SIRT1 Regulates TSH Release Figure 3. Identification of PIP5Kc as a SIRT1-binding protein. (A) PIP5Ks were identified as SIRT1-associated protein by immunoprecipitation with anti-FLAG antibodies from HEK293T cells expressing FLAG-SIRT1. A full-length SIRT1 and inactive form of SIRT1 (H355A) interacted with PIP5K (+). (B) Immunoprecipitation analysis of endogenous SIRT1 and endogenous PIP5Ka, PIP5Kb, PIP5Kc, or PIP4Kb in HEK293T cells. (C) Mapping the interaction interface of SIRT1 with PIP5Kc. A schematic representation of the SIRT1 interaction site (right panel; positive (+), negative (2)). doi:10.1371/journal.pone.0011755.g003 SIRT1-overexpressing pituitary cells. The specificities of RNAi against PIP5Ks were examined; RNAi against each PIP5K showed the specific knockdown effect to each target (Fig. 5E). We confirmed that PIP2 production was significantly decreased in the PIP5K-RNAi knockdown pituitary cells (Fig. 5F). Basal-level TSH secretion was reduced by the knockdown of PIP5Kc, whereas neither PIP5Ka-RNAi nor PIP5Kb-RNAi knockdown showed such an effect (Fig. 5G). Strikingly, the knockdown of PIP5Kc completely abolished the effect of SIRT1 overexpression on the enhancement of TSH secretion (Fig. 5H). This result demonstrates that SIRT1 and PIP5Kc work in the same pathway, and supports our concept that SIRT1 regulates TSH secretion via the deacetylation of PIP5Kc. SIRT1 KO mice show high acetylated-PIP5Kc, low PIP2 and low TSH Secretion To confirm that SIRT1 regulates TSH secretion in vivo via modulating PIP5Kc acetylation level and its lipid kinase activity, we investigated SIRT1 KO mice. First, we examined the level of acetylated PIP5Kc in the pituitary gland of SIRT1 KO mice. The level of acetylated PIP5Kc was higher in SIRT1 KO mice than that in wild-type (WT) mice (Fig. 6A). The PIP2 levels in the brain were lower in the SIRT1 KO mice than in the WT mice (Fig. 6B). SIRT1 KO mice have smaller pituitary than wild-type mice [39]. Thus, we examined the number of TSHb cells in SIRT1 KO mice, before investigating the TSH release. Unexpectedly, the number of TSHb cells was higher in the pituitary glands of SIRT1 KO when compared to normal (Fig. 6C). Consistent with this finding, the total TSH protein content in SIRT1 KO pituitary was significantly elevated (Fig. 6D). Despite the increased pituitary TSH contents, the amount of secreted TSH from SIRT1 KO pituitary glands was significantly lower than that from WT pituitary glands (Fig. 6E). The difference was more remarkable when the plasma TSH level was normalized with the number of pituitary TSHb cells (Fig. 6F). There was no difference in the TSH mRNA levels in the thyrotropes of SIRT1 KO and WT mice (Fig. 6G). Taken together, these findings support our model that SIRT1 regulates TSH release through modulating PIP5Kc activity (Fig. 7). Discussion In the present study, we showed that SIRT1 is abundantly expressed in TSH-producing cells in the anterior pituitary gland. Hypothalamic–pituitary–thyroid (HPT) axis regulates energy expenditure, oxygen consumption, and fuel metabolism [40]. Thyroid hormones (triiodothyronine [T3] and thyroxine [T4]) negatively regulate the HPT axis [41,42], and influence adipose tissue metabolism and cholesterol homeostasis [43]. In addition, the combination of serum TSH and tissue insulin sensitivity has important effects on serum lipid parameters in type 2 diabetes [44]. SIRT1 is also involved in insulin sensitivity and lipid metabolism [5]. Under fasting conditions, SIRT1 has a negative effect on insulin sensitivity [45]. Under fed conditions, activation of SIRT1 improves insulin sensitivity [46,47]. Thus it is reasonable to speculate that the SIRT1-PIP5Kc pathway described in this study represents a key event in the process of energy metabolism. The longevity response to calorie restriction (CR) is actively regulated by nutrient-sensing pathways involving the kinase target of rapamycin (TOR) [48], AMP kinase [49,50] and insulin/ insulin-like growth factor (IGF-1) signalling [51] in lower organisms and mice. Sirtuins is also thought to be linked to CR PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11755