SIRT1 Regulates TSH Release Figure 5. SIRT1 regulates TSH secretion via PIP5Kc (K265/K268). (A) Western blot analysis of PIP5Kc in the nerve tissues of mice. (B) Distribution of SIRT1 and PIP5Kc in fractionated pituitary gland of mice. SIRT1 and PIP5Kc were found to be present in cytosolic fractions rather than nuclear fractions. The first lane contains crude extract prior to separation. Nucleoporin, a nuclear marker; actin, a cytosolic fraction marker. (C) Double immunostaining with SIRT1 (red) and PIP5Kc (green) in the anterior pituitary gland of rats. Scale bar, 10 mm. (D) Co-immunoprecipitation analysis of SIRT1 and PIP5Kc in the cytosolic fraction of the pituitary gland of mice. Rabbit IgG was used as the negative control. (E) Western blot analysis of each PIP5K in PIP5Ks RNAi knockdown cells. (F) TLC analysis of [32P]PIP2 was extracted from PIP5Kc RNAi pituitary cells metabolically labelled (for 24 h) with [32P]orthophosphate. The PIP2 level was corrected for the PC level (n = 5). (G) Amounts of TSH secreted in each PIP5Ks RNAi knockdown cells (n = 16). (H) Amounts of TSH secreted in cells overexpressing SIRT1 and PIP5Kc RNAi (n = 16). Empty vectors and random RNAi constructs were used as controls. Statistical analyses were performed using Fisher’s PLSD test. *P,0.05 versus control. Values represent mean6SEM (% of control: F–H). doi:10.1371/journal.pone.0011755.g005 of TSH from pituitary thyrotropes. K265 and K268 of PIP5Kc correspond to R215 and R218 of PIP5Kb in human, respectively. The region from Y209 to R215 in PIP5Kb is a phosphatidic acid (PA)-binding site, and the kinase activity of PIP5Kb is completely lost when basic residues in this site are substituted with acidic residues [56]. This loss of function is thought to be caused by the alteration of the electrostatic interactions in the pocket composed of the PA-binding site and the surrounding residues. Since acetylation of lysine residues causes the alteration of electrical charge, the acetylation-deacetylation cycle we describe can easily be understood to regulate the kinase activity of PIP5Kc. Although our work points to SIRT1 as the deacetylase responsible for activating PIP5Kc, we have no information of which protein acetyltransferase is involved in the inactivating (acetylation) reaction. We also visualized the vesicle of EGFP-TSHb on exocytosis in primary culture of pituitary cells by using TIRF microscopy. Because the TIRF microscope only visualizes vesicles just beneath the membrane, the traffic event is mainly reflecting exocytosis [27,57,58]. The number of disappeared TSH-containing granules within 5 s was increased in the SIRT1-overexpressing cells compared to the control cells (Fig. 2C and 2D), confirming the activation of vesicle trafficking. In accordance with our findings, previous work has shown that the absence of PIP5Kc causes a reduction of the readily releasable vesicle pool and a delay of fusion pore expansion indicating a defect in vesicle priming in chromaffin cells [33]. Our assay system revealed that PIP5Kc knockdown inhibited PIP2 production and TSH secretion (Fig. 5G and 5H). Produced PI(4,5)P2 produced by PIP5Kc is an important regulator of synaptic vesicle trafficking in nerve terminal [59]. Although SIRT1 has a many substrates, the SIRT1-PIP5Kc pathway is important for intracellular trafficking and secretion in the thyrotropes. We also visualized the vesicle of EGFP-growth hormone (GH) on exocytosis in GH3 pituitary cells by using as TIRF microscopy (data not shown). Consistent with the observation of TSH-containing granules trafficking, the numbers of disappearing GH-containing granules was increased in the SIRT1-overexpressing cells (data not shown). These findings showed that the SIRT1-PIP5Kc secretion pathway is shared by thyrotropes and other pituitary endocrine cells. The physiological properties of the SIRT1 KO mouse are also consistent with the model proposed here. The levels of PIP5Kc acetylation were elevated in SIRT1 KO mice and the TSH secretion levels were lower (Fig. 6D). SIRT1 KO mice have smaller pituitary than wild-type mice, however differentiation of the anterior pituitary occurs normally [39]. GH levels and insulin- like growth factor (IGF)-1 are not affected in SIRT1 KO mice [39]. In neuron-specific SIRT1 KO mice, the production and PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11755