SIRT1 Regulates TSH Release Figure 4. Activation of PIP5Kc by deacetylation. (A, C) Effects of NAM on the acetylation of PIP5Ks (A) and PIP5Kc K265/268R (C). The level of acetylated-lysine/PIP5K level was determined. Statistical analyses were performed using Student’s t test (n = 3). *P,0.05 versus the non-NAM-treated group. (B) Sequence alignment of PIP5Ks. PIP5Ka (amino acids 205–226), PIP5Kb (246–267), and PIP5Kc (255–276) are shown. The light blue columns indicate candidates for deacetylation site by SIRT1. (D) In vitro deacetylation of PIP5Ks by SIRT1. Each Flag-PIP5Ks were purified from HEK293T cells after treatment with 5 mM NAM and was incubated with full-length recombinant SIRT1 or SIRT1 (H355A) in the presence of NAD+ for 3 h at 30uC. The acetylated-lysine/PIP5K level was determined. Statistical analyses were performed using Fisher’s PLSD test or Student’s t test (n = 3). *P,0.05 versus control. (E) TLC analysis of [32P]PI(4,5)P2 produced by acetylated PIP5Kc incubated with recombinant SIRT1 or SIRT1 (H355A). The PI(4,5)P2 level was measured using the ImageJ software (n = 3). Statistical analyses were performed using Fisher’s PLSD test. *P,0.05 versus control. (F) Western blot analysis of SIRT1 in HEK293 cells and SIRT1 RNAi cells. Actin was used as the control. (G) TLC analysis of [32P]PIP2 that was extracted from SIRT1 KD HEK 293T cells metabolically labelled (for 24 h) with [32P]orthophosphate. The PIP2 level was corrected for the PC level (n = 5). PC, phosphatidylcholine; SM, sphingomyelin; PS, phosphatidylserine; PA, phosphatidic acid; PI, phosphatidylinositol; and PIP, phosphatidylinositol phosphate. Values represent mean6SEM (A–D and F). doi:10.1371/journal.pone.0011755.g004 and autophagy to prolong lifespan with TOR pathway [52]. Resveratrol, an activator of sirtuins, antagonizes the mTOR/S6K pathway [53]. In addition, sirtuins and TOR have opposing effects on autophagy independently from each other. Thus sirutins and the downstream signalling pathway form a complex network. It will be important to study to address whether SIRT1 in the pituitary also controls the other endocrine axes and other downstream target involving aging and lifespan. In thyrotropes, SIRT1 was found to be distributed in the cytosol, suggesting a role of SIRT1 in the cytoplasm (Fig. 5B). In fact, we confirmed the colocalization and binding between PIP5Kc and SIRT1 in the pituitary gland (Fig. 5C). SIRT1 has been shown to shuttle between the cytoplasm and nucleus [54]. SIRT1 is localized in cytoplasm of pancreatic a cells and endothelial cells, and localized in nuclei in liver, muscle, and white adipose tissue [9,55]. Consistent with our findings that SIRT1 and PIP5Kc were localized in the cytoplasm of pituitary gland cells, SIRT1 and endothelial nitric oxide synthase (eNOS) also colocalize and coprecipitate in cytoplasm of endothelial cells [55]. SIRT1 deacetylates eNOS for stimulating the activity and increases endothelial nitric oxide (NO) [55]. This result suggests SIRT1 has some targets in the cytoplasm as well as in the nucleus. Our findings indicate that SIRT1 deacetylates acetylated PIP5Kc at K265/K268, thereby activating TSH secretion from the pituitary gland (Fig. 7). The deacetylated form of PIP5Kc is more enzymatically active and the PIP2 synthesized the secretion PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11755