SIRT1 Regulates TSH Release Figure 6. High Ac-PIP5Kc, low PIP2, and low TSH secretion in pituitary glands obtained from SIRT1 KO mice. (A) Levels of acetylated PIP5Kc in the pituitary of SIRT1 KO mice and WT mice. Endogenous PIP5Kc was immunoprecipitated. The acetylated-lysine/PIP5Kc level was measured (n = 3). (B) TLC analysis of PIP2 levels in the brain of SIRT1 KO mice and WT mice (WT: n = 6, KO: n = 6). (C) The number of TSHb- immunopositive cells in SIRT1 KO mice and WT mice (n = 5). (D) Pituitary concentrations of TSH in SIRT1 KO mice and WT mice (n = 10). (E) Levels of TSH secreted by the pituitary cells in the organ culture over 24 h in SIRT1 KO mice and WT mice (n = 7). (F) Plasma TSH levels per TSH- immnunopositive cell in SIRT1 KO mice and WT mice (n = 5). (G) mRNA levels of anterior pituitary hormone in SIRT1 KO mice and WT mice. RNA levels were determined using RT-PCR analysis. GAPDH was used as a loading control (n = 3). Values represent mean6SEM. Statistical analyses were performed using Student’s t test or Mann-Whitney U test. *P,0.05 versus WT. doi:10.1371/journal.pone.0011755.g006 secretion of GH but not other pituitary hormones is reduced [11]. The production of all pituitary hormones is regulated at least in part by signals from the hypothalamus. TSH-releasing hormone (TRH) secreted by the hypothalamus is the main stimulator of TSH synthesis and secretion [60,61]. It is generally considered that low serum TSH levels are attributable to the inhibition of TSH synthesis in the pituitary, which depletes the existing reserves of the hormone (exhausting the TSH ‘‘stock’’), without changing the rate of secretion (‘‘flow’’). However, in this study, SIRT1 regulates release (related to ‘‘flow’’, not ‘‘stock’’) of TSH, suggesting that the physiological regulation of the pituitary-thyroid axis is dependent on regulated secretion. The activity of SIRT1 is thought to be modulated by the nutritional status of the cell [62]. Recently, the expression of SIRT1 regulates oncogenic viral protein HPV7 [63] and locally acting IGF-1 isoform (mIGF-1), not circulating IGF-1 isoform [64]. Thus the activity and expression of SIRT1 changes topically and temporally. The core circadian rhythm regulator protein circadian locomotor output cycles kaput (CLOCK) is a histone acetyltransferase, and its activity is counterbalanced by NAD+ and SIRT1 [65]. The circadian rhythm modulates metabolic activities on a long-term basis, while intracellular traffic regulation of TSH by SIRT1 might provide short-term control of metabolism dependent on nutrient availability. Materials and Methods Study subjects SIRT1 KO mice were generated on a CD1 and 129/J mixed genetic background, and genotyped using a polymerase chain PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11755