Stat binding sites but not Stat3 are required for fasting-induced transcription of Agouti-related protein mRNA

Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor Signal Transducer and Activator of Transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide which stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a "default" state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.