xenobioticsand endogenous compounds in the hepatocyte has been achievedthrough intensive research conducted in the past few years.These studies have lead to the identification of specificmembrane transporters located in the basolateral andcanalicular membrane domains of the hepatocyte. The organicanion-transporting polypeptide (OATP), present in thebasolateral membrane of the hepatocyte, is responsible for thetranslocation of xenobiotics from the sinusoidal space into thehepatocyte. Once inside the cell, unconjugated neutral, anionicand cationic xenobiotics can be secreted into bile by themultidrug-resistance P-glycoprotein 1 (MDR1). Conjugatedxenobiotics (e.g. glucuronides and glutathione conjugates) aresecreted into bile by the canalicular multispecific organicanion transporter (cMOAT). Other transporters play keyphysiological roles, including the basolateral uptake of bilesalts (sodium-taurocholate cotransporter, NTCP) and thesecretion into bile of conjugated and unconjugated bile salts(bile salt export pump, BSEP) and phospholipids (MDR2).Experimental approaches used to investigate the role of thebasolateral and canalicular transporters in the hepatocyte haveincluded both in vivo and in vitro models. Animalmodels lacking canalicular transporters include the`hyperbilirubinemic' rats (Groningen-Yellow (GY), Eisaihyperbilirubinemic (EHB) and TR<sup>-</sup> rats), which aredeficient in the cMOAT protein, and `knock-out' mice, lackingeither the MDR1 or MDR2 transporter. Although no animal modelsare currently available for the study of basolateraltransporters, their function has been conveniently investigatedthrough heterologous expression in Xenopus laevis oocytesand also with basolateral membrane vesicles isolated fromhepatocytes. The total number of basolateral and canaliculartransport proteins present in the hepatocyte is still unknown,but current knowledge indicates that there are at least fourpresent in the basolateral membrane and five in the canaliculardomain. The present review focuses on the current knowledgeabout the most relevant hepatocyte transporters involved in theuptake of foreign and endogenous compounds from the sinusoidalspace and in their active secretion into bile. The first partof the review deals with the basolateral (sinusoidal) transportof organic anions, and the major basolateral transporters (e.g.NTCP, OATP) are described here, both in terms of their knownbiochemistry and physiology. In the second part of the review,the canalicular (apical) transport of organic anions isdiscussed and the biochemistry and physiological role of MDR1,MDR2, cMOAT and BSEP is described in detail. The concludingremarks point out areas of research that need to be addressedin order to answer important questions that still remainunanswered in this important field of study.     

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.     

Gender‐dimorphic regulation of DJ1 and its interactions with metabolic proteins in streptozotocin‐induced diabetic rats

Regulation of DJ1 is associated with a number of human diseases. To determine the involvement of DJ1 in progression of diabetes in a gender‐dependent manner, we investigated its tissue‐specific expression in streptozotocin (STZ)‐induced diabetic male and female rats in this study. In animal experiments, females showed greater susceptibility towards developing diabetes because of lower insulin secretion and higher blood glucose levels as compared to male diabetic rats upon exposure to STZ. Immunoblotting confirmed sexually dimorphic regulation of DJ1 in various metabolic tissues such as the liver, pancreas and skeletal muscle. Immunofluorescence analysis revealed the location as well as reinforced the gender‐dependent expression of DJ1 in hepatic tissue. Co‐immunoprecipitation assay identified several interacting proteins with DJ1 whose functions were shown to be involved in various metabolic pathways viz. antioxidative and stress defence system, protein and methionine metabolism, nitrogen metabolism, urea metabolism, etc. Using GeneMANIA, a predictive web interface for gene functions, we showed for the first time that DJ1 may regulate T1DM via the JNK1 pathway, suggesting DJ1 interacts with other proteins from various metabolic pathways. We anticipate that the current data will provide insights into the aetiology of T1DM.     

Enhanced brain disposition and effects of Δ9-tetrahydrocannabinol in P-glycoprotein and breast cancer resistance protein knockout mice

The ABC transporters P-glycoprotein (P-gp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis Δ(9)-tetrahydrocannabinol (THC) has affinity for P-gp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that mice devoid of Abcb1 and Abcg2 retain higher brain THC levels and are more sensitive to cannabinoid-induced hypothermia than wild-type (WT) mice. Abcb1a/b (-/-), Abcg2 (-/-) and wild-type (WT) mice were injected with THC before brain and blood were collected and THC concentrations determined. Another cohort of mice was examined for THC-induced hypothermia by measuring rectal body temperature. Brain THC concentrations were higher in both Abcb1a/b (-/-) and Abcg2 (-/-) mice than WT mice. ABC transporter knockout mice exhibited delayed elimination of THC from the brain with the effect being more prominent in Abcg2 (-/-) mice. ABC transporter knockout mice were more sensitive to THC-induced hypothermia compared to WT mice. These results show P-gp and Bcrp prolong the brain disposition and hypothermic effects of THC and offer a novel mechanism for both genetic vulnerability to the psychoactive effects of cannabis and drug interactions between CNS therapies and cannabis.     

cGMP transport by vesicles from human and mouse erythrocytes

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.     

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.     

Evaluation of the microsomal glutathione S-transferase 3 (MGST3) locus on 1q23 as a Type 2 diabetes susceptibility gene in Pima Indians

Elevation of plasma glucose concentration may induce generation of oxygen-free radicals, which can play an important role in the progression of diabetes and/or development of its complications. Various glutathione transferases utilize the availability of reduced glutathione for the cellular defense against oxygen-free radicals. One such enzyme is microsomal glutathione S-transferase 3 encoded by MGST3, which maps to chromosome 1q23, a region linked to Type 2 diabetes mellitus (T2DM) in Pima Indians, Caucasian, and Chinese populations. We investigated the MGST3 gene as a potential susceptibility gene for T2DM by screening this locus for single nucleotide polymorphisms (SNPs) in diabetic and non-diabetic Pima Indians. We also measured the skeletal muscle MGST3 mRNA level by Real-Time (RT) PCR and its relationship with insulin action in non-diabetic individuals. We identified 25 diallelic variants, most of which, based on their genotypic concordance, could be divided into three distinct linkage disequilibrium (LD) groups. We genotyped unique representative SNPs in selected diabetic and non-diabetic Pima Indians and found no evidence for association with T2DM. Furthermore, inter-individual variation of skeletal muscle MGST3 mRNA was not correlated with differences in insulin action in non-diabetic subjects. We conclude that alterations of MGST3 are unlikely to contribute to T2DM or differences in insulin sensitivity in the Pima Indians.     

Regulation of the expression of cholesterol transporters by lipid-lowering drugs ezetimibe and pemafibrate in rat liver and intestine

Ezetimibe and pemafibrate are lipid-lowering drugs and promote reverse cholesterol transport. However, it is unknown whether cholesterol is mainly excreted by hepatobiliary excretion or by non-biliary transintestinal cholesterol efflux (TICE). We evaluated the effects of ezetimibe and pemafibrate on hepatic and intestinal cholesterol transporter regulation in Sham-operated rats, and examined the effects of these drugs on TICE in bile duct-ligated rats. Seven-week-old male Sprague-Dawley rats were treated as follows for two weeks: 1) Sham, Sham operation; 2) BDL, bile duct ligation; 3) E-Sham, Sham + ezetimibe; 4) E-BDL, BDL + ezetimibe; 5) P-Sham, Sham + pemafibrate; and 6) P-BDL, BDL + pemafibrate. Blood, liver, jejunum, and feces were collected 72 h post-surgery. Hepatic cholesterol levels were decreased in P-Sham and E-Sham, and were lower in E-BDL and P-BDL than in BDL. Fecal cholesterol levels increased in E-Sham and P-Sham compared with Sham, and were higher in E-BDL and P-BDL than in BDL. In liver, Abcg5 mRNA showed induction in E-Sham, Abcg5 and Abca1 mRNA were induced in P-Sham, Abcg5 mRNA was reduced in E-BDL, and Abca1 mRNA was increased in P-BDL. In jejunum, Abcg5 mRNA was induced in E-Sham. Abcg8 mRNA was induced in E-Sham and P-Sham. NPC1L1 mRNA showed reduced expression in P-Sham and P-BDL. SR-B1 mRNA was reduced in P-Sham, and the expression decreased in P-BDL. LDL receptor mRNA was induced in BDL and P-BDL. Ezetimibe and pemafibrate may promote TICE by increasing Abcg5/g8, while pemafibrate may inhibit intestinal cholesterol absorption by decreasing SR-B1 and NPC1L1.     

Protective effect of heme oxygenase induction in ethinylestradiol‐induced cholestasis

Estrogen‐induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase‐1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol‐induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down‐regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up‐regulated Mrp3 (348%, P ≤ 0.05). Heme pre‐treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up‐regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol‐treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme‐induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up‐regulation (Mrp2/Mrp4) or down‐regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2‐dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol‐induced cholestasis.