Dynamic Localization of Glucokinase and Its Regulatory Protein in Hypothalamic Tanycytes Magdiel Salgado 1. , Estefanı´a Tarifen ˜ o-Saldivia 1. , Patricio Ordenes 1 , Carola Milla ´n 3 , Marı´a Jose ´ Yan ˜ ez 1 , Paula Llanos 1 , Marcos Villagra 1 , Roberto Elizondo-Vega 1 , Fernando Martı´nez 1 , Francisco Nualart 1 , Elena Uribe 2 , Marı´a de los Angeles Garcı ´a-Robles 1 * 1 Departamento de Biologı ´a Celular, Facultad de Ciencias Biolo ´ gicas, Universidad de Concepcio ´ n, Concepcio ´ n, Chile, 2 Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Ciencias Biolo ´ gicas, Universidad de Concepcio ´ n, Concepcio ´ n, Chile, 3 Facultad de Artes Liberales, Universidad Adolfo Iban ˜ ez, Vin ˜ a del Mar, Chile Abstract Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic b cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S 0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a K i 0.2 mM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose. Citation: Salgado M, Tarifen ˜ o-Saldivia E, Ordenes P, Milla ´n C, Yan ˜ ez MJ, et al. (2014) Dynamic Localization of Glucokinase and Its Regulatory Protein in Hypothalamic Tanycytes. PLoS ONE 9(4): e94035. doi:10.1371/journal.pone.0094035 Editor: Julie A. Chowen, Hosptial Infantil Universitario Nin ˜o Jesu ´ s, CIBEROBN, Spain Received November 6, 2013; Accepted March 11, 2014; Published April 16, 2014 Copyright: ß 2014 Salgado et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FONDECYT grant 1100705(to MAG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mgarcia@udec.cl . These authors contributed equally to this work. Introduction Specific brain regions, such as the hypothalamus and brain stem, are able to detect and respond to changes in glucose concentration, triggering neuroendocrine responses that regulate feeding behavior [1]. In the hypothalamus, specifically in the arcuate nucleus (AN), neurons are capable of responding to changes in glucose and lactate concentrations [2,3]. AN neurons are in close contact with ependymal cells, known as tanycytes, which are the main glial cell present in the basal hypothalamus [4,5]. Their proximal pole is located within the ventricular wall, and their long cellular process is projected towards the ventrome- dial hypothalamus [6,7]. There are four types of tanycytes: a1, a2, b1 and b2 [8]. a2 and b1 tanycytes are localized in the lower lateral wall of the third ventricle; they have extended cell processes that contact the neurons in the AN, in particular neuropeptide Y (NPY) neurons [5,9,10] and pro-opiomelanocortin (POMC) neurons [11]. b2 tanycytes form the cerebrospinal fluid (CSF)-median eminence (ME) barrier, and their extended processes contact vessels devoid of the blood–brain barrier and are sometimes in direct contact with microvessels present in the ME [12]. We have demonstrated that these cells express proteins, including glucose transporter (GLUT2), glucokinase (GK) and ATP-sensitive K + (K + ATP ) channels, involved in the b cell glucose sensing mechanism in the pancreas, indicating their possible involvement in hypothala- mus-mediated glucosensing [6,13]. Notably, GLUT2 is located in the proximal pole of tanycytes, which are in direct contact with the CSF in the third ventricle [6]. GK was localized mainly in the tanycyte nuclei in the euglycemic condition [13]. We have also demonstrated that glucose induces a rise in intracellular Ca 2+ in tanycyte cultures, which depended on ATP generated by the uptake and metabolism of glucose [14]. Independent study in brain slices has shown that selective stimulation of tanycyte cell bodies by glucose and also non-metabolizable analogs of glucose evokes robust ATP-mediated Ca 2+ responses [15]. Nevertheless, both studies showed that Ca 2+ waves were dependent on ATP release and subsequent activation of P2Y1 receptors. In addition, intracerebroventricular infusion of ATP stimulates neurons localized in the lateral hypothalamic area [16], dorsomedial hypothalamic nucleus [17] and ventromedial nucleus VMN [18]. Similarly, we have demonstrated that tanycytes release lactate in response to glucose [19]. ATP or lactate release by tanycytes may modulate neuronal activity in hypothalamic areas associated with feeding behavior, including the arcuate nucleus (AN) and VMN, which are in close contact with these cells. Moreover, extracellular PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e94035