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Background & Aims: Inhibition of cholesterol 7α-hydroxylase (CYP7A1) by bile acids and inflammatory cytokines provides an important mechanism to protect hepatocytes from bile acid toxicity during cholestasis. Transforming growth factor β1 (TGFβ1) released by hepatic stellate cells during chronic liver injury plays a critical role in liver inflammation and fibrogenesis. The objective of this study is to investigate the role of TGFβ1 in hepatic bile acid synthesis. Methods: mRNA expressions in primary human hepatocytes and HepG2 cells were measured by quantitative real-time polymerase chain reaction. Reporter assay, glutathione-S-transferase pull-down assay, adenovirus-mediated gene transduction, and chromatin immunoprecipitation assay were used to study the mechanism of TGFβ1 regulation of CYP7A1 gene transcription. Results: TGFβ1 inhibited the mRNA expression of CYP7A1 and bile acid synthesis in HepG2 cells and primary human hepatocytes. Mothers against decapentaplegic homolog (Smad3) inhibited both CYP7A1 promoter activity and mRNA expression by inhibiting DNA-binding activity of hepatocyte nuclear factor 4α (HNF4alpha). The histone deacetylase (HDAC) inhibitor Tricostatin A partially blocked the TGFβ1 inhibition of CYP7A1 mRNA expression, whereas TGFβ1 decreased histone 3 acetylation in the CYP7A1 chromatin. TGFβ1 treatment and adenovirus Smad3 reduced HNF4α binding but increased the recruitment of Smad3, HDAC1, and a repressor mSin3A to the CYP7A1 chromatin. Conclusions: This study provides the first evidence that TGFβ1 represses CYP7A1 gene transcription in human hepatocytes by a mechanism involving Smad3-dependent inhibition of HNF4α and HDAC remodeling of CYP7A1 chromatin. The TGFβ1/Smad3 signaling may reduce bile acid synthesis in the liver and prevent hepatocyte injury in cholestatic liver disease.

Insulin promotes hepatic apolipoprotein B100 (apoB100) degradation, whereas insulin resistance is a major cause of hepatic apoB100/triglyceride overproduction in type 2 diabetes. The cellular trafficking receptor sortilin 1 (Sort1) was recently identified to transport apoB100 to the lysosome for degradation in the liver and thus regulate plasma cholesterol and triglyceride levels. Genetic variation of SORT1 was strongly associated with cardiovascular disease risk in humans. The major goal of this study is to investigate the effect and molecular mechanism of insulin regulation of Sort1. Results showed that insulin induced Sort1 protein, but not mRNA, in AML12 cells. Treatment of PI3K or AKT inhibitors decreased Sort1 protein, whereas expression of constitutively active AKT induced Sort1 protein in AML12 cells. Consistently, hepatic Sort1 was down-regulated in diabetic mice, which was partially restored after the administration of the insulin sensitizer metformin. LC-MS/MS analysis further revealed that serine phosphorylation of Sort1 protein was required for insulin induction of Sort1 in a casein kinase 2-dependent manner and that inhibition of PI3K signaling or prevention of Sort1 phosphorylation accelerated proteasome-dependent Sort1 degradation. Administration of a PI3K inhibitor to mice decreased hepatic Sort1 protein and increased plasma cholesterol and triglyceride levels. Adenovirus-mediated overexpression of Sort1 in the liver prevented PI3K inhibitor-induced Sort1 down-regulation and decreased plasma triglyceride but had no effect on plasma cholesterol in mice. This study identified Sort1 as a novel target of insulin signaling and suggests that Sort1 may play a role in altered hepatic apoB100 metabolism in insulin-resistant conditions. Background: Sortilin 1 mediates lysosome-dependent apoB100 degradation in hepatocytes. Results: Blocking insulin/PI3K/AKT signaling caused posttranslational hepatic sortilin 1 degradation. Conclusion: Insulin resistance is an underlying cause of decreased hepatic Sort1 in diabetes. Significance: This study suggests a new mechanism underlying altered hepatic apoB100 metabolism in type 2 diabetes.

Bile acids are synthesized from cholesterol in the liver. Some cytochrome P450 (CYP) enzymes play key roles in bile acid synthesis. Bile acids are physiological detergent molecules, so are highly cytotoxic. They undergo enterohepatic circulation and play important roles in generating bile flow and facilitating biliary secretion of endogenous metabolites and xenobiotics and intestinal absorption of dietary fats and lipid-soluble vitamins. Bile acid synthesis, transport, and pool size are therefore tightly regulated under physiological conditions. In cholestasis, impaired bile flow leads to accumulation of bile acids in the liver, causing hepatocyte and biliary injury and inflammation. Chronic cholestasis is associated with fibrosis, cirrhosis, and eventually liver failure. Chronic cholestasis also increases the risk of developing hepatocellular or cholangiocellular carcinomas. Extensive research in the last two decades has shown that bile acids act as signaling molecules that regulate various cellular processes. The bile acid-activated nuclear receptors are ligand-activated transcriptional factors that play critical roles in the regulation of bile acid, drug, and xenobiotic metabolism. In cholestasis, these bile acid-activated receptors regulate a network of genes involved in bile acid synthesis, conjugation, transport, and metabolism to alleviate bile acid-induced inflammation and injury. Additionally, bile acids are known to regulate cell growth and proliferation, and altered bile acid levels in diseased conditions have been implicated in liver injury/regeneration and tumorigenesis. We will cover the mechanisms that regulate bile acid homeostasis and detoxification during cholestasis, and the roles of bile acids in the initiation and regulation of hepatic inflammation, regeneration, and carcinogenesis.

Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7α-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4α and peroxisome proliferator-activated receptor γ coactivator-1α trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor γ-coactivator-1α but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes.

Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4α (HNF4α) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptorγ co-activator 1α (PGC-1α) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4α binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4α. Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4α and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4α and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4α and PGC-1α. Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4α, SRC-1, and PGC-1α. PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs.

Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.

Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7 alpha-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4 alpha (HNF4 alpha, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4 alpha. PXR also interacted with peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha), which interacted with HNF4 alpha and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4 alpha and reduced PGC-1 alpha interaction with HNF4 alpha. Chromatin immunoprecipitation assay showed that PXR, HNF4 alpha, and PGC-1 alpha bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1 alpha from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4 alpha and blocks PGC-1 alpha activation with HNF4 alpha and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis.

Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.

We focus with significant interest on the report by Lv et al ,1 which was related to comparison of Tripterygium wilfordii Hook F (TwHF) with methotrexate (MTX) in the treatment of active rheumatoid arthritis. Lv et al found that TwHF monotherapy was not inferior to, and MTX+TwHF was better than, MTX monotherapy in patients with active RA. However, we think that there are still several critical questions in this research, and one of them should be treated carefully in revealing the results of this trial. First, the trial was an open-label study. Patients were aware of the corresponding treatment with …

Obesity and diabetes are associated with hepatic triglyceride overproduction and hypertriglyceridemia. Recent studies have found that the cellular trafficking receptor sortilin 1 (Sort1) inhibits hepatic apolipoprotein B secretion and reduces plasma lipid levels in mice, and its hepatic expression was negatively associated with plasma lipids in humans. This study investigated the regulation of hepatic Sort1 under diabetic conditions and by lipid-lowering fish oil and fenofibrate. Results showed that hepatic Sort1 protein, but not mRNA, was markedly lower in Western diet-fed mice. Knockdown of hepatic Sort1 increased plasma triglyceride in mice. Feeding mice a fish oil-enriched diet completely restored hepatic Sort1 levels in Western diet-fed mice. Fenofibrate also restored hepatic Sort1 protein levels in Western diet-fed wild type mice, but not in peroxisome proliferator-activated receptor α (PPARα) knock-out mice. PPARα ligands did not induce Sort1 in hepatocytes in vitro. Instead, fish oil and fenofibrate reduced circulating and hepatic fatty acids in mice, and n-3 polyunsaturated fatty acids prevented palmitate inhibition of Sort1 protein in HepG2 cells. LC/MS/MS analysis revealed that Sort1 phosphorylation at serine 793 was increased in obese mice and in palmitate-treated HepG2 cells. Mutations that abolished phosphorylation at Ser-793 increased Sort1 stability and prevented palmitate inhibition of Sort1 ubiquitination and degradation in HepG2 cells. In summary, therapeutic strategies that prevent posttranslational hepatic Sort1 down-regulation in obesity and diabetes may be beneficial in improving dyslipidemia.

SHP, an orphan nuclear hormone receptor, functions as a corepressor and antagonizes coactivator recruitment to target transcription factors with which it interacts (1, 2). Molecular and genetic studies have suggested that SHP also plays a role in obesity and diabetes (2-6). Supporting this, deletion or overexpression of SHP gene in mice disrupts lipid and glucose homeostasis (7-10). Congenic C57BL/6 SHP−/− mice show a more insulin resistant phenotype in response to a western diet (WestD), but are protected against development of hepatic steatosis and obesity (7, 11). The protection against steatosis was attributed to enhancement of hepatic β-oxidation and reduced expression of fatty acid synthetic genes. Gene expression profiling revealed that PPARγ was markedly down-regulated in SHP−/− mouse liver regardless of type of diets, suggesting that the effect of SHP on PPARγ expression is not a secondary complication of fat accumulation. PPARγ expression is low in normal livers, but is strongly induced in models of nonalcoholic fatty liver diseases (NAFLD). Given its role in adipogenesis, this induction has been considered a key factor contributing to fat accumulation in the livers of animal models and human patients associated with obesity and diabetes (12-16). Indeed, adenoviral overexpression of PPARγ in the liver is sufficient to drive marked steatosis (17-19), and liver specific deletion of PPARγ strongly decreases steatosis in the ob/ob background (13) and high fat-fed mice (14, 15, 18). One proposed mechanism for PPARγ-dependent fat accumulation in ob/ob mice is direct induction of fat specific factor 27 (Fsp27), a lipid droplet binding protein promoting lipid accumulation in adipocytes (20). As observed in white adipose tissue, deletion of Fsp27 decreases the size of lipid droplets in liver and increases lipolysis and fatty acid oxidation due to increased accessibility of lipolytic enzymes and increased availability of non esterified free fatty acids (FFA) without affecting FFA uptake, triglyceride (TG) export, and de novo TG synthesis (20, 21). Thus, decreased PPARγ expression in SHP−/− liver (7, 11) may protect the mice from development of hepatic steatosis at least in part by decreasing Fsp27 expression. Hes6 is a novel member of the family of mammalian homologues of Drosophila hairy and enhancer of split, and was initially identified as an inhibitor of its relative Hes1, a basic helix-loop-helix transcription factor regulating neuronal and muscle differentiation negatively (22). Like SHP, Hes6 alone cannot bind directly to DNA, but represses target gene transcription through protein-protein interaction. Intriguingly, Hes6 was recently identified as a repressor of hepatic PPARγ expression via inhibition of HNF4α transactivation and its expression is directly regulated by HNF4α, which is one of the direct targets of SHP-mediated repression (2, 23). These results suggest that Hes6 may function as a mediator in the regulation of fatty acid synthesis by SHP. In the present study, we discovered a novel transcriptional cascade consisting of RAR, Hes6, HNF4α, and PPARγ in the regulation of hepatic fat mobilization. In the proposed transcriptional cascade, SHP and atRA coordinately regulates transcription of Hes6, and Hes6 subsequently suppresses Pparγ2 expression via repression of HNF4α transcriptional activity. We confirmed the physiologic effects of atRA and RAR on fat mobilization via the cascade using mouse models of fatty liver disease.

Bile acid synthesis is the major pathway for catabolism of cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the bile acid biosynthetic pathway in the liver and plays an important role in regulating lipid, glucose and energy metabolism. Transgenic mice overexpressing CYP7A1 (CYP7A1-tg mice) were resistant to high-fat diet (HFD)-induced obesity, fatty liver, and diabetes. However the mechanism of resistance to HFD-induced obesity of CYP7A1-tg mice has not been determined. In this study, metabolomic and lipidomic profiles of CYP7A1-tg mice were analyzed to explore the metabolic alterations in CYP7A1-tg mice that govern the protection against obesity and insulin resistance by using ultra-performance liquid chromatography-coupled with electrospray ionization quadrupole time-of-flight mass spectrometry combined with multivariate analyses. Lipidomics analysis identified seven lipid markers including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and ceramides that were significantly decreased in serum of HFD-fed CYP7A1-tg mice. Metabolomics analysis identified 13 metabolites in bile acid synthesis including taurochenodeoxycholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, and tauro-β-muricholic acid (T-β-MCA) that differed between CYP7A1-tg and wild-type mice. Notably, T-β-MCA, an antagonist of the farnesoid X receptor (FXR) was significantly increased in intestine of CYP7A1-tg mice. This study suggests that reducing 12α-hydroxylated bile acids and increasing intestinal T-β-MCA may reduce high fat diet-induced increase of phospholipids, sphingomyelins and ceramides, and ameliorate diabetes and obesity. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Objective: To investigate the effects of Chinese Traditional Setting-up Exercise on Blood Glucose, Heart rate and vital capacity of healthy undergraduate. Methods: 200 undergraduate volunteers were selected according to the diagnostic standards and inclusion criteria. They were randomly and equally allocated into the two groups. The case group practiced the Chinese Traditional Setting-up Exercise and the control group practiced the 8th Broadcast Gymnastic Exercise, three times a week and last for 4 weeks. The three biological indexes were measured half an hour before and after the exercise. From t test (single variable), correlation (two variables) to multi-variables, such as Principal component analysis, Heat map and Random forest, we presented a systematical methods to understand the data. Results: There were significant differences before and after the exercise in both two groups. Chinese Traditional Setting-up Exercise performed better than the 8th Broadcast Gymnastic Exercise in reducing BG, balancing HR and enlarging VC. They are interacted with each other to separate control group from case group with a accuracy of nearly 80 %. The most important is BG, the less important is HR and VC is with least importance. Conclusion: Systematically statistical methods could discover inside rules behind the data. It is found that “Chinese Traditional Setting-up Exercise” performed better than “8th Broadcast Gymnastic Exercise” in reducing BG, balancing HR and enlarging VC of undergraduate students, so the “Chinese Traditional Setting-up Exercise” was suitable for popularizing among universities.

Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.

Prompt and accurate diagnosis of acute ischemic stroke is critical to seek acute therapy. In traditional Chinese medicine (TCM) science, there is a comprehensive system of diagnosis and medical care of acute ischemic stroke. Here we introduce a two-level model for the analysis of TCM syndrome of acute ischemic stroke. Owing to the limitation of sample size and imbalance, we focused on the analysis of wind-phlegm collateral obstruction syndrome (Feng Tan Yu Zu Zheng). Firstly, a Support-Vector-Machine- (SVM-) based diagnostic model was set up through selection of core symptoms. After pairwise undersampling, we improved the performance of prediction and generated the core symptoms-based diagnostic model of wind-phlegm collateral obstruction syndrome. Next, Pathway Pattern-based method and MetaDrug platform were used to shed light on the molecular basis of the significance of core symptoms in three complementary aspects: symptom-gene-pathway multilayer correlation network, enriched pathways, and most relevant interaction network. The integration of diagnostic model and molecular mechanism analysis creates an interesting perspective for better understanding the syndrome. The two-level model would provide a new opportunity for the study of TCM syndromes.

Bile acids concentration in liver is tightly regulated to prevent cell damage. Previous studies have demonstrated that deregulation of bile acid homeostasis can lead to cholestatic liver disease. Recently, we have shown that ER-bound transcription factor Crebh is a downstream effector of hepatic Cb1r signaling pathway. In this study, we have investigated the effect of alcohol exposure on hepatic bile acid homeostasis and elucidated the mediatory roles of Cb1r and Crebh in this process. We found that alcohol exposure or Cb1r-agonist 2-AG treatment increases hepatic bile acid synthesis and serum ALT, AST levels in vivo alongwith significant increase in Crebh gene expression and activation. Alcohol exposure activated Cb1r, Crebh, and perturbed bile acid homeostasis. Overexpression of Crebh increased the expression of key bile acid synthesis enzyme genes via direct binding of Crebh to their promoters, whereas Cb1r knockout and Crebh-knockdown mice were protected against alcohol-induced perturbation of bile acid homeostasis. Interestingly, insulin treatment protected against Cb1r-mediated Crebh-induced disruption of bile acid homeostasis. Furthermore, Crebh expression and activation was found to be markedly increased in insulin resistance conditions and Crebh knockdown in diabetic mice model (db/db) significantly reversed alcohol-induced disruption of bile acid homeostasis. Overall, our study demonstrates a novel regulatory mechanism of hepatic bile acid metabolism by alcohol via Cb1r-mediated activation of Crebh, and suggests that targeting Crebh can be of therapeutic potential in ameliorating alcohol-induced perturbation of bile acid homeostasis.

Obesity, diabetes, and metabolic syndromes are increasingly recognized as health concerns worldwide. Overnutrition and insulin resistance are the major causes of diabetic hyperglycemia and hyperlipidemia in humans. Studies in the past decade provide evidence that bile acids are not just biological detergents facilitating gut nutrient absorption, but also important metabolic regulators of glucose and lipid homeostasis. Pharmacological alteration of bile acid metabolism or bile acid signaling pathways such as using bile acid receptor agonists or bile acid binding resins may be a promising therapeutic strategy for the treatment of obesity and diabetes. On the other hand, bile acid signaling is complex, and the molecular mechanisms mediating the bile acid effects are still not completely understood. This paper will summarize recent advances in our understanding of bile acid signaling in regulation of glucose and lipid metabolism, and the potentials of developing novel therapeutic strategies that target bile acid metabolism for the treatment of metabolic disorders.

Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.

Aldo-keto reductase 1B7 (AKR1B7) is proposed to play a role in detoxification of by-products of lipid peroxidation. In this article, we show that activation of the nuclear receptor farnesoid X receptor (FXR) induces AKR1B7 expression in the liver and intestine, and reduces the levels of malondialdehyde (MDA), the end product of lipid peroxidation, in the intestine but not in the liver. To determine whether AKR1B7 regulates MDA levels in vivo, we overexpressed AKR1B7 in the liver. Overexpression of AKR1B7 in the liver had no effect on hepatic or plasma MDA levels. Interestingly, hepatic expression of AKR1B7 significantly lowered plasma glucose levels in both wild-type and diabetic db/db mice, which was associated with reduced hepatic gluconeogenesis. Hepatic expression of AKR1B7 also significantly lowered hepatic triglyceride and cholesterol levels in db/db mice. These data reveal a novel function for AKR1B7 in lipid and glucose metabolism and suggest that AKR1B7 may not play a role in detoxification of lipid peroxides in the liver. AKR1B7 may be a therapeutic target for treatment of fatty liver disease associated with diabetes mellitus.

Obesity and diabetes are closely associated with impaired lipid and glucose homeostasis, which significantly increases the risk of coronary heart disease.1 Obesity often results from a Western lifestyle with overnutrition and a lack of exercise. Visceral fat accumulation due to increased caloric intake is known to cause organ insulin resistance.2 It is believed that insulin resistance and overnutrition directly contribute to abnormal adiposity and hepatic very low density lipoprotein (VLDL) synthesis and thus lead to nonalcoholic fatty liver disease and dyslipidemia.3 Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the bile acid biosynthetic pathway that converts cholesterol into bile acids and plays an important role in regulating cholesterol and bile acid homeostasis. The role of CYP7A1 in regulating cholesterol homeostasis has been well established. Patients with mutations in the CYP7A1 gene developed gallstones and premature atherosclerosis,4 whereas Cyp7a1 transgenic (Cyp7a1-tg) mice were protected from atherogenic diet–induced atherosclerosis.5 Bile acids are essential for the biliary secretion of cholesterol and phospholipids and the intestinal absorption of fats and nutrients. Bile acids also are versatile signaling molecules that activate nuclear receptors and cell signaling pathways, and they play critical roles in the regulation of lipid, glucose, and energy metabolism.6–8 The critical role of bile acids in the regulation of triglyceride (TG) metabolism has been recognized for many years. The administration of a bile acid sequestrant or ileal resection depletes the bile acid pool and increases serum TG levels. 9 On the other hand, increasing the bile acid pool by the administration of chenodeoxycholic acid reduces plasma TGs.10 Recent studies have revealed that bile acids may have a beneficial effect by improving obesity, insulin sensitivity, and whole body lipid and glucose homeostasis in mice.11, 12 Bile acids may also negatively regulate serum TG levels by activating the nuclear receptor farnesoid X receptor (FXR), which induces small heterodimer partner (SHP) to inhibit sterol response element binding protein-1c (SREBP-1c) expression and its target genes in lipogenesis.11 It has been reported that FXR represses hepatic expression of the gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), in the liver.13 Activation of FXR significantly improved insulin sensitivity, hyperglycemia, and hyperlipidemia in db/db mice.12 Bile acids also prevent diet-induced obesity in mice by activating the membrane bile acid receptor TGR5 to promote energy expenditure in brown adipocytes.14 In this study, we show that overexpression of CYP7A1 in the liver prevents high-fat diet (HFD)–induced obesity, fatty liver, and insulin resistance in mice. These results underscore the importance of bile acid homeostasis in the regulation of whole body lipid, glucose, and energy homeostasis.

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7α-hydroxylase ( CYP7A1 ) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a ∼ 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4alpha (HNF4alpha) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptorgamma co-activator 1alpha (PGC-1alpha) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4alpha binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4alpha. Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4alpha and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4alpha and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4alpha and PGC-1alpha. Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4alpha, SRC-1, and PGC-1alpha. PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs.

Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7alpha-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4alpha and peroxisome proliferator-activated receptor gamma coactivator-1alpha trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor gamma-coactivator-1alpha but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes.

The Dongsheng uranium deposit in the Ordos basin, Northern China occurs in fluvial sandstones in the Zhiluo Formation, Middle Jurassic. Geologic characteristics of the deposit such as depositional environments of host sandstones, the distribution of ore bodies, uranium minerals, the mineralization age etc. are described in this paper. A “Multi-stage Uranium Mineralization” model is proposed for uranium ore-formation of the deposit. The mineralization process can be divided into four main stages, namely the preconcentration during diagenesis, the interlayer infiltration mineralization during Late Jurassic to Paleocene time, the reconcentration in Miocene, and the last stage of ore preservation exerted by reducing fluids after Eocene time.