My lifelong dedication to bile acid research.

It is a great honor to be invited to write a reflections article on my scientific journey and lifelong bile acid research for the Journal of Biological Chemistry, in which I am proud to have published 24 articles. I have also published 21 articles in the Journal of Lipid Research, another journal of the American Society of Biochemistry and Molecular Biology. I begin my reflections from my early education in Taiwan, my coming to America for graduate study, and continue with my postdoctoral training in cytochrome P450 research, and my lifelong bile acid research career at Northeast Ohio Medical University. I have witnessed and helped in the transformation of this rural not so visible medical school to a well-funded leader in liver research. Writing this reflections article on my long and rewarding journey in bile acid research brings back many good memories. I am proud of my scientific contributions and attribute my academic success to hard work, perseverance, good mentoring, and networking. I hope these reflections of my academic career would help inspire young investigators to pursue an academic career in biochemistry and metabolic diseases.

My lifelong dedication to bile acid research.

It is a great honor to be invited to write a reflection of my lifelong bile acid research for the Journal of Biological Chemistry, the premier biochemistry journal in which I am proud to have published 24 manuscripts. I published 21 manuscripts in the Journal of Lipid Research, also a journal of American Society of Biochemistry and Molecular Biology. I started my reflection from my early education in Taiwan, my coming to America for graduate study, my postdoctoral training in cytochrome P450 research, and my lifelong bile acid research career at the not so "visible" Northeast Ohio Medical University. I have witnesses and help to transform this sleepy rural medical school to a well-funded powerhouse in liver research. Writing this reflection of my long, exciting, and rewarding journey in bile acid research brought back many good memories. I am proud of my scientific contribution. I attribute my lifelong academic success to working hard, perseverance, good mentoring, and networking. I hope that this reflection of my academic career may provide guidance to younger investigators who are pursuing academic teaching and research and might inspire the next generation of researchers in biochemistry and metabolic diseases.

Aromatic Amino Acids Promote Lipid Metabolism Disorders by Increasing Hepatic Bile Acid Synthesis.

BACKGROUND: Obesity is a progressive metabolic disease that begins with lipid metabolism disorders. Aromatic amino acids (AAAs), including tryptophan, phenylalanine, and tyrosine, have diverse biological activities as nutrients. However, the underlying mechanisms by which AAAs affect lipid metabolism are unclear. OBJECTIVES: This study was designed to investigate the possible roles and underlying molecular mechanisms of AAA in the pathogenesis of lipid metabolism disorders. METHODS: We added an AAA mixture to the high-fat diet (HFD) of mice. Glucose tolerance test was recorded. Protein expression of hepatic bile acid (BA) synthase and mRNA expression of BA metabolism-related genes were determined. Hepatic BA profiles and gut microbial were also determined in mice. RESULTS: The results showed that AAA significantly increased body weight and white adipose tissue, aggravated liver injury, impaired glucose tolerance and intestinal integrity, and significantly increased hepatic BA synthesis by inhibiting intestinal farnesoid X receptor (FXR). Moreover, AAA increased the content of total BA in the liver and altered the hepatic BA profile, with elevated levels of lithocholic acid, glycochenodeoxycholic acid, and glycoursodeoxycholic acid. AAA markedly increased the levels of proteins involved in BA synthesis (cholesterol 7α-hydroxylase and oxysterol 7α-hydroxylase) and inhibited the intestinal FXR. Gut microbial composition also changed, reducing the abundance of some beneficial bacteria, such as Parvibacter and Lactobacillus. CONCLUSIONS: Under HFD conditions, AAAs stimulate BA synthesis in both the classical and alternative pathways, leading to aggravation of liver injury and fat deposition. Excessive intake of AAA disrupts BA metabolism and contributes to the development of lipid metabolism disorders, suggesting that AAA may be a causative agent of lipid metabolism disorders.

Specific gut microbiome's role in skin pigmentation: insights from SCARB1 mutants in Oujiang colour common carp.

AIMS: Beyond the pivotal roles of the gut microbiome in initiating physiological processes and modulating genetic factors, a query persists: Can a single gene mutation alter the abundance of the gut microbiome community? Not only this, but the intricate impact of gut microbiome composition on skin pigmentation has been largely unexplored. METHODS AND RESULTS: Based on these premises, our study examines the abundance of lipase-producing gut microbes about differential gene expression associated with bile acid synthesis and lipid metabolism-related blood metabolites in red (whole wild) and white (whole white wild and SCARB1-/- mutant) Oujiang colour common carp. Following the disruption of the SCARB1 gene in the resulting mutant fish with white body colour (SCARB1-/-), there is a notable decrease in the abundance of gut microbiomes (Bacillus, Staphylococcus, Pseudomonas, and Serratia) associated with lipase production. This reduction parallels the downregulation seen in wild-type white body colour fish (WW), as contrasting to the wild-type red body colour fish (WR). Meanwhile, in SCARB1-/- fish, there was a downregulation noted not only at the genetic and metabolic levels but also a decrease in lipase-producing bacteria. This consistency with WW contrasts significantly with WR. Similarly, genes involved in the bile acid synthesis pathway, along with blood metabolites related to lipid metabolism, exhibited downregulation in SCARB1-/- fish. CONCLUSIONS: The SCARB1 knockout gene blockage led to significant alterations in the gut microbiome, potentially influencing the observed reduction in carotenoid-associated skin pigmentation. Our study emphasizes that skin pigmentation is not only impacted by genetic factors but also by the gut microbiome. Meanwhile, the gut microbiome's adaptability can be rapidly shaped and may be driven by specific single-gene variations.

In vitro screening of understudied PFAS with a focus on lipid metabolism disruption.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many industrial applications. Exposure to PFAS is associated with several health risks, including a decrease in infant birth weight, hepatoxicity, disruption of lipid metabolism, and decreased immune response. We used the in vitro cell models to screen six less studied PFAS [perfluorooctane sulfonamide (PFOSA), perfluoropentanoic acid (PFPeA), perfluoropropionic acid (PFPrA), 6:2 fluorotelomer alcohol (6:2 FTOH), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and 8:2 fluorotelomer sulfonic acid (8:2 FTSA)] for their capacity to activate nuclear receptors and to cause differential expression of genes involved in lipid metabolism. Cytotoxicity assays were run in parallel to exclude that observed differential gene expression was due to cytotoxicity. Based on the cytotoxicity assays and gene expression studies, PFOSA was shown to be more potent than other tested PFAS. PFOSA decreased the gene expression of crucial genes involved in bile acid synthesis and detoxification, cholesterol synthesis, bile acid and cholesterol transport, and lipid metabolism regulation. Except for 6:2 FTOH and 8:2 FTSA, all tested PFAS downregulated PPARA gene expression. The reporter gene assay also showed that 8:2 FTSA transactivated the farnesoid X receptor (FXR). Based on this study, PFOSA, 6:2 FTSA, and 8:2 FTSA were prioritized for further studies to confirm and understand their possible effects on hepatic lipid metabolism.

Tailoring FXR Modulators for Intestinal Specificity: Recent Progress and Insights.

While FXR has shown promise in regulating bile acid synthesis and maintaining glucose and lipid homeostasis, undesired side effects have been observed in clinical trials. To address this issue, the development of intestinally restricted FXR modulators has gained attention as a new avenue for drug design with the potential for safer systematic effects. Our review examines all currently known intestinally restricted FXR ligands and provides insights into the steps taken to enhance intestinal selectivity.

Stigmasterol Protects Against Steatohepatitis Induced by High-Fat and High-Cholesterol Diet in Mice by Enhancing the Alternative Bile Acid Synthesis Pathway.

BACKGROUND: Hepatic cholesterol accumulation is a significant risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis. However, the precise mechanism by which stigmasterol (STG) mitigates this process remains unclear. OBJECTIVES: This study aimed to investigate the potential mechanism underlying the protective effect of STG in mice with NAFLD progressing to steatohepatitis while being fed a high-fat and high-cholesterol (HFHC) diet. METHODS: Male C57BL/6 mice were fed an HFHC diet for 16 wk to establish the NAFLD model. Subsequently, the mice received STG or a vehicle via oral gavage while continuing the HFHC diet for an additional 10 wk. The study evaluated hepatic lipid deposition and inflammation as well as the expression of key rate-limiting enzymes involved in the bile acid (BA) synthesis pathways. BAs in the colonic contents were quantified using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: Compared with the vehicle control group, STG significantly reduced hepatic cholesterol accumulation (P < 0.01) and suppressed the gene expression of NLRP3 inflammasome and interleukin-18 (P < 0.05) in the livers of HFHC diet-fed mice. The total fecal BA content in the STG group was nearly double that of the vehicle control group. Additionally, the administration of STG increased the concentrations of representative hydrophilic BAs in the colonic contents (P < 0.05) along with the upregulation of gene and protein expression of CYP7B1 (P < 0.01). Furthermore, STG enhanced the α-diversity of the gut microbiota and partially reversed the alterations in the relative abundance of the gut microbiota induced by the HFHC diet. CONCLUSIONS: STG mitigates steatohepatitis by enhancing the alternative pathway for BA synthesis.

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.

Tissue-specific mechanisms of bile acid homeostasis and activation of FXR-FGF19 signaling in preterm and term neonatal pigs.

The tissue-specific molecular mechanisms involved in perinatal liver and intestinal farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling are poorly defined. Our aim was to establish how gestational age and feeding status affect bile acid synthesis pathway, bile acid pool size, ileal response to bile acid stimulation, genes involved in bile acid-FXR-FGF19 signaling and plasma FGF19 in neonatal pigs. Term (n = 23) and preterm (n = 33) pigs were born via cesarean section at 100% and 90% gestation, respectively. Plasma FGF19, hepatic bile acid and oxysterol profiles, and FXR target gene expression were assessed in pigs at birth and after a bolus feed on day 3 of life. Pig ileal tissue explants were used to measure signaling response to bile acids. Preterm pigs had smaller, more hydrophobic bile acid pools, lower plasma FGF19, and blunted FXR-mediated ileal response to bile acid stimulation than term pigs. GATA binding protein 4 (GATA-4) expression was higher in jejunum than ileum and was higher in preterm than term pig ileum. Hepatic oxysterol analysis suggested dominance of the alternative pathway of bile acid synthesis in neonates, regardless of gestational age and persists in preterm pigs after feeding on day 3. These results highlight the tissue-specific molecular basis for the immature enterohepatic bile acid signaling via FXR-FGF19 in preterm pigs and may have implications for disturbances of bile acid homeostasis and metabolism in preterm infants.NEW & NOTEWORTHY Our results show that the lower hepatic bile acid synthesis and ileum FXR-FGF19 pathway responsiveness to bile acids contribute to low-circulating FGF19 in preterm compared with term neonatal pigs. The molecular mechanism explaining immature or low-ileum FXR-FGF19 signaling may be linked to developmental patterning effects of GATA-4.

Liver transplantation in an infant with cerebrotendinous xanthomatosis, cholestasis, and rapid evolution of liver failure.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a disorder of bile acid (BA) metabolism due to biallelic mutations in CYP27A1. The deposition of cholesterol and cholestanol in multiple tissues results, manifesting as neurologic disease in adults or older children. Neonatal cholestasis (NC) as a presentation of CTX is rare; it may self-resolve or persist, evolving to require liver transplantation (LT). METHODS: We present in the context of similar reports an instance of CTX manifest as NC and requiring LT. RESULTS: A girl aged 4mo was evaluated for NC with normal serum gamma-glutamyl transpeptidase activity. An extensive diagnostic work-up, including liver biopsy, identified no etiology. Rapid progression to end-stage liver disease required LT aged 5mo. The explanted liver showed hepatocyte loss and micronodular cirrhosis. Bile salt export pump (BSEP), encoded by ABCB11, was not demonstrable immunohistochemically. Both severe ABCB11 disease and NR1H4 disease-NR1H4 encodes farsenoid-X receptor, necessary for ABCB11 transcription-were considered. However, selected liver disorder panel sequencing and mass-spectrometry urinary BA profiling identified CTX, with homozygosity for the predictedly pathogenic CYP27A1 variant c.646G > C p.(Ala216Pro). Variation in other genes associated with intrahepatic cholestasis was not detected. Immunohistochemical study of the liver-biopsy specimen found marked deficiency of CYP27A1 expression; BSEP expression was unremarkable. Aged 2y, the girl is free from neurologic disease. CONCLUSIONS: Bile acid synthesis disorders should be routinely included in the NC/"neonatal hepatitis" work-up. The mutually supportive triple approach of BA profiling, immunohistochemical study, and genetic analysis may optimally address diagnosis in CTX, a treatable disease with widely varying presentation.

Congenital Bile Acid Synthesis Defect Type 3 With Severe Neonatal Cholestasis.

Congenital bile acid synthesis defect type 3 is a rare metabolic liver disease with only eight patients reported in literature. We describe clinical, pathological and molecular features for a ninth patient. A 4-month-old infant presented to us with conjugated hyperbilirubinemia. His liver biopsy revealed giant cell change, steatosis, and activity with diffuse fibrosis. Immunostaining with bile salt export pump showed preserved canalicular pattern and γ-glutamyl transferase 1 staining showed unusual complete membranous pattern. Genetic workup revealed homozygous single base pair duplication in exon 3 of the CYP7B1 gene. He succumbed to liver disease at 7 months of age.

Bile Acid Synthesis Disorders in Japan: Long-Term Outcome and Chenodeoxycholic Acid Treatment.

BACKGROUND: We encountered 7 Japanese patients with bile acid synthesis disorders (BASD) including 3ß-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase (3ß-HSD) deficiency (n = 3), Δ4-3-oxosteroid 5ß-reductase (5ß-reductase) deficiency (n = 3), and oxysterol 7α-hydroxylase deficiency (n = 1) over 21 years between 1996 and 2017. AIM: We aimed to clarify long-term outcome in the 7 patients with BASD as well as long-term efficacy of chenodeoxycholic acid (CDCA) treatment in the 5 patients with 3ß-HSD deficiency or 5ß-reductase deficiency. METHODS: Diagnoses were made from bile acid and genetic analyses. Bile acid analysis in serum and urine was performed using gas chromatography-mass spectrometry. Clinical and laboratory findings and bile acid profiles at diagnosis and most recent visit were retrospectively obtained from medical records. Long-term outcome included follow-up duration, treatments, growth, education/employment, complications of treatment, and other problems. RESULTS: Medians with ranges of current patient ages and duration of CDCA treatment are 10 years (8 to 43) and 10 years (8 to 21), respectively. All 7 patients, who had homozygous or compound heterozygous mutations in the HSD3B7, SRD5B1, or CYP7B1 gene, are currently in good health without liver dysfunction. In the 5 patients with CDCA treatment, hepatic function gradually improved following initiation. No adverse effects were noted. CONCLUSIONS: We concluded that CDCA treatment is effective in 3ß-HSD deficiency and 5ß-reductase deficiency, as cholic acid has been in other countries. BASD carry a good prognosis following early diagnosis and initiation of long-term CDCA treatment.

α-globulin-rich rice cultivar, low glutelin content-1 (LGC-1), decreases serum cholesterol concentration in exogenously hypercholesterolemic rats.

BACKGROUND: Rice α-globulin has been reported to have serum cholesterol-lowering activity in rats. However, it is still unclear whether α-globulin exerts this effect when taken as one of the dietary components. In the present study, we investigated the effect of two cultivars of rice, low glutelin content (LGC)-1 and LGC-Jun, on reducing serum cholesterol in exogenously hypercholesterolemic (ExHC) rats. LGC-1 is enriched in α-globulin (10.6 mg g-1 rice flour, which is an approximately 1.5 times higher α-globulin content than in Koshihikari a predominant rice cultivar in Japan), whereas LGC-Jun is a globulin-negative cultivar. METHODS: ExHC rats, the model strain of diet-induced hypercholesterolemia, were fed 50% LGC-1 or LGC-Jun and 0.5% cholesterol-containing diets for 2 weeks, followed by measurement of cholesterol metabolism parameters in serum and tissues. RESULTS: Serum cholesterol and non-high-density lipoprotein cholesterol levels were significantly lower in the LGC-1 group compared to the LGC-Jun group. Cholesterol intestinal absorption markers, hepatic and serum levels of campesterol and ß-sitosterol, and lymphatic cholesterol transport were not different between the two groups. Levels of 7α-hydroxycholesterol, an intermediate of bile acid synthesis, showed a downward trend in the livers of rats that were fed LGC-1 (P = 0.098). There was a significant decrease in the hepatic mRNA expression of Cyp7a1 (a synthetic enzyme for 7α-hydroxycholesterol) in the LGC-1 group compared to the LGC-Jun group. CONCLUSION: Dietary LGC-1 significantly decreased serum cholesterol levels in ExHC rats. The possible mechanism for the cholesterol-lowering activity of LGC-1 is partial inhibition of bile acid and cholesterol synthesis in the liver. © 2021 Society of Chemical Industry.

The effectiveness of correcting abnormal metabolic profiles.

Inborn errors of metabolism cause disease because of accumulation of a metabolite before the blocked step or deficiency of an essential metabolite downstream of the block. Treatments can be directed at reducing the levels of a toxic metabolite or correcting a metabolite deficiency. Many disorders have been treated successfully first in a single patient because we can measure the metabolites and adjust treatment to get them as close as possible to the normal range. Examples are drawn from Komrower's description of treatment of homocystinuria and the author's trials of treatment in bile acid synthesis disorders (3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase deficiency and Δ4 -3-oxosteroid 5ß-reductase deficiency), neurotransmitter amine disorders (aromatic L-amino acid decarboxylase [AADC] and tyrosine hydroxylase deficiencies), and vitamin B6 disorders (pyridox(am)ine phosphate oxidase deficiency and pyridoxine-dependent epilepsy [ALDH7A1 deficiency]). Sometimes follow-up shows there are milder and more severe forms of the disease and even variable clinical manifestations but by measuring the metabolites we can adjust the treatment to get the metabolites into the normal range. Biochemical measurements are not subject to placebo effects and will also show if the disorder is improving spontaneously. The hypothesis that can then be tested for clinical outcome is whether getting metabolite(s) into a target range leads to an improvement in an outcome parameter such as abnormal liver function tests, hypokinesia, epilepsy control etc. The metabolite-guided approach to treatment is an example of personalized medicine and is a better way of determining efficacy for disorders of variable severity than a randomized controlled clinical trial.

Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes.

Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.

The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry.

The SCP2 (sterol carrier protein 2)-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the ß-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-thiolase (type-1) as well as its human homolog are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-thiolase (type-1) is equally extensive as in other thiolase dimers; however, it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insights into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.

Ganoderma lucidum spore ethanol extract attenuates atherosclerosis by regulating lipid metabolism via upregulation of liver X receptor alpha.

CONTEXT: Ganoderma lucidum (Leyss.ex Fr.) Karst (Ganodermataceae) is a fungus that has been used in traditional Chinese medicine. OBJECTIVE: This is the first investigation of the lipid-lowering and anti-atherosclerotic effects of Ganoderma lucidum spore ethanol extract (EEG) in hyperlipidemic rabbits. MATERIALS AND METHODS: Fifty-four Japanese rabbits were randomly divided into six groups (n = 9): control, model, atorvastatin and three EEG groups (6, 24 and 96 mg/kg/day, p.o.). Control group was administered a normal diet and other groups were administered a high-fat diet to induce hyperlipidaemia and atherosclerosis for 14 weeks. During this time, lipid profiles were recorded; lipid testing and histopathological examination of aorta and liver were conducted. LXRα and its downstream genes expression in the liver and small intestine were examined. The effect of EEG on macrophage cholesterol efflux and ABCA1/G1 expression was observed under silenced LXRα expression. RESULTS: EEG reduced serum cholesterol (20.33 ± 3.62 mmol/L vs 34.56 ± 8.27 mmol/L for the model group) and LDL-C, reduced the area of arterial plaques (24.8 ± 10% vs 53.9 ± 15.2% for the model group) and Intima/Medium thickness ratio, increased faecal bile acid content, upregulated LXRα, CYP7A1, ABCA1/G1, ABCG5/G8 expression in the liver, small intestine and macrophages. After silencing LXRα in macrophages, the ability of EEG to promote cholesterol efflux was inhibited. DISCUSSION AND CONCLUSION: EEG exert lipid-lowering and anti-atherosclerotic effects via upregulating expression of LXRα and downstream genes associated with reverse cholesterol transport and metabolism. However, whether PPARα/γ are involved in the up-regulation of LXR expression by EEG remains to be elucidated.

CYP7A1 expression in hepatocytes is retained with upregulated fibroblast growth factor 19 in pediatric biliary atresia.

AIM: Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of fibroblast growth factor (FGF) 19 is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. Fibroblast growth factor 19 is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA). METHODS: We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls. RESULTS: CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA HET, and its serum concentration was elevated. Fibroblast growth factor receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET. CONCLUSIONS: This is the first study to measure the CYP7A1 mRNA levels in human BA HET. Fibroblast growth factor 19 was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF19 pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.

Ganoderma Lucidum Polysaccharide Peptide Alleviates Hepatoteatosis via Modulating Bile Acid Metabolism Dependent on FXR-SHP/FGF.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) encompasses a series of pathologic changes ranging from steatosis to steatohepatitis, which may progress to cirrhosis and hepatocellular carcinoma. The purpose of this study was to determine whether ganoderma lucidum polysaccharide peptide (GLPP) has therapeutic effect on NAFLD. METHODS: Ob/ ob mouse model and ApoC3 transgenic mouse model were used for exploring the effect of GLPP on NAFLD. Key metabolic pathways and enzymes were identified by metabolomics combining with KEGG and PIUmet analyses and key enzymes were detected by Western blot. Hepatosteatosis models of HepG2 cells and primary hepatocytes were used to further confirm the therapeutic effect of GLPP on NAFLD. RESULTS: GLPP administrated for a month alleviated hepatosteatosis, dyslipidemia, liver dysfunction and liver insulin resistance. Pathways of glycerophospholipid metabolism, fatty acid metabolism and primary bile acid biosynthesis were involved in the therapeutic effect of GLPP on NAFLD. Detection of key enzymes revealed that GLPP reversed low expression of CYP7A1, CYP8B1, FXR, SHP and high expression of FGFR4 in ob/ob mice and ApoC3 mice. Besides, GLPP inhibited fatty acid synthesis by reducing the expression of SREBP1c, FAS and ACC via a FXR-SHP dependent mechanism. Additionally, GLPP reduced the accumulation of lipid droplets and the content of TG in HepG2 cells and primary hepatocytes induced by oleic acid and palmitic acid. CONCLUSION: GLPP significantly improves NAFLD via regulating bile acid synthesis dependent on FXR-SHP/FGF pathway, which finally inhibits fatty acid synthesis, indicating that GLPP might be developed as a therapeutic drug for NAFLD.

Attenuated Accumulation of Novel Fluorine (19F)-Labeled Bile Acid Analogues in Gallbladders of Fibroblast Growth Factor-15 (FGF15)-Deficient Mice.

Our work has focused on defining the utility of fluorine (19F)-labeled bile acid analogues and magnetic resonance imaging (MRI) to identify altered bile acid transport in vivo. In the current study, we explored the ability of this approach to differentiate fibroblast growth factor-15 (FGF15)-deficient from wild-type (WT) mice, a potential diagnostic test for bile acid diarrhea, a commonly misdiagnosed disorder. FGF15 is the murine homologue of human FGF19, an intestinal hormone whose deficiency is an underappreciated cause of bile acid diarrhea. In a pilot and three subsequent pharmacokinetic studies, we treated mice with two 19F-labeled bile acid analogues, CA-lys-TFA and CA-sar-TFMA. After oral dosing, we quantified 19F-labeled bile acid analogue levels in the gallbladder, liver, small and large intestine, and plasma using liquid chromatography mass spectrometry (LC-MS/MS). Both 19F bile acid analogues concentrated in the gallbladders of FGF15-deficient and WT mice, attaining peak concentrations at approximately 8.5 h after oral dosing. However, analogue levels in gallbladders of FGF15-deficient mice were several-fold less compared to those in WT mice. Live-animal 19F MRI provided agreement with our LC-MS/MS-based measures; we detected robust CA-lys-TFA 19F signals in gallbladders of WT mice but no signals in FGF15-deficient mice. Our finding that 19F MRI differentiates FGF15-deficient from WT mice provides additional proof-of-concept for the development of 19F bile acid analogues and 19F MRI as a clinical test to diagnose bile acid diarrhea due to FGF19 deficiency and other disorders.