Monitoring FoxO1 Localization in Chemically Identified Neurons

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Abstract
The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action ofCrerecombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons followingin vivoadministration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, ourFoxO1GFPmouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.