title: Monitoring FoxO1 localization in chemically identified neurons.
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 of Cre recombinase. 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 following in vivo administration 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, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.
authors.affiliation: Division of Hypothalamic Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9077, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Fukuda
authors.forename: Juli E
authors.initials: JE
authors.lastname: Jones
authors.forename: David
authors.initials: D
authors.lastname: Olson
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hill
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Lee
authors.forename: Laurent
authors.initials: L
authors.lastname: Gautron
authors.forename: Michelle
authors.initials: M
authors.lastname: Choi
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Zigman
authors.forename: Bradford B
authors.initials: BB
authors.lastname: Lowell
authors.forename: Joel K
authors.initials: JK
authors.lastname: Elmquist
mesh_terms: D000818:Animals; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007031:Hypothalamus / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007328:Insulin / Q000378:metabolism; D020738:Leptin / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D005230:Fatty Acids, Nonesterified; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D007328:Insulin; D020738:Leptin; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt