A high-capacity assay for PPARγ ligand regulation of endogenous aP2 expression in 3T3-L1 cells
| Autor: | Linda J. Kelly, Chhabi Biswas, Joel P. Berger, G. Marie Thompson, Deirdre Trainor, Carl LaCerte |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Ribosomal Proteins
Cellular differentiation Peroxisome Proliferator-Activated Receptors Drug Evaluation Preclinical Biophysics Biology Fatty Acid-Binding Proteins Sensitivity and Specificity Biochemistry Fatty acid-binding protein Mice Transactivation 3T3-L1 Cells Adipocytes Animals RNA Messenger Receptor Molecular Biology Messenger RNA Oligonucleotide Nucleic Acid Hybridization food and beverages Cell Differentiation Cell Biology Diabetes Mellitus Type 2 Adipogenesis Thiazolidinediones RNA extraction Carrier Proteins DNA Probes |
| Zdroj: | Analytical Biochemistry. 330:21-28 |
| ISSN: | 0003-2697 |
| Popis: | A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) gamma agonists. PPARgamma plays a critical role in adipogenesis and PPARgamma agonists have been shown to induce adipocyte differentiation. Here, PPARgamma ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARgamma agonists. The potencies of PPARgamma agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARgamma transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARgamma ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARgamma modulators. |
| Databáze: | OpenAIRE |
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