Abstract
Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.
Highlights
The p21Waf1/CIP1 protein is a cyclin-dependent protein kinase (CDK)3 inhibitor that binds to cyclinCDK complexes, as well as free CDKs, to inhibit cell cycle progression
We found that shortterm food deprivation potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei
Two types of reporter mice were used in this study to monitor endogenous p21 expression, one that enables p21 expression to be visualized throughout the whole mouse and the other that enables specific tissues of the mouse to be interrogated
Summary
To confirm Cre-mediated excision of the stop codon, DNA was isolated from livers and PCR analysis was performed with four primers (A-C above and D: 5Ј-CTA ACG ACG TCG TTC ATC TTG T-3Ј). Laser Capture Microdissection—Brains were dissected from p21ϩ/ϩ or p21FLuc/FLuc mice, sliced coronally into three sections using a coronal brain tissue matrix, immediately frozen in optimal cutting temperature (OCT, TissueTek) compound, and stored at Ϫ80 °C. Metabolic Analysis—Plasma was isolated from 5-week-old male p21ϩ/ϩ and p21FLuc/FLuc littermates that had been allowed to feed ad libitum and from the same mice 1 week later after a 24 h fast. Livers were isolated after 24 h of fasting, fixed, frozen in OCT, and 5 m sections were analyzed to determine efficiency of viral infection by quantification of the percent of GFP-positive cells within 5 fields of view
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