Transgenic mice lacking FGF15/19-SHP phosphorylation display altered bile acids and gut bacteria, promoting nonalcoholic fatty liver disease.

Dysregulated bile acid (BA)/lipid metabolism and gut bacteria dysbiosis are tightly associated with the development of obesity and non-alcoholic fatty liver disease (NAFLD). The orphan nuclear receptor, Small Heterodimer Partner (SHP/NR0B2), is a key regulator of BA/lipid metabolism, and its gene-regulating function is markedly enhanced by phosphorylation at Thr-58 mediated by a gut hormone, fibroblast growth factor-15/19 (FGF15/19). To investigate the role of this phosphorylation in whole-body energy metabolism, we generated transgenic SHP-T58A knock-in mice. Compared with wild-type (WT) mice, the phosphorylation-defective SHP-T58A mice gained weight more rapidly with decreased energy expenditure and increased lipid/BA levels. This obesity-prone phenotype was associated with the upregulation of lipid/BA synthesis genes and downregulation of lipophagy/ß-oxidation genes. Mechanistically, defective SHP phosphorylation selectively impaired its interaction with LRH-1, resulting in de-repression of SHP/LRH-1 target BA/lipid synthesis genes. Remarkably, BA composition and selective gut bacteria which are known to impact obesity, were also altered in these mice. Upon feeding a high-fat diet, fatty liver developed more severely in SHP-T58A mice compared to WT mice. Treatment with antibiotics substantially improved the fatty liver phenotypes in both groups but had greater effects in the T58A mice so that the difference between the groups was largely eliminated. These results demonstrate that defective phosphorylation at a single nuclear receptor residue can impact whole-body energy metabolism by altering BA/lipid metabolism and gut bacteria, promoting complex metabolic disorders like NAFLD. Since posttranslational modifications generally act in gene- and context-specific manners, the FGF15/19-SHP phosphorylation axis may allow more targeted therapy for NAFLD.

Transcriptional Control of Trpm6 by the Nuclear Receptor FXR.

Farnesoid x receptor (FXR) is a nuclear bile acid receptor that belongs to the nuclear receptor superfamily. It plays an essential role in bile acid biosynthesis, lipid and glucose metabolism, liver regeneration, and vertical sleeve gastrectomy. A loss of the FXR gene or dysregulations of FXR-mediated gene expression are associated with the development of progressive familial intrahepatic cholestasis, tumorigenesis, inflammation, and diabetes mellitus. Magnesium ion (Mg2+) is essential for mammalian physiology. Over 600 enzymes are dependent on Mg2+ for their activity. Here, we show that the Trpm6 gene encoding a Mg2+ channel is a direct FXR target gene in the intestinal epithelial cells of mice. FXR expressed in the intestinal epithelial cells is absolutely required for sustaining a basal expression of intestinal Trpm6 that can be robustly induced by the treatment of GW4064, a synthetic FXR agonist. Analysis of FXR ChIP-seq data revealed that intron regions of Trpm6 contain two prominent FXR binding peaks. Among them, the proximal peak from the transcription start site contains a functional inverted repeat 1 (IR1) response element that directly binds to the FXR-RXRα heterodimer. Based on these results, we proposed that an intestinal FXR-TRPM6 axis may link a bile acid signaling to Mg2+ homeostasis.

NPC1L1 Deficiency Suppresses Ileal Fibroblast Growth Factor 15 Expression and Increases Bile Acid Pool Size in High-Fat-Diet-Fed Mice.

Niemann-Pick C1-like 1 (NPC1L1) mediates intestinal uptake of dietary and biliary cholesterol and is the target of ezetimibe, a cholesterol absorption inhibitor used to treat hypercholesterolemia. Genetic deletion of NPC1L1 or ezetimibe treatment protects mice from high-fat diet (HFD)-induced obesity; however, the molecular mechanisms responsible for this therapeutic benefit remain unknown. A major metabolic fate of cholesterol is its conversion to bile acids. We found that NPC1L1 knockout (L1-KO) mice fed an HFD had increased energy expenditure, bile acid pool size, and fecal bile acid excretion rates. The elevated bile acid pool in the HFD-fed L1-KO mice was enriched with tauro-ß-muricholic acid. These changes in the L1-KO mice were associated with reduced ileal mRNA expression of fibroblast growth factor 15 (FGF15) and increased hepatic mRNA expression of cholesterol 7α-hydroxylase (Cyp7A1) and mitochondrial sterol 27-hydroxylase (Cyp27A1). In addition, mRNA expression of the membrane bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) and type 2 iodothyronine deiodinase (Dio2) were elevated in brown adipose tissue of L1-KO mice, which is known to promote energy expenditure. Thus, altered bile acid homeostasis and signaling may play a role in protecting L1-KO mice against HFD-induced obesity.

Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro.

The well-established 3D organoid culture method enabled efficient expansion of cholangiocyte-like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal-invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD- and EHBD-tissue. We demonstrate successful organoid-initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional-specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP-derived BCOs and gallbladder-derived BCOs expressing gallbladder-specific genes. Moreover, BCOs have limited hepatocyte-fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid-initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte-like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid-initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine.

Genetic and environmental influences on serum oxylipins, endocannabinoids, bile acids and steroids.

Lipid bioactivity is a result of direct action and the action of lipid mediators including oxylipins, endocannabinoids, bile acids and steroids. Understanding the factors contributing to biological variation in lipid mediators may inform future approaches to understand and treat complex metabolic diseases. This research aims to determine the contribution of genetic and environmental influences on lipid mediators involved in the regulation of inflammation and energy metabolism. This study recruited 138 monozygotic (MZ) and dizygotic (DZ) twins aged 18-65 years and measured serum oxylipins, endocannabinoids, bile acids and steroids using liquid chromatography mass-spectrometry (LC-MS). In this classic twin design, the similarities and differences between MZ and DZ twins are modelled to estimate the contribution of genetic and environmental influences to variation in lipid mediators. Heritable lipid mediators included the 12-lipoxygenase products 12-hydroxyeicosatetraenoic acid [0.70 (95% CI: 0.12,0.82)], 12-hydroxyeicosatetraenoic acid [0.73 (95% CI: 0.30,0.83)] and 14­hydroxy-docosahexaenoic acid [0.51 (95% CI: 0.07,0.71)], along with the endocannabinoid docosahexaenoy-lethanolamide [0.52 (95% CI: 0.15,0.72)]. For others such as 13-hydroxyoctadecatrienoic acid and lithocholic acid the contribution of environment to variation was stronger. With increased understanding of lipid mediator functions in health, it is important to understand the factors contributing to their variance. This study provides a comprehensive analysis of lipid mediators and extends pre-existing knowledge of the genetic and environmental influences on the human lipidome.

The spectrum of Progressive Familial Intrahepatic Cholestasis diseases: Update on pathophysiology and emerging treatments.

The Progressive Familial Intrahepatic Cholestasis (PFIC) disease spectrum encompasses a variety of genetic diseases that affect the bile production and the secretion of bile acids. Typically, the first presentation of these diseases is in early childhood, frequently followed by a severe course necessitating liver transplantation before adulthood. Except for transplantation, treatment modalities have been rather limited and frequently only aim at the symptoms of cholestasis, such as cholestatic pruritus. In recent years, progress has been made in understanding the pathophysiology of these diseases and new treatment modalities have been emerging. Herewith we summarize the latest developments in the field and formulate the current key questions and opportunities for further progress.

Alterations in bile acid metabolizing gut microbiota and specific bile acid genes as a precision medicine to subclassify NAFLD.

Multiple mechanisms for the gut microbiome contributing to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) have been implicated. Here, we aim to investigate the contribution and potential application for altered bile acids (BA) metabolizing microbes in NAFLD by post hoc analysis of whole metagenome sequencing (WMS) data. The discovery cohort consisted of 86 well-characterized patients with biopsy-proven NAFLD and 38 healthy controls. Assembly-based analysis was performed to identify BA-metabolizing microbes. Statistical tests, feature selection, and microbial coabundance analysis were integrated to identify microbial alterations and markers in NAFLD. An independent validation cohort was subjected to similar analyses. NAFLD microbiota exhibited decreased diversity and microbial associations. We established a classifier model with 53 differential species exhibiting a robust diagnostic accuracy [area under the receiver-operator curve (AUC) = 0.97] for detecting NAFLD. Next, eight important differential pathway markers including secondary BA biosynthesis were identified. Specifically, increased abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH), 3α-hydroxysteroid dehydrogenase (baiA), and bile acid-coenzyme A ligase (baiB) was detected in NAFLD. Furthermore, 10 of 50 BA-metabolizing metagenome-assembled genomes (MAGs) from Bacteroides ovatus and Eubacterium biforme were dominant in NAFLD and interplayed as a synergetic ecological guild. Importantly, two subtypes of patients with NAFLD were observed according to secondary BA metabolism potentials. Elevated capability for secondary BA biosynthesis was also observed in the validation cohort. These bacterial BA-metabolizing genes and microbes identified in this study may serve as disease markers. Microbial differences in BA-metabolism and strain-specific differences among patients highlight the potential for precision medicine in NAFLD treatment.

Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvß6 upregulation following liver injury.

Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. Using microdissection and next-generation RNA-Seq, we identified 5 genes, including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2, that were most upregulated in proliferating BECs after acute injury. Immunohistochemical analyses confirmed robust upregulation of integrin αvß6 (ITGß6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of the Itgb6 gene attenuated BEC proliferation after acute bile duct injury. Macrophage depletion or Ccr2 deficiency impaired ITGß6 expression and BEC proliferation. In vitro experiments revealed that bile acid-activated monocytes promoted BEC proliferation through ITGß6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGß6, which work together to promote BEC proliferation and therefore represent potential therapeutic targets for cholangiopathies.

Protein Phosphatase 1 Regulatory Subunit 3B Genotype at rs4240624 Has a Major Effect on Gallbladder Bile Composition.

The protein phosphatase 1 regulatory subunit 3B (PPP1R3B) gene is a target of farnesoid X receptor (FXR), which is a major regulator of bile acid metabolism. Both PPP1R3B and FXR have been suggested to take part in glycogen metabolism, which may explain the association of PPP1R3B gene variants with altered hepatic computed tomography attenuation. We analyzed the effect of PPP1R3B rs4240624 variant on bile acid composition in individuals with obesity. The study cohort consisted of 242 individuals from the Kuopio Obesity Surgery Study (73 men, 169 women, age 47.6 ± 9.0 years, body mass index 43.2 ± 5.4 kg/m2) with PPP1R3B genotype and liver RNA sequencing (RNA-seq) data available. Fasting plasma and gallbladder bile samples were collected from 50 individuals. Bile acids in plasma did not differ based on the PPP1R3B rs4240624 genotype. However, the concentration of total bile acids (109 ± 55 vs. 35 ± 19 mM; P = 1.0 × 10-5) and all individual bile acids (also 7α-hydroxy-4-cholesten-3-one [C4]) measured from bile were significantly lower in those with the AG genotype compared to those with the AA genotype. In addition, total cholesterol (P = 0.011) and phospholipid (P = 0.001) levels were lower in individuals with the AG genotype, but cholesterol saturation index did not differ, indicating that the decrease in cholesterol and phospholipid levels was secondary to the change in bile acids. Liver RNA-seq data demonstrated that expression of PPP1R3B, tankyrase (TNKS), Homo sapiens chromosome 8 clone RP11-10A14.5 (AC022784.1 [LOC157273]), Homo sapiens chromosome 8 clone RP11-375N15.1 (AC021242.1), and Homo sapiens chromosome 8, clone RP11-10A14 (AC022784.6) associated with the PPP1R3B genotype. In addition, genes enriched in transmembrane transport and phospholipid binding pathways were associated with the genotype. Conclusion: The rs4240624 variant in PPP1R3B has a major effect on the composition of gallbladder bile. Other transcripts in the same loci may be important mediators of the variant effect.

Lactobacillus casei YRL577 ameliorates markers of non-alcoholic fatty liver and alters expression of genes within the intestinal bile acid pathway.

Non-alcoholic fatty liver disease (NAFLD) has become the main cause of end-stage liver disease. Probiotics have the potential effect of alleviating NAFLD. The aim of this study was to explore functional probiotics and their underlying mechanisms. The bile salt hydrolase (BSH) activity in thirty-four strains was determined in vitro. Then, C57BL/6 mice were used to explore the effects of probiotics on NAFLD. Body weight and food intake were measured, and serum lipid concentrations, oxidative stress and proinflammatory cytokines levels were determined using commercial kits. The expressions of intestinal bile acid pathway genes were evaluated via real-time PCR. The results showed that Lactobacillus casei YRL577 and L. paracasei X11 had higher BSH activity. L. casei YRL577 significantly reduced liver weight and liver index and could regulate the levels of lipid metabolism, oxidative stress and proinflammatory cytokines as compared with L. paracasei X11. Furthermore, the results indicated that L. casei YRL577 up-regulated the mRNA levels of farnesoid X receptor and fibroblast growth factor 15, whereas down-regulated the mRNA level of apical Na-dependent bile acid transporter. These findings suggested that L. casei YRL577 modified genes in the intestinal bile acid pathway which might contribute to the alleviation of NAFLD.

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates.

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7ß-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3ß-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7ß-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7ß-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.

A Villin-Driven Fxr Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an n-6-Enriched High-Fat Diet.

A diet high in n-6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by n-6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low n-6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (n-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the Fxrα1 isoform under the control of the intestine-specific Villin promoter (Fxrα1TG) were fed the CNTL or n-6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the n-6HFD supports higher weight gain in both WT and FxrαTG littermates; increases the expression of Fxrα1/2, and peroxisome proliferator-activated receptor-γ1 (Pparγ1) in the small intestine, Fxrα1/2 in the colon, and cytochrome P4507A1 (Cyp7a1) and small heterodimer protein (Shp) in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and ß-muricholic (ßMCA) acid in the cecum. Intestinal overexpression of the Fxra1TG augments expression of Shp and ileal bile acid-binding protein (Ibabp) in the small intestine and Ibabp in the proximal colon. Conversely, it antagonizes n-6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of Cyp7a1; and expression of Pparγ in the small intestine. We conclude that intestinal Fxrα1 overexpression represses hepatic de novo BA synthesis and protects against n-6HFD-induced accumulation of human-specific primary bile acids in the cecum.

Hepatic Bile Acid Reuptake in the Rat Depends on Bile Acid Conjugation but Not on Agonistic Properties towards FXR and TGR5.

Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) are the two known bile acid (BA) sensitive receptors and are expressed in the intestine and liver as well as in extra-enterohepatic tissues. The physiological effects of extra-enterohepatic FXR/TRG5 remain unclear. Further, the extent BAs escape liver reabsorption and how they interact with extra-enterohepatic FXR/TGR5 is understudied. We investigated if hepatic BA reuptake differed between BAs agonistic for FXR and TGR5 compared to non-agonists in the rat. Blood was collected from the portal vein and inferior caval vein from anesthetized rats before and 5, 20, 30, and 40 min post stimulation with sulfated cholecystokinin-8. Plasma concentrations of 20 different BAs were assessed by liquid chromatography coupled to mass spectrometry. Total portal vein BA AUC was 3-4 times greater than in the vena cava inferior (2.7 ± 0.6 vs. 0.7 ± 0.2 mM x min, p < 0.01, n = 8) with total unconjugated BAs being 2-3-fold higher than total conjugated BAs (AUC 8-10 higher p < 0.05 for both). However, in both cases, absolute ratios varied greatly among different BAs. The average hepatic reuptake of BAs agonistic for FXR/TGR5 was similar to non-agonists. However, as the sum of non-agonist BAs in vena portae was 2-3-fold higher than the sum agonist (p < 0.05), the peripheral BA pool was composed mostly of non-agonist BAs. We conclude that hepatic BA reuptake varies substantially by type and does not favor FXR/TGR5 BAs agonists.

Gender differences in the bile acid profiles of APP/PS1 transgenic AD mice.

Alzheimer's disease (AD) is a neurodegenerative disease and presents in the accumulation of amyloid and neurofibrillary tangle. The association between modulations of gut symbiotic microbes with neurological disease via bidirectional gut-brain axis has been well documented. Bile acid (BA) pools in the enterohepatic circulation could be valuable for probing complex biochemical interactions between host and their symbiotic microbiota. Herein we investigated the levels of 28 BAs in several compartments in enterohepatic circulation (including jejunal, ileum, cecum, colon and feces, plasma and liver tissue) by employing an APP/PS1 induced transgenic AD mouse model. We found that BA profiles in AD mice were gender specific. We observed decreased levels of taurine-conjugated primary BAs (TUDCA, TCA, T-α-MCA and T-ß-MCA) and increased levels of secondary BA (iso-DCA) in plasma and liver extracts for female AD transgenic mice. In contrast, increased levels of TDCA in liver extracts and decreased levels of T-ß-MCA in jejunal content were noted in male AD mice. These observations suggested that perturbations of BA profiles in AD mice displayed clear gender variations. Our study highlighted the roles of gut microbiota on neurodegenerative disease, which could be gender specific.

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.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Loss of Thymine DNA Glycosylase Causes Dysregulation of Bile Acid Homeostasis and Hepatocellular Carcinoma.

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver.

In Vitro Functional Characterization and in Silico Prediction of Rare Genetic Variation in the Bile Acid and Drug Transporter, Na+-Taurocholate Cotransporting Polypeptide (NTCP, SLC10A1).

Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1) is a key hepatic uptake transporter for bile acids and drugs and is the main functional receptor for hepatitis B and D viruses. Next-generation sequencing has revealed that a large number of rare SLC10A1 variants exist in the population. Little data exist regarding head-to-head comparison of in silico algorithms to predict functional effects of pharmacogenetic variants when compared to direct in vitro functional assessment. This study aimed at characterizing rare SLC10A1 variants in vitro and to assess the performance of seven in silico algorithms to predict the observed functional impacts. Thirty-five previously uncharacterized, rare, missense SLC10A1 variants were transiently expressed in human embryonic kidney 293 type T (HEK293T) cells. NCTP protein expression as well as uptake of substrates taurocholic acid (TCA) and rosuvastatin were assessed. Substrate-specific effects were observed for NTCP G191R, with TCA and rosuvastatin transport observed at 89 and 8% of wild-type (WT) uptake, respectively. Significantly reduced transport of TCA and rosuvastatin was observed for 19 variants (p < 0.05), with seven variants displaying decreased protein expression and marked reduction in transport of both substrates (0-13% of WT uptake, p < 0.0001). Performance of in silico algorithms to predict in vitro uptake, assessed using the area under the receiver operating characteristic curves (AUCROC), ranged from 0.69 to 0.97 and 0.72 to 0.84 for TCA and rosuvastatin uptake, respectively. In conclusion, we identified rare variants with significantly reduced NTCP expression and function. We demonstrated that no algorithm performed robustly enough to replace functional study in vitro, particularly given the broad substrate specificity of many pharmacogenes.

Picroside II protects against cholestatic liver injury possibly through activation of farnesoid X receptor.

BACKGROUD: Cholestasis, accompanied by the accumulation of bile acids in body, may ultimately cause liver failure and cirrhosis. There have been limited therapies for cholesteric disorders. Therefore, development of appropriate therapeutic drugs for cholestasis is required. Picroside II is a bioactive component isolated from Picrorhiza scrophulariiflora Pennell, its mechanistic contributions to the anti-cholestasis effect have not been fully elucidated, especially the role of picroside II on bile acid homeostasis via nuclear receptors remains unclear. PURPOSE: This study was designed to investigate the hepatoprotective effect of picroside II against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury and elucidate the mechanisms in vivo and in vitro. METHODS: The ANIT-induced cholestatic mouse model was used with or without picroside II treatment. Serum and bile biochemical indicators, as well as liver histopathological changes were examined. siRNA, Dual-luciferase reporter, quantitative real-time PCR and Western blot assay were used to demonstrate the farnesoid X receptor (FXR) pathway in the anti-cholestasis effects of picroside II in vivo and in vitro. RESULTS: Picroside II exerted hepatoprotective effect against ANIT-induced cholestasis by impaired hepatic function and tissue damage. Picroside II increased bile acid efflux transporter bile salt export pump (Bsep), uptake transporter sodium taurocholate cotransporting polypeptide (Ntcp), and bile acid metabolizing enzymes sulfate transferase 2a1 (Sult2a1) and UDP-glucuronosyltransferase 1a1 (Ugt1a1), whereas decreased the bile acid synthesis enzymes cholesterol 7α-hydroxylase (Cyp7a1) and oxysterol 12α-hydroxylase (Cyp8b1). In addition, expression of FXR and the target gene Bsep was increased, whereas aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARα) and their corresponding target genes were not significantly influenced by picroside II under cholestatic conditions. Furthermore, regulation of transporters and enzymes involved in bile acid homeostasis by picroside II were abrogated by FXR silencing in mouse primary cultured hepatocytes. Dual-luciferase reporter assay performed in HepG2 cells demonstrated FXR activation by picroside II. CONCLUSION: Our findings demonstrate that picroside II exerts protective effect on ANIT-induced cholestasis possibly through FXR activation that regulates the transporters and enzymes involved in bile acid homeostasis. Picroside II might be an effective approach for the prevention and treatment of cholestatic liver diseases.

Improvements in Metabolic Syndrome by Xanthohumol Derivatives Are Linked to Altered Gut Microbiota and Bile Acid Metabolism.

SCOPE: Two hydrogenated xanthohumol (XN) derivatives, α,ß-dihydro-XN (DXN) and tetrahydro-XN (TXN), improved parameters of metabolic syndrome (MetS), a critical risk factor of cardiovascular disease (CVD) and type 2 diabetes, in a diet-induced obese murine model. It is hypothesized that improvements in obesity and MetS are linked to changes in composition of the gut microbiota, bile acid metabolism, intestinal barrier function, and inflammation. METHODS AND RESULTS: To test this hypothesis, 16S rRNA genes were sequenced and bile acids were measured in fecal samples from C57BL/6J mice fed a high-fat diet (HFD) or HFD containing XN, DXN or TXN. Expression of genes associated with epithelial barrier function, inflammation, and bile acid metabolism were measured in the colon, white adipose tissue (WAT), and liver, respectively. Administration of XN derivatives decreases intestinal microbiota diversity and abundance-specifically Bacteroidetes and Tenericutes-alters bile acid metabolism, and reduces inflammation. In WAT, TXN supplementation decreases pro-inflammatory gene expression by suppressing macrophage infiltration. Transkingdom network analysis connects changes in the microbiota to improvements in MetS in the host. CONCLUSION: Changes in the gut microbiota and bile acid metabolism may explain, in part, the improvements in obesity and MetS associated with administration of XN and its derivatives.