Biliary Phospholipid Research Articles

Tetraparesis following thoracic spine surgery in a patient with Klippel–Feil syndrome and ABCB4 mutation: a case report

BackgroundKlippel–Feil syndrome is a rare condition described in 1912 by Maurice Klippel and André Feil. It is defined as a congenital cervical fusion of at least two vertebrae, associated with a classical triad of clinical signs: short neck, low posterior hairline, and limited range of movement. However, Klippel–Feil syndrome manifests with a vast spectrum of phenotypes, ranging from no symptoms to complete triad, with or without other associated malformations. Most commonly, CCF results from sporadic mutations, even though autosomal recessive, autosomal dominant, or even X-linked inheritance can be detected. The ATP-binding cassette subfamily B member 4 is only expressed in the liver and is involved in biliary phospholipid secretion. The clinical spectrum includes various hepatobiliary pathologies, including low phospholipid-associated cholelithiasis, and has never been associated with musculoskeletal anomalies.Case presentationA 55-year-old male Caucasian patient presenting with low phospholipid-associated cholelithiasis syndrome with ATP-binding cassette subfamily B member 4 mutation and liver cirrhosis was referred to our clinic for a liver transplant. A period of 6 months before, the patient underwent a T7–T9 posterior fixation for a T8 osteoporotic fracture. Postoperatively, he was tetraparetic, whereas he was neurologically intact before the operation. At admission to our hospital, he was still tetraparetic and presented with clinical signs of cervical myelopathy. Moreover, he suffered a limitation of cervical range of motion in all directions, short neck, and low posterior hairline. Imaging showed multiple cervical and thoracic vertebral bodies fusion, as well as cervical spine stenosis. Based on the available data, we diagnosed a type 3 Klippel–Feil syndrome according to Samartzis’ classification.ConclusionsThe heterogeneity of KFS and the various potential hereditary links that are known indicate that it is important to highlight all potential cases related to known genetic defects. At present, no association between ATP-binding cassette subfamily B member 4 mutation and congenital cervical fusions has been reported. The other important clinical focus of this case is the appearance of spontaneous tetraparesis after thoracic spine surgery. This mechanism remains unclear, but considering different spinal anatomy it might have been due to difficult intubation and patient’s positioning during his previous operation.

Outcomes of 38 patients with PFIC3: Impact of genotype and of response to ursodeoxycholic acid therapy

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease caused by biallelic variations in ABCB4. Data reporting on the impact of genotype and of response to ursodeoxycholic acid (UDCA) therapy on long-term outcomes are scarce. We retrospectively describe a cohort of 38 patients with PFIC3 with a median age at last follow-up of 19.5 years (range 3.8-53.8). Twenty patients presented with symptoms before 1 year of age. Thirty-one patients received ursodeoxycholic acid (UDCA) therapy resulting in serum liver test improvement in 20. Twenty-seven patients had cirrhosis at a median age of 8.1 years of whom 18 received a liver transplant at a median age of 8.5 years. Patients carrying at least one missense variation were more likely to present with positive (normal or decreased) canalicular MDR3 expression in the native liver and had prolonged native liver survival (NLS; median 12.4 years [range 3.8-53.8]). In contrast, in patients with severe genotypes (no missense variation), there was no detectable canalicular MDR3 expression, symptom onset and cirrhosis occurred earlier, and all underwent liver transplantation (at a median age of 6.7 years [range 2.3-10.3]). The latter group was refractory to UDCA treatment, whereas 87% of patients with at least one missense variation displayed an improvement in liver biochemistry in response to UDCA. Biliary phospholipid levels over 6.9% of total biliary lipid levels predicted response to UDCA. Response to UDCA predicted NLS. Patients carrying at least one missense variation, with positive canalicular expression of MDR3 and a biliary phospholipid level over 6.9% of total biliary lipid levels were more likely to respond to UDCA and to exhibit prolonged NLS. In this study, data show that genotype and response to ursodeoxycholic acid therapy predicted native liver survival in patients with PFIC3 (progressive familial intrahepatic cholestasis type 3). Patients carrying at least one missense variation, with positive (decreased or normal) immuno-staining for canalicular MDR3, and a biliary phospholipid level over 6.9% of total biliary lipids were more likely to respond to ursodeoxycholic acid therapy and to exhibit prolonged native liver survival.

Preclinical evaluation of Hibiscus cannabinus Linn. in the treatment of urolithiasis and cholelithiasis

ObjectiveTo investigate the therapeutic effect of Hibiscus cannabinus Linn. (H. cannabinus) leaves on cholelithiasis and urolithiasis. MethodsThe study evaluated the effect of aqueous leaf extract of H. cannabinus on thiouracil and cholesterol cholic acid diet induced cholelithiasis in BALB/c mice and ethylene glycol induced urolithiasis in Wistar rats. Three doses of aqueous extract (40, 80, and 160 mg/kg) were selected to evaluate the effectiveness in cholelithiasis in mice; another three doses of aqueous extract (400, 800, and 1600 mg/kg) were administered for evaluating the effect on urolithiasis in rats. Biochemical parameters such as biliary cholesterol, biliary phospholipid, and bile acid were determined in cholelithiasis model. Similarly, 24-hour urine output, urinary parameters such as creatinine, uric acid, protein, urea, presence of calcium oxalate crystals, red blood cells (RBCs), and pyuria were determined in urolithiasis model. ResultsStatistically significant differences were noted in the biliary and urinary parameters after administrating three test doses of H. cannabinus aqueous extract (P < 0.05). ConclusionH. cannabinus was found to be effective against high fat lithogenic diet urolithiasis and cholelithiasis.

Effects of Biliary Phospholipids on Cholesterol Crystallization and Growth in Gallstone Formation.

The prevalence of cholesterol gallstone disease is increasing, primarily due to the global epidemic of obesity associated with insulin resistance, and this trend leads to a considerable healthcare, financial, and social burden worldwide. Although phospholipids play an essential role in maintaining cholesterol solubility in bile through both mixed micelles and vesicles, little attention has been paid to the impact of biliary phospholipids on the pathogenesis of cholesterol gallstone formation. A reduction or deficiency of biliary phospholipids results in a distinctly abnormal metastable physical-chemical state of bile predisposing to supersaturation with cholesterol. Changes in biliary phospholipid concentrations influence cholesterol crystallization by yielding both liquid crystalline and "anhydrous" crystalline metastable intermediates, evolving into classical parallelogram-shaped cholesterol monohydrate crystals in supersaturated bile. As a result, five distinct crystallization pathways, A-E, have been defined, mainly based on the prime habits of liquid and solid crystals in the physiological or pathophysiological cholesterol saturation of gallbladder and hepatic bile. This review concisely summarizes the chemical structures and physical-chemical properties of biliary phospholipids and their physiological functions in bile formation and cholesterol solubility in bile, as well as comprehensively discusses the latest advances in the role of biliary phospholipids in cholesterol crystallization and growth in gallstone formation, largely based on the findings from clinical and animal studies and in vitro experiments. The insights gleaned from uncovering the cholelithogenic mechanisms are expected to form a fundamental framework for investigating the hitherto elusive events in the earliest stage of cholesterol nucleation and crystallization. This may help to identify better measures for early diagnosis and prevention in susceptible subjects and effective treatment of patients with gallstones.

S3198 Was It Always in the Genes? ABCB4 and Its Variable Presentations

Introduction: Progressive familial intrahepatic cholestasis (PFIC), low phospholipid-associated cholestasis (LPAC), and autosomal dominant intrahepatic cholestasis of pregnancy (ICP) are rare genetic disorders that should be considered in patients with elevated alkaline phosphatase and unrevealing primary work ups. We present a case of a 36-year-old female with a history of ICP who presented with elevated ALP, eventually found to have a nonspecific variant in the ABCB4 gene, concerning for Type 3 PFIC. Case Description/Methods: A 36-year-old female with a history of ICP treated with Ursodiol, current oral contraceptive use (OCP), and remote cholecystectomy presented to a gastroenterologist for asymptomatic ALP elevation. Initial labs revealed ALP 279 U/L, bile acids 9.9 μmol/L, and gamma-glutamyl transferase (GGT) 283U/L. Magnetic resonance cholangiopancreatography (MRCP) was unrevealing. Given no signs of hepatic dysfunction, portal hypertension, or steatosis on fibroscan, liver biopsy was obtained, which was unremarkable. Due to persistent elevation in ALP without a clear etiology and history of ICP, genetic testing was pursued. Genetic testing revealed a heterozygous variant of uncertain significance in the ABCB4 gene that is associated with PFIC type 3. OCPs were discontinued due to an association of ICP and OCP induced cholestasis. Ursodiol was restarted given high rates of recurrent cholelithiasis in patients with PFIC. Discussion: Type 3 PFIC, LPAC, and ICP are rare genetic disorders linked to unique mutations in the ABCB4 gene responsible for ALP elevation. The ABCB4 gene may manifest later in life as cholestasis, jaundice, or pruritus. Mutations in the ABCB4 gene can cause a reduction in biliary phospholipid concentration, leading to sludge, cholesterol stones, and microlithiasis. Type 3 PFIC has a unique presentation associated with elevated GGT, unseen in type 1 or 2. LPAC should be considered in patients presenting with cholelithiasis before the age of 40, recurrent cholithialsis after cholecystectomy, ICP, and family history of cholelithiasis. The variant of uncertain significance in our patient’s ABCB4 gene appeared to be a unifying diagnostic factor given her history of ICP, cholelithaisis at age 18 years requiring cholecystectomy, and persistent new elevation in ALP. She ultimately met criteria for Type 3 PFIC, LPAC and ICP. Given the rapidly evolving nature of medical genetics, reclassification and evaluation of genetic mutations requires follow up.

Nutritional assessment in patients with progressive familial intrahepatic cholestasis type 3

Rationale: Progressive familial intrahepatic cholestasis (PFIC) is a group of genetic diseases affecting the hepatocellular transport and bile formation. In PFIC3 there is a defect in the ABCB4 gene, which leads to impaired biliary phospholipid secretion, damaged hepatocytes and bile ducts, and heterogeneous clinical presentation. Growth impairment is often described in children with cholestatic liver diseases. The aim of our study was to assess the nutritional status in patients with PFIC3.

Pharmacological and Metabolic Significance of Bile Acids in Retinal Diseases.

Bile acids (BAs) are amphipathic sterols primarily synthesized from cholesterol in the liver and released in the intestinal lumen upon food intake. BAs play important roles in micellination of dietary lipids, stimulating bile flow, promoting biliary phospholipid secretion, and regulating cholesterol synthesis and elimination. Emerging evidence, however, suggests that, aside from their conventional biological function, BAs are also important signaling molecules and therapeutic tools. In the last decade, the therapeutic applications of BAs in the treatment of ocular diseases have gained great interest. Despite the identification of BA synthesis, metabolism, and recycling in ocular tissues, much remains unknown with regards to their biological significance in the eye. Additionally, as gut microbiota directly affects the quality of circulating BAs, their analysis could derive important information on changes occurring in this microenvironment. This review aims at providing an overview of BA metabolism and biological function with a focus on their potential therapeutic and diagnostic use for retinal diseases.

Mechanism of Taurohyodeoxycholate-induced Biliary Phospholipid Efflux -Understanding the Function of the ABCB4 Enhancer for Developing Therapeutic Agents against Bile Salt-induced Liver Injury

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Depletion of hepatic forkhead box O1 does not affect cholelithiasis in male and female mice

Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium‐taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B–mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP‐binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 −/− mice. Stimulatory effects of NTCP inhibition were maintained in Sr‐b1 −/− mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high‐density lipoprotein–derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

New treatments/targets for primary biliary cholangitis.

SummaryPrimary biliary cholangitis (PBC) is an autoimmune, cholestatic, chronic liver disease that ultimately progresses towards cirrhosis and liver failure if untreated. While ursodeoxycholic acid has been established as standard of care for PBC in the last few decades, significant advances in second-line treatment options have recently been made and new therapeutic developments are currently under evaluation. The purpose of this article is to provide the clinician with an overview of the current treatment options and future opportunities for patients with PBC.

A homozygous ABCB4 mutation causing an LPAC syndrome evolves into cholangiocarcinoma

Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. In the present study, we reported a case of a 63-year-old woman, who presented a biliary pain beginning at the age of 30, recurrent after cholecystectomy, along with “comet-tail shadows” revealed by ultrasonography thus, fulfilling the diagnosis of LPAC. This disease evolved into a cholangiocarcinoma. To understand the molecular basis of this phenotype, we performed the ABCB4 gene sequencing, followed by in silico analysis and Q-RT-PCR assay. The results displayed a homozygous missense sequence variation (c.140G > A, p.Arg47Gln), predicted as pathogenic according to MutPred. Accordingly, this gave rise to a decreased hepatic ABCB4 mRNA level and structural alterations of the mutated protein. Eventually, we reported, here, the first description of an ABCB4 missense mutation (p.Arg47Gln) at homozygous state in a Tunisian LPAC syndrome. An elucidation of its functional consequences was performed. Besides, this case suggests that the delayed diagnosis of LPAC syndrome and the lack of UDCA treatment may contribute in the development of complications, such as cholangiocarcinoma.

Lack of phospholipids in bile enhances cholesterol cholelithogenesis in the ATP‐binding cassette transporter B4 (Abcb4) knockout mice

BackgroundBiliary phospholipids play a key role in transporting cholesterol (Ch) by forming unilamellar and multilamellar vesicles that display a significantly higher ability to solubilize Ch in bile compared to simple and mixed micelles. The ATP‐binding cassette transporter B4 (ABCB4) is responsible for hepatic phospholipid secretion and its deletion in the liver causes a lack of phospholipids in bile of mice. Our aim is to explore whether targeted deletion of the Abcb4 gene in the liver leads to more rapid Ch crystallization and gallstone formation in Abcb4 knockout (KO) mice fed even a low Ch diet compared to wild‐type (WT) mice.MethodsThe critical role of biliary phospholipids in Ch crystallization and gallstone formation was investigated by physical‐chemical methods in male Abcb4 KO vs WT mice fed a chow diet (on day 0) and a special diet containing 0.1% Ch, 15% butterfat and 0.25% cholic acid for 4 wk.ResultsOn the chow diet (day 0), biliary phospholipids are 18.1±0.3 mM in pooled gallbladder bile of WT mice. However, no phospholipids are detected in Abcb4 KO mice fed either the chow or the special diet. After 1 wk of feeding the special diet, phase diagram analysis finds that lipid composition of pooled gallbladder bile of Abcb4 KO mice is located in crystallization region A. Moreover, many needle‐like Ch crystals, typical Ch monohydrate crystals, sandy stones and small gallstones (0.1–0.2 mm in diameter), but not liquid crystals, are detected in bile by phase contrast and polarizing light microscopy. This shows that lack of phospholipids in bile produces anhydrous crystalline metastable intermediates that rapidly evolve to Ch monohydrate crystals from supersaturated bile. In contrast, lipid composition of pooled gallbladder bile of WT mice is still within the micellar zone, indicating that bile is unsaturated with Ch. After 4 wk of feeding the special diet, lipid composition of pooled gallbladder bile of Abcb4 KO mice still stays in crystallization region A, with many larger gallstones (0.3–0.6 mm in diameter) being formed in the gallbladder. Notably, lipid composition of pooled gallbladder bile of WT mice enters crystallization region C, as analyzed by phase diagram. Thus, many liquid crystals (i.e., unilamellar and multilamellar vesicles) and classical solid plate‐like Ch monohydrate crystals, but not gallstones, are found in the gallbladder of WT mice. These results indicate that solid Ch crystals are formed through the liquid crystalline pathway in WT mice.ConclusionsLack of biliary phospholipids caused by the Abcb4 deletion in the mouse liver is a critical risk factor for Ch gallstone disease by significantly reducing Ch solubility in bile through disrupting the liquid crystalline pathway, whereas it leads to more rapid Ch crystallization via the anhydrous crystalline pathway. Our findings provide novel insight into the pathophysiological mechanisms of why Ch gallstones are rapidly formed in patients with the ABCB4 mutations, thereby leading to low phospholipid‐associated cholelithiasis, a rare biliary disease caused by a single‐gene mutation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Mechanisms for Protection of Hepatocytes against Bile Salt Cytotoxicity.

Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice

Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Ten-week-old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry. Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study. Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. The proliferation of liver cells was also reduced, which contributes to long-term correction of the phenotype. Further studies are required to evaluate whether this approach can be applied to patients with PFIC3.

Hydrophilic bile acids prevent liver damage caused by lack of biliary phospholipid in Mdr2−/− mice

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3), severe liver diseases associated with genetic defects in the biliary bile acid transporter bile salt export pump (BSEP; ABCB11) or phosphatidylcholine transporter multidrug resistance protein 3 (MDR3; ABCB4), respectively. Mdr2-/- mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the nonmicelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep-/- mice could protect Mdr2-/- mice from progressive liver damage. We generated double-KO (DKO: Bsep-/- and Mdr2-/- ) mice. Their bile acid composition resembles that of Bsep-/- mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBAs), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2-/- littermates. The livers of DKO mice have gene expression profiles very similar to Bsep-/- mice, with 4,410 of 6,134 gene expression changes associated with the Mdr2-/- mutation being suppressed. Feeding with THBAs partially alleviates liver damage in the Mdr2-/- mice. Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2-/- (PFIC3) mutation.

Cytoprotective effects of biliary phospholipids –Looking back on my academic life–

Cytoprotective effects of biliary phospholipids –Looking back on my academic life–

Na+‐taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice

Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+‐Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5‐diethoxycarbonyl‐1.4‐dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1‐G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC‐fed, Atp8b1‐G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC‐fed and Atp8b1‐G308V mice. NTCP‐inhibition increased plasma BS levels from 604±277 to 1746±719 μm in DDC‐fed mice, 432±280 to 762±288 μm in Atp8b1‐G308V mice and from 522±130 to 3625±378 μm in BDL mice. NTCP‐inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP‐inhibition reduced biliary BS output in DDC‐fed and Atp8b1‐G308V mice by ∼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP‐inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP‐inhibition were well tolerated. NTCP‐inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).

8-Methoxypsoralen disrupts MDR3-mediated phospholipids efflux and bile acid homeostasis and its relevance to hepatotoxicity.

Since its discovery in 1987, multidrug resistance 3 P-glycoprotein (MDR3) had recognized to play a crucial role in the translocation of phospholipids from the inner to outer leaflets of bile canalicular membranes. An increasing number of reports suggest that drug-mediated functional disruption of MDR3 is responsible for drug-induced cholestasis. 8-Methoxypsoralen (8-MOP) is used clinically to treat psoriasis, vitiligo and other skin disorders. However, psoralens safety for long-term use is a concern. In the current study, we evaluate 8-MOP's potential hepatotoxicity and effects on bile formation. Sprague Dawley (SD) rats were treated daily 200mg/kg or 400mg/kg of 8-MOP orally for 28 days. The result showed a prominent decrease in biliary phospholipids output, which associated with the down-regulation of MDR3. Elevated bile acid serum level and increased biliary bile acid outputs were observed in 8-MOP-treated groups. The disturbance of bile acid homeostasis was associated with changes in enzymes and proteins involved in bile acid synthesis, regulation and transport. Human liver cell line L02 was used to determine on the mRNA and protein levels of MDR3. Cells treated with 8-MOP reveled a decrease in fluorescent PC (phosphatidylcholine) secretion into the pseudocanaliculi (formed between adjacent cells) compared with untreated cells. Our investigation represent the first evidence that 8-MOP can induce cholestatic liver injury by disturbing MDR3-mediated phospholipids efflux and bile acid homeostasis.

Intraduodenal infusion of cyanidin-3-glucoside transiently promotes triglyceride excretion into bile in rats

Flavonoids purportedly have a role in improving lipid metabolism. In our preliminary study, highly concentrated flavonoid metabolites appeared in bile juice in rats, which also contains various lipids. Biliary flavonoid metabolites generally have amphiphilic properties, may influence lipid solubility, and possibly contribute to the improvement of dyslipidemia. However, the influence of biliary flavonoid metabolites on the biliary lipid profile is not well known. Therefore, we hypothesized that the amphiphilic property of biliary flavonoid metabolites alters biliary lipid profiles. To estimate the influence of flavonoids on the biliary lipid profile, we laparotomized rats under anesthesia, intraduodenally injected them with cyanidin-3-glucoside chloride (C3G) or quercetin, and analyzed their biliary metabolite concentrations for 2 hours. Concentrations of C3G and quercetin metabolites peaked at 30 minutes after the injection; those of quercetin were 6 to 10 times higher than those of C3G throughout the sampling period up to 2 hours. Biliary triglyceride (TG) concentrations were higher in the C3G group at 30 and 45 minutes; biliary cholesterol and phospholipid concentrations were lower in the quercetin group at 30 minutes than those in the control group. Hepatic TG content after the 2-hour sampling was lower in the C3G group than in the control group. These results suggest that C3G, but not quercetin, may transiently promote TG excretion into bile, with a reduction in hepatic TG content. This C3G effect may be involved in improvement of TG metabolism.