Rick Havinga

Enterohepatic circulation in the rat

Several compounds, in particular bile acids, undergo enterohepatic circulation (EHC). Limited data are available on the pathophysiologic aspects of this circulation. In the present study we describe a surgical technique in rats that allows a long-term, reversible interruption of the EHC and monitoring of peripheral blood levels without direct surgical intervention. This technique excludes the effects of anesthesia and surgical trauma. The models validity has been tested extensively. We used this animal model to investigate acute and chronic effects of interruption and subsequent restoration of the EHC on (a) nutritional status, (b) plasma cholesterol levels and hepatic cholesterol synthesis, and (c) biliary bile acid, phospholipid, and cholesterol excretion. Interruption of the EHC resulted in an increased food intake and enhanced fecal energy loss, caused by a less efficient intestinal absorption. Plasma cholesterol concentrations declined immediately after the interruption of the EHC, but returned to almost control values during bile diversion. A marked overshoot followed the subsequent restoration of the EHC. Hepatic cholesterol synthesis showed a five-fold increase after 8 days of bile diversion but returned to control values within 2 days after restoration of the EHC. After interruption of the EHC, bile acid, phospholipid, and cholesterol excretion decreased sharply but stabilized after 3 h at 7.6%, 20%, and 23%, respectively, of their initial values. Bile acid output, representing hepatic synthesis, slowly increased over 4 days, but never exceeded 13% of its value during intact EHC. Subsequent restoration of the EHC could rapidly reverse the observed effects of the interruption. The animal model described in the present study is an excellent tool in studying the acute and chronic effects of disturbances of the EHC.

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine‐conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)‐induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco‐conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol‐3 kinase pathway (PI3K) and nuclear factor‐κB were inhibited using LY 294002 and adenoviral overexpression of dominant‐negative IκB, respectively. The role of p38 and extracellular signal‐regulated protein kinase mitogen‐activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin‐D. Apoptosis was determined by measuring caspase‐3, ‐9, and ‐8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase‐8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA‐induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF‐κB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA‐induced mitochondria‐controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563–1573.)

Peroxisome proliferator-activated receptor alpha (PPARalpha)-mediated regulation of multidrug resistance 2 (Mdr2) expression and function in mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls expression of genes involved in lipid metabolism and is activated by fatty acids and hypolipidaemic fibrates. Fibrates induce the hepatic expression of murine multidrug resistance 2 ( Mdr2 ), encoding the canalicular phospholipid translocator. The physiological role of PPARalpha in regulation of Mdr2 and other genes involved in bile formation is unknown. We found no differences in hepatic expression of the ATP binding cassette transporter genes Mdr2, Bsep (bile salt export pump), Mdr1a / 1b, Abca1 and Abcg5 / Abcg8 (implicated in cholesterol transport), the bile salt-uptake systems Ntcp (Na(+)-taurocholate co-transporting polypeptide gene) and Oatp1 (organic anion-transporting polypeptide 1 gene) or in bile formation between wild-type and Ppar alpha((-/-)) mice. Upon treatment of wild-type mice with ciprofibrate (0.05%, w/w, in diet for 2 weeks), the expression of Mdr2 (+3-fold), Mdr1a (+6-fold) and Mdr1b (+11-fold) mRNAs was clearly induced, while that of Oatp1 (-5-fold) was reduced. Mdr2 protein levels were increased, whereas Bsep, Ntcp and Oatp1 were drastically decreased. Exposure of cultured wild-type mouse hepatocytes to PPARalpha agonists specifically induced Mdr2 mRNA levels and did not affect expression of Mdr1a / 1b. Altered transporter expression in fibrate-treated wild-type mice was associated with a approximately 400% increase in bile flow: secretion of phospholipids and cholesterol was increased only during high-bile-salt infusions. No fibrate effects were observed in Ppar alpha((-/-)) mice. In conclusion, our results show that basal bile formation is not affected by PPARalpha deficiency in mice. The induction of Mdr2 mRNA and Mdr2 protein levels by fibrates is mediated by PPARalpha, while the induction of Mdr1a / 1b in vivo probably reflects a secondary phenomenon related to chronic PPARalpha activation.

The uncoupling of biliary lipid from bile acid secretion by organic anions in the rat

A number of organic anions has been shown to inhibit biliary phospholipid and cholesterol secretion without affecting bile acid secretion. However, the mechanism of this uncoupling phenomenon is still unclear. This study shows a comparison of the effects of ampicillin (18 mumol/100g body wt), sulfated taurolithocholic acid (0.2 and 1.0 mumol/100 g body wt), and indocyanine green (0.6 mumol/100 g body wt) in control and Groningen Yellow-Wistar rats with chronic (8 days) biliary drainage. Groningen Yellow rats have a hereditary defect in hepatobiliary transport of various organic anions. Bile secretion, but not hepatic uptake, of the three organic anions was strongly impaired in Groningen Yellow rats compared with controls. Ampicillin and sulfated taurolithocholic acid caused a strong uncoupling in control rats but had no effect or a much smaller effect on lipid secretion in Groningen Yellow rats. Indocyanine green did not affect lipid secretion, in either control or in Groningen Yellow rats. Gel-filtration chromatography of bile showed a specific coelution of ampicillin and sulfated taurolithocholic acid with the bile acid fraction, whereas indocyanine green coeluted with the phospholipid/cholesterol fraction. This study concludes that the uncoupling by ampicillin and sulfated taurolithocholic acid occurs after their secretion into bile and is caused by interaction of these compounds with bile acids. It is hypothesized that this interaction inhibits the capacity of bile acids to induce secretion of phospholipids and cholesterol into the bile.

TWO PATHWAYS FOR BILIARY COPPER EXCRETION IN THE RAT - THE ROLE OF GLUTATHIONE

To evaluate the role of glutathione in biliary copper excretion, we studied this process in control Wistar rats and in mutant Wistar rats (GY rats), in which the secretion of glutathione into bile is deficient. For comparison, biliary zinc excretion was determined simultaneously. In spite of the markedly reduced bile flow (-45%) in GY rats, biliary output rates of endogenous copper were virtually identical in GY and control rats. In contrast, zinc output was drastically reduced in GY rats compared to controls (-80%). Biliary excretion patterns after intravenous administration of copper, in doses ranging from 65 to 2265 nmol/100 g/body wt, showed a distinct rapid and slow phase in control rats. In GY rats, on the other hand, the rapid phase in copper excretion was absent but the slow phase appeared to be unaffected. Pretreatment of rats with diethylmaleate to deplete hepatic and biliary glutathione abolished the rapid phase of copper excretion in control rats, while the slow phase remained unaffected. No significant effect of diethylmaleate on the hepatic handling of exogenous copper was observed in GY rats. The maximal capacity of the slow copper excretion pathway was 40-45 nmol/hr/100 g body wt, both in control and GY rats; the capacity of rapid excretion pathway depended on the administered copper load. Intravenous injection of copper induced the biliary excretion of a substantial amount of zinc in control rats, but not in GY rats. These results indicate the existence of at least two distinct biliary excretory pathways for copper in the rat, i.e. a slow and a rapid pathway, with a glutathione dependency of the latter only. The basal excretion of (endogenous) copper, in contrast to that of zinc, can proceed independently of glutathione excretion. However, glutathione appears to be involved in the rapid secretion of excess copper.

ATP binding cassette transporter gene expression in rat liver progenitor cells

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. Methods: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF ϕ 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF ϕ 13 cells by flow cytometry. Results: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF ϕ 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF ϕ 13 cells. Conclusion: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.

Bile secretion of cadmium, silver, zinc and copper in the rat. Involvement of various transport systems.

In the present study we compared, in vivo in rats, the hepatobiliary transport of monovalent (silver:Ag) and divalent metals (zinc:Zn; cadmium:Cd) with that of copper (Cu). Cu can have two oxidation states in vivo, i.e. Cu(I) and Cu(II). Studies were performed in normal Wistar (NW) rats and mutant GY Wistar rats. The latter express defective canalicular ATP-dependent glutathione-conjugate transport (cMOAT); reduced glutathione (GSH) is virtually absent in bile of these mutants. Cd (400 nmol/100g body wt, i.v.) was rapidly secreted into bile in NW rats concommitant with a 4-fold increase in biliary GSH secretion. In contrast, biliary Cd concentrations remained below detection limits in GY rats. Injection of Zn (1500 nmol/100g body wt) did not affect Zn secretion in GY rats and resulted only in a very small increase in NW rats (recovery < 2%). The biliary secretion pattern of Ag (800 nmol/100g body wt, i.v.) was highly similar to that of Cu (260 nmol/100g body wt). A biphasic pattern composed of a rapid and slow phase was observed in NW rats for both metals with a recovery of 48.5 +/- 10.6% and 44.9 +/- 8.4% of the dose for Ag and Cu, respectively. In GY rats, the rapid phase of both Ag and Cu secretion was absent and recoveries were 23.2 +/- 3.6% and 19.7 +/- 3.2%, respectively. When Ag and Cu were administered simultaneously, the recoveries of Ag and Cu were decreased in NW and GY rats when compared to single administration. Our data indicate that divalent and monovalent metals are secreted into bile via different transport systems in the rat. The absence of Cd and Zn secretion into bile of GY rats after their i.v. administration suggest a role of cMOAT in their biliary elimination. Cu and Ag probably share common transport systems for hepatobiliary removal, being in part dependent on the presence of either GSH in bile or cMOAT activity or on both. The GSH-independent portion of transport, i.e. the slow phase, may be mediated by the newly identified Cu transporting P-type ATPase (cCOP).

Lipoproteins and liposomes as in vivo cholesterol vehicles in the rat: preferential use of cholesterol carried by small unilamellar liposomes for the formation of muricholic acids

Hepatic cholesterol metabolism was studied in rats with a permanent biliary drainage. Three cholesterol vehicles were used to discriminate between metabolic pathways of cholesterol in the liver. [3H]Cholesterol was administered intravenously associated with rat serum lipoproteins, multilamellar (MLV) or small unilamellar (SUV) liposomes. The liposomes were made from cholesterol, sphingomyelin and phosphatidylserine in a 5:4:1 molar ratio. Initial blood elimination differed markedly for the three vehicles: 15 min after injection the 3H radioactivity content of blood for MLV, SUV and lipoprotein was 3, 50 and 54% of the injected dose, respectively. After about 30 min, MLV-cholesterol label started to reappear in the blood, probably after processing of the vehicle by the Kupffer cells. For all vehicles about 80% of the cholesterol label had been excreted in bile after 120 h, predominantly as bile acids. Initial biliary excretion was highest for lipoproteins (5.7% at 1 h), followed by MLV and SUV (1.3 and 1.2%, respectively). No differences in the radioactivity of excreted bile acids were detectable between the three vehicles at 12 h after injection. However, at 1 h the radioactivity in the muricholic acid fraction was markedly increased, as compared to the other bile acids after injection of SUV-cholesterol, but not after injection of MLV- or lipoprotein-cholesterol. Also, the glycine/taurine conjugation ratio of bile acids was increased for SUV-cholesterol at 1 h as compared to that for the other two vehicles. Since SUV appear to donate their cholesterol to a pool which preferentially supplies cholesterol for muricholic acid synthesis, we conclude that more than one cholesterol pool exists in the hepatocytes from which cholesterol can be recruited for bile acid synthesis. Zonal heterogeneity might be responsible for the observed differences.

Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats

BACKGROUND/AIMSnFluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids.nnnMETHODSnWe analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR- rats with a selective canalicular transport defect of non-bile acid organic anions.nnnRESULTSnCLF and LLF, which like their natural nonsulfated bile acid congeners are expected to be handled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR- rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate.nnnCONCLUSIONnThe close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.

Effect of ML-236B (compactin) on biliary excretion of bile salts and lipids, and on bile flow, in the rat.

To assess the importance of de novo cholesterol synthesis for bile salt formation, the effects of ML-236B (an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase) on biliary excretion of bile salts and lipids were studied in rats with permanent catheters in bile duct, heart and duodenum. In rats having their bile diverted continuously for 8 days, duodenal administration of ML-236B (50 mg/kg) caused an immediate transient choleresis, which subsided after 2 h. Concomitant with the choleresis concentrations of bile salt, phospholipid and cholesterol fell, but this decrease was maintained for 6 h. Consequently, ML-236B inhibited biliary output salts and lipids from the second till the sixth hour after injection. The kinetics of biliary excretion of intravenously injected [14C]taurocholate were not affected by ML-236B administration. In rats having their biliary catheter connected to the duodenal catheter, or in rats with prolonged bile diversion but treated with mevalonolactone, ML-236B again caused a transient choleresis (having subsided after 2 h), but now did not affect biliary excretion of bile salts and lipids. It is concluded that (1) ML-236B causes a transient bile salt-independent choleresis, (2) ML-236B depresses excretion of bile salts and lipids by blocking mevalonate synthesis and not by blocking the bile salt or lipid transport, (3) biliary excretions of phospholipids and cholesterol partly depend on excretion of bile salt, and (4) in rats with a prolonged total bile diversion newly formed mevalonate is a major substrate for bile salt synthesis.