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.

Changes in thyroid hormone action profoundly affect both metabolism and food intake in man. Hypothyroidism results in obesity, reduced energy expenditure (EE), and hypophagia, while hyperthyroidism causes weight loss, increased EE, and hyperphagia. We discovered a novel AMPK signaling pathway that phosphorylates a specific thyroid hormone receptor (THRB2) and suppresses the thyroid axis and food intake, establishing this pathway as a central regulator of the hypothalamus and suggesting it may be dysfunctional in human obesity.