Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium-dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na+ -taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na+ -taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium-dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.

Symptom patterns in the daily life of PSC patients.

BACKGROUND & AIMS: Patients with primary sclerosing cholangitis (PSC) may suffer from complaints such as pruritus, right upper abdominal quadrant pain (RUQ-A) and fatigue. However, the severity of these complaints, daily and/or seasonal patterns and other factors of influence in PSC are largely unknown. The aim of this study is to assess daily symptoms and patterns thereof in PSC patients in their natural setting. METHODS: A mobile application was designed according to the experience sampling method. Push notifications with a response time of max 4 h were sent during tiers of 3 months. Questions comprised VAS scales on degree of pruritus, fatigue, RUQ-A, time of the day these symptoms were worst, as well as time of intake of medication. Linear mixed modelling was used to identify patient- and external factors associated with pruritus, fatigue and RUQ-A pain. RESULTS: A total of 6713 questionnaires were completed by 137 patients. Fatigue was the most prevalent symptom among PSC patients being reported in a striking 71% of measurements, followed by pruritus (38%). Both increased during the day and were associated with longer disease duration. A highly significant correlation between pruritus and day temperature was observed (ρ = -0.14, p = .000), and itch was generally worse during winter (p = .000). Patient preference for the tool was high. CONCLUSION: Pruritus and fatigue are prevalent symptoms in the daily life of PSC patients and show a distinct diurnal pattern. This may have implications for efficient dosing of anti-pruritic agents. The level of pruritus is highly correlated with day temperature, which may have several implications.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia.

Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state.

The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.

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.

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).

Effect of ileal bile acid transporter inhibitor GSK2330672 on pruritus in primary biliary cholangitis: a double-blind, randomised, placebo-controlled, crossover, phase 2a study.

BACKGROUND: Up to 70% of patients with primary biliary cholangitis develop pruritus (itch) during the course of their disease. Treatment of pruritus in primary biliary cholangitis is challenging and novel therapies are needed. Ursodeoxycholic acid, the standard first-line treatment for primary biliary cholangitis, is largely ineffective for pruritus. We investigated the efficacy and safety of GSK2330672, a selective inhibitor of human ileal bile acid transporter (IBAT), in patients with primary biliary cholangitis with pruritus. METHODS: We conducted this phase 2a, double-blind, randomised, placebo-controlled, crossover trial in two UK medical centres. Following 2 weeks of open placebo run-in, patients were randomly assigned in a 1:1 ratio with a block size of 4 to receive GSK2330672 or placebo twice daily during two consecutive 14-day treatment periods in a crossover sequence. The treatment periods were followed by a 14-day single-blinded placebo follow-up period. The primary endpoints were safety of GSK2330672, assessed using clinical and laboratory parameters, and tolerability as rated by the Gastrointestinal Symptom Rating Scale. The secondary endpoints were changes in pruritus scores measured using the 0 to 10 numerical rating scale (NRS), primary biliary cholangitis-40 (PBC-40) itch domain score and 5-D itch scale, changes in serum total bile acids and 7 alpha hydroxy-4-cholesten-3-one (C4), and changes in the pharmacokinetic parameters of ursodeoxycholic acid and its conjugates. The trial was registered with ClinicalTrials.gov, number NCT01899703. FINDINGS: Between March 10, 2014, and Oct 7, 2015, we enrolled 22 patients. 11 patients were assigned to receive intervention followed by placebo (sequence 1), and 11 patients were assigned to receive placebo followed by intervention (sequence 2). One patient assigned to sequence 2 withdrew consent prior to receiving randomised therapy. One patient did not attend the placebo follow-up period, but was included in the final analysis. GSK2330672 treatment for 14 days was safe with no serious adverse events reported. Diarrhoea was the most frequent adverse event during treatment with GSK2330672 (seven with GSK2330672 vs one with placebo) and headache was the most frequent adverse event during treatment with placebo (seven with placebo vs six with GSK2330672). After GSK2330672 treatment, the percentage changes from baseline itch scores were -57% (95% CI -73 to -42, p<0·0001) in the NRS, -31% (-42 to -20, p<0·0001) in the PBC-40 itch domain and -35% (-45 to -25, p<0·0001) in the 5-D itch scale. GSK2330672 produced significantly greater reduction from baseline than the double-blind placebo in the NRS (-23%, 95% CI -45 to -1; p=0·037), PBC-40 itch domain, (-14%, -26 to -1; p=0·034), and 5-D itch scale (-20%, -34 to -7; p=0·0045). After GSK2330672 treatment, serum total bile acid concentrations declined by 50% (95% CI -37 to -61, p<0·0001) from 30 to 15 µM, with a significant 3·1-times increase (95% CI 2·4 to 4·0, p<0·0001) in serum C4 concentrations from 7·9 to 24·7ng/mL. INTERPRETATION: In patients with primary biliary cholangitis with pruritus, 14 days of ileal bile acid transporter inhibition by GSK2330672 was generally well tolerated without serious adverse events, and demonstrated efficacy in reducing pruritus severity. GSK2330672 has the potential to be a significant and novel advance for the treatment of pruritus in primary biliary cholangitis. Diarrhoea, the most common adverse event associated with GSK2330672 treatment, might limit the long-term use of this drug. FUNDING: GlaxoSmithKline and National Institute for Health Research.

Intestinal Farnesoid X Receptor Controls Transintestinal Cholesterol Excretion in Mice.

BACKGROUND & AIMS: The role of the intestine in the maintenance of cholesterol homeostasis increasingly is recognized. Fecal excretion of cholesterol is the last step in the atheroprotective reverse cholesterol transport pathway, to which biliary and transintestinal cholesterol excretion (TICE) contribute. The mechanisms controlling the flux of cholesterol through the TICE pathway, however, are poorly understood. We aimed to identify mechanisms that regulate and stimulate TICE. METHODS: We performed studies with C57Bl/6J mice, as well as with mice with intestine-specific knockout of the farnesoid X receptor (FXR), mice that express an FXR transgene specifically in the intestine, and ABCG8-knockout mice. Mice were fed a control diet or a diet supplemented with the FXR agonist PX20606, with or without the cholesterol absorption inhibitor ezetimibe. Some mice with intestine-specific knockout of FXR were given daily injections of fibroblast growth factor (FGF)19. To determine fractional cholesterol absorption, mice were given intravenous injections of cholesterol D5 and oral cholesterol D7. Mice were given 13C-acetate in drinking water for measurement of cholesterol synthesis. Bile cannulations were performed and biliary cholesterol secretion rates were assessed. In a separate set of experiments, bile ducts of male Wistar rats were exteriorized, allowing replacement of endogenous bile by a model bile. RESULTS: In mice, we found TICE to be regulated by intestinal FXR via induction of its target gene Fgf15 (FGF19 in rats and human beings). Stimulation of this pathway caused mice to excrete up to 60% of their total cholesterol content each day. PX20606 and FGF19 each increased the ratio of muricholate:cholate in bile, inducing a more hydrophilic bile salt pool. The altered bile salt pool stimulated robust secretion of cholesterol into the intestinal lumen via the sterol-exporting heterodimer adenosine triphosphate binding cassette subfamily G member 5/8 (ABCG5/G8). Of note, the increase in TICE induced by PX20606 was independent of changes in cholesterol absorption. CONCLUSIONS: Hydrophilicity of the bile salt pool, controlled by FXR and FGF15/19, is an important determinant of cholesterol removal via TICE. Strategies that alter bile salt pool composition might be developed for the prevention of cardiovascular disease. Transcript profiling: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=irsrayeohfcntqx&acc=GSE74101.

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity.

BACKGROUND/AIMS: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective. METHODS: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription. RESULTS: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible. CONCLUSION: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.

Role of the bicarbonate-responsive soluble adenylyl cyclase in cholangiocyte apoptosis in primary biliary cholangitis; a new hypothesis.

Primary biliary cholangitis (PBC) is a chronic fibrosing cholangiopathy characterized by an autoimmune stereotype and defective biliary bicarbonate secretion due to down-regulation of anion exchanger 2 (AE2). Despite the autoimmune features, immunosuppressants are ineffective while two bile acid-based therapies (ursodeoxycholic acid and obeticholic acid) have been shown to improve biochemical and histological features of cholestasis and long-term prognosis. However, the etiology and pathogenesis of PBC is largely unknown. Recently, it has been shown that microRNA-506 (miR-506) on chromosome X is up-regulated in PBC cholangiocytes and suppresses AE2 expression, which sensitizes cholangiocytes to bile salt-induced apoptosis by activating soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. In this review, we discuss the experimental evidence for the emerging role of the miR-506-AE2-sAC axis in PBC pathogenesis. We further hypothesize that the initial disease trigger induces an X-linked epigenetic change, leading to a female-biased activation of the miR-506-AE2-sAC axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

Knockout of the primary sclerosing cholangitis-risk gene Fut2 causes liver disease in mice.

The etiopathogenesis of primary sclerosing cholangitis is unknown. Genetic variants of fucosyltransferase 2 (FUT2) have been identified in genome-wide association studies as risk factors for primary sclerosing cholangitis. We investigated the role of Fut2 in murine liver pathophysiology by studying Fut2-/- mice. Fut2-/- mice were viable and fertile, had lower body weight than wild-type (wt) littermates and gray fur. Half of the Fut2-/- mice showed serum bile salt levels 40 times higher than wt (Fut2-/-high ), whereas the remainder were normocholanemic (Fut2-/-low ). Fut2-/- mice showed normal serum liver tests, bile flow, biliary bile salt secretion, fecal bile salt loss, and expression of major hepatocellular bile salt transporters and cytochrome P450 7a1, the key regulator of bile salt synthesis, indicating that elevated serum bile salts in Fut2-/-high mice were not explained by cholestasis. Fut2-/-high mice, but not Fut2-/-low mice, were sensitive to hydrophobic bile salt feeding (0.3% glycochenodeoxycholate); they rapidly lost weight and showed elevation of serum liver tests (alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase) and areas of liver parenchymal necrosis. Histomorphological evaluation revealed the presence of paraportal shunting vessels, increased numbers of portal vascular structures, wall thickening of some portal arteries, and periductal fibrosis in Fut2-/-high mice more than Fut2-/-low mice and not wt mice. Unconjugated bilirubin and ammonia were or tended to be elevated in Fut2-/-high mice only. Portosystemic shunting was demonstrated by portal angiography, which disclosed virtually complete portosystemic shunting in Fut2-/-high mice, discrete portosystemic shunting in Fut2-/-low mice, and no shunting in wt littermates. CONCLUSION: Liver pathology in Fut2-/- mice is dominated by consequences of portosystemic shunting resulting in microcirculatory disturbances, mild (secondary) periductal fibrosis, and sensitivity toward human bile salt toxicity. (Hepatology 2017;66:542-554).

Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. CONCLUSION: NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4.

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the ß-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.

Enteroendocrine cells are a potential source of serum autotaxin in men.

OBJECTIVE: Serum autotaxin (ATX) activity is significantly increased in cholestatic patients. Our study aimed to unravel the source(s) of ATX in cholestasis. MATERIALS AND METHODS: ATX activity and protein were measured in sera of healthy (n=33) and cholestatic patients (n=152), including women with intrahepatic cholestasis of pregnancy. ATX mRNA and protein expression were analyzed in various tissues from mice and men. Induction of ATX activity was assessed in mouse models of extrahepatic (bile duct ligation) and intrahepatic cholestasis (Atp8b1(G308V/G308V), 0.1% cholate-supplemented diet). ATX clearance in cholestatic and control mice was assessed after intravenous injection of recombinant ATX. Human hepatic clearance was estimated by comparing ATX activity in portal and hepatic vein serum. RESULTS: Serum ATX activity and ATX protein concentration tightly correlated under all conditions in patients and controls (p<0.0001). In humans Atx mRNA was highly expressed in small intestine, whereas in mice Atx was expressed mainly in brain and placenta but not in small intestine. Extensive ATX protein expression was identified in human, but not murine intestinal enteroendocrine cells. In murine models of cholestasis and cholestatic pregnancy plasma ATX activity was only mildly elevated (up to 2.1-fold). Atx tissue expression and rATX clearance after parenteral administration did not differ between cholestatic and control mice. CONCLUSION: Serum ATX activity during cholestasis and itch is enhanced by increased protein concentration rather than enzymatic induction. In mice, clearance of ATX is not affected by cholestasis. Small intestinal ATX expression by enteroendocrine cells might represent an important source of cholestasis-induced serum ATX activity in men.

The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator.

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.

Bile acid receptor agonists INT747 and INT777 decrease oestrogen deficiency-related postmenopausal obesity and hepatic steatosis in mice.

Menopause is often followed by obesity and, related to this, non-alcoholic fatty liver disease (NAFLD). Two bile acid (BA) receptors, farnesoid X receptor (FXR) and G-protein-coupled receptor TGR5, have emerged as putative therapeutic targets for obesity and NAFLD. AIM OF THIS STUDY: to evaluate the efficacy of selective agonists INT747/obeticholic acid (FXR) and INT777 (TGR5) as novel treatments for the metabolic effects of oestrogen deficiency. Ovariectomized (OVX) or sham-operated (SHAM) mice were fed a high-fat diet (HFD) for 5weeks. During the last 4weeks two groups of OVX and SHAM mice received either INT747- or INT777-supplemented HFD. OVX mice had significantly higher bodyweight gain than SHAM mice, which was attenuated by INT747- or INT777-treatment. No significant changes in food intake or physical activity were found. OVX mice had significantly lower energy expenditure than SHAM mice; INT747- and INT777-treated OVX mice had intermediate energy expenditure. Liver triglyceride and cholesterol content was significantly increased in OVX compared to SHAM mice, which was normalized by INT747- or INT777-treatment. Significant changes in metabolic gene expression were found in liver (Cpt1, Acox1), muscle (Ucp3, Pdk4, Cpt1, Acox1, Fasn, Fgf21), brown adipocytes (Dio2) and white adipocytes (c/EBPα, Pparγ, Adipoq). For the first time, expression of FXR and induction of its target gene Pltp1 was shown in skeletal muscle. BA receptor agonists are suitable therapeutics to correct postmenopausal metabolic changes in an OVX mouse model. Potential mechanisms include increased energy expenditure and changes in expression patterns of key metabolic genes in liver, muscle and adipose tissues.

ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.

UNLABELLED: ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ∼65% was conjugated and ∼35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na(+) -taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels. CONCLUSIONS: ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome. (Hepatology 2016;64:161-174).

The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic acid and reduced expression of anion exchanger 2 (AE2) on canalicular membranes and small bile ducts underline the importance of bicarbonate transportation in its disease mechanism. Soluble adenylyl cyclase (sAC; ADCY10) is an evolutionarily conserved bicarbonate sensor that regulates apoptosis, barrier function and TNF signaling. Key Messages: The biliary epithelium defends against the toxic bile by bicarbonate secretion and by maintaining a tight barrier. Passive diffusion of weak acid conjugates (e.g. bile salts and other toxins) across plasma membrane is pH-dependent. Reduced AE2 expression results in both reduced bicarbonate secretion and accumulation of bicarbonate in the cells. Increased intracellular bicarbonate leads to increased sAC activity, which regulates bile salt-induced apoptosis. Reduced bicarbonate secretion causes more bile salts to enter cells, which further increase sAC activity by releasing intracellular Ca2+ store. In vitro studies demonstrate that inhibition of sAC not only corrects sensitization to bile salt-induced apoptosis as a result of AE2 down-regulation but also prevents bile salt-induced apoptosis altogether. Targeting sAC is also likely to slow down disease progression by strengthening the barrier function of biliary epithelia and by reducing oxidative stress as a result of chronic inflammation. CONCLUSIONS: sAC is a potential therapeutic target for PBC. More in vitro and in vivo studies are needed to understand how sAC regulates bile salt-induced apoptosis and to establish its therapeutic value in PBC and other cholestatic cholangiopathies.