Food-Restricted and Dehydrated-Induced Anorexic Rats Present Differential TRH Expression in Anterior and Caudal PVN. Role of Type 2 Deiodinase and Pyroglutamyl Aminopeptidase II

T H Y R O I D - T R H - T S H Food-Restricted and Dehydrated-Induced Anorexic Rats Present Differential TRH Expression in Anterior and Caudal PVN. Role of Type 2 Deiodinase and Pyroglutamyl Aminopeptidase II E. Alvarez-Salas, C. Aceves, B. Anguiano, R. M. Uribe, C. García-Luna, E. Sánchez, and P. de Gortari Neurofisiología Molecular (E.A.-S., C.G.-L., P.de.G.), Neuroendocrinología Molecular (E.S.), Escuela de Dietética y Nutrición ISSSTE, México D.F., México (E.A.-S., C.G.-L.), Dirección de Neurociencias, Instituto Nacional de Psiquiatría Ramón de la Fuente, México D.F., México; Instituto de Neurobiología (C.A., B.A.), Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, 76230, México; Instituto de Biotecnología (R.M.U.), Universidad Nacional Autónoma de México, A.P. 510-3, Mor 62210 Cuernavaca, Morelos, México TRH synthesized in hypothalamic paraventricular nucleus (PVN) regulates thyroid axis function and is also implicated in anorexigenic effects. Under energy deficit, animals present decreased PVN TRH expression and release, low TSH levels, and increased appetite. Dehydration-induced anorexia (DIA) model allows insight into underlying mechanisms of feeding regulation. Animals drinking a 2.5% NaCl solution for 7 d present body weight reduction; despite their negative energy balance, they avoid food and have increased PVN TRH expression and TSH serum levels. These findings support an inhibiting role of PVN TRH in feeding control. We compared TRH expression by in situ hybridization in PVN subdivisions of 7-d dehydrated male rats to those of a pair-fed group (forced food-restricted) with similar metabolic changes than DIA, but motivated to eat, and to controls. We measured peripheral deiodinase activities, and expression and activity of medial basal hypothalamic type 2 deiodinase and pyroglutamyl-aminopeptidase II, to understand their regulating role in PVN TRH changes between food restriction and anorexia. TRH mRNA levels increased in anterior (aPVN) and medial-caudal subdivisions in DIA rats, whereas it decreased in medial PVN in both experimental groups. We confirmed the nonhypophysiotropic nature of aPVN TRHergic cells by injecting ip fluorogold tracer. Findings support a subspecialization of TRHergic hypophysiotrophic cells that responded differently between anorexic and food-restricted animals; also, that aPVN TRH participates in food intake regulation. Increased type 2 deiodinase activity seemed responsible for low medial PVN TRH synthesis, whereas increased medial basal hypothalamic pyroglutamyl-aminopeptidase II activity in DIA rats might counteract their high TRH release. (Endocrinology 153: 4067– 4076, 2012) egulation of hypothalamic-pituitary-thyroid (HPT) axis function by TRH is extensively studied. Cell bodies synthesizing hypophysiotropic TRH are confined to medial and periventricular parvocellular subdivisions of hypothalamic paraventricular nucleus (PVN); axons of these cells project to the median eminence (ME) where the peptide is released into the pitu- R itary portal blood. Acting through its receptor TRH-R1 in the thyrotropes, TRH activates synthesis and release of TSH that, in turn, induces the secretion of thyroid hormones (TH) T3, T4, from the thyroid to the peripheral blood. By controlling TH serum concentration, hypothalamic TRH indirectly regulates energy utilization and basal metabolic rate. ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/en.2011-2011 Received November 18, 2011. Accepted June 1, 2012. First Published Online June 19, 2012 Abbreviations: AgRP, Agouti-related protein; aPVN, anterior PVN; ARC, arcuate hypothalamic nucleus; AVP, vasopressin; BAT, brown adipose tissue; CART, cocaine and amphetamine-regulated transcript; cPVN, medial-caudal PVN; D1 and D2, type 1 and 2 deiodinase; DIA, dehydration-induced anorexia; DTT, dithiothreitol; FFR, forced food-restricted; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; HPT, hypothalamic-pituitary-thyroid; ISH, in situ hybridization; MBH, medial basal hypothalamus; ME, median eminence; mPVN, medial PVN; NPY, neuropeptide Y; PPII, pyroglutamyl aminopeptidase II; PVN, paraventricular nucleus; TH, thyroid hormone; TRH-␤NA, TRH-␤-naphtylamide. Endocrinology, August 2012, 153(8):4067– 4076 endo.endojournals.org 4067 4068 Alvarez-Salas et al. TRH Increases in Anterior PVN during Anorexia Anterior part of PVN (aPVN) contains TRH-expressing neurons that are not involved in hypophysiotropic functions of the peptide (1); aPVN TRH neurons possibly regulate homeostatic and behavioral functions at hypothalamic and forebrain levels such as locomotion, thermoregulation, and food intake (2). Both, medial (mPVN) and anterior subsets of PVN TRHergic neurons receive afferent connections from arcuate hypothalamic nucleus (ARC) a region involved in regulation of food intake and fuel expenditure (3–5), supporting that PVN TRH expressing cells are implicated in both aspects of energy homeostasis. Evidence suggesting an involvement of TRH in appetite regulation is the fact that its icv injection reduces food consumption in both ad libitum or food-restricted animals (6 – 8). Additionally, PVN TRH expression is positively modulated by anorexigenic signals [leptin, ␣-MSH, cocaine, and amphetamineregulated transcript (CART)] and negatively by orexigenic peptides [neuropeptide Y (NPY), agouti-related protein (AgRP)] (9). During caloric restriction low circulating leptin levels decelerate HPT axis function (10, 11) and increase food intake (12); TRH release from medial basal hypothalamus (MBH) also decreases in parallel to serum TSH and TH levels. Adaptation of HPT axis is advantageous to the survival of fasted animals by saving their energy deposits. Animals subjected to dehydration-induced anorexia (DIA) spontaneously reduce their food intake and body weight despite their negative energy balance. In contrast to fasting or food restriction, DIA rats have increased PVN TRH mRNA expression (by RT-PCR) and higher serum TSH content compared with those of a pair-fed group (forced food-restricted FFR) (13) even though both present similar metabolic changes (reduced leptin and increased corticosterone serum levels), up-regulation of ARC orexigenic, and decreased anorexigenic peptide expression (14); in contrast to the food-avoiding behavior of DIA, FFR animals would eat if food was offered. Increased PVN TRH mRNA expression of DIA group returns to levels observed in FFR rats if an antagonist of CRH-2 receptor is injected into the PVN (15). The antagonist also decreases TSH serum content but only attenuates food-avoiding behavior of anorexic rats, suggesting that at least some part of the medial hypophysiotropic TRHergic cells are activated in DIA, but that a different subset of neurons not having a neuroendocrine function should be responsible for the aberrant behavior displayed by those animals; this also suggests that in a (...truncated)