Dietary Hyodeoxycholic Acid Exerts Hypolipidemic Effects by Reducing Farnesoid X Receptor Antagonist Bile Acids in Mouse Enterohepatic Tissues

Mice were fed a control diet or a diet supplemented with hyodeoxycholic acid, the most abundant bile acid contained in pig bile, for 4 weeks, after which their serum and livers were collected. The contents of total fatty acids of serum and liver cholesteryl esters, and of liver triglycerides, were reduced following the administration of the hyodeoxycholic acid‐supplemented diet, which was mainly due to the reductions in the contents of monounsaturated fatty acids. Free cholesterol contents in the serum and liver were not changed by hyodeoxycholic acid administration. Hyodeoxycholic acid administration reduced the gene expression levels of sterol regulatory element binding protein 1c, acetyl‐CoA carboxylase, fatty acid synthase, and stearoyl‐CoA desaturase‐1. Hyodeoxycholic acid administration markedly changes the ratio of FXR‐antagonist/FXR‐agonist bile acids in the enterohepatic tissues of the mice (1.13 and 7.60 in hyodeoxycholic acid and control diet groups, respectively). Our findings demonstrate that hyodeoxycholic acid administration exerts the hypolipidemic effect in mice, in which downregulations of de novo lipogenesis and desaturation of saturated fatty acids are suggested to play important roles. In addition, regulation of FXR activation through the selective modification of the enterohepatic bile acid pool may be involved in the hypolipidemic effect of hyodeoxycholic acid administration.     

Muricholic Acids Promote Resistance to Hypercholesterolemia in Cholesterol-Fed Mice

Background and aims: Hypercholesterolemia is a major risk factor for atherosclerosis and cardiovascular diseases. Although resistant to hypercholesterolemia, the mouse is a prominent model in cardiovascular research. To assess the contribution of bile acids to this protective phenotype, we explored the impact of a 2-week-long dietary cholesterol overload on cholesterol and bile acid metabolism in mice. Methods: Bile acid, oxysterol, and cholesterol metabolism and transport were assessed by quantitative real-time PCR, western blotting, GC-MS/MS, or enzymatic assays in the liver, the gut, the kidney, as well as in the feces, the blood, and the urine. Results: Plasma triglycerides and cholesterol levels were unchanged in mice fed a cholesterol-rich diet that contained 100-fold more cholesterol than the standard diet. In the liver, oxysterol-mediated LXR activation stimulated the synthesis of bile acids and in particular increased the levels of hydrophilic muricholic acids, which in turn reduced FXR signaling, as assessed in vivo with Fxr reporter mice. Consequently, biliary and basolateral excretions of bile acids and cholesterol were increased, whereas portal uptake was reduced. Furthermore, we observed a reduction in intestinal and renal bile acid absorption. Conclusions: These coordinated events are mediated by increased muricholic acid levels which inhibit FXR signaling in favor of LXR and SREBP2 signaling to promote efficient fecal and urinary elimination of cholesterol and neo-synthesized bile acids. Therefore, our data suggest that enhancement of the hydrophilic bile acid pool following a cholesterol overload may contribute to the resistance to hypercholesterolemia in mice. This work paves the way for new therapeutic opportunities using hydrophilic bile acid supplementation to mitigate hypercholesterolemia.     

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.     

Ursodeoxycholic Acid Suppresses Lipogenesis in Mouse Liver: Possible Role of the Decrease in β-Muricholic Acid,a Farnesoid X Receptor Antagonist

The farnesoid X receptor (FXR) is a major nuclear receptor of bile acids; its activation suppresses sterol regulatory element-binding protein 1c (SREBP1c)-mediated lipogenesis and decreases the lipid contents in the liver. There are many reports showing that the administration of ursodeoxycholic acid (UDCA) suppresses lipogenesis and reduces the lipid contents in the liver of experimental animals. Since UDCA is not recognized as an FXR agonist, these effects of UDCA cannot be readily explained by its direct activation of FXR. We observed that the dietary administration of UDCA in mice decreased the expression levels of SREBP1c and its target lipogenic genes. Alpha- and β-muricholic acids (MCA) and cholic acid (CA) were the major bile acids in the mouse liver but their contents decreased upon UDCA administration. The hepatic contents of chenodeoxycholic acid and deoxycholic acid (DCA) were relatively low but were not changed by UDCA. UDCA did not show FXR agonistic or antagonistic potency in in vitro FXR transactivation assay. Taking these together, we deduced that the above-mentioned change in hepatic bile acid composition induced upon UDCA administration might cause the relative increase in the FXR activity in the liver, mainly by the reduction in the content of β-MCA, a farnesoid X receptor antagonist, which suggests a mechanism by which UDCA suppresses lipogenesis and decreases the lipid contents in the mouse liver.     

The New Therapeutic Approaches in the Treatment of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease which is characterized by extremely complex pathogenetic mechanisms and multifactorial etiology. Some of the many pathophysiological mechanisms involved in the development of NAFLD include oxidative stress, impaired mitochondrial metabolism, inflammation, gut microbiota, and interaction between the brain-liver-axis and the regulation of hepatic lipid metabolism. The new therapeutic approaches in the treatment of NAFLD are targeting some of these milestones along the pathophysiological pathway and include drugs like agonists of peroxisome proliferator-activated receptors (PPARs), glucagon-like peptide-1 (GLP-1) agonists, sodium/glucose transport protein 2 (SGLT2) inhibitors, farnesoid X receptor (FXR) agonists, probiotics, and symbiotics. Further efforts in biomedical sciences should focus on the investigation of the relationship between the microbiome, liver metabolism, and response to inflammation, systemic consequences of metabolic syndrome.     

Enhancement of Farnesoid X Receptor Inhibits Migration,Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

(1) Background: Bladder cancer is a malignant tumor mainly caused by exposure to environmental chemicals, with a high recurrence rate. NR1H4, also known as Farnesoid X Receptor (FXR), acts as a nuclear receptor that can be activated by binding with bile acids, and FXR is highly correlated with the progression of cancers. The aim of this study was to verify the role of FXR in bladder cancer cells. (2) Methods: A FXR overexpressed system was established to investigate the effect of cell viability, migration, adhesion, and angiogenesis in low-grade TSGH8301 and high-grade T24 cells. (3) Results: After FXR overexpression, the ability of migration, adhesion, invasion and angiogenesis of bladder cancer cells declined significantly. Focal adhesive complex, MMP2, MMP9, and angiogenic-related proteins were decreased, while FXR was overexpressed in bladder cancer cells. Moreover, FXR overexpression reduced vascular endothelial growth factor mRNA and protein expression and secretion in bladder cancer cells. After treatment with the proteosome inhibitor MG132, the migration, adhesion and angiogenesis caused by FXR overexpression were all reversed in bladder cancer cells. (4) Conclusions: These results may provide evidence on the role of FXR in bladder cancer, and thus may improve the therapeutic efficacy of urothelial carcinoma in the future.     

Microbiome and PCOS: State-of-Art and Future Aspects

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disease. The hypothesis that alterations in the microbiome are involved in the genesis of PCOS has been postulated. Aim of this review is to summarize the available literature data about the relationship between microbiome and PCOS. A search on PubMed and Medline databases was performed from inception to November 20Most of evidence has focused on the connection of intestinal bacteria with sex hormones and insulin-resistance: while in the first case, a relationship with hyperandrogenism has been described, although it is still unclear, in the second one, chronic low-grade inflammation by activating the immune system, with increased production of proinflammatory cytokines which interfere with insulin receptor function, causing IR (Insulin Resistance)/hyperinsulinemia has been described, as well as the role of gastrointestinal hormones like Ghrelin and peptide YY (PYY), bile acids, interleukin-22 and Bacteroides vulgatus have been highlighted. The lower genital tract microbiome would be affected by changes in PCOS patients too. The therapeutic opportunities include probiotic, prebiotics and synbiotics, as well as fecal microbiota transplantation and the use of IL-22, to date only in animal models, as a possible future drug. Current evidence has shown the involvement of the gut microbiome in PCOS, seen how humanized mice receiving a fecal transplant from women with PCOS develop ovarian dysfunction, immune changes and insulin resistance and how it is capable of disrupting the secondary bile acid biosynthesis. A future therapeutic approach for PCOS may involve the human administration of IL-22 and bile acid glycodeoxycholic acid.     

八肽胆囊收缩素对大鼠滑膜细胞株RSC-364 TNF受体基因表达的影响

目的探讨CCK8对RSC364细胞株TNF受体基因表达的影响。方法采用RTPCR法检测不同浓度的八肽胆囊收缩素(CCK8)对在TNF-α诱导下的大鼠滑膜细胞RSC364TNF受体(Tumornecrosisfactorreceptor,TNFR)mRNA表达的影响。结果CCK8在10-6mol/L和10-7mol/L浓度时可抑制TNF-α诱导的TNFR2mRNA在RSC364细胞的表达,且表现了一定的剂量和时间依赖性,CCK受体拮抗剂丙谷胺则可抑制此作用。结论CCK8可抑制TNF-α诱导的大鼠滑膜细胞RSC364株TNFR2基因的表达。     

Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay

The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7α-hydroxylase, which in turn regulates bile acid production and cholesterol excretion. To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library. Three human FXR cDNA, designated FXR₂₀, FXR₃₃, and FXR₅₃ cDNA, were subcloned and ligated into a pET28a expression vector. Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography. Approximately 5 mg of FXR₃₃ (1–182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR₅₃ (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity. The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N¹-labeled anti-His antibody. From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR₃₃ and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 μM). The ligand-binding domain of FXR (FXR₃₃) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR₅₃) or the DNA-binding domain of FXR (FXR₂₀).     

Pectin Penta-Oligogalacturonide Suppresses Intestinal Bile Acids Absorption and Downregulates the FXR-FGF15 Axis in High-Cholesterol Fed Mice

Haw pectin penta‐oligogalacturonide (HPPS), purified from the hydrolysates of haw pectin, has important role in decreasing hepatic cholesterol accumulation and promoting bile acids (BA) excretion in the feces of mice fed a high‐cholesterol diet (HCD). However, the mechanism is not clear. This study aims to investigate the effects of HPPS on BA reabsorption in ileum and biosynthesis in liver of mice. Results showed that HPPS increased fecal BA output by approximately 110%, but decreased ileal BA and the total BA pool size by approximately 47 and 36%, respectively, compared to HCD. Studies of molecular mechanism revealed that HPPS significantly decreased the mRNA and protein levels of farnesoid X receptor (FXR) in the small intestine of mice and inactivated the fibroblast growth factor 15 (FXR‐FGF15) axis, which increased the mRNA and protein levels of CYP7A1 by approximately 204 and 104%, respectively, compared to HCD. Interestingly, the mRNA and protein levels of apical sodium‐dependent bile acid transporter (ASBT) in the small intestine were approximately 128 and 73% higher in HPPS‐fed mice than those in HCD‐fed mice, respectively. However, no significant difference was detected for ASBT expression between HCD group and BA sequestrant cholestyramine group. These findings indicate that HPPS can suppress intestinal BA reabsorption and promoting hepatic BA biosynthesis. We speculated that HPPS could be ASBT competitive inhibitor rather than BA sequestrant in inhibiting BA reabsorption in ileum and improving cholesterol metabolism.     

Development and in vitro Profiling of Dual FXR/LTA4H Modulators

Designed polypharmacology presents as an attractive strategy to increase therapeutic efficacy in multi-factorial diseases by a directed modulation of multiple involved targets with a single molecule. Such an approach appears particularly suitable in non-alcoholic steatohepatitis (NASH) which involves hepatic steatosis, inflammation and fibrosis as pathological hallmarks. Among various potential pharmacodynamic mechanisms, activation of the farnesoid X receptor (FXRa) and inhibition of leukotriene A4 hydrolase (LTA4Hi) hold promise to counteract NASH according to preclinical and clinical observations. We have developed dual FXR/LTA4H modulators as pharmacological tools, enabling evaluation of this polypharmacology concept to treat NASH and related pathologies. The optimized FXRa/LTA4Hi exhibits well-balanced dual activity on the intended targets with sub-micromolar potency and is highly selective over related nuclear receptors and enzymes rendering it suitable as tool to probe synergies of dual FXR/LTA4H targeting.     

Dual Farnesoid X Receptor/Soluble Epoxide Hydrolase Modulators Derived from Zafirlukast

The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non-alcoholic steatohepatitis (NASH). Their simultaneous modulation in vivo has demonstrated a triad of anti-NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti-asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver-related metabolic diseases.     

Effect of Peucedanum japonicum Thunb on the expression of obesity-related genes in mice on a high-fat diet

The present study describes the effect of Peucedanum japonicum Thunb (PJT) intake on the expression of obesity-related genes in mice fed a high-fat diet. To explore the mechanism underlying the effect of PJT, This study focused on the expression of genes, especially those related to obesity and metabolism syndrome, in the liver and adipose tissues. In agreement with our previous observations, intake of 10 % PJT for 4 weeks significantly reduced serum triglyceride (TG), leptin, abdominal fat, and adipocyte size. PJT also significantly increased fecal excretion of TG, decreased that of bile acid, and tended to increase the fecal excretion of total cholesterol. Microarray analysis was used to monitor changes in 324 metabolic syndrome-related genes in the liver. Statistically significant upregulation of PPP1R10, RORC, and PBEF1 genes and downregulation of DUSP1, INSIG2, and SERPINA12 genes were noted and confirmed by real-time RT-PCR. These changes were indicative of increased fatty acid oxidation in the maintenance of lipid homeostasis and insulin sensitivity in the livers of PJT-fed mice. PJT increased the expression of PPARγ, FXRα, DGAT1, and ATGL genes, suggesting an enhancement of adipocyte differentiation and normalization of functionality of adipose tissue.     

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.     

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.     

Heat-Inactivated Akkermansia muciniphila Improves Gut Permeability but Does Not Prevent Development of Non-Alcoholic Steatohepatitis in Diet-Induced Obese Ldlr−/−.Leiden Mice

The development of non-alcoholic steatohepatitis (NASH) has been associated with alterations in gut microbiota composition and reduced gut barrier function. Akkermansia muciniphila is a gut microbe that is thought to have health-promoting properties, including the ability to improve gut barrier function and host metabolism, both when administered live and after heat-inactivation. We questioned whether heat-inactivated A. muciniphila may reduce NASH development. Ldlr−/−.Leiden mice, a translational, diet-induced model for NASH, were fed a NASH-inducing high-fat diet (HFD) supplemented with heat-inactivated A. muciniphila. After 28 weeks, effects of the treatment on obesity and associated metabolic dysfunction in the gut (microbiota composition and permeability), adipose tissue, and liver were studied relative to an untreated HFD control. Treatment with heat-inactivated A. muciniphila did not affect body weight or adiposity and had no effect on plasma lipids, blood glucose, or plasma insulin. Heat-inactivated A. muciniphila had some minor effects on mucosal microbiota composition in ileum and colon and improved gut barrier function, as assessed by an in vivo functional gut permeability test. Epidydimal white adipose tissue (WAT) hypertrophy and inflammation were not affected, but heat-inactivated A. muciniphila did reduce hypertrophy in the mesenteric WAT which is in close proximity to the intestine. Heat-inactivated A. muciniphila did not affect the development of NASH or associated fibrosis in the liver and did not affect circulating bile acids or markers of liver fibrosis, but did reduce PRO-C4, a type IV collagen synthesis marker, which may be associated with gut integrity. In conclusion, despite beneficial effects in the gut and mesenteric adipose tissue, heat-inactivated A. muciniphila did not affect the development of NASH and fibrosis in a chronic disease setting that mimics clinically relevant disease stages.     

Bile Acid Signaling in Inflammatory Bowel Disease

Inflammatory bowel disease is a chronic, idiopathic and complex condition, which most often manifests itself in the form of ulcerative colitis or Crohn’s disease. Both forms are associated with dysregulation of the mucosal immune system, compromised intestinal epithelial barrier, and dysbiosis of the gut microbiome. It has been observed for a long time that bile acids are involved in inflammatory disorders, and recent studies show their significant physiological role, reaching far beyond being emulsifiers helping in digestion of lipids. Bile acids are also signaling molecules, which act, among other things, on lipid metabolism and immune responses, through several nuclear and membrane receptors in hepatocytes, enterocytes and cells of the immune system. Gut microbiota homeostasis also seems to be affected, directly and indirectly, by bile acid metabolism and signaling. This review summarizes recent advances in the field of bile acid signaling, studies of inflamed gut microbiome, and the therapeutic potential of bile acids in the context of inflammatory bowel disease.