Identification of metabolism-related key genes as potential biomarkers for pathogenesis of immune thrombocytopenia.

Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction. A biomarker is a biological entity that contributes to disease pathogenesis and reflects disease activity. Metabolic alterations are reported to be associated with the occurrence of various diseases. As metabolic biomarkers for ITP have not been identified. This study aimed to identify metabolism-related differentially expressed genes as potential biomarkers for pathogenesis of ITP using bioinformatic analyses.The microarray expression data of the peripheral blood mononuclear cells were downloaded from the Gene Expression Omnibus database (GSE112278 download link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112278 ). Key module genes were intersected with metabolism-related genes to obtain the metabolism-related key candidate genes. The hub genes were screened based on the degree function in the coytoscape sofware. The key ITP-related genes were subjected to functional enrichment analysis. Immune infiltration analysis was performed using a single-sample gene set enrichment analysis algorithm to evaluate the differential infiltration levels of immune cell types between ITP patient and control. Molecular subtypes were identified based on the expression of hub genes. The expression of hub genes in the ITP patients was validated using quantitative real-time polymerase chain reaction analysis. This study identified five hub genes (ADH4, CYP7A1, CYP1A2, CYP8B1, and NR1H4), which were be associated with the pathogenesis of ITP, and two molecular subtypes of ITP. Among these hub genes, CYP7A1 and CYP8B1 involved in cholesterol metabolism,were further verified in clinical samples.

Therapeutic Potential of Danyankang Capsule in High-Fat Diet-Induced Cholelithiasis and Its Impact on Liver FXR Signaling and Gut Microbiota.

Cholelithiasis, commonly known as gallstones, represents a prevalent hepatobiliary disorder. This study aimed to elucidate the therapeutic role and mechanism of Danyankang capsulein treating cholelithiasis induced by a high-fat diet in C57BL/6 mice. The therapeutical potential of Danyankang was assessed through biochemical analyses, histopathological examinations, protein detection, and 16S rDNA sequencing. A high-fat diet resulted in cholelithiasis manifestation in mice, with discernable abnormal serum biochemical indices and disrupted biliary cholesterol homeostasis. Danyankang treatment notably ameliorated liver inflammation symptoms and rectified serum and liver biochemical abnormalities. Concurrently, it addressed biliary imbalances. Elevated expressions of toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-κB)/pNF-κB, HMGCR, CYP7A1, and CYP8B1 observed at the inception of cholelithiasis, were notably reduced upon Danyankang administration. Furthermore, 16S rDNA analysis revealed a decline in species number and diversity of the intestinal flora in cholelithiasis-treated mice, while the decline was reversed with Danyankang treatment. Danyankang capsules reduced the abundance of Verrucomicrobiota and increased the abundance of Actinobacteriota and Proteobacteria. In conclusion, the present study demonstrates that Danyankang exerts potent therapeutic efficacy against high-fat diet-induced cholelithiasis. This beneficial outcome is potentially linked to the inhibition of the TLR4/pNF-κB and SHP/CYP7A1/CYP8B1 signaling pathways, as well as the enhancement of intestinal flora species abundance.

[Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells].

Objective: To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 µmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). Results: Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 µmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. Conclusion: After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.

CYP8B1 downregulation mediates the metabolic effects of vertical sleeve gastrectomy in mice.

BACKGROUND AND AIMS: Although the benefits of vertical sleeve gastrectomy (VSG) surgery are well known, the molecular mechanisms by which VSG alleviates obesity and its complications remain unclear. We aim to determine the role of CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1) in mediating the metabolic benefits of VSG. APPROACH AND RESULTS: We found that expression of CYP8B1, a key enzyme in controlling the 12α-hydroxylated (12α-OH) bile acid (BA) to non-12α-OH BA ratio, was strongly downregulated after VSG. Using genetic mouse models of CYP8B1 overexpression, knockdown, and knockout, we demonstrated that overexpression of CYP8B1 dampened the metabolic improvements associated with VSG. In contrast, short hairpin RNA-mediated CYP8B1 knockdown improved metabolism similar to those observed after VSG. Cyp8b1 deficiency diminished the metabolic effects of VSG. Further, VSG-induced alterations to the 12α-OH/non-12α-OH BA ratio in the BA pool depended on CYP8B1 expression level. Consequently, intestinal lipid absorption was restricted, and the gut microbiota (GM) profile was altered. Fecal microbiota transplantation from wild type-VSG mice (vs. fecal microbiota transplantation from wild-type-sham mice) improved metabolism in recipient mice, while there were no differences between mice that received fecal microbiota transplantation from knockout-sham and knockout-VSG mice. CONCLUSIONS: CYP8B1 is a critical downstream target of VSG. Modulation of BA composition and gut microbiota profile by targeting CYP8B1 may provide novel insight into the development of therapies that noninvasively mimic bariatric surgery to treat obesity and its complications.

Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.

Gut microbiota depletion aggravates bile acid-induced liver pathology in mice with a human-like bile acid composition.

Cyp2c70-deficient mice have a human-like bile acid (BA) composition due to their inability to convert chenodeoxycholic acid (CDCA) into rodent-specific muricholic acids (MCAs). However, the hydrophobic BA composition in these animals is associated with liver pathology. Although Cyp2c70-ablation has been shown to alter gut microbiome composition, the impact of gut bacteria on liver pathology in Cyp2c70-/- mice remains to be established. Therefore, we treated young-adult male and female wild-type (WT) and Cyp2c70-/- mice with antibiotics (AB) with broad specificity to deplete the gut microbiota and assessed the consequences on BA metabolism and liver pathology. Female Cyp2c70-/- mice did not tolerate AB treatment, necessitating premature termination of the experiment. Male Cyp2c70-/- mice did tolerate AB but showed markedly augmented liver pathology after 6 weeks of treatment. Dramatic downregulation of hepatic Cyp8b1 expression (-99%) caused a reduction in the proportions of 12α-hydroxylated BAs in the circulating BA pools of AB-treated male Cyp2c70-/- mice. Interestingly, the resulting increased BA hydrophobicity strongly correlated with various indicators of liver pathology. Moreover, genetic inactivation of Cyp8b1 in livers of male Cyp2c70-/- mice increased liver pathology, while addition of ursodeoxycholic acid to the diet prevented weight loss and liver pathology in AB-treated female Cyp2c70-/- mice. In conclusion, depletion of gut microbiota in Cyp2c70-/- mice aggravates liver pathology at least in part by increasing the hydrophobicity of the circulating BA pool. These findings highlight that the potential implications of AB administration to cholestatic patients should be evaluated in a systematic manner.

Fucogalactan Sulfate (FS) from Laminaria japonica Regulates Lipid Metabolism in Diet-Induced Humanized Dyslipidemia Mice via an Intestinal FXR-FGF19-CYP7A1/CYP8B1 Pathway.

Our previous study found that fucogalactan sulfate (FS) from Laminaria japonica exhibited significant hypolipidemic effects. To further elucidate the mechanism, we first constructed a dyslipidemia mouse model with humanized gut microbiota and proved the main differential metabolic pathway involved bile acid metabolism. Then, we evaluated the beneficial effects of FS on dyslipidemia in this model mice, which revealed that oral FS administration reduced serum cholesterol levels and mitigated liver fat accumulation. Gut microbiota and microbiome analysis showed FS increased the abundance of Ruminococcaceae_NK4A214_group, GCA-900066755, and Eubacterium, which were positively associated with the fecal DCA, ß-MCA, and HDCA. Further investigation demonstrated that FS inhibited the hepatic farnesoid X receptor (FXR), while activating the intestinal FXR-FGF19 pathway, leading to suppression of CYP7A1 and CYP8B1, as well as potentially reduced bile acid synthesis and lipid absorption. Overall, FS regulated lipid metabolism in diet-induced humanized dyslipidemia mice via the bile acid-mediated intestinal FXR-FGF19-CYP7A1/CYP8B1 pathway.

Discovery biomarker to optimize obeticholic acid treatment for non-alcoholic fatty liver disease.

The response rate to obeticholic acid (OCA), a potential therapeutic agent for non-alcoholic fatty liver disease, is limited. This study demonstrated that upregulation of the alternative bile acid synthesis pathway increases the OCA treatment response rate. The hepatic transcriptome and bile acid metabolite profile analyses revealed that the alternative bile acid synthesis pathway (Cyp7b1 and muricholic acid) in the OCA-responder group were upregulated compared with those in the OCA-non-responder group. Intestinal microbiome analysis also revealed that the abundances of Bacteroidaceae, Parabacteroides, and Bacteroides, which were positively correlated with the alternative bile acid synthesis pathway, were higher in the OCA-responder group than in the non-responder group. Pre-study hepatic mRNA levels of Cyp8b1 (classic pathway) were downregulated in the OCA-responder group. The OCA response rate increased up to 80% in cases with a hepatic Cyp7b1/Cyp8b1 ratio ≥ 5.0. Therefore, the OCA therapeutic response can be evaluated based on the Cyp7b1/Cyp8b1 ratio or the alternative/classic bile acid synthesis pathway activity.

The Placenta-A New Source of Bile Acids during Healthy Pregnancy? First Results of a Gene Expression Study in Humans and Mice.

Bile acids (BAs) are natural ligands for several receptors modulating cell activities. BAs are synthesized via the classic (neutral) and alternative (acidic) pathways. The classic pathway is initiated by CYP7A1/Cyp7a1, converting cholesterol to 7α-hydroxycholesterol, while the alternative pathway starts with hydroxylation of the cholesterol side chain, producing an oxysterol. In addition to originating from the liver, BAs are reported to be synthesized in the brain. We aimed at determining if the placenta potentially represents an extrahepatic source of BAs. Therefore, the mRNAs coding for selected enzymes involved in the hepatic BA synthesis machinery were screened in human term and CD1 mouse late gestation placentas from healthy pregnancies. Additionally, data from murine placenta and brain tissue were compared to determine whether the BA synthetic machinery is comparable in these organs. We found that CYP7A1, CYP46A1, and BAAT mRNAs are lacking in the human placenta, while corresponding homologs were detected in the murine placenta. Conversely, Cyp8b1 and Hsd17b1 mRNAs were undetected in the murine placenta, but these enzymes were found in the human placenta. CYP39A1/Cyp39a1 and cholesterol 25-hydroxylase (CH25H/Ch25h) mRNA expression were detected in the placentas of both species. When comparing murine placentas and brains, Cyp8b1 and Hsd17b1 mRNAs were only detected in the brain. We conclude that BA synthesis-related genes are placentally expressed in a species-specific manner. The potential placentally synthesized BAs could serve as endocrine and autocrine stimuli, which may play a role in fetoplacental growth and adaptation.

Deletion of hepatic growth hormone receptor (GHR) alters the mouse gut microbiota by affecting bile acid metabolism.

Both growth hormone (GH) and gut microbiota play significant roles in diverse physiological processes, but the crosstalk between them is poorly understood. Despite the regulation of GH by gut microbiota, study on GH's influence on gut microbiota is limited, especially on the impacts of tissue specific GH signaling and their feedback effects on the host. In this study, we profiled gut microbiota and metabolome in tissue-specific GHR knockout mice in the liver (LKO) and adipose tissue (AKO). We found that GHR disruption in the liver rather than adipose tissue affected gut microbiota. It changed the abundance of Bacteroidota and Firmicutes at phylum level as well as abundance of several genera, such as Lactobacillus, Muribaculaceae, and Parasutterella, without affecting α-diversity. Moreover, the impaired liver bile acid (BA) profile in LKO mice was strongly associated with the change of gut microbiota. The BA pools and 12-OH BAs/non-12-OH BAs ratio were increased in the LKO mice, which was due to the induction of CYP8B1 by hepatic Ghr knockout. Consequently, the impaired BA pool in cecal content interacted with gut bacteria, which in turn increased the production of bacteria derived acetic acid, propionic acid, and phenylacetic acid that were possible to participate in the impaired metabolic phenotype of the LKO mice. Collectively, our findings suggested that the liver GH signaling regulates BA metabolism by its direct regulation on CYP8B1, which is an important factor influencing gut microbiota. Our study is significant in exploring gut microbiota modification effects of tissue-specific GH signaling as well as its involvement in gut microbiota-host interaction.

UHPLC-Orbitrap-Fusion-TMS-Based Metabolomics Study of Phenylpropionamides in the Seed of Cannabis sativa L. against Alzheimer's Disease.

Phenylpropionamides in the seed of Cannabis sativa L. (PHS) have a protective effect on neuroinflammation and antioxidant activity. In this study, the UHPLC-Orbitrap-fusion-TMS-based metabolomics approach was used to analyze the serum samples and identify potential biomarkers in Streptozotocin (STZ) induced Alzheimer's disease (AD) rats. The results revealed that primary bile acid biosynthesis and taurine and hypotaurine metabolism were significantly correlated with STZ-induced AD rats. In addition, the key enzymes in these two pathways were verified at the protein level. The levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD), cysteamine (2-aminoethanethiol) dioxygenase (ADO), 7α-hydroxylase (CYP7A1), and sterol 12α-hydroxylase (CYP8B1) were the key enzymes affecting the two pathways in AD rats compared with the control group (CON). Furthermore, after a high-dose group of phenylpropionamides in the seed of Cannabis sativa L. (PHS-H) was administrated, the levels of CDO1, CSAD, CYP7A1, and CYP8B1 were all callback. These findings demonstrate for the first time that the anti-AD effect of PHS is associated with the regulation of primary bile acid biosynthesis and taurine and hypotaurine metabolism in STZ-induced AD rats.

Pyridine-containing substrate analogs are restricted from accessing the human cytochrome P450 8B1 active site by tryptophan 281.

The human oxysterol 12α-hydroxylase cytochrome P450 8B1 (CYP8B1) is a validated drug target for both type 2 diabetes and nonalcoholic fatty liver disease, but effective selective inhibitors are not yet available. Herein, steroidal substrate-mimicking compounds with a pyridine ring appended to the C12 site of metabolism were designed as inhibitors, synthesized, and evaluated in terms of their functional and structural interactions with CYP8B1. While the pyridine nitrogen was intended to coordinate the CYP8B1 active site heme iron, none of these compounds elicited shifts in the CYP8B1 Soret absorbance consistent with this type of interaction. However, when CYP8B1 was cocrystallized with the pyridine-containing compound with the 3-keto-Δ4 steroid backbone most similar to the endogenous substrate, it was apparent that this ligand was bound in a channel leading to the active site, instead of near the heme iron. Inspection of this structure suggested that tryptophan 281 directly above the heme might restrict active site binding of potential inhibitors with this design. This hypothesis was supported when a CYP8B1 W281F mutation did allow all three compounds to coordinate the heme iron as designed. These results indicated that the design of next-generation CYP8B1 inhibitors should be compatible with the low-ceiling tryptophan immediately above the heme iron.

Haploinsufficiency of CYP8B1 associates with increased insulin sensitivity in humans.

BACKGROUNDCytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.METHODSTo determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.RESULTSMutation carriers had lower plasma 12α-hydroxylated/non-12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.CONCLUSIONOur findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.FUNDINGBiomedical Research Council/National Medical Research Council of Singapore.

The metabolic genomic atlas reveals potential drivers and clinically relevant insights into the etiology of esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers globally, with a poor prognosis and ambiguous therapy target. As a hallmark of cancer, metabolism reprogramming plays a critical role in the development of ESCC; however, the genomic alterations underlying this reconfiguration are still largely unknown. Methods: We have comprehensively studied the metabolic genomic variations in an integrated ESCC cohort of 490 patients and characterized the somatic alterations associated with various metabolic pathways. Results: The somatic mutations and copy number alterations (CNAs) occurred heterogeneously in all patients. Using CNA-based clustering, we stratified patients into three clusters and Cluster3 with more deletions marked for worse prognosis. Our findings revealed detailed genetic alterations in components of metabolic pathways and highlighted the role of metal ion channel transporters and non-neuronal/neuronal synapse systems in the development of ESCC. We found a subset of potential metabolic drivers and functionally validated RYR2, MGST3, and CYP8B1 involved in the ESCC-associated malignancy. Another key finding was that we identified 27 metabolic genes with genomic alterations that could serve as independent prognostic factors and figured out two genetic panels that could stratify patients into distinct prognostic groups. Conclusion: Collectively, our study provided a deep insight into the metabolic landscape in ESCC, extending our understanding of the metabolic reconfiguration underlying the genomic basis of ESCC. Furthermore, our findings revealed potential prognostic factors of ESCC, which are expected to contribute to the accurate determination of the prognosis in the clinic.

Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal.

Although disrupted bile acid (BA) homeostasis is implicated in inflammatory bowel disease (IBD), the role of hepatic BA metabolism in the pathogenesis of colitis is poorly understood. Here, we found that cholic acid (CA) levels were increased in patients and mice. Cytochrome P450 8B1 (CYP8B1), which synthesizes CA, was induced in livers of colitic mice. CA-treated or liver Cyp8b1-overexpressing mice developed more severe colitis with compromised repair of the mucosal barrier, whereas Cyp8b1-knockout mice were resistant to colitis. Mechanistically, CA inhibited peroxisome proliferator-activated receptor alpha (PPARα), resulting in impeded fatty acid oxidation (FAO) and impaired Lgr5+ intestinal stem cell (ISC) renewal. A PPARα agonist restored FAO and improved Lgr5+ ISC function. Activation of the farnesoid X receptor (FXR) suppressed liver CYP8B1 expression and ameliorated colitis in mice. This study reveals a connection between the hepatic CYP8B1-CA axis and colitis via regulating intestinal epithelial regeneration, suggesting that BA-based strategies might be beneficial in IBD treatment.

Daytime restricted feeding promotes circadian desynchrony and metabolic disruption with changes in bile acids profiles and gut microbiota in C57BL/6 Male Mice.

Fasting/feeding cycles regulate clock-lipid-bile acid (BA) metabolic homeostasis, and gut microbiota also participates in connecting circadian rhythms with BA metabolism. To investigate the cyclical nature of microbial-metabolism-host interactions, sixty male C57BL/6 mice were randomized into three feeding regimens with a chow diet: 24 h ad libitum (AC), 12 h nighttime feeding (NC) or 12 h daytime feeding (DC). Five weeks later, the mice were sacrificed at six-hour intervals over 24 hours. Daytime feeding abolished hepatic rhythmic expressions of Per1, Cry1/2 and Rev-erbα or changed the acrophase of Clock, Bmal1 and Per2, also the rhythmic expression of genes Hsl, Fas, Acc, Srebp-1c in lipid homeostasis and Cyp7a1, Cyp7b1, Cyp8b1, Lrh-1 and Shp in bile acid metabolism compared with their ad libitum and dark-fed companions. Furthermore, daytime feeding upregulated the levels of fecal primary BA, secondary BA and unconjugated BA at ZT0 and decreased their levels at ZT12. Meanwhile, daytime feeding altered the diversity of gut microbiota and microbiota compositions, with obviously higher abundance of Firmicutes and F/B ratio, and significantly lower abundance of Verrucomicrobia, as well as altered fluctuations of Akkermansia, Lactobacillus and Parabacteroides. In conclusion, shifting food intake to the rest phase caused a desynchronization in the liver between circadian rhythm and metabolism, as well as abnormal circadian variations in fecal BA profiles and gut microbiota.

The structure and characterization of human cytochrome P450 8B1 supports future drug design for nonalcoholic fatty liver disease and diabetes.

Human cytochrome P450 8B1 (CYP8B1) is involved in conversion of cholesterol to bile acids. It hydroxylates the steroid ring at C12 to ultimately produce the bile acid cholic acid. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. Herein, the human CYP8B1 protein was generated and used to identify and characterize interactions with a series of azole inhibitors, which tend to be poorly selective P450 inhibitors. Structurally related miconazole, econazole, and tioconazole bound with submicromolar dissociation constants and were effective inhibitors of the native reaction. CYP8B was cocrystallized with S-tioconazole to yield the first X-ray structure. This inhibitor bound in the active site with its azole nitrogen coordinating the heme iron, consistent with inhibitor binding and inhibition assay data. Additionally, the CYP8B1 active site was compared with similar P450 enzymes to identify features that may facilitate the design of more selective inhibitors. Selective inhibitors should promote a better understanding of the role of CYP8B1 inhibition in normal physiology and disease states and provide a possible treatment for nonalcoholic fatty liver disease and type 2 diabetes.

Loss of Hepatic Small Heterodimer Partner Elevates Ileal Bile Acids and Alters Cell Cycle-related Genes in Male Mice.

Small heterodimer partner (Shp) regulates several metabolic processes, including bile acid levels, but lacks the conserved DNA binding domain. Phylogenetic analysis revealed conserved genetic evolution of SHP, FXR, CYP7A1, and CYP8B1. Shp, although primarily studied as a downstream target of Farnesoid X Receptor (Fxr), has a distinct hepatic role that is poorly understood. Here, we report that liver-specific Shp knockout (LShpKO) mice have impaired negative feedback of Cyp7a1 and Cyp8b1 on bile acid challenge and demonstrate that a single copy of the Shp gene is sufficient to maintain this response. LShpKO mice also exhibit elevated total bile acid pool with ileal bile acid composition mimicking that of cholic acid-fed control mice. Agonistic activation of Fxr (GW4064) in the LShpKO did not alter the elevated basal expression of Cyp8b1 but lowered Cyp7a1 expression. We found that deletion of Shp led to an enrichment of distinct motifs and pathways associated with circadian rhythm, copper ion transport, and DNA synthesis. We confirmed increased expression of metallothionein genes that can regulate copper levels in the absence of SHP. LShpKO livers also displayed a higher basal proliferation that was exacerbated specifically with bile acid challenge either with cholic acid or 3,5-diethoxycarbonyl-1,4-dihydrocollidine but not with another liver mitogen, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Overall, our data indicate that hepatic SHP uniquely regulates certain proliferative and metabolic cues.

Long-term oral administration of Epimedii Folium induced cholestasis in mice by interfering with bile acid transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedii Folium (EF) is a common traditional Chinese medicine that functions as a tonifying kidney yang to strengthen bones and muscles and dispel wind dampness (limb pain, lethargy, nausea, anorexia, and loose stools). Several studies have reported the potential risk of cholestatic liver damage from EF use; however, there have been few investigations of EF-induced cholestasis, particularly the underlying mechanisms. AIMS OF THE STUDY: The purpose of this study was to evaluate the risk of EF-induced cholestasis in vivo and to explore the mechanisms of action. MATERIALS AND METHODS: ICR mice were orally administered a water extract of EF (WEF) in doses of 6.5 and 19.5 g/kg/day for 14 weeks. Liver-to-body weight ratios, body weight, histopathological examination, and biochemical analyses were performed to assess WEF-induced cholestasis in the mice. Genes associated with bile acid (BA) metabolism and transport, including sodium taurocholate cotransporting polypeptide (NTCP), cytochrome P450 8B1 (CYP8B1), bile-salt export pump (BSEP), multidrug resistance P-glycoproteins 1 (MDR1), and farnesoid X receptor (FXR), were measured at the transcript and protein levels to investigate the potential mechanisms through which cholestasis is aroused by EF. RESULTS: After administration of WEF for 14 weeks, mice in the high-dose WEF group showed poor health with an increased liver-to-body weight ratio as well as higher serum aminotransferase, alkaline phosphatase, direct bilirubin, and total BA levels. Compared with the control group, mRNA expression of NTCP and cholesterol 7a-hydroxylase (CYP7A1) increased, and levels of BSEP, MDR1, multidrug resistance-associated protein 2, and multidrug resistance-associated protein 3 decreased in the WEF-treated group. NTCP, BSEP, MDR1, and CYP8B1 showed similar mRNA and protein expression trends. CONCLUSION: We demonstrated that the long-term oral administration of WEF causes cholestatic liver injury in mice, which is consistent with reported clinical cases. Furthermore, we found that the destruction of BA metabolism and transport is involved in WEF-induced cholestasis. The fine-scale molecular mechanisms of WEF-induced cholestasis and the active compounds of EF need further study.

Insulin Prevents Hypercholesterolemia by Suppressing 12α-Hydroxylated Bile Acids.

BACKGROUND: The risk of cardiovascular disease in type 1 diabetes remains extremely high, despite marked advances in blood glucose control and even the widespread use of cholesterol synthesis inhibitors. Thus, a deeper understanding of insulin regulation of cholesterol metabolism, and its disruption in type 1 diabetes, could reveal better treatment strategies. METHODS: To define the mechanisms by which insulin controls plasma cholesterol levels, we knocked down the insulin receptor, FoxO1, and the key bile acid synthesis enzyme, CYP8B1. We measured bile acid composition, cholesterol absorption, and plasma cholesterol. In parallel, we measured markers of cholesterol absorption and synthesis in humans with type 1 diabetes treated with ezetimibe and simvastatin in a double-blind crossover study. RESULTS: Mice with hepatic deletion of the insulin receptor showed marked increases in 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol. This phenotype was entirely reversed by hepatic deletion of FoxO1. FoxO1 is inhibited by insulin and required for the production of 12α-hydroxylated bile acids, which promote intestinal cholesterol absorption and suppress hepatic cholesterol synthesis. Knockdown of Cyp8b1 normalized 12α-hydroxylated bile acid levels and completely prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor (n=5-30), as well as mouse models of type 1 diabetes (n=5-22). In parallel, the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and low-density lipoprotein cholesterol in patients with type 1 diabetes as well as, or better than, the cholesterol synthesis inhibitor, simvastatin (n=20). CONCLUSIONS: Insulin, by inhibiting FoxO1 in the liver, reduces 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol levels. Thus, type 1 diabetes leads to a unique set of derangements in cholesterol metabolism, with increased absorption rather than synthesis. These derangements are reversed by ezetimibe, but not statins, which are currently the first line of lipid-lowering treatment in type 1 diabetes. Taken together, these data suggest that a personalized approach to lipid lowering in type 1 diabetes may be more effective and highlight the need for further studies specifically in this group of patients.