1. 1 Role of hypothalamic Foxo1 in the regulation of food intake and energy homeostasis Min-Seon Kim1,6, Youngmi K Pak2,6, Pil-Geum Jang2,6, Cherl Namkoong2, Yon-Sik Choi2, Jong-Chul Won1,Kyung-Sup Kim3, Seung-Whan Kim1,2, Hyo- Soo Kim4, Joong-Yeol Park1, Young-Bum Kim5 & Ki-Up Lee1 Kyunghee University College of Medicine Department of Internal Medicine R3 Ha Sung Hyung 1

2. 2 Background 2

3. 3 Leptin and insulin signal adiposity to the brain and inhibit feeding, in part by suppressing the expression of the orexigenic neuropeptide Y (Npy) and Agouti-related protein (Agrp) and enhancing the expression of anorexigenic proopiomelanocortin (Pomc) in the hypothalamic arcuate nucleus (ARC). Nature 404, 661–671 (2000). 3

4. 4 Background Phosphatidylinositol-3-kinase (PI3K)/Akt pathway in hypothalamic neurons has been implicated in the regulation of food intake and energy homeostasis. Nature 413, 794–795 (2001). Diabetes 52, 227–231 (2003). Activation of Akt phosphorylates Foxo1, leading to its nuclear exclusion and proteosomal degradation, and thereby inhibiting its action. Natl. Acad. Sci. USA 100, 11285–11290 (2003). Foxo1 (forkhead transcriptional factor subfamily) meditates… the metabolic actions of insulin, hepatic gluconeogenesis, skeletal muscle glucose disposal, adipocyte differentiation and pancreatic b-cell growth 4

5. 5 Hypothalamic Foxo1 has an important role in the regulation of food intake and body weight. 5

6. 6 RESULT 1.Hypothalamic Foxo1 affects food intake and body weight. 2.Insulin and leptin inhibit hypothalamic Foxo1 activity. 3.Foxo1 stimulates the transcriptional activity of Npy. 4.Foxo1 regulates transcription of Agrp and Pomc. 6

7. 7 1.Hypothalamic Foxo1 affects food intake and body weight 7

8. 8 Bilaterally injected the ARC of mice with - adenoviruses that expressed constitutively active Foxo1-3A (Foxo1-3A-Ad, 1011 pfu) - adenoviruses expressing green fluorescent protein (GFP-Ad) Figure 1 Hypothalamic Foxo1 expression affects food intake and body weight. 8

9. 9 Foxo1 siRNA to suppress the endogenous expression of Foxo1 in the ARC. The expression of both endogenous Foxo1 mRNA and Foxo1 protein were reduced in C2C12 mouse myocytes by 65–80% at 24 h and 72 h after the transfection of two Foxo1 siRNAs (siF1 or siF2) that were targeted to different Foxo1 sites Figure 1 Hypothalamic Foxo1 expression affects food intake and body weight. 9

10. 10 2. Insulin and leptin inhibit hypothalamic Foxo1 activity 10

11. 11 Figure 2 Inhibitory effects of insulin and leptin on hypothalamic Foxo1 expression. - ICV administration of leptin (0.2 nmol) or insulin (12 nmol) decreased hypothalamic Foxo1 protein expression.(Fig.2-a) - Intraperitoneal administration of leptin (15 nmol) or insulin (600 nmol) reduced hypothalamic Foxo1 expression.(Fig.2-b) 11

12. 12 Figure 2 Inhibitory effects of insulin and leptin on hypothalamic Foxo1 expression. The i.c.v. administration of the PI3K inhibitor LY294002 (1 nmol) before the administration of insulin or leptin significantly attenuated these hormones’ effects on hypothalamic Foxo1. 12

13. 13 인슐린 인슐린 수용체 전사활성의 제어 탈 인산화 ? 핵 외 이행 인산화 13

14. 14 Figure 2 Inhibitory effects of insulin and leptin on hypothalamic Foxo1 expression. Leptin and insulin induce anorexia when administered i.c.v. ? insulin- and leptin-induced Foxo1 suppression mediates the anorexigenic effects of these hormones. Leptin (0.2 nmol) or insulin (12 nmol) i.c.v. to mice expressing Foxo1-3A or GFP in the ARC. 14

15. 15 3. Foxo1 stimulates the transcriptional activity of Npy 15

16. 16 Figure 3 Foxo1 binds to the Npy promoter. The mRNA expression of both Foxo1 and Npy in the ARC was higher in mice expressing Foxo1-3A than in mice expressing GFP.(Fig.3-a) Conversely, both ARC Foxo1 and Npy mRNA expression decreased in mice injected with Foxo1 siRNA.(Fig.3-b) 16

17. 17 Figure 3 Foxo1 binds to the Npy promoter. Four putative Foxo1-binding sites, which are known as insulin responsive elements (IREs), in the 1-kb promoter region of the human NPY gene.(Fig.3-c) A competition EMSA showed that Foxo1 binds to the region between nucleotides 705 and 816 upstream of the Npy coding sequence that includes the IRE2 and IRE3 sites.(Fig.3-d) 17

18. 18 Figure 3 Foxo1 binds to the Npy promoter. Cold IRE2 and IRE3 or Foxo1 antibody inhibited the binding of nuclear proteins to radiolabeled IREs, confirming that Foxo1 binds to these IRE sites. Chromatin immunoprecipitation (ChIP) assay also demonstrated that endogenous Foxo1 binds to this promoter region. 18

19. 19 Figure 4 Foxo1 stimulates Npy transcriptional activity. 19

20. 20 4. Foxo1 regulates transcription of Agrp and Pomc 20

21. 21 Figure 5 Effect of Foxo1 on transcriptional activity of Agrp and Pomc. 21

22. 22 DISCUSSION 22

23. 23 Foxo1 P Npy Food intake PI3K/Akt Insulin,Leptin Food intake - ? IRE2,IRE3 Npy POMC Agrp ARC STAT3 Foxo1 P Npy Food intake PI3K/Akt Insulin,Leptin Food intake - ? IRE2,IRE3 Npy ARC STAT3 Foxo1 P Npy Food intake PI3K/Akt Insulin,Leptin Food intake - ? IRE2,IRE3 Npy ARC STAT3 Foxo1 P Npy Food intake PI3K/Akt Insulin,Leptin Food intake - ? IRE2,IRE3 Npy ARC STAT3 23

24. 24 DISCUSSION Because Foxo1 is widely expressed throughout the hypothalamus, it may also be involved in the regulation of physiological effectors other than Npy, Agrp and Pomc. Hypothalamic Npy and Agrp expression increased 5- to 15- fold during a fast, but Foxo1 expression increased Npy and Agrp expression by 2- to 4-fold.  May be as yet unidentified factors that mediate fast-induced increases in Npy and Agrp expression. 24

25. 25 CONCLUSION The hypothalamic PI3K-Akt-Foxo1 signaling pathway mediates the effects of insulin and leptin on the transcriptional regulation of Npy, Agrp and Pomc. Therefore, we suggest that “Hypothalamic Foxo1 is an important player in the regulation of food intake and energy homeostasis.” 25