Hypothalamic regulation by thyroid hormone receptor phosphorylation

三种主要的甲状腺激素受体(Thr)亚型，由两个基因座(ThrA和Thrb)表达，分别是 存在于哺乳动物中：THRA1、THRB1和THRB2。甲状腺激素(TH)通过THR调节食物 人体摄取代谢与下丘脑-垂体-甲状腺(HPT)轴我们的实验室长期以来一直是 对THRB2亚型作为下丘脑-垂体-甲状腺(HPT)轴的中央调节因子感兴趣，因为它 独特而有限的表达模式。我们最近发现THRB2被TH和TH高度磷酸化 N-末端丝氨酸处的AMPK(S101-小鼠，S102-人)，在其他THR中没有发现。基座 关于携带该磷酸化突变的小鼠的摄食量增加、肥胖和TH抵抗 (S101a)，我们假设AMPK依赖的THRB2 S101磷酸化途径在 下丘脑抑制食物摄取和HPT轴。我们还假设这一功能障碍 途径导致瘦素抵抗、食物摄入量增加和肥胖(图1)。三个密切相关的目标 具体目标1：功能上共同定位下丘脑THRB2和AMPK在摄食中的作用。两者都有 弓状核(ARC)和腹内侧核(VMN)以THRB和AMPK依赖的方式调节摄食。 为了开始在功能上定位THRB2在下丘脑的作用，THRB2将从POMC中移除 使用Thrb2漂白的小鼠和细胞特异性CRE驱动程序，在VMN的ARC和SF-1神经元中的神经元。牢房- 为了了解AMPKa2的S在摄食和呼吸中的作用，还将在相同的神经元中进行AMPKa2的特异性KO。 在p-THRB2途径中。雄性和雌性小鼠都将被研究，因为雌性小鼠的THRB2表达较高 HA标记的THRB2小鼠。特定目标2：确定THRB2磷酸化在摄食和调节中的作用 下丘脑瘦素信号。以前的研究已经清楚地表明，瘦素调节HPT和HPT 轴心和进食。鉴于S101a小鼠表现出瘦素，p-THRB2是瘦素作用的潜在介体 抵抗。开发了一种磷酸特异性抗体来进一步探索这一途径，并将在一段时间内用于- 摄食-禁食过程中THRB2磷酸化的过程研究。此外，一种仿磷剂 建立了S101D Ki小鼠模型，以确定这种变化是否对饮食诱导的肥胖具有保护作用。特定的 目的3：明确THRB2在禁食过程中对TRH的调节作用。而禁食诱导的抑制 HPT轴是通过减少TRH在室旁核(PVN)的表达而介导的，其机制尚不清楚 在低TH电平的设置中保持HPT轴抑制。令人惊讶的是，S101a小鼠对 禁食诱导的TRH抑制，提示THRB2 S101的磷酸化在Low的感知中起关键作用 TH级别。这一目标将通过靶向确定禁食诱导的和TH依赖的TRH抑制的位置 弓状核和室旁核的神经元。通过对THRB2的研究，将进一步探讨其抑制机制。 磷酸化是调节HPT轴和食物摄入量的共同机制。

Three major thyroid hormone receptor (THR) isoforms, expressed from two genetic loci (Thra and Thrb), are present in mammals: THRA1, THRB1, and THRB2. Thyroid hormone (TH) acting through THRs regulate food intake metabolism and the hypothalamic-pituitary-thyroid (HPT) axis in man. Our laboratories have long been interested in the THRB2 isoform as a central regulator of the hypothalamic-pituitary-thyroid (HPT) axis, given its unique and limited-expression pattern. We recently found that THRB2 is heavily phosphorylated by both TH and AMP kinase (AMPK) at an N-terminal serine site (S101-mouse, S102-human), not found in other THRs. Based on increased food intake, obesity, and TH resistance in mice carrying a mutation of this phosphorylation site (S101A), we hypothesize that an AMPK-dependent THRB2 S101 phosphorylation pathway in the hypothalamus suppresses food intake and the HPT axis. We also hypothesize that dysfunction of this pathway results in leptin resistance, increased food intake, and obesity (Fig. 1). Three closely related aims are proposed: Specific Aim 1: Functionally co-localize hypothalamic THRB2 and AMPK action on feeding. Both the arcuate (ARC) and ventromedial nucleus (VMN) regulate feeding in a THRB- and AMPK-dependent manner. To begin to functionally localize THRB2 action in the hypothalamus, THRB2 will be removed from the POMC neurons in the ARC and SF-1 neurons in the VMN using Thrb2 floxed mice and cell-specific Cre drivers. Cell- specific KO of AMPKa2 in the same neurons will also be performed to understand AMPKa2’s role in feeding and in the p-THRB2 pathway. Both male and female mice will be studied, given higher THRB2 expression in female HA-tagged THRB2 mice. Specific Aim 2: Determine the role of THRB2 phosphorylation in feeding and mediating hypothalamic leptin signaling. Previous studies have clearly demonstrated that leptin regulates both the HPT axis and feeding. A potential mediator of leptin action is p-THRB2, given that S101A mice demonstrate leptin resistance. A phospho-specific antibody was developed to probe this pathway further and will be used in a time- course study of THRB2 phosphorylation during the fed-fasting transition. Furthermore, a phosphomimetic S101D KI mouse model was generated to determine if this change protects against diet-induced obesity. Specific Aim 3: Define the locus of TRH regulation by THRB2 during fasting. While fasting-induced suppression of the HPT axis is mediated by reduced TRH expression in the paraventricular nucleus (PVN), it remains unknown how HPT axis suppression is maintained in the setting of low TH levels. Strikingly, S101A mice display resistance to fasting-induced TRH suppression, suggesting that THRB2 S101 phosphorylation is critical in the sensing of low TH levels. This aim will determine the locus of fasting-induced and TH-dependent TRH suppression by targeting neurons in the ARC and PVN. The mechanism of suppression will be explored further by studying if THRB2 phosphorylation is a common mechanism regulating the HPT axis and food intake.