RESUMEN
OBJECTIVE: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. METHODS: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. RESULTS: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. CONCLUSIONS: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders.
Asunto(s)
Adipocitos Marrones/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Desacopladores/farmacología , Proteína Desacopladora 1/metabolismo , Adipocitos Marrones/metabolismo , Animales , Fármacos Antiobesidad/química , Respiración de la Célula , Células HEK293 , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estabilidad Proteica , Bibliotecas de Moléculas Pequeñas/química , Desacopladores/química , Proteína Desacopladora 1/genéticaRESUMEN
Activated Transcription Factor-2 (ATF-2) is important during development of and during injury to the brain. Both Jun N-terminal Kinases (JNKs) and p38 Mitogen-Activated Protein Kinases (p38MAPKs) may phosphorylate ATF-2, but the contribution of these two pathways in cells has never been investigated. We have assayed endogenous p38MAPK activity in SK-N-MC and SH-SY5Y human neuroblastoma cells for activation of a GAL4/ATF-2 fusionprotein, by means of titrations of transfected expression plasmids and by using the p38MAPK inhibitor SB203580. It was found that basal activation of ATF-2 was independent of p38MAPK and that whereas MAPK kinase-3 (MKK3) was a weak inducer of ATF-2 activation, it was a potent activator of the stress activated transcription factor CHOP. In contrast, ATF-2 was very potently activated by the JNK pathway activator MAPK kinase kinase-1 (MEKK1). Thus, kinases downstream of MEKK1 appear relevant, but it is unlikely that p38MAPKs contribute quantitatively to activation of ATF-2 in these cells.