Search

Hypothalamic-pituitary-thyroid (HPT) axis activity is important for energy homeostasis, and is modified by stress. Maternal separation (MS) alters the stress response and predisposes to metabolic disturbances in the adult. We therefore studied the effect of MS on adult HPT axis activity. Wistar male and female pups were separated from their mothers 3 h/d during postnatal day (PND)2–PND21 (MS), or left nonhandled (NH). Open field and elevated plus maze tests revealed increased locomotion in MS males and anxiety-like behavior in MS females. At PND90, MS females had increased body weight gain, Trhexpression in the hypothalamic paraventricular nucleus, and white adipose tissue mass. MS males had increased expression of TRH-degrading enzyme in tanycytes, reduced TSH and T3, and enhanced corticosterone serum concentrations. MS stimulated brown adipose tissue deiodinase 2 activity in either sex. Forty-eight hours of fasting (PND60) augmented serum corticosterone levels similarly in MS or NH females but more in MS than in NH male rats. MS reduced the fasting-induced drop in hypothalamic paraventricular nucleus-Trhexpression of males but not of females and abolished the fasting-induced increase in Trhexpression in both sexes. Fasting reduced serum concentrations of TSH, T4, and T3, less in MS than in NH males, whereas in females, TSH decreased in MS but not in NH rats, but T4and T3decreased similarly in NH and MS rats. In conclusion, MS produced long-term changes in the activity of the HPT axis that were sex specific; response to fasting was partially blunted in males, which could affect their adaptive response to negative energy balance.

Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of β2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN.

Thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2) has multiple, but transient, homeostatic functions in the brain. It is hydrolyzed in vitro by pyroglutamyl peptidase II (PPII), a narrow specificity ectoenzyme with a preferential localization in the brain, but evidence that PPII controls TRH communication in the brain in vivo is scarce. We therefore studied in male Wistar rats the distribution of PPII mRNA in the septum and the consequence of PPII inhibition on the analeptic effect of TRH injected into the medial septum. Twelve to 14% of cell profiles expressed PPII mRNA in the medial septum-diagonal band of Broca; in this region the specific activity of PPII was relatively high. Twenty to 35% of PPII mRNA-labeled profiles were positive for TRH-receptor 1 (TRH-R1) mRNA. The intramedial septum injection of TRH reduced, in a dose-dependent manner, the duration of ethanol-induced loss of righting reflex (LORR). Injection of the PPII inhibitor pGlu-Asn-Pro-7-amido-4-methylcoumarin into the medial septum enhanced the effect of TRH. The injection of a phosphinic TRH analog, a higher-affinity inhibitor of PPII, diminished the duration of LORR by itself. In contrast, the intraseptal injection of pGlu-Asp-Pro-NH2, a peptide that did not inhibit PPII activity, or an inhibitor of prolyl oligopeptidase did not change the duration of LORR. We conclude that in the medial septum PPII activity may limit TRH action, presumably by reducing the concentration of TRH in the extracellular fluid around cells coexpressing PPII and TRH-R1.

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivoby cold exposure or suckling, and in vitroby 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

The impact of hormones on the efficacy of antisense oligodeoxynucleotides (ASOs) is a poorly analyzed subject. We designed, based on the identification of potentially favorable local elements of mRNA secondary structure, eight phosphorothioate ASOs to knock down the expression of an ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in primary cultures of adenohypophysis. Two of the PPII ASOs were very efficient, sequence-specific, and target-specific. Because the expression of PPII is upregulated by 3,3',5'-triiodo-L-thyronine (T3), we studied the impact of varying the protocol of PPII induction on the knockdown efficacy. Hormone removal at transfection increased markedly the ability of (1) PPII ASOs to reduce PPII mRNA levels or PPII activity in adenohypophyseal cells or in C6 rat glioma cells and (2) a thyrotropin-releasing hormone (TRH) receptor-1 (TRH-R1) ASO to reduce TRH-R1 mRNA levels in adenohypophyseal cells. There was no effect of hormone removal on transfection efficacy and no correlation between target mRNA levels and ASO efficacy. These data demonstrated that ASO efficacy could depend on T3 levels; this might be due to regulation of a step generally critical for ASO efficiency.

Uribe RM, Joseph-Bravo P, Charli J-L. Pups removal enhances thyrotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus. Eur J Endocrinol 1–60. ISSN 0804–4643Previous studies have shown that lactation and suckling alter thyrotropin-releasing hormone (TRH) biosynthesis in hypothalamic paraventricular neurons. The amounts of paraventricular TRH mRNA and mediobasal hypothalamus (MBH) TRH were determined following removal of the pups to examine whether paraventricular TRH neuron activity is altered during the transition from lactation to estrous cycle. Paraventricular TRH mRNA and MBH TRH levels were determined by Northern blot analysis and radioimmunoassay, respectively. We had shown previously that after an 8-h withdrawal of the pups at mid-lactation the MBH TRH and paraventricular TRH mRNA levels are not modified. This condition was compared to one where pups were removed for 56 h, finding a significant decrease (46%, p < 0.005) of MBH TRH and a significant increase (156%, p < 0.02) of paraventricular TRH mRNA. The effect observed in the paraventricular TRH mRNA was correlated negatively with the serum corticosterone levels, a potential negative regulator of paraventricular TRH mRNA. The results were similar if a 1-h suckling period was introduced 8 h after withdrawal of the pups to induce a transient increase of corticosterone levels. The pattern of TRH mRNA was specific to the paraventricular nucleus because there was no enhancement in the preoptic area-anterior hypothalamus. In summary, our data suggest that TRH biosynthesis in paraventricular neurons is slowly adjusted after withdrawal of the pups, possibly to prepare TRH neurons to the new secretory demands of the estrous cycle.JL Charli, Instituto de Biotecnologia, UNAM, AP 510-3, Cuernavaca, Mor. 62271, México

Pyroglutamyl peptidase II (PPII) is a neuronal ectoenzyme responsible for thyrotropin releasing hormone (TRH) degradation at the synaptic cleft. PPII, heterogeneously distributed in different brain regions and adenohypophysis, is regulated under various endocrine conditions where TRH is involved in thyrotropin or prolactin regulation but only at the adenohypophyseal level. TRH can downregulate PPII activity in cultured adenohypophyseal cells. TRH present in extrahypothalamic brain areas has been postulated to serve as a neuromodulator and levels of this peptide increase in amygdala, hippocampus and cortex after electrical stimulation (kindling or electroshock). To study whether brain PPII could be regulated in conditions that stimulate TRHergic neurons, TRH and PPII activity were determined during the development of amygdaloid kindling in the rat. TRH levels increased from stage II to V in amygdala and hippocampus in the ipsi- and contralateral side to stimulation. In n. accumbens a decrease, compared to sham was observed at stage II, but levels raised through stage V. In contrast, PPII activity was increased at stage II, in amygdala of both sides and in hippocampus, frontal cortex, n. accumbens and hypothalamus of the contralateral side; levels decreased at stage V to sham values in most structures (except amygdala and hippocampus where the activity was 30% below controls). These results suggest that PPII activity in the central nervous system can be regulated in conditions known to affect TRHergic neurons.