pathways in liver in response to fasting by interacting with and deacetylating PGC-1a, a key transcriptional regulator of glucose production [6]. SIRT1 plays other roles in stabilizing genomic DNA and proteins [7,8]. Recently, evidence has accumulated that SIRT1 could be involved in the endocrine system. In pancreas, SIRT1 modulates serum glucose levels by regulating pancreatic insulin through repressing the expression of mitochondrial uncoupling protein 2 (UCP2) [9]. In agreement with these findings, SIRT1 knockout (KO) mice show impaired glucose-stimulated insulin secretion [10]. Neuron-specific SIRT1 KO mice display reduced growth hormone level, which results in the impairment of body growth [11]. SIRT1 transgenic mice have reduced levels of blood cholesterol and adipokines, and are more metabolically active than littermate controls [12]. SIRT1 is expressed in the anorexigenic proopiomelanocortin (POMC) neurons [13], and hypothalamic SIRT1 mediates the up-regulation of the S6K pathway [14]. Despite the accumulation of evidence for the importance of SIRT1 in endocrine system, highly basic questions have remained to be addressed such as whether SIRT1 regulates metabolism in the brain and pituitary gland. Hormones and neurotransmitters are transported as secretory granules by diverse cellular processes. Long-distance transport of these vesicles is regulated by kinesin-driven transport [15,16] and posttranslational modifications, such as tyrosination [17] and polyglutamylation [18]. When the vesicles come to the terminal points, the process is passed to the exocytotic mechanisms [19,20,21]. Phosphatidylinositol plays critical roles in this exocytotic step [22]. The phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) interacts with the some proteins involved in the exocytotic machinery, such as the Ca2+-dependent activator protein for secretion (CAPS) which is a priming factor, and synaptotagmin which is a Ca2+ sensor [23]. How SIRT1 is involved in the exocytosis machineries is poorly understood. In the present study, we investigated how SIRT1 regulates hormone release. We identify the SIRT1 substrate, and provide evidence for a regulatory mechanism of SIRT1- and SIRT1 target-dependent hormone release. Results Abundant expression of SIRT1 in pituitary thyrotropes In previous work, SIRT1 KO shows the decrease in the plasma level of thyroid hormone [24]. The report suggests that SIRT1 is involved in the regulatory axis of thyroid activity or function. We thus first investigated where SIRT1 was predom- inantly expressed in the whole body. To this end, we performed Western blot analyses with a commercially available anti-SIRT1 antibody on lysates obtained from a variety of adult murine tissues. These assays revealed that the expression level of the SIRT1 protein in the pituitary glands was highest among that in many other adult murine tissues (Fig. 1A). The SIRT1 expression in other tissues was detected as previous reported when the expose time was elongated (data not shown). The SIRT1 expression in the pituitary gland was also highest when compared to that in other brain tissues (Fig. 1B). We next performed immunohistochemical analysis of sagittal slices of rat brain. The immunohistochemical analysis revealed strong expression of SIRT1 in the anterior pituitary (Fig. 1C). We tested the specificity of the antibody used in Fig. 1A–C by performing Western blotting with SIRT1 KO pituitary sample as a negative control (Fig. 1D). As SIRT1 signal was not detected in the SIRT1 KO tissue sample using the antibody, the antibody has an excellent specificity (Fig. 1D). SIRT1 Regulates TSH Release Pituitary gland is a critical component of the endocrine system that is present in all vertebrates, and pituitary hormones control homeostasis of the whole body [25]. The anterior pituitary secretes thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), adrenocorticotropic hormone (ACTH), growth hormone (GH), and prolactin (PRL). We used immunohistochemistry to examine the distribution of SIRT1 among hormone-secreting pituitary cells in mice and humans. SIRT1 was most frequently localized in TSH-positive cells, but barely observable in ACTH-, FSH-, LH-, GH-, and PRL-positive cells in mice (Fig. 1E) and humans (Fig. 1F). We examined human pituitary glands that were obtained during the autopsies of 20 cadavers, ranging in age from 20 to 103 years. SIRT1-positive cells were mostly found to be localized in TSH-positive cells in the case of all the cadavers (data not shown). These results show that mammalian SIRT1 is highly abundant in thyrotropes. Although normally a nuclear protein, the SIRT1 protein was in the cytoplasm of thyrotropes (Fig. 1E and 1F), a distribution similar to that reported for alpha cells of the pancreatic islets, another type of endocrine cells [9]. SIRT1-mediated positive regulation of TSH secretion We next examined whether SIRT1 regulates TSH secretion by manipulating SIRT1 expression level in pituitary cells. SIRT1- expression plasmids or SIRT1-RNAi was introduced by electro- poration into primary cultures of rat anterior pituitary. The cultured cells expressed either increased (overexpression) or decreased (knockdown) levels of SIRT1 protein compared to the controls (Fig. 2A). SIRT1 overexpression and knockdown led to an increase and decrease, respectively, in the amount of TSH secreted into the medium (Fig. 2B). To test the effect of SIRT1 on the release of secretory granules, we used rat pituitary cells transfected with an expression vector encoding enhanced green fluorescent protein (EGFP) fused in frame with TSHb. TSH is a 28k-Da heterodimer composed of covalently linked a and b subunits. The a subunit is common to TSH, FSH, and LH, whereas the b subunit is specific to the TSH molecule [26]. We observed, in real-time, the motion of individual EGFP-TSHb granules undergoing exocytosis in living cells by using total internal reflection fluorescence (TIRF) microscopy—a method that enables fluorescence excitation within a closely restricted domain situated close to the plasma membrane [21,27]. Given the characteristic of TIRF microscopy, the granule disappearance represents exocytosis of EGFP-TSHb or returning of vesicles to cytoplasm via kiss-and-run recycling system. Thus the disappearance of granules is a result of vesicle trafficking. We recorded sequential images every 5 s (Video S1–S3) and counted the number of EGFP-positive granules that disappeared during the 5 s intervals (surrounded by red circles in Fig. 2C). The number of vanishing granules was increased in the SIRT1-overexpressing cells, and conversely decreased in the SIRT1-RNAi knockdown cells compared to the control cells (Fig. 2D). Hence, the trafficking of TSH-containing vesicles was more active in the SIRT1- overexpressing cells, and lesser in SIRT1-RNAi knockdown cells, compared to the control cells. These findings show that SIRT1 activates TSH release in thyrotropes. Identification of PIP5Kc as a SIRT1-binding protein To determine the molecular mechanisms that regulate TSH release by SIRT1, we sought out the potential targets that could physically associate with SIRT1. It is known that the catalytically inactive form of protein kinase binds more avidly to its substrates than the native form of enzyme, so substrates are more effectively pulled down with the mutant enzyme compared with the wild-type PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11755