Animals ; Humans ; Oxazines ; metabolism ; Phenylpropionates ; metabolism ; Receptors, Cytoplasmic and Nuclear ; agonists ; antagonists & inhibitors ; Thiazolidinediones ; metabolism ; Transcription Factors ; agonists ; antagonists & inhibitors

OBJECTIVETo develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library.

METHODScDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid.

RESULTSAfter optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65.

CONCLUSIONA stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

Base Sequence ; Cell Line ; DNA Primers ; DNA, Complementary ; DNA-Binding Proteins ; agonists ; chemistry ; genetics ; Humans ; Hypolipidemic Agents ; analysis ; Plasmids ; Receptors, Cytoplasmic and Nuclear ; agonists ; chemistry ; genetics ; Reproducibility of Results ; Transcription Factors ; agonists ; chemistry ; genetics ; Transfection

Activation of D1-like dopamine receptors by psychostimulants, such as amphetamine, upregulates the expression of immediate early gene and opioid peptide gene in the striatum. The genomic changes are regulated by phosphorylated transcription factors via complicated intracellular events. To evaluate temporal expression of the transcription factors by dopaminergic stimulation, the D1-like dopamine agonist, amphetamine or SKF82958, was systematically delivered. As intracellular markers in response to the agonist, phosphorylated cAMP response element-binding protein (pCREB), Fos-related antigens (FRA) and FosB immunoreactivity (IR) was compared at 20 and 120 min time points in the selected areas of the striatum. Semi-quantitative immunocytochemistry showed that amphetamine (5 mg/kg, i.p.) significantly increased pCREB-IR at 20 min, sustained up to 60 min and decreased at 120 min after the infusion. Like amphetamine, the full D1 agonist, SKF82958 (0.5 mg/kg, s.c.), also increased pCREB-IR at 20 min, but not at 120 min after the infusion in the dorsal striatum (caudoputaman, CPu) and shell of ventral striatum (nucleus accumbens, NAc). In contrast, FRA- and FosB-IR induced by SKF82958 was significantly increased at 120 min, but not at 20 min after the administration. These data indicate that SKF82958 mimics induction of CREB phosphorylation by amphetamine and differentially regulates temporal induction of pCREB, and FRA and FosB expression in the striatum.
Amphetamine ; Animals ; Basal Ganglia ; Cyclic AMP Response Element-Binding Protein* ; Dopamine Agonists* ; Dopamine* ; Immunohistochemistry ; Opioid Peptides ; Phosphorylation ; Proto-Oncogene Proteins c-fos ; Rats* ; Receptors, Dopamine ; Transcription Factors

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).
Anilides/*pharmacology ; Bile/metabolism ; Cells, Cultured ; Cholesterol/*metabolism ; Cholesterol Esters/metabolism ; DNA-Binding Proteins/agonists/*metabolism ; Enzyme Inhibitors/pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Hepatocytes/*drug effects/metabolism ; Humans ; Lipid Metabolism/drug effects/genetics ; Macrophages/*drug effects/metabolism ; Models, Biological ; Oleic Acids/*pharmacology ; Receptors, Cytoplasmic and Nuclear/agonists/*metabolism ; Sterol O-Acyltransferase/*antagonists & inhibitors/physiology ; Transcription Factors/agonists/*metabolism

To determine whether the PPARalpha agonist fenofibrate regulates obesity and lipid metabolism with sexual dimorphism, we examined the effects of fenofibrate on body weight, white adipose tissue (WAT) mass, circulating lipids, and the expression of PPARalpha target genes in both sexes of high fat diet-fed C57BL/6J mice. Both sexes of mice fed a high-fat diet for 14 weeks exhibited increases in body weight, visceral WAT mass, as well as serum triglycerides and cholesterol, although these effects were more pronounced among males. Feeding a high fat diet supplemented with fenofibrate (0.05% w/w) reduced all of these effects significantly in males except serum cholesterol level. Females on a fenofibrate-enriched high fat diet had reduced serum triglyceride levels, albeit to a smaller extent compared to males, but did not exhibit decreases in body weight, WAT mass, and serum cholesterol. Fenofibrate treatment resulted in hepatic induction of PPAR alpha target genes encoding enzymes for fatty acid beta-oxidation, the magnitudes of which were much higher in males compared to females, as evidenced by results for acyl-CoA oxidase, a first enzyme of the beta-oxidation system. These results suggest that observed sexually dimorphic effects on body weight, WAT mass and serum lipids by fenofibrate may involve sexually related elements in the differential activation of PPARalpha.
Adipose Tissue/drug effects/metabolism ; Animals ; Body Composition/drug effects ; Body Weight/drug effects ; Diet ; Dietary Fats/pharmacology ; Female ; Gene Expression Regulation/drug effects ; Lipids/blood/*metabolism ; Liver/drug effects/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/*metabolism/pathology ; Organ Weight/drug effects ; Procetofen/*pharmacology ; Receptors, Cytoplasmic and Nuclear/*agonists ; *Sex Characteristics ; Time Factors ; Transcription Factors/*agonists

Brown fat is a specialized fat depot that can increase energy expenditure and produce heat. After the recent discovery of the presence of active brown fat in human adults and novel transcription factors controlling brown adipocyte differentiation, the field of the study of brown fat has gained great interest and is rapidly growing. Brown fat expansion and/or activation results in increased energy expenditure and a negative energy balance in mice and limits weight gain. Brown fat is also able to utilize blood glucose and lipid and results in improved glucose metabolism and blood lipid independent of weight loss. Prolonged cold exposure and beta adrenergic agonists can induce browning of white adipose tissue. The inducible brown adipocyte, beige adipocyte evolving by thermogenic activation of white adipose tissue have different origin and molecular signature from classical brown adipocytes but share the characteristics of high mitochondria content, UCP1 expression and thermogenic capacity when activated. Increasing browning may also be an efficient way to increase whole brown fat activity. Recent human studies have shown possibilities that findings in mice can be reproduced in human, making brown fat a good candidate organ to treat obesity and its related disorders.
Adipocytes ; Adipocytes, Brown ; Adipose Tissue, Brown* ; Adipose Tissue, White ; Adrenergic beta-Agonists ; Adult ; Animals ; Blood Glucose ; Energy Metabolism ; Glucose ; Hot Temperature ; Humans ; Metabolism ; Mice ; Mitochondria ; Obesity* ; Transcription Factors ; Weight Gain ; Weight Loss

To study the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in lung cancer cells, and to testify if the PPAR-gamma agonists can inhibit human lung cancer cell growth through induction of apoptosis, PPAR-gamma was detected in two lung cancer cell lines by RT-PCR and immunohistochemistry, the inhibition of human lung cancer cell growth was investigated by MTT and cell counts, and the apoptosis was assessed by TUNEL. The results showed that: (1) PPAR-gamma expressed on two lung cancer cell lines; (2) PPAR-gamma activated by ligands could inhibit human lung cancer cell growth remarkably; (3) PPAR-gamma agonists could induce apoptosis to inhibit lung cancer cell growth. It was concluded that PPAR-gamma expressed in lung cancer cell can be activated by ligands and can inhibit lung cancer cell growth through induction of apoptosis.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; pathology ; Carcinoma, Small Cell ; pathology ; Cell Division ; drug effects ; Gene Expression Regulation, Neoplastic ; Humans ; Ligands ; Lung Neoplasms ; pathology ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Receptors, Cytoplasmic and Nuclear ; agonists ; metabolism ; Transcription Factors ; agonists ; metabolism ; Tumor Cells, Cultured

Estrogen Receptor (ERalpha) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERalpha for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERalpha modulator. We cloned the ERalpha LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Eralpha(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERalpha(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERalpha modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 micromol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 micromol/L. Using this screening model, we discovered four ERalpha agonists from 2000 natural and synthetic compounds.
3T3-L1 Cells ; Animals ; Chimera ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; Estrogen Receptor Modulators ; chemistry ; isolation & purification ; Estrogen Receptor alpha ; agonists ; Genes, Reporter ; genetics ; Genistein ; chemistry ; isolation & purification ; HeLa Cells ; Humans ; Luciferases ; genetics ; metabolism ; Mice ; Models, Chemical ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Transfection