Bile Acid Output Research Articles

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis

Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-β-muricholic acid and taurocholic acid. Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Oleanolic acid attenuates obstructive cholestasis in bile duct-ligated mice, possibly via activation of NRF2-MRPs and FXR antagonism

Obstructive cholestasis is characterized by impairment of hepatic canalicular bile efflux and there are no clinically effective drugs to cure except surgeries. Previously we revealed that oleanolic acid (OA) protected against lithocholic acid (LCA)-induced intrahepatic cholestasis in mice. Cholestasis caused by LCA is characterized by segmental bile duct obstruction, whether OA possesses the beneficial effect on completed obstructive cholestasis induced by bile duct ligation (BDL) remains unknown. In this study, we demonstrated that BDL-induced mice liver pathological change, and increase in serum levels of ALT, AST and ALP were all significantly reduced by OA (20mg/kg, i.p.). Meanwhile, OA also lowered total bilirubin and total bile acids levels in serum, as well as total bile acids level in liver, in contrast, urinary total bile acids output was remarkably up-regulated by OA. Gene expression analysis showed that OA caused significant increased mRNA expression of MRP3 and MRP4 located at hepatic basolateral membrane, and restoration of MRP2 and BSEP located at hepatic cannalicular membrane. Furthermore, significant NRF2 protein accumulation in nucleus was also observed in OA treated mice. In mice primary cultured hepatocytes, the effects of OA on MRP2, MRP3 and MRP4 expression were directly proved to be mediated via NRF2 activation, and BSEP downregulation induced by OA was in part due to FXR antagonism. Luciferase assay performed in Hep G2 cells also illustrated that OA was a partial FXR antagonist. Taken together, we conclude that OA attenuates obstructive cholestasis in BDL mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Impaired uptake of conjugated bile acids and hepatitis b virus pres1‐binding in na+‐taurocholate cotransporting polypeptide knockout mice

The Na+-taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1−/− hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1−/− mice, explained by increased Asbt and Ostα/β expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1−/− hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1-/- mice. Conclusion: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation. (Hepatology 2015;62:207–219)

Chronic activation of FXR in transgenic mice caused perinatal toxicity and sensitized mice to cholesterol toxicity.

The nuclear receptor farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4, or NR1H4) is highly expressed in the liver and intestine. Previous reports have suggested beneficial functions of FXR in the homeostasis of bile acids, lipids, and glucose, as well as in promoting liver regeneration and inhibiting carcinogenesis. To investigate the effect of chronic FXR activation in vivo, we generated transgenic mice that conditionally and tissue specifically express the activated form of FXR in the liver and intestine. Unexpectedly, the transgenic mice showed several intriguing phenotypes, including partial neonatal lethality, growth retardation, and spontaneous liver toxicity. The transgenic mice also displayed heightened sensitivity to a high-cholesterol diet-induced hepatotoxicity but resistance to the gallstone formation. The phenotypes were transgene specific, because they were abolished upon treatment with doxycycline to silence the transgene expression. The perinatal toxicity, which can be rescued by a maternal vitamin supplement, may have resulted from vitamin deficiency due to low biliary bile acid output as a consequence of inhibition of bile acid formation. Our results also suggested that the fibroblast growth factor-inducible immediate-early response protein 14 (Fn14), a member of the proinflammatory TNF family, is a FXR-responsive gene. However, the contribution of Fn14 induction in the perinatal toxic phenotype of the transgenic mice remains to be defined. Because FXR is being explored as a therapeutic target, our results suggested that a chronic activation of this nuclear receptor may have an unintended side effect especially during the perinatal stage.

Mechanism underlying an elevated serum bile acid level in chronic renal failure patients.

Bile acids play an important role in the digestion of dietary lipids. Bile acid metabolism is regulated by the digestive system. The kidney is an important organ of the urinary system and is believed to play a minor role in bile acid excretion; however, many recent studies have reported an increased serum bile acid level and alterations in bile acid homeostasis in both clinical and animal model studies on chronic renal failure. The existing research findings on the mechanisms underlying this phenomenon were mostly derived from animal model studies, but clinical investigations have been limited. Kidney tissues and serum and urine samples from CRF patients and normal controls were studied. We found increased serum bile acid levels and decreased urine bile acid output levels in chronic renal failure patients. Mesangial cell and endothelial cell proliferation, glomerular sclerosis, renal interstitial fibrosis, and intrarenal vascular sclerosis were observed based on hematoxylin-eosin and Masson trichrome staining pathology analysis. Scatter diagram and Pearson correlation analysis showed that in chronic renal failure patients, the estimated glomerular filtration rate and serum bile acid level were interrelated. Reverse transcription polymerase chain reaction and Western blotting results indicated that reabsorption and secretion of bile acid at the apical surface of the proximal renal tubular did not contribute to the elevated serum BA level. The increase in plasma bile acid is due to decreased bile acid filtration through the kidneys in CRF patients.

Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE*3Leiden.CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (−37%; −46%, P < 0.001) and TGs (−36%; −39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (−48%; −58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (−71%; −88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (−89%; −98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. In addition, alirocumab improves plaque morphology.

Bile acid flux through portal but not peripheral veins inhibits CYP7A1 expression without involvement of ileal FGF19 in rabbits.

It was proposed that CYP7A1 expression is suppressed through the gut-hepatic signaling pathway fibroblast growth factor (FGF) 15/19-fibroblast growth factor receptor 4, which is initiated by activation of farnesoid X receptor in the intestine rather than in the liver. The present study tested whether portal bile acid flux alone without ileal FGF19 could downregulate CYP7A1 expression in rabbits. A rabbit model was developed by infusing glycodeoxycholic acid (GDCA) through the splenic vein to bypass ileal FGF19. Study was conducted in four groups of rabbits: control; bile fistula + bovine serum albumin solution perfusion (BF); BF + GDCA (by portal perfusion); and BF + GDCA-f (by femoral perfusion). Compared with only BF, BF + GDCA (6 h portal perfusion) suppressed CYP7A1 mRNA, whereas BF + GDCA-f (via femoral vein) with the same perfusion rate of GDCA did not show inhibitory effects. Meanwhile, there was a decrease in ileal FGF19 expression and portal FGF19 protein levels, but an equivalent increase in biliary bile acid outputs in both GDCA perfusion groups. This study demonstrated that portal bile acid flux alone downregulated CYP7A1 expression with diminished FGF19 expression and protein levels, whereas the same bile acid flux reaching the liver through the hepatic artery via femoral vein had no inhibitory effect on CYP7A1. We propose that bile acid flux through the portal venous system may be a kind of "intestinal factor" that suppresses CYP7A1 expression.

Portal bile acid perfusion bypasses ileal FGF19 and suppresses hepatic CYP7A1 expression in rabbits (605.6)

This study challenged the hypothesis that CYP7A1 expression is regulated by a gut‐hepatic signaling pathway FGF15/19‐FGFR4 initiated from the ileum but not the liver. We would like to show that increase of portal bile acid flux alone should be able to suppress CYP7A1 expression. Methods: We developed a rabbit model to infuse glycodeoxycholic acid (GDCA) via the splenic vein to bypass ileal FGF19. Four study groups of rabbits (n=8/group): Control; bile fistula (BF); BF+GDCA (by portal perfusion); and BF+ GDCA‐f (by femoral perfusion). Results: Portal GDCA perfusion in the BF+GDCA group for 6 hours resulted in significant reduction in CYP7A1 mRNA but femoral perfusion in the BF+GDCA‐f group showed no change with the same perfusion rate of GDCA. In both GDCA perfusion groups, there was no increase in ileal FGF19 expression nor portal FGF19 protein levels, but similar increase in biliary bile acid outputs. Conclusions: This study demonstrated that portal bile acid flux alone inhibited CYP7A1 expression without increase of FGF19 in the ileum. However, the same bile acid flux through the liver via the hepatic artery (femoral perfusion) did not suppress the CYP7A1 expression. We propose that bile acid flux through the portal venous system may be the so called “intestinal factor” that suppresses CYP7A1 expression.Grant Funding Source: VA Merit Review Grant

Depicting a novel regulatory Bcl2/SHP/lncRNA H19 network in bile acid homeostasis (605.12)

Objective: The aim of this study is to investigate a novel function of the anti‐apoptotic protein Bcl2 in bile acid (BA) homeostasis. Methods: Bcl2 was overexpressed using adenoviruses in male C57BL6 livers. After two weeks, BA homeostasis, liver morphology, fibrosis, and inflammation were analyzed. RNA‐seq and GC/MS were used to identify changes in gene expression and metabolites, respectively. Results: Hepatic overexpression of Bcl2 largely induced serum BA and FGF15 levels and decreased total BA pool size and fecal BA output. Severe hepatocyte necrosis, fibrosis, and Kupffer cell activation were observed, which was accompanied by a marked induction of long non‐coding RNA H19. RNA‐seq identified significant downregulation of genes in bile acid synthesis and transport, but upregulation of genes in collagen formation and inflammatory response by Bcl2. The increases in TCA cycle and lipid metabolites were the most drastic metabolomics changes. Using a series of cell based biochemistry and molecular biology approaches, we found that Bcl2 induced rapid nuclear receptor SHP protein degradation via activation of the caspase 8‐caspase 3 pathway. Furthermore, bile acid directly induced H19 expression in cultured primary hepatocytes that was independent of Bcl2 activation by bile acid. Importantly, Bcl2 and H19 were both dramatically increased in human cirrhotic livers and in mouse cholestatic liver fibrosis models. The dysregulation of the Bcl2/SHP/H19 network resulted in the disruption of bile acid homeostasis which contributed to the development of cholestatic liver fibrosis. Conclusions: Our results uncovered a unique metabolic regulatory network that integrated Bcl2 and SHP with lncRNA H19 to control BA homeostasis.Grant Funding Source: Supported by DK080440 (L.W).

Effects of chicken-liver hydrolysates on lipid metabolism in a high-fat diet

The contents of free hydrophobic amino acids, taurine and carnosine/anserine were elevated after hydrolyzing chicken livers by pepsin and compared to dried chicken livers. Chicken-liver-hydrolysates (CLHs) exhibited in vitro inhibitory lipase activity and bile-acid binding ability (p<0.05). Forty-eight male hamsters were assigned randomly to the following groups: (1) chow diet; (2) high-fat diet (HFD); (3) HFD and 100mgCLH/kgBW; (4) HFD and 200mgCLH/kgBW; (5) HFD and 400mgCLH/kgBW; (6) HFD and 200mgcarnosine/kgBW. CLHs alleviated (p<0.05) serum oxidative stress and improved (p<0.05) the serum lipid profile in the high-fat dietary groups; meanwhile, improved (p<0.05) antioxidant abilities and decreased (p<0.05) lipid accumulation, oxidative stress and TNF-α/IL-1β levels in the livers. These benefits might result from regulations of lipid homeostasis and increased faecal bile-acid outputs (p<0.05). Hence, lipid-homeostasis and antioxidant abilities of CLHs in the high-fat dietary habit were demonstrated and were similar to pure carnosine.

The effect of Macrotyloma uniflorum seed on bile lithogenicity against diet induced cholelithiasis on mice.

Background:The seeds of Macrotyloma uniflorum Lam. (Family Fabaceae) contain extractable total and tannins with reported hepatoprotective, hypocholesterolemic and antioxidant activity. In this study, dietary M. uniflorum seed, methanolic and acetone extracts (ME and AE) were examined for their bile-antilithogenic potential.Materials and Methods:Mice fed with 1% cholesterol and 0.5% cholic acid lithogenic (LG) diet for 8 weeks resulted in cholesterol super saturation in gallbladder bile, which promotes the formation of cholesterol gallstones (CGSs).Results:AE reduced the CGS incidence by 60.21%, and serum total cholesterol, triglyceride (TG), very low density lipoprotein (LDL) and LDL compared to control animals. Seed extracts at 300 mg/kg dose markedly reduced biliary cholesterol (BC) and decreased bile salt content. The ratio of BC to phospholipid which was 2.64 in the LG diet group was reduced to 1.57–1.35 in the M. uniflorum seed extracts treated groups. Liver cholesterol and TG were decreased significantly by feeding of ME and AE at 300 mg/kg dose. AE significantly reversed the changes in apolipoproteins A-I and C-II level disturbed by LG diet.Conclusions:M. uniflorum seed exerted antilithogenic influence by decreasing the cholesterol hyper-secretion into bile and increasing the bile acid output, thus decreasing the formation of LG bile in mice. The effect was maximum in the AE as it also reduced papillary proliferation of gallbladder and fatty degeneration of the liver. The potential antilithogenic effect of the AE of M. uniflorum may be due to antioxidant property of its rich total polyphenol and tannins content.

Bile acid sequestration normalizes plasma cholesterol and reduces atherosclerosis in hypercholesterolemic mice. No additional effect of physical activity

AimsBile acid sequestrants (BAS) and physical activity (RUN) decrease incidence of cardiovascular events. Both treatments are often prescribed, yet it is not known whether their beneficial effects are additive. We assessed the effects of BAS treatment alone and in combination with RUN on cholesterol metabolism, heart function and atherosclerotic lesion size in hypercholesterolemic mice. MethodsMale Ldlr-deficient mice remained either sedentary (CONTROL), were treated with Colesevelam HCl (BAS), had access to a running wheel (RUN), or were exposed to BAS and RUN (BAS RUN). All groups were fed a high cholesterol diet for 12 weeks. Then, feces, bile and plasma were collected. Atherosclerotic lesion size was determined in the aortic arch and heart function by echocardiography. ResultsBAS RUN ran more than RUN (6.4 ± 1.4 vs. 3.5 ± 1.0 km/day, p < 0.05). BAS and BAS RUN displayed ∼3-fold reductions in plasma cholesterol levels (p < 0.001), ∼2.5-fold increases in fecal neutral sterol (p < 0.001) and bile acid (p = 0.01) outputs, decreases in biliary secretions of cholesterol (∼6-fold, p < 0.0001) and bile acids (∼2-fold, p < 0.001) vs. CONTROL while no significant effects were observed in RUN. Compared to CONTROL, lesion size decreased by 78% in both BAS and BAS RUN, (p < 0.0001). ConclusionBAS reduce atherosclerosis in Ldlr-deficient mice, coinciding with a switch from body cholesterol accumulation to cholesterol loss. RUN slightly modulated atherosclerotic lesion formation but the combination of BAS and RUN had no clear additive effects in this respect.

Role of plant stanol derivatives in the modulation of cholesterol metabolism and liver gene expression in mice

The present study was to evaluate the cholesterol-lowering effect of two novel plant stanol derivatives and its potential molecular mechanism in hyper-cholesterol mice induced by a high-cholesterol diet. Results showed that oral administration of plant stanyl hemisuccinate (2×, 5×) and plant stanyl sorbitol succinate (2×, 5×) effectively attenuated the serum total cholesterol and low density lipoprotein cholesterol levels, while had no effect on the serum triacylglycerol and high density lipoprotein cholesterol. And plant stanol derivatives decreased liver cholesterol concentration and increased faecal cholesterol output. Meanwhile, both plant stanyl hemisuccinate and plant stanyl sorbitol succinate could remarkably promote liver X receptor alpha (LXRα) expression, and increased cholesterol 7α-hydroxylase (CYP7A1) expression and faecal total bile acid output to varying degrees. These results suggested two novel plant stanol derivatives possessed hypocholesterolemic effect, and the cholesterol-lowering action of plant stanol derivatives may be through activating the potential LXRα-CYP7A1-bile acid excretion pathway.

Alleviative effects of deep-seawater drinking water on hepatic lipid accumulation and oxidation induced by a high-fat diet

BackgroundHepatic steatosis is defined as excessive amounts of triglyceride and other fats inside liver cells and has become an emergent liver disease in developed and developing countries. MethodsDeep seawater (DSW)300, DSW900, and DSW1500 drinking waters were formulated via a combination of reverse osmosis and electrodialysis. Hamsters on a high-fat diet were assigned to drink the following solutions: (1) normal distilled water, (2) DSW300, (3) DSW900, or (4) DSW1500. Serum, liver, and fecal biochemical values, expression of hepatic genes related to fatty-acid homeostasis, as well as liver antioxidative levels were measured after a 6-week feeding period. Additionally, hematoxylin and eosin staining was used to investigate the liver histopathology. ResultsSerum/liver lipids, liver sizes, liver malondialdehyde content, and serum aspartate aminotransferase and alanine aminotransferase of high-fat diet hamsters were reduced (p < 0.05) by drinking DSW, while daily fecal lipid and bile acid outputs were increased (p < 0.05). DSW drinking water maintained (p < 0.05) higher liver glutathione and Trolox equivalent antioxidant capacity levels. Although hepatic sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme gene expression were not (p > 0.05) altered, DSW drinking water upregulated (p < 0.05) hepatic peroxisome proliferator-activated receptor-alpha, retinoid X receptor alpha, and uncoupling protein-2 gene expression in high-fat diet hamsters. The lipid droplets in livers were also reduced in DSW-drinking-water groups as compared to those only drinking distilled water. ConclusionDSW shows a preventive effect on development of hepatosteatosis induced by a high-fat diet.

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.

Loss of A1 Adenosine Receptor Attenuates Alpha-naphthylisothiocyanate-Induced Cholestatic Liver Injury in Mice

Cholestasis has limited therapeutic options and is associated with high morbidity and mortality. The A(1) adenosine receptor (A(1)AR) was postulated to participate in the pathogenesis of hepatic fibrosis induced by experimental extrahepatic cholestasis; however, the contribution of A(1)AR to intrahepatic cholestatic liver injury remains unknown. Here, we found that mice lacking A(1)AR were resistant to alpha-naphthyl isothiocyanate (ANIT)-induced liver injury, as evidenced by lower serum liver enzyme levels and reduced extent of histological necrosis. Bile acid accumulation in liver and serum was markedly diminished in A(1)AR(-/-) mice compared with wild-type (WT) mice. However, biliary and urinary outputs of bile acids were significantly enhanced in A(1)AR(-/-) mice. In the liver, mRNA expression of genes related to bile acid transport (Bsep and Mdr2) and hydroxylation (Cyp3a11) was increased in A(1)AR(-/-) mice. In the kidney, A(1)AR deficiency prevented the decrease of glomerular filtration rate caused by ANIT. Treatment of WT mice with A(1)AR antagonist DPCPX also protected against ANIT hepatotoxicity. Our results indicated that lack of A(1)AR gene protects mice from ANIT-induced cholestasis by enhancing toxic biliary constituents efflux through biliary excretory route and renal elimination system and suggested a potential role of A(1)AR as therapeutic target for the treatment of intrahepatic cholestasis.

Study on hepatotoxicity of aqueous extracts of Polygonum multiflorum in rats after 28-day oral administration-analysis on correlation of cholestasis

To observe the liver injury degree of SD rats after 28-day administration of aqueous extracts of Polygonum multiflorum (AEPM) and the correlation with cholestasis mechanism. Adult SD rats were orally administered with 30, 60 g x kg(-1) of APEM once every day for 28 d. After 28 d, the general condition of rats such as weight were observed, liver function-related indicators were detected. Bile was collected to determine total bile acid output, flow rate and density and changes in major compositions. Their livers were weighed then sent for histopathological examination. AEPM did not change the general conditions and weights of rats. From the results of the related indicators of liver function and cholestasis, AEPM did not change the contents of ALT and AST in serum, but high dose of AEPM can increase the contents of ALP, GGT and TBA in serum (P < 0.05, P < 0.01) and decrease the content of TBIL in serum (P < 0.05). And the contents of GGT in serum of low dose rats were increased (P < 0.05). The bile flow was not changed by AEPM, but bile compositions of high dose male rats were obviously changed (TG increase, TBIL decrease, TBA decrease). The weights of liver and ratio of liver of the high dose rats were increased but showed no statistical significance. Pathologic examination displayed that there were only small pieces of necrosis in livers of several rats, without any severe disease. AEPM can obviously injure bile duct epithelial cells, intervene liver cell functions and change bile compositions in rats, thus it is proved to induce cholestasis without severe liver injury.

Abstract 282: Murine Abcb1 Functions as an Efflux Transporter for Bile Salts After Chronic Administration of a Western Diet

Rationale: Removal of bile salts from the liver is the final step of the reverse cholesterol transport pathway. We studied the contribution of Abcb1 (P-glycoprotein), in bile acid efflux. Although a number of endogenous substrates have been postulated for Abcb1 based on in vitro evidence, studies using animal models have not supported these claims. Recent studies in mice demonstrated that in the absence of the Bile Salt Efflux Pump (Bsep), Abcb1 is required for removal of bile salts, especially when challenged with a cholic acid containing diet. To date, no study using atherogenic diets has demonstrated the role of Abcb1 in the removal of bile salts in the presence of functional Bsep. Methods: We fed male mice lacking both isoforms of Abcb1 (Abcb1a -/- /1b -/- ) and wild-type controls a diet providing either 25% or 45% of the kcal from fat, supplemented with either normal chow or high levels of cholesterol (0.02% w/w or 0.2% w/w respectively) for nine weeks; n=5 per group. On the tenth week, we assessed the efflux of cholesterol, phospholipid and bile acids to the gallbladder. Enzymatic assays were used to measure cholesterol and phospholipid, the pool of bile acids was quantified by HPLC to determine the concentrations of the six most prevalent murine bile acids. Results: Abcb1 knockout mice have a &gt;30% reduction in the moles of bile salt normalized to phospholipid relative to wild type mice after administration of diets containing either elevated fat or cholesterol (p&lt;0.05). Neither the efflux of phospholipid, nor the molar composition of the six bile acids was affected by deletion of Abcb1. Conclusions: We conclude that Abcb1 is a secondary efflux mechanism required for the removal of bile acids after consumption of diets rich in fat and/or cholesterol. Although Abcb1 knockout mice have reduced total bile acids in the gallbladder, the molar ratio of the specific bile acids is the same as in the wild type mice. These data suggest that Abcb1 effluxes the six bile acids in a non-specific manner, unlike Bsep which preferentially effluxes hydrophobic bile acids. The lack of specificity demonstrated by Abcb1 is desirable for a low- affinity secondary efflux mechanism, which supplements Bsep activity in bile acid output. Acknowledgments: Canadian Institutes of Health Research, Michael Smith Foundation for Health Research

Voluntary wheel running increases bile acid as well as cholesterol excretion and decreases atherosclerosis in hypercholesterolemic mice

ObjectiveRegular physical activity decreases the risk for atherosclerosis but underlying mechanisms are not fully understood. We questioned whether voluntary wheel running provokes specific modulations in cholesterol turnover that translate into a decreased atherosclerotic burden in hypercholesterolemic mice. MethodsMale LDLR-deficient mice (8 weeks old) had either access to a voluntary running wheel for 12 weeks (RUN) or remained sedentary (CONTROL). Both groups were fed a western-type/high cholesterol diet. Running activity and food intake were recorded. At 12 weeks of intervention, feces, bile and plasma were collected to determine fecal, biliary and plasma parameters of cholesterol metabolism and plasma cytokines. Atherosclerotic lesion size was determined in the aortic root. ResultsRUN weighed less (∼13%) while food consumption was increased by 17% (p=0.004). Plasma cholesterol levels were decreased by 12% (p=0.035) and plasma levels of pro-atherogenic lipoproteins decreased in RUN compared to control. Running modulated cholesterol catabolism by enhancing cholesterol turnover: RUN displayed an increased biliary bile acid secretion (68%, p=0.007) and increased fecal bile acid (93%, p=0.009) and neutral sterol (33%, p=0.002) outputs compared to control indicating that reverse cholesterol transport was increased in RUN. Importantly, aortic lesion size was decreased by ∼33% in RUN (p=0.033). ConclusionVoluntary wheel running reduces atherosclerotic burden in hypercholesterolemic mice. An increased cholesterol turnover, specifically its conversion into bile acids, may underlie the beneficial effect of voluntary exercise in mice.

Addition of Methionine to Rice Protein Affects Hepatic Cholesterol Output Inducing Hypocholesterolemia in Rats Fed Cholesterol-Free Diets

To elucidate whether a low methionine (Met)/glycine (Gly) ratio is responsible for the hypocholesterolemic effect of rice protein (RP), the effects of adding Met to RP, increasing its Met/Gly ratio, on hepatic cholesterol output was investigated in rats fed cholesterol-free diets. The hepatic secretion of cholesterol into bile or circulation was measured from isolated perfused livers of 7-week-old male Wistar rats fed RP (cultivar Koshihikari) with a lower Met/Gly ratio and methionine-supplemented RP with a higher Met/Gly ratio (RP-M) matched with casein (CAS). RP-M produced a significant hypocholesterolemic effect, whereas the effect of RP on plasma cholesterol level was comparable to that of CAS. Hepatic accumulation of total lipids, cholesterol, and phospholipids was higher in RP-M rats than in RP rats, again following a pattern similar to that in CAS rats. The hepatic total and very-low-density lipoprotein (VLDL)-cholesterol secretions into circulation were effectively decreased by RP, but not by RP-M. Bile flow and biliary outputs of bile acids, cholesterol, and phospholipids were significantly stimulated by RP-M, causing an increase in fecal sterol excretion compared with CAS and RP. Neither biliary output nor fecal excretion of bile acids was affected by RP compared with CAS. The present results demonstrate that the plasma cholesterol-lowering effect of RP cannot be merely ascribed to the low Met/Gly ratio of RP in growing rats fed cholesterol-free diets. Results suggest that the hypocholesterolemic response induced by RP-M with a higher Met/Gly ratio primarily contributes to the stimulation of hepatic cholesterol for elimination via biliary secretion rather than the inhibition of hepatic cholesterol release via VLDL into circulation.