Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the KX2-391 impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction. The hypothalamic-pituitary-thyroid (HPT) axis is of prime importance for the central regulation of KX2-391 appetite and energy metabolism (1, 2). The hypothalamic nucleus arcuatus receives peripheral satiety signals such as leptin or insulin and reacts by releasing melanocyte-stimulating hormone (MSH). MSH activates cAMP-response element (CRE)-binding protein (CREB) in neurons of the paraventricular nucleus (PVN) and thereby controls the expression of TRH, resulting in TSH secretion from the pituitary gland. Thus, dysfunction of hypothalamic MSH signaling in humans or mice causes a severe obesity-diabetes syndrome (3,C11). Regulation of transcriptional activity is a key control mechanism within the HPT axis and CREB-mediated CRE activation has been shown to play a major role in hypothalamic gene transcription (12,C14). Because of its impact on energy metabolism, dysregulation of the HPT axis has frequently been implicated in severe metabolic disorders including type 2 diabetes mellitus (T2DM) (15,C18). Glucose is the key player in diabetes and obesity and, similar to MSH, induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. Besides being a pivotal energy carrier, glucose-derived ATP is an important regulator KX2-391 of intracellular signaling in hypothalamic neurons. In analogy to pancreatic -cells, ATP inhibits ATP-sensitive K+ (KATP) channels and thus depolarizes hypothalamic neurons (19,C24). Furthermore, ATP dampens AMP-activated protein kinase (AMPK) activity in hypothalamic neurons, thereby relieving CREB-regulated transcriptional coactivator 2 (CRTC-2), an important transcriptional coactivator of CREB, from tonic phosphorylation and inhibition by AMPK (25). Elevating extracellular glucose concentrations from 0.5mM to 15.0mM was found to enhance CRTC-2 activity due to AMPK inhibition and expression of the insulin receptor substrate-2 in hypothalamic cells (25), thus establishing the first link between hypothalamic glucose sensing and CREB/CRTC-2-dependent gene regulation. KX2-391 Beyond this possible association of glucose and hypothalamic insulin signaling (25), there is also some evidence suggesting that glucose affects leptin-induced signal transduction in hypothalamic cells (26). Thus, at present, it appears that in hypothalamic cells glucose promotes gene transcription via CRTC-2 and affects key players of the HPT axis, such as leptin and insulin. However, direct effects of physiological relevant fluctuations in brain glucose levels on basal or MSH-induced CRE activity or hypothalamic TRH expression have Wisp1 not been shown yet. In the present study, we investigated mHypoA-2/10-CRE cells that express a wide range of hypothalamic markers and resemble PVN neurons with regard to melanocortin sensitivity and TRH production and in addition, stable express a CRE-dependent reporter construct (27,C33). We dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription and hypothalamic TRH expression, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. These findings significantly advance our knowledge of the impact of glucose on hypothalamic signaling and might reveal a new association between hyperglycaemia and hyperthyroidism. Materials and Methods Materials Cell culture reagents were obtained from Invitrogen and TurboFect from Fermentas. The anti-pro-TRH (M-166) antiserum was from Santa Cruz Biotechnology. The p-AMPK (2535s) and p-CREB (9198s) antiserum were purchased from Cell Signaling, the CRTC-2 (sc-46272) or CREB (sc-240) antiserum and fructose-1,6-bisphosphate (F-1,6-BP) was from Santa Cruz Biotechnology. The peroxidase-conjugated antimouse or antirabbit antibody, both raised in goat, the histone-3 antiserum and D-glucose, 2-deoxy-D-glucose (2-DG), and tolbutamide from Sigma-Aldrich. 2-[1,2-3H(N)]-2-DG was from PerkinElmer. The firefly luciferase substrate, the signal transducers and activator of transcription (STAT), and nuclear factor of activated t cells (NFAT) reporter gene construct pGL4.47.