Alterations of the bile microbiome in primary sclerosing cholangitis.

BACKGROUND: Patients with primary sclerosing cholangitis (PSC) display an altered colonic microbiome compared with healthy controls. However, little is known on the bile duct microbiome and its interplay with bile acid metabolism in PSC. METHODS: Patients with PSC (n=43) and controls without sclerosing cholangitis (n=22) requiring endoscopic retrograde cholangiography were included prospectively. Leading indications in controls were sporadic choledocholithiasis and papillary adenoma. A total of 260 biospecimens were collected from the oral cavity, duodenal fluid and mucosa and ductal bile. Microbiomes of the upper alimentary tract and ductal bile were profiled by sequencing the 16S-rRNA-encoding gene (V1-V2). Bile fluid bile acid composition was measured by high-performance liquid chromatography mass spectrometry and validated in an external cohort (n=20). RESULTS: The bile fluid harboured a diverse microbiome that was distinct from the oral cavity, the duodenal fluid and duodenal mucosa communities. The upper alimentary tract microbiome differed between PSC patients and controls. However, the strongest differences between PSC patients and controls were observed in the ductal bile fluid, including reduced biodiversity (Shannon entropy, p=0.0127) and increase of pathogen Enterococcus faecalis (FDR=4.18×10-5) in PSC. Enterococcus abundance in ductal bile was strongly correlated with concentration of the noxious secondary bile acid taurolithocholic acid (r=0.60, p=0.0021). CONCLUSION: PSC is characterised by an altered microbiome of the upper alimentary tract and bile ducts. Biliary dysbiosis is linked with increased concentrations of the proinflammatory and potentially cancerogenic agent taurolithocholic acid.

Liver infiltrating T cells regulate bile acid metabolism in experimental cholangitis.

BACKGROUND & AIMS: T cells are central mediators of liver inflammation and represent potential treatment targets in cholestatic liver disease. Whereas emerging evidence shows that bile acids (BAs) affect T cell function, the role of T cells for the regulation of BA metabolism is unknown. In order to understand this interplay, we investigated the influence of T cells on BA metabolism in a novel mouse model of cholangitis. METHODS: Mdr2-/- mice were crossed with transgenic K14-OVAp mice, which express an MHC class I restricted ovalbumin peptide on biliary epithelial cells (Mdr2-/-xK14-OVAp). T cell-mediated cholangitis was induced by the adoptive transfer of antigen-specific CD8+ T cells. BA levels were quantified using a targeted liquid chromatography-mass spectrometry-based approach. RESULTS: T cell-induced cholangitis resulted in reduced levels of unconjugated BAs in the liver and significantly increased serum and hepatic levels of conjugated BAs. Genes responsible for BA synthesis and uptake were downregulated and expression of the bile salt export pump was increased. The transferred antigen-specific CD8+ T cells alone were able to induce these changes, as demonstrated using Mdr2-/-xK14-OVAp recipient mice on the Rag1-/- background. Mechanistically, we showed by depletion experiments that alterations in BA metabolism were partly mediated by the proinflammatory cytokines TNF and IFN-γ in an FXR-dependent manner, a process that in vitro required cell contact between T cells and hepatocytes. CONCLUSION: Whereas it is known that BA metabolism is dysregulated in sepsis and related conditions, we have shown that T cells are able to control the synthesis and metabolism of BAs, a process which depends on TNF and IFN-γ. Understanding the effect of lymphocytes on BA metabolism will help in the design of combined treatment strategies for cholestatic liver diseases. LAY SUMMARY: Dysregulation of bile acid metabolism and T cells can contribute to the development of cholangiopathies. Before targeting T cells for the treatment of cholangiopathies, it should be determined whether they exert protective effects on bile acid metabolism. Herein, we demonstrate that T cell-induced cholangitis resulted in decreased levels of harmful unconjugated bile acids. T cells were able to directly control synthesis and metabolism of bile acids, a process which was dependent on the proinflammatory cytokines TNF and IFN-γ. Understanding the effect of lymphocytes on bile acid metabolism will help in the design of combined treatment strategies for cholestatic liver diseases.

Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice.

Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species.

Diabetes prevalence in NZO females depends on estrogen action on liver fat content.

In humans and rodents, risk of metabolic syndrome is sexually dimorphic, with an increased incidence in males. Additionally, the protective role of female gonadal hormones is ostensible, as prevalence of type 2 diabetes mellitus (T2DM) increases after menopause. Here, we investigated the influence of estrogen (E2) on the onset of T2DM in female New Zealand obese (NZO) mice. Diabetes prevalence (defined as blood glucose levels >16.6 mmol/l) of NZO females on high-fat diet (60 kcal% fat) in week 22 was 43%. This was markedly dependent on liver fat content in week 10, as detected by computed tomography. Only mice with a liver fat content >9% in week 10 plus glucose levels >10 mmol/l in week 9 developed hyperglycemia by week 22. In addition, at 11 wk, diacylglycerols were elevated in livers of diabetes-prone mice compared with controls. Hepatic expression profiles obtained from diabetes-prone and -resistant mice at 11 wk revealed increased abundance of two transcripts in diabetes-prone mice: Mogat1, which catalyzes the synthesis of diacylglycerols from monoacylglycerol and fatty acyl-CoA, and the fatty acid transporter Cd36. E2 treatment of diabetes-prone mice for 10 wk prevented any further increase in liver fat content and reduced diacylglycerols and the abundance of Mogat1 and Cd36, leading to a reduction of diabetes prevalence and an improved glucose tolerance compared with untreated mice. Our data indicate that early elevation of hepatic Cd36 and Mogat1 associates with increased production and accumulation of triglycerides and diacylglycerols, presumably resulting in reduced hepatic insulin sensitivity and leading to later onset of T2DM.

Cold-Induced Lipoprotein Clearance in Cyp7b1-Deficient Mice.

Brown adipose tissue (BAT) has emerged as an appealing therapeutic target for cardio metabolic diseases. BAT is a heat-producing organ and upon activation substantially lowers hyperlipidemia. In response to cold exposure, not only the uptake of lipids into BAT is increased but also the Cyp7b1-mediated synthesis of bile acids (BA) from cholesterol in the liver is triggered. In addition to their role for intestinal lipid digestion, BA act as endocrine signals that can activate thermogenesis in BAT. When exposed to cold temperatures, Cyp7b1 -/- mice have compromised BAT function along with reduced fecal bile acid levels. Here, we aim to evaluate the role of Cyp7b1 for BAT-dependent lipid clearance. Using metabolic studies with radioactive tracers, we show that in response to a cold stimulus, BAT-mediated clearance of fatty acids derived from triglyceride-rich lipoproteins (TRL), and their remnants are reduced in Cyp7b1 -/- mice. The impaired lipid uptake can be explained by reduced BAT lipoprotein lipase (LPL) levels and compromised organ activity in Cyp7b1 -/- mice, which may be linked to impaired insulin signaling. Overall, our findings reveal that alterations of systemic lipoprotein metabolism mediated by cold-activated BAT are dependent, at least in part, on CYP7Β1.

Increased concentrations of conjugated bile acids are associated with osteoporosis in PSC patients.

Primary sclerosing cholangitis (PSC) is an idiopathic cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of intra- and extrahepatic bile ducts. Osteoporosis is a frequent comorbidity in PSC, and we could previously demonstrate that IL17-dependent activation of bone resorption is the predominant driver of bone loss in PSC. Since we additionally observed an unexpected heterogeneity of bone mineral density in our cohort of 238 PSC patients, the present study focused on a comparative analysis of affected individuals with diagnosed osteoporosis (PSCOPO, n = 10) or high bone mass (PSCHBM, n = 7). The two groups were not distinguishable by various baseline characteristics, including liver fibrosis or serum parameters for hepatic function. In contrast, quantification of serum bile acid concentrations identified significant increases in the PSCOPO group, including glycoursodeoxycholic acid (GUDCA), an exogenous bile acid administered to both patient groups. Although cell culture experiments did not support the hypothesis that an increase in circulating bile levels is a primary cause of PSC-associated osteoporosis, the remarkable differences of endogenous bile acids and GUDCA in the serum of PSCOPO patients strongly suggest a yet unknown impairment of biliary metabolism and/or hepatic bile acid clearance in this patient subgroup, which is independent of liver fibrosis.

Oxysterol 7-α Hydroxylase (CYP7B1) Attenuates Metabolic-Associated Fatty Liver Disease in Mice at Thermoneutrality.

Ambient temperature is an important determinant of both the alternative bile acid synthesis pathway controlled by oxysterol 7-α hydroxylase (CYP7B1) and the progression of metabolic-associated fatty liver disease (MAFLD). Here, we investigated whether CYP7B1 is involved in the etiology of MAFLD under conditions of low and high energy expenditure. For this, Cyp7b1-/- and wild type (WT) mice were fed a choline-deficient high-fat diet and housed either at 30 °C (thermoneutrality) or at 22 °C (mild cold). To study disease phenotype and underlying mechanisms, plasma and organ samples were analyzed to determine metabolic parameters, immune cell infiltration by immunohistology and flow cytometry, lipid species including hydroxycholesterols, bile acids and structural lipids. In WT and Cyp7b1-/- mice, thermoneutral housing promoted MAFLD, an effect that was more pronounced in CYP7B1-deficient mice. In these mice, we found higher plasma alanine aminotransferase activity, hyperlipidemia, hepatic accumulation of potentially harmful lipid species, aggravated liver fibrosis, increased inflammation and immune cell infiltration. Bile acids and hydroxycholesterols did not correlate with aggravated MAFLD in Cyp7b1-/- mice housed at thermoneutrality. Notably, an up-regulation of lipoprotein receptors was detected at 22 °C but not at 30 °C in livers of Cyp7b1-/- mice, suggesting that accelerated metabolism of lipoproteins carrying lipotoxic molecules counteracts MAFLD progression.

Endothelial Lipase Is Involved in Cold-Induced High-Density Lipoprotein Turnover and Reverse Cholesterol Transport in Mice.

The physiologic activation of thermogenic brown and white adipose tissues (BAT/WAT) by cold exposure triggers heat production by adaptive thermogenesis, a process known to ameliorate hyperlipidemia and protect from atherosclerosis. Mechanistically, it has been shown that thermogenic activation increases lipoprotein lipase (LPL)-dependent hydrolysis of triglyceride-rich lipoproteins (TRL) and accelerates the generation of cholesterol-enriched remnants and high-density lipoprotein (HDL), which promotes cholesterol flux from the periphery to the liver. HDL is also subjected to hydrolysis by endothelial lipase (EL) (encoded by LIPG). Genome-wide association studies have identified various variants of EL that are associated with altered HDL cholesterol levels. However, a potential role of EL in BAT-mediated HDL metabolism has not been investigated so far. In the present study, we show that in mice, cold-stimulated activation of thermogenic adipocytes induced expression of Lipg in BAT and inguinal WAT but that loss of Lipg did not affect gene expression of thermogenic markers. Furthermore, in both wild type (WT) and Lipg-deficient mice, activation of thermogenesis resulted in a decline of HDL cholesterol levels. However, cold-induced remodeling of the HDL lipid composition was different between WT and Lipg-deficient mice. Notably, radioactive tracer studies with double-labeled HDL indicated that cold-induced hepatic HDL cholesterol clearance was lower in Lipg-deficient mice. Moreover, this reduced clearance was associated with impaired macrophage-to-feces cholesterol transport. Overall, these data indicate that EL is a determinant of HDL lipid composition, cholesterol flux, and HDL turnover in conditions of high thermogenic activity.

Inulin Supplementation Disturbs Hepatic Cholesterol and Bile Acid Metabolism Independent from Housing Temperature.

Dietary fibers are fermented by gut bacteria into the major short chain fatty acids (SCFAs) acetate, propionate, and butyrate. Generally, fiber-rich diets are believed to improve metabolic health. However, recent studies suggest that long-term supplementation with fibers causes changes in hepatic bile acid metabolism, hepatocyte damage, and hepatocellular cancer in dysbiotic mice. Alterations in hepatic bile acid metabolism have also been reported after cold-induced activation of brown adipose tissue. Here, we aim to investigate the effects of short-term dietary inulin supplementation on liver cholesterol and bile acid metabolism in control and cold housed specific pathogen free wild type (WT) mice. We found that short-term inulin feeding lowered plasma cholesterol levels and provoked cholestasis and mild liver damage in WT mice. Of note, inulin feeding caused marked perturbations in bile acid metabolism, which were aggravated by cold treatment. Our studies indicate that even relatively short periods of inulin consumption in mice with an intact gut microbiome have detrimental effects on liver metabolism and function.

Assessment of Uptake and Biodistribution of Radiolabeled Cholesterol in Mice Using Gavaged Recombinant Triglyceride-rich Lipoprotein Particles (rTRL).

The here described method can be used to estimate the uptake of orally provided cholesterol in mice. Briefly, mice are gavaged with radiolabeled cholesterol and 4 h later, organ distribution of the radiolabel is determined by liquid scintillation counting. The method has been applied successfully to determine dietary cholesterol handling of mice housed at different ambient temperatures.

Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis.

Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism.

Thermogenic adipocytes promote HDL turnover and reverse cholesterol transport.

Brown and beige adipocytes combust nutrients for thermogenesis and through their metabolic activity decrease pro-atherogenic remnant lipoproteins in hyperlipidemic mice. However, whether the activation of thermogenic adipocytes affects the metabolism and anti-atherogenic properties of high-density lipoproteins (HDL) is unknown. Here, we report a reduction in atherosclerosis in response to pharmacological stimulation of thermogenesis linked to increased HDL levels in APOE*3-Leiden.CETP mice. Both cold-induced and pharmacological thermogenic activation enhances HDL remodelling, which is associated with specific lipidomic changes in mouse and human HDL. Furthermore, thermogenic stimulation promotes HDL-cholesterol clearance and increases macrophage-to-faeces reverse cholesterol transport in mice. Mechanistically, we show that intravascular lipolysis by adipocyte lipoprotein lipase and hepatic uptake of HDL by scavenger receptor B-I are the driving forces of HDL-cholesterol disposal in liver. Our findings corroborate the notion that high metabolic activity of thermogenic adipocytes confers atheroprotective properties via increased systemic cholesterol flux through the HDL compartment.

Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development.

Brown adipose tissue (BAT) combusts high amounts of fatty acids, thereby lowering plasma triglyceride levels and reducing obesity. However, the precise role of BAT in plasma cholesterol metabolism and atherosclerosis development remains unclear. Here we show that BAT activation by ß3-adrenergic receptor stimulation protects from atherosclerosis in hyperlipidemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism that unlike hyperlipidemic Apoe(-/-) and Ldlr(-/-) mice expresses functional apoE and LDLR. BAT activation increases energy expenditure and decreases plasma triglyceride and cholesterol levels. Mechanistically, we demonstrate that BAT activation enhances the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT, subsequently accelerating the hepatic clearance of the cholesterol-enriched remnants. These effects depend on a functional hepatic apoE-LDLR clearance pathway as BAT activation in Apoe(-/-) and Ldlr(-/-) mice does not attenuate hypercholesterolaemia and atherosclerosis. We conclude that activation of BAT is a powerful therapeutic avenue to ameliorate hyperlipidaemia and protect from atherosclerosis.

A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids.

Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79-92% in liver, 76-98% in adipose tissue, 93-104% in feces and 62-79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation.