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

FDA-drug screening identifies deptropine inhibiting hepatitis E virus involving the NF-κB-RIPK1-caspase axis

Hepatitis E virus (HEV) infection is the leading cause of acute hepatitis worldwide and can develop into chronic infection in immunocompromised patients, promoting the development of effective antiviral therapies. In this study, we performed a screening of a library containing over 1,000 FDA-approved drugs. We have identified deptropine, a classical histamine H1 receptor antagonist used to treat asthmatic symptoms, as a potent inhibitor of HEV replication. The anti-HEV activity of deptropine appears dispensable of the histamine pathway, but requires the inhibition on nuclear factor-κB (NF-κB) activity. This further activates caspase mediated by receptor-interacting protein kinase 1 (RIPK1) to restrict HEV replication. Given deptropine being widely used in the clinic, our results warrant further evaluation of its anti-HEV efficacy in future clinical studies. Importantly, the discovery that NF-κB-RIPK1-caspase pathway interferes with HEV infection reveals new insight of HEV-host interactions

Developments in bile salt based therapies: A critical overview

Bile acids, amphipathic molecules known for their facilitating role in fat absorption, are also recognized as signalling molecules acting via nuclear and membrane receptors. Of the bile acid-activated receptors, the Farnesoid X Receptor (FXR) and the G protein-coupled bile acid receptor-1 (Gpbar1 or TGR5) have been studied most extensively. Bile acid signaling is critical in the regulation of bile acid metabolism itself, but it also plays a significant role in glucose, lipid and energy metabolism. Activation of FXR and TGR5 leads to reduced hepatic bile salt load, improved insulin sensitivity and glucose regulation, increased energy expenditure, and anti-inflammatory effects. These beneficial effects render bile acid signaling an interesting therapeutic target for the treatment of diseases such as cholestasis, non-alcoholic fatty liver disease, and diabetes. Here, we summarize recent findings on bile acid signaling and discuss potential and current limitations of bile acid receptor agonist and modulators of bile acid transport as future therapeutics for a wide-spectrum of diseases

Mechanistic insights into the inhibition of NTCP by myrcludex B

Background & aims: The sodium taurocholate co-transporting polypeptide (NTCP) is the entry receptor for the hepatitis B and delta virus (HBV/HDV) and the main hepatic uptake transporter of conjugated bile acids. Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, blocks HBV/HDV infection and inhibits NTCP-mediated bile acid uptake. In humans this increases systemic bile acid levels, which remain elevated for hours even after Myrcludex B is cleared from the circulation. Here, we investigated the dynamics of Myrcludex B-induced NTCP-mediated bile acid transport inhibition in mice and if/how the duration of this effect relates to NTCP protein turnover. Methods: Plasma bile acids were determined in Myrcludex B-treated OATP1a/1b-deficient mice. In vitro, plasma membrane-resident NTCP was labeled with biotin or fluorescein isothiocyanate (FITC)-labeled Myrcludex B and traced in time using hNTCP-overexpressing U2OS cells. Förster resonance energy transfer by fluorescent lifetime imaging microscopy was used to investigate whether Myrcludex B can transfer to newly synthesized NTCP. Results: Conjugated bile salt levels in plasma peaked 4 h after subcutaneous Myrcludex B administration. After 24 h, plasma bile salt levels were completely normalized, in line with restored NTCP-mediated bile acid transport in vitro. Biotin-labeled NTCP disappeared faster than Myrcludex B-FITC, with almost 40% of FITC signal remaining after 24 h. FITC fluorescence lifetime was strongly decreased upon expression of DY547-labeled acyl carrier protein-tagged NTCP, demonstrating transfer of pre-bound Myrcludex B-FITC to newly formed NTCP. Conclusions: The dynamics of NTCP protein turnover and Myrcludex B-induced plasma bile salt elevations are similar, suggesting that the Myrcludex B:NTCP interaction is very long-lived. Nevertheless, Myrcludex B is not completely degraded together with NTCP and can transfer to newly synthesized NTCP. Lay summary: The experimental drug Myrcludex B binds the sodium taurocholate co-transporting polypeptide (NTCP), the viral entry receptor for the hepatitis B and D virus (HBV/HDV), and thereby prevents infection, but also inhibits hepatic bile salt uptake leading to transiently elevated bile salt levels. This study describes that while the normalization of plasma bile salt levels likely depends on the protein turnover rate of NTCP, Myrcludex B partly escapes co-degradation with NTCP by transferring from one NTCP molecule to another. This is of importance to the HBV/HDV research field as it provides a potential explanation for the distinct kinetics and dose-dependence of Myrcludex B's effects on viral infection versus bile salt transport

Intestinal explant barrier chip: Long-term intestinal absorption screening in a novel microphysiological system using tissue explants

The majority of intestinal in vitro screening models use cell lines that do not reflect the complexity of the human intestinal tract and hence often fail to accurately predict intestinal drug absorption. Tissue explants have intact intestinal architecture and cell type diversity, but show short viability in static conditions. Here, we present a medium throughput microphysiological system, Intestinal Explant Barrier Chip (IEBC), that creates a dynamic microfluidic microenvironment and prolongs tissue viability. Using a snap fit mechanism, we successfully incorporated human and porcine colon tissue explants and studied tissue functionality, integrity and viability for 24 hours. With a proper distinction of transcellular over paracellular transport (ratio >2), tissue functionality was good at early and late timepoints. Low leakage of FITC-dextran and preserved intracellular lactate dehydrogenase levels indicate maintained tissue integrity and viability, respectively. From a selection of low to high permeability drugs, 6 out of 7 properly ranked according to their fraction absorbed. In conclusion, the IEBC is a novel screening platform benefitting from the complexity of tissue explants and the flow in microfluidic chips

The progress of intestinal epithelial models from cell lines to gut-on-chip

Over the past years, several preclinical in vitro and ex vivo models have been developed that helped to understand some of the critical aspects of intestinal functions in health and disease such as inflammatory bowel disease (IBD). However, the translation to the human in vivo situation remains problematic. The main reason for this is that these approaches fail to fully reflect the multifactorial and complex in vivo environment (e.g., including microbiota, nutrition, and immune response) in the gut system. Although conventional models such as cell lines, Ussing chamber, and the everted sac are still used, increasingly more sophisticated intestinal models have been developed over the past years including organoids, InTESTine™ and microfluidic gut-on-chip. In this review, we gathered the most recent insights on the setup, advantages, limitations, and future perspectives of most frequently used in vitro and ex vivo models to study intestinal physiology and functions in health and disease

Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity

Background & Aims: Bile acids are important metabolic signaling molecules. Bile acid receptor activation promotes body weight loss and improves glycemic control. The incretin hormone GLP-1 and thyroid hormone activation of T4 to T3 have been suggested as important contributors. Here, we identify the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as target to prolong postprandial bile acid signaling. Methods: Organic anion transporting polypeptide (OATP)1a/1b KO mice with or without reconstitution with human OATP1B1 in the liver were treated with the NTCP inhibitor Myrcludex B for 3.5 weeks after the onset of obesity induced by high fat diet-feeding. Furthermore, radiolabeled T4 was injected to determine the role of NTCP and OATPs in thyroid hormone clearance from plasma. Results: Inhibition of NTCP by Myrcludex B in obese Oatp1a/1b KO mice inhibited hepatic clearance of bile acids from portal and systemic blood, stimulated GLP-1 secretion, reduced body weight, and decreased (hepatic) adiposity. NTCP inhibition did not affect hepatic T4 uptake nor lead to increased thyroid hormone activation. Myrcludex B treatment increased fecal energy output, explaining body weight reductions amongst unaltered food intake and energy expenditure. Conclusions: Pharmacologically targeting hepatic bile acid uptake to increase bile acid signaling is a novel approach to treat obesity and induce GLP1- secretion

Role of the bile acid transporter SLC10A1 in liver targeting of the lipid-lowering thyroid hormone analog eprotirome

The thyroid hormone (TH) analog eprotirome (KB2115) was developed to lower cholesterol through selective activation of the TH receptor (TR) b1 in the liver. Interestingly, eprotirome shows low uptake in nonhepatic tissues, explaining its lipid-lowering action without adverse extrahepatic thyromimetic effects. Clinical trials have shown marked decreases in serum cholesterol levels. We explored the transport of eprotirome across the plasma membrane by members of three TH transporter families: monocarboxylate transporters MCT8 and MCT10; Na-independent organic anion transporters 1A2, 1B1, 1B3, 1C1, 2A1, and 2B1; and Na-dependent organic anion transporters SLC10A1 to SLC10A7. Cellular transport was studied in transfected COS1 cells using [14C]eprotirome and [125I]TH analogs. Of the 15 transporters tested initially, the liver-specific bile acid transporter SLC10A1 showed the highest eprotirome uptake (greater than a sevenfold induction after 60 minutes) as well as TRb1-mediated transcriptional activity. Uptake of eprotirome by SLC10A1 was Na+ dependent and saturable with a Michaelis constant of 8 mM. Eprotirome transport was inhibited by known substrates for SLC10A1 (e.g., cholate and taurocholate), and by TH analogs such as triiodothyropropionic acid and triiodothyroacetic acid. However, no significant SLC10A1-mediated transport was observed of these [125I]TH analogs. We also studied the plasma disappearance and biliary excretion of [14C]eprotirome injected in control and Slc10a1 knockout mice. Although eprotirome is also transported by mouse Slc10a1, the pharmacokinetics of eprotirome were not affected by Slc10a1 deficiency. In conclusion, we have demonstrated that the liver-specific bile acid transporter SLC10A1 effectively transports eprotirome. However, Slc10a1 does not appear to be critical for the liver targeting of this TH analog inmice. Therefore, the importance of SLC10A1 for liver uptake of eprotirome in humans remains to be elucidated. (Endocrinology 158: 3307-3318, 2017)

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines

<div><p>Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the <i>in-vitro</i> maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.</p></div

DMF-culturing does not improve the polarization of the HepaRG cells.

<p><b>(A-B)</b> Staining of hepatic transporters OATP1a1(green) and MRP2 (red) with DAPI counter-staining for the nuclei (blue). <b>(C)</b> Visualization of CFDA (green) with DAPI counter-staining of the nuclei (blue), scale bar = 50μm.</p