StarD5 levels of expression correlate with onset and progression of steatosis and liver fibrosis.

Insufficient expression of steroidogenic acute regulatory-related lipid transfer protein 5 (StarD5) on liver cholesterol/lipid homeostasis is not clearly defined. The ablation of StarD5 was analyzed in mice on a normal or Western diet (WD) to determine its importance in hepatic lipid accumulation and fibrosis compared with wild-type (WT) mice. Rescue experiments in StarD5-/- mice and hepatocytes were performed. In addition to increased hepatic triglyceride (TG)-cholesterol levels, global StarD5-/- mice fed a normal diet displayed reduced plasma triglycerides and liver very low-density lipoprotein (VLDL) secretion as compared with WT counterparts. Insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) scoring were elevated, demonstrating developing insulin resistance (IR). WD-fed StarD5-/- mice upregulated WW domain containing transcription regulator 1 (TAZ or WWTR1) expression with accelerated liver fibrosis when compared with WD-fed WT mice. Suppression of oxysterol 7α-hydroxylase (CYP7B1) coupled with chronic accumulation of toxic oxysterol levels correlated with presentation of fibrosis. "Hepatocyte-selective" StarD5 overexpression in StarD5-/- mice restored expression, reduced hepatic triglycerides, and improved HOMA-IR. Observations in two additional mouse and one human metabolic dysfunction-associated steatotic liver disease (MASLD) model were supportive. The downregulation of StarD5 with hepatic lipid excess is a previously unappreciated physiological function appearing to promote lipid storage for future needs. Conversely, lingering downregulation of StarD5 with prolonged lipid-cholesterol excess accelerates fatty liver's transition to fibrosis; mediated via dysregulation in the oxysterol signaling pathway.NEW & NOTEWORTHY We have found that deletion of the cholesterol transport protein StarD5 in mice leads to an increase in insulin resistance and lipid accumulation due to the upregulation of lipid synthesis and decrease VLDL secretion from the liver. In addition, deletion of StarD5 increased fibrosis when mice were fed a Western diet. This represents a novel pathway of fibrosis development in the liver.

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease.

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3ß-hydroxy-5-cholesten-(25R)26-oic acid (3ßHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3ßHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3ßHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1-/- mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3ßHCA levels were maintained at basal levels in ND-fed Cyp7b1-/- mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1-/- mice developed insulin resistance (IR) with subsequent 26HC/3ßHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1-/- mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3ßHCA accumulation. The results suggest 26HC/3ßHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3ßHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.

Coffee modulates insulin-hepatocyte nuclear factor-4α-Cyp7b1 pathway and reduces oxysterol-driven liver toxicity in a nonalcoholic fatty liver disease mouse model.

Oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic pathway" of cholesterol metabolism. Previously, we demonstrated that an inability to upregulate CYP7B1 in the setting of insulin resistance leads to the accumulation of cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) that initiate and promote hepatocyte injury; followed by an inflammatory response. The current study demonstrates that dietary coffee improves insulin resistance and restores Cyp7b1 levels in a well-characterized Western diet (WD)-induced nonalcoholic fatty liver disease (NAFLD) mouse model. Ingestion of a WD containing caffeinated (regular) coffee or decaffeinated coffee markedly reduced the serum ALT level and improved insulin resistance. Cyp7b1 mRNA and protein levels were preserved at normal levels in mice fed the coffee containing WD. Additionally, coffee led to upregulated steroid sulfotransferase 2b1 (Sult2b1) mRNA expression. In accordance with the response in these oxysterol metabolic genes, hepatocellular 26HC levels were maintained at physiologically low levels. Moreover, the current study provided evidence that hepatic Cyp7b1 and Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, hepatocyte nuclear factor (HNF)-4α. We conclude coffee achieves its beneficial effects through the modulation of insulin resistance. Both decaffeinated and caffeinated coffee had beneficial effects, demonstrating caffeine is not fundamental to this effect. The effects of coffee feeding on the insulin-HNF4α-Cyp7b1 signaling pathway, whose dysregulation initiates and contributes to the onset and progression of NASH as triggered by insulin resistance, offer mechanistic insight into approaches for the treatment of NAFLD.NEW & NOTEWORTHY This study demonstrated dietary coffee prevented the accumulation of hepatic oxysterols by maintaining Cyp7b1/Sult2b1 expression in a diet-induced NAFLD mice model. Lowering liver oxysterols markedly reduced inflammation in the coffee-ingested mice. Caffeine is not fundamental to this effect. In addition, this study showed Cyp7b1/Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, HNF4α. The insulin-HNF4α-Cyp7b1/Sult2b1 signaling pathway, which directly correlates to the onset of NASH triggered by insulin resistance, offers insight into approaches for NAFLD treatment.

Progression to Cirrhosis Leads to Improvement in Atherogenic Milieu.

INTRODUCTION: The prevalence of coronary artery disease (CAD) is high among patients with cirrhosis; however, the impact of it on cardiovascular disease (CVD) is not known. The aim of the current study was to evaluate CVD events in patients with cirrhosis and impact of cirrhosis on biomarkers of atherogenesis. METHODS: The study included 682 patients with decompensated cirrhosis referred for liver transplantation (LT) evaluation between 2010 and 2017. All patients were followed until they experienced a CVD event, non-cardiac death, liver transplantation or last follow-up. To evaluate mechanistic link, patients with NASH cirrhosis were propensity matched 1:2 to non-cirrhosis NASH patients and biomarkers of atherogenic risk were compared. RESULTS: The composite CVD outcome occurred in 23(3.4%) patients after a median follow-up period of 585 days (IQR 139, 747). A strong association between presence of any CAD and CVD event was noted (HR = 6.8, 95% CI 2.9, 15.9) that was independent of age, gender, BMI, and MELD score. In competing risk model, the combined rate of LT and non-cardiac was significantly higher when compared to the rate of CVD events. Marker of insulin resistance and inflammation-related markers were similar in patients with and without cirrhosis. Patients with cirrhosis were more likely to have reduced VLDL, sdLDL-C, LDL-C, and triglycerides. Interestingly, patients with cirrhosis had an increase in serum HDL-2, the anti-atherogenic lipoprotein, and adiponectin, a protective serum adipokine. CONCLUSION: The risk of CVD events in patients with cirrhosis is low and may potentially be due to improvement in markers of atherogenic risk.

Insulin resistance dysregulates CYP7B1 leading to oxysterol accumulation: a pathway for NAFL to NASH transition.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

Cholangiocyte-Derived Exosomal Long Noncoding RNA H19 Promotes Hepatic Stellate Cell Activation and Cholestatic Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) represents the primary driving force to promote the progression of chronic cholestatic liver diseases. We previously reported that cholangiocyte-derived exosomal long noncoding RNA-H19 (lncRNA-H19) plays a critical role in promoting cholestatic liver injury. However, it remains unclear whether cholangiocyte-derived lncRNA-H19 regulates HSC activation, which is the major focus of this study. Both bile duct ligation (BDL) and Mdr2 knockout (Mdr2-/- ) mouse models were used. Wild-type and H19maternalΔExon1/+ (H19KO) mice were subjected to BDL. Mdr2-/- H19maternalΔExon1/+ (DKO) mice were generated. Exosomes isolated from cultured mouse and human cholangiocytes or mouse serum were used for in vivo transplantation and in vitro studies. Fluorescence-labeled exosomes and flow cytometry were used to monitor exosome uptake by hepatic cells. Collagen gel contraction and bromodeoxyuridine assays were used to determine the effect of exosomal-H19 on HSC activation and proliferation. Mouse and human primary sclerosing cholangitis (PSC)/primary biliary cholangitis (PBC) liver samples were analyzed by real-time PCR, western blot analysis, histology, and immunohistochemistry. The results demonstrated that hepatic H19 level was closely correlated with the severity of liver fibrosis in both mouse models and human patients with PSC and PBC. H19 deficiency significantly protected mice from liver fibrosis in BDL and Mdr2-/- mice. Transplanted cholangiocyte-derived H19-enriched exosomes were rapidly and preferentially taken up by HSCs and HSC-derived fibroblasts, and promoted liver fibrosis in BDL-H19KO mice and DKO mice. H19-enriched exosomes enhanced transdifferentiation of cultured mouse primary HSCs and promoted proliferation and matrix formation in HSC-derived fibroblasts. Conclusion: Cholangiocyte-derived exosomal H19 plays a critical role in the progression of cholestatic liver fibrosis by promoting HSC differentiation and activation and represents a potential diagnostic biomarker and therapeutic target for cholangiopathies.

StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

Alterations in Skin Microbiomes of Patients With Cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis have intestinal dysbiosis and are prone to itching and skin or soft-tissue infections. The skin microbiome, and its relationship with intestinal microbiome, have not been characterized. We investigated alterations in skin microbiota of patients with cirrhosis and their association with intestinal microbiota and modulators of itch. METHODS: We collected skin swabs at 7 sites and blood and stool samples from 20 healthy individuals (control subjects; mean age, 59 years) and 50 patients with cirrhosis (mean age, 61 years; mean model for end-stage disease score, 12; 20 with decompensation). Skin and stool samples were analyzed by 16s rRNA sequencing and serum samples were analyzed by liquid chromatography and mass spectrometry for levels of bile acids (BAs) and by an ELISA for autotaxin (an itch modulator). Participants were analyzed by the visual analog itch scale (VAS, 0-10,10 = maximum intensity). Data were compared between groups (cirrhosis vs control subjects, with vs without decompensation, VAS 5 or higher vs less than 5). Correlation networks between serum levels of BAs and skin microbiomes were compared between patients with cirrhosis with vs without itching. RESULTS: The composition of microbiomes at all skin sites differed between control subjects and patients with cirrhosis and between patients with compensated vs decompensated cirrhosis. Skin microbiomes of patients with cirrhosis (especially those with decompensation) contained a higher relative abundance of Gammaproteobacteria, Streptococaceae, and Staphylococcaceae, and fecal microbiomes contained a higher relative abundance of Gammaproteobacteria, than control subjects. These bacterial taxa were associated with serum levels of autotaxin and BAs, which were higher in patients with VAS scores ≥5. Based on network statistics, microbial and BA interactions at all sites were more complex in patients with greater levels of itching in the shin, the most common site of itch. CONCLUSIONS: We identified alterations in skin microbiome of patients with cirrhosis (in Gammaproteobacteria, Streptococcaceae, and Staphylococcaceae)-especially in patients with decompensation; fecal microbiomes of patients with cirrhosis had a higher relative abundance of Gammaproteobacteria than control subjects. These specific microbial taxa are associated with itching intensity and itch modulators, such as serum levels of BAs and autotaxin.

C/EBP homologous protein-induced loss of intestinal epithelial stemness contributes to bile duct ligation-induced cholestatic liver injury in mice.

Impaired intestinal barrier function promotes the progression of various liver diseases, including cholestatic liver diseases. The close association of primary sclerosing cholangitis (PSC) with inflammatory bowel disease highlights the importance of the gut-liver axis. It has been reported that bile duct ligation (BDL)-induced liver fibrosis is significantly reduced in C/EBP homologous protein knockout (CHOP-/- ) mice. However, the underlying mechanisms remain unclear. In the current study, we demonstrate that BDL induces striking and acute hepatic endoplasmic reticulum (ER) stress responses after 1 day, which return to normal after 3 days. No significant hepatocyte apoptosis is detected 7-14 days following BDL. However, the inflammatory response is significantly increased after 7 days, which is similar to what we found in human PSC liver samples. BDL-induced loss of stemness in intestinal stem cells (ISCs), disruption of intestinal barrier function, bacterial translocation, activation of hepatic inflammation, M2 macrophage polarization and liver fibrosis are significantly reduced in CHOP-/- mice. In addition, intestinal organoids derived from CHOP-/- mice contain more and longer crypt structures than those from wild-type (WT) mice, which is consistent with the upregulation of stem cell markers (leucine-rich repeat-containing G-protein-coupled receptor 5, olfactomedin 4, and SRY [sex determining region Y]-box 9) and in vivo findings that CHOP-/- mice have longer villi and crypts as compared to WT mice. Similarly, mRNA levels of CD14, interleukin-1ß, tumor necrosis factor-alpha, and monocyte chemotactic protein-1 are increased and stem cell proliferation is suppressed in the duodenum of patients with cirrhosis. CONCLUSION: Activation of ER stress and subsequent loss of stemness of ISCs plays a critical role in BDL-induced systemic inflammation and cholestatic liver injury. Modulation of the ER stress response represents a potential therapeutic strategy for cholestatic liver diseases as well as other inflammatory diseases. (Hepatology 2018;67:1441-1457).

Cholangiocyte-derived exosomal long noncoding RNA H19 promotes cholestatic liver injury in mouse and humans.

Cholestatic liver injury is an important clinical problem with limited understanding of disease pathologies. Exosomes are small extracellular vesicles released by a variety of cells, including cholangiocytes. Exosome-mediated cell-cell communication can modulate various cellular functions by transferring a variety of intracellular components to target cells. Our recent studies indicate that the long noncoding RNA (lncRNA), H19, is mainly expressed in cholangiocytes, and its aberrant expression is associated with significant down-regulation of small heterodimer partner (SHP) in hepatocytes and cholestatic liver injury in multidrug resistance 2 knockout (Mdr2-/- ) mice. However, how cholangiocyte-derived H19 suppresses SHP in hepatocytes remains unknown. Here, we report that cholangiocyte-derived exosomes mediate transfer of H19 into hepatocytes and promote cholestatic injury. Hepatic H19 level is correlated with severity of cholestatic injury in both fibrotic mouse models, including Mdr2-/- mice, a well-characterized model of primary sclerosing cholangitis (PSC), or CCl4 -induced cholestatic liver injury mouse models, and human PSC patients. Moreover, serum exosomal-H19 level is gradually up-regulated during disease progression in Mdr2-/- mice and patients with cirrhosis. H19-carrying exosomes from the primary cholangiocytes of wild-type (WT) mice suppress SHP expression in hepatocytes, but not the exosomes from the cholangiocytes of H19-/- mice. Furthermore, overexpression of H19 significantly suppressed SHP expression at both transcriptional and posttranscriptional levels. Importantly, transplant of H19-carrying serum exosomes of old fibrotic Mdr2-/- mice significantly promoted liver fibrosis (LF) in young Mdr2-/- mice. CONCLUSION: Cholangiocyte-derived exosomal-H19 plays a critical role in cholestatic liver injury. Serum exosomal H19 represents a noninvasive biomarker and potential therapeutic target for cholestatic diseases. (Hepatology 2018).

Antibiotic-Associated Disruption of Microbiota Composition and Function in Cirrhosis Is Restored by Fecal Transplant.

Patients with cirrhosis are often exposed to antibiotics that can lead to resistance and fungal overgrowth. The role of fecal microbial transplant (FMT) in restoring gut microbial function is unclear in cirrhosis. In a Food and Drug Administration-monitored phase 1 clinical safety trial, patients with decompensated cirrhosis on standard therapies (lactulose and rifaximin) were randomized to standard-of-care (SOC, no antibiotics/FMT) or 5 days of broad-spectrum antibiotics followed by FMT from a donor enriched in Lachnospiraceae and Ruminococcaceae. Microbial composition (diversity, family-level relative abundances), function (fecal bile acid [BA] deconjugation, 7α-dehydroxylation, short-chain fatty acids [SCFAs]), and correlations between Lachnospiraceae, Ruminococcaceae, and clinical variables were analyzed at baseline, postantibiotics, and 15 days post-FMT. FMT was well tolerated. Postantibiotics, there was a reduced microbial diversity and autochthonous taxa relative abundance. This was associated with an altered fecal SCFA and BA profile. Correlation linkage changes from beneficial at baseline to negative after antibiotics. All of these parameters became statistically similar post-FMT to baseline levels. No changes were seen in the SOC group. CONCLUSION: In patients with advanced cirrhosis on lactulose and rifaximin, FMT restored antibiotic-associated disruption in microbial diversity and function. (Hepatology 2018; 00:000-000).

Alterations in gut microbial function following liver transplant.

Liver transplantation (LT) improves daily function and ameliorates gut microbial composition. However, the effect of LT on microbial functionality, which can be related to overall patient benefit, is unclear and could affect the post-LT course. The aims were to determine the effect of LT on gut microbial functionality focusing on endotoxemia, bile acid (BA), ammonia metabolism, and lipidomics. We enrolled outpatient patients with cirrhosis on the LT list and followed them until 6 months after LT. Microbiota composition (Shannon diversity and individual taxa) and function analysis (serum endotoxin, urinary metabolomics and serum lipidomics, and stool BA profile) and cognitive tests were performed at both visits. We enrolled 40 patients (age, 56 ± 7 years; mean Model for End-Stage Liver Disease score, 22.6). They received LT 6 ± 3 months after enrollment and were re-evaluated 7 ± 3 months after LT with a stable course. A significant improvement in cognition with increase in microbial diversity, increase in autochthonous and decrease in potentially pathogenic taxa, and reduced endotoxemia were seen after LT compared with baseline. Stool BAs increased significantly after LT, and there was evidence of greater bacterial action (higher secondary, oxo and iso-BAs) after LT although the levels of conjugated BAs remained similar. There was a reduced serum ammonia and corresponding rise in urinary phenylacetylglutamine after LT. There was an increase in urinary trimethylamine-N-oxide, which was correlated with specific changes in serum lipids related to cell membrane products. The ultimate post-LT lipidomic profile appeared beneficial compared with the profile before LT. In conclusion, LT improves gut microbiota diversity and dysbiosis, which is accompanied by favorable changes in gut microbial functionality corresponding to BAs, ammonia, endotoxemia, lipidomic, and metabolomic profiles. Liver Transplantation 24 752-761 2018 AASLD.

The role of long noncoding RNA H19 in gender disparity of cholestatic liver injury in multidrug resistance 2 gene knockout mice.

The multidrug resistance 2 knockout (Mdr2-/- ) mouse is a well-established model of cholestatic cholangiopathies. Female Mdr2-/- mice develop more severe hepatobiliary damage than male Mdr2-/- mice, which is correlated with a higher proportion of taurocholate in the bile. Although estrogen has been identified as an important player in intrahepatic cholestasis, the underlying molecular mechanisms of gender-based disparity of cholestatic injury remain unclear. The long noncoding RNA H19 is an imprinted, maternally expressed, and estrogen-targeted gene, which is significantly induced in human fibrotic/cirrhotic liver and bile duct-ligated mouse liver. However, whether aberrant expression of H19 accounts for gender-based disparity of cholestatic injury in Mdr2-/- mice remains unknown. The current study demonstrated that H19 was markedly induced (∼200-fold) in the livers of female Mdr2-/- mice at advanced stages of cholestasis (100 days old) but not in age-matched male Mdr2-/- mice. During the early stages of cholestasis, H19 expression was minimal. We further determined that hepatic H19 was mainly expressed in cholangiocytes, not hepatocytes. Both taurocholate and estrogen significantly activated the extracellular signal-regulated kinase 1/2 signaling pathway and induced H19 expression in cholangiocytes. Knocking down H19 not only significantly reduced taurocholate/estrogen-induced expression of fibrotic genes and sphingosine 1-phosphate receptor 2 in cholangiocytes but also markedly reduced cholestatic injury in female Mdr2-/- mice. Furthermore, expression of small heterodimer partner was substantially inhibited at advanced stages of liver fibrosis, which was reversed by H19 short hairpin RNA in female Mdr2-/- mice. Similar findings were obtained in human primary sclerosing cholangitis liver samples. CONCLUSION: H19 plays a critical role in the disease progression of cholestasis and represents a key factor that causes the gender disparity of cholestatic liver injury in Mdr2-/- mice. (Hepatology 2017;66:869-884).

The role of sphingosine 1-phosphate receptor 2 in bile-acid-induced cholangiocyte proliferation and cholestasis-induced liver injury in mice.

Bile duct obstruction is a potent stimulus for cholangiocyte proliferation, especially for large cholangiocytes. Our previous studies reported that conjugated bile acids (CBAs) activate the protein kinase B (AKT) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways through sphingosine 1-phosphate receptor (S1PR) 2 in hepatocytes and cholangiocarcinoma cells. It also has been reported that taurocholate (TCA) promotes large cholangiocyte proliferation and protects cholangiocytes from bile duct ligation (BDL)-induced apoptosis. However, the role of S1PR2 in bile-acid-mediated cholangiocyte proliferation and cholestatic liver injury has not been elucidated. Here, we report that S1PR2 is the predominant S1PR expressed in cholangiocytes. Both TCA- and sphingosine-1-phosphate (S1P)-induced activation of ERK1/2 and AKT were inhibited by JTE-013, a specific antagonist of S1PR2, in cholangiocytes. In addition, TCA- and S1P-induced cell proliferation and migration were inhibited by JTE-013 and a specific short hairpin RNA of S1PR2, as well as chemical inhibitors of ERK1/2 and AKT in mouse cholangiocytes. In BDL mice, expression of S1PR2 was up-regulated in whole liver and cholangiocytes. S1PR2 deficiency significantly reduced BDL-induced cholangiocyte proliferation and cholestatic injury, as indicated by significant reductions in inflammation and liver fibrosis in S1PR2 knockout mice. Treatment of BDL mice with JTE-013 significantly reduced total bile acid levels in serum and cholestatic liver injury. CONCLUSION: This study suggests that CBA-induced activation of S1PR2-mediated signaling pathways plays a critical role in obstructive cholestasis and may represent a novel therapeutic target for cholestatic liver diseases. (Hepatology 2017;65:2005-2018).

Continued Alcohol Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis.

BACKGROUND: Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS: Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS: Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS: Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Conjugated bile acid-activated S1P receptor 2 is a key regulator of sphingosine kinase 2 and hepatic gene expression.

UNLABELLED: Bile acids are important hormones during the feed/fast cycle, allowing the liver to coordinately regulate nutrient metabolism. How they accomplish this has not been fully elucidated. Conjugated bile acids activate both the ERK1/2 and AKT signaling pathways via sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes and in vivo. Here, we report that feeding mice a high-fat diet, infusion of taurocholate into the chronic bile fistula rat, or overexpression of the gene encoding S1PR2 in mouse hepatocytes significantly upregulated hepatic sphingosine kinase 2 (SphK2) but not SphK1. Key genes encoding nuclear receptors/enzymes involved in nutrient metabolism were significantly downregulated in livers of S1PR2(-/-) and SphK2(-/-) mice. In contrast, overexpression of the gene encoding S1PR2 in primary mouse hepatocytes differentially increased SphK2, but not SphK1, and mRNA levels of key genes involved in nutrient metabolism. Nuclear levels of sphingosine-1-phosphate, an endogenous inhibitor of histone deacetylases 1 and 2, as well as the acetylation of histones H3K9, H4K5, and H2BK12 were significantly decreased in hepatocytes prepared from S1PR2(-/-) and SphK2(-/-) mice. CONCLUSION: Both S1PR2(-/-) and SphK2(-/-) mice rapidly developed fatty livers on a high-fat diet, suggesting the importance of conjugated bile acids, S1PR2, and SphK2 in regulating hepatic lipid metabolism.

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

UNLABELLED: Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/"duct-like" structures, which was blocked by treatment with JTE-013. CONCLUSION: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS.

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.

Colonic inflammation and secondary bile acids in alcoholic cirrhosis.

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1ß, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1ß, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.

The role of CCAAT enhancer-binding protein homologous protein in human immunodeficiency virus protease-inhibitor-induced hepatic lipotoxicity in mice.

UNLABELLED: Human immunodeficiency virus (HIV) protease inhibitors (HIV PIs) are the core components of highly active antiretroviral therapy, which has been successfully used in the treatment of HIV-1 infection in the past two decades. However, benefits of HIV PIs are compromised by clinically important adverse effects, such as dyslipidemia, insulin resistance, and cardiovascular complications. We have previously shown that activation of endoplasmic reticulum (ER) stress plays a critical role in HIV PI-induced dys-regulation of hepatic lipid metabolism. HIV PI-induced hepatic lipotoxicity is closely linked to the up-regulation of CCAAT enhancer binding protein homologous protein (CHOP) in hepatocytes. To further investigate whether CHOP is responsible for HIV PI-induced hepatic lipotoxicity, C57BL/6J wild-type (WT) or CHOP knockout (CHOP(-/-) ) mice or the corresponding primary mouse hepatocytes were used in this study. Both in vitro and in vivo studies indicated that HIV PIs (ritonavir and lopinavir) significantly increased hepatic lipid accumulation in WT mice. In contrast, CHOP(-/-) mice showed a significant reduction in hepatic triglyceride accumulation and liver injury, as evidenced by hematoxylin and eosin and Oil Red O staining. Real-time reverse-transcriptase polymerase chain reaction and immunoblotting data showed that in the absence of CHOP, HIV PI-induced expression of stress-related proteins and lipogenic genes were dramatically reduced. Furthermore, tumor necrosis factor alpha and interleukin-6 levels in serum and liver were significantly lower in HIV PI-treated CHOP(-/-) mice, compared to HIV PI-treated WT mice. CONCLUSION: Taken together, these data suggest that CHOP is an important molecular link of ER stress, inflammation, and hepatic lipotoxicity, and that increased expression of CHOP represents a critical factor underlying events leading to hepatic injury. (HEPATOLOGY 2013).