Development of a Ligand Screening Tool Using Full-Length Human Peroxisome Proliferator-Activated Receptor-Expressing Cell Lines to Ameliorate Metabolic Syndrome.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and include three subtypes (PPARα, PPARδ, and PPARγ). They regulate gene expression in a ligand-dependent manner. PPARα plays an important role in lipid metabolism. PPARγ is involved in glucose metabolism and is a potential therapeutic target in Type 2 diabetes. PPARδ ligands are candidates for the treatment of metabolic disorders. Thus, the detection of PPAR ligands may facilitate the treatment of various diseases. In this study, to identify PPAR ligands, we engineered reporter cell lines that can be used to quantify PPARγ and PPARδ activity. We evaluated several known ligands using these reporter cell lines and confirmed that they are useful for PPAR ligand detection. Furthermore, we evaluated extracts of approximately 200 natural resources and found various extracts that enhance reporter gene activity. Finally, we identified a main alkaloid of the Evodia fruit, evodiamine, as a PPARγ activator using this screening tool. These results suggest that the established reporter cell lines may serve as a useful cell-based screening tool for finding PPAR ligands to ameliorate metabolic syndromes.

Discovery of peroxisome proliferator-activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Vitamin D Metabolite, 25-Hydroxyvitamin D, Regulates Lipid Metabolism by Inducing Degradation of SREBP/SCAP.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that control lipid homeostasis. SREBP activation is regulated by a negative feedback loop in which sterols bind to SREBP cleavage-activating protein (SCAP), an escort protein essential for SREBP activation, or to insulin-induced genes (Insigs) (endoplasmic reticulum [ER] anchor proteins), sequestering the SREBP-SCAP-Insig complex in the ER. We screened a chemical library of endogenous molecules and identified 25-hydroxyvitamin D (25OHD) as an inhibitor of SREBP activation. Unlike sterols and other SREBP inhibitors, 25OHD impairs SREBP activation by inducing proteolytic processing and ubiquitin-mediated degradation of SCAP, thereby decreasing SREBP levels independently of the vitamin D receptor. Vitamin D supplementation has been proposed to reduce the risk of metabolic diseases, but the mechanisms are unknown. The present results suggest a previously unrecognized molecular mechanism of vitamin D-mediated lipid control that might be useful in the treatment of metabolic diseases.

SOX6 attenuates glucose-stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice.

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.