Research reportIntracerebroventricular administration of α-melanocyte stimulating hormone increases phosphorylation of CREB in TRH- and CRH-producing neurons of the hypothalamic paraventricular nucleus
Introduction
Responses to starvation not only include the shift from a carbohydrate-based to a fat-based metabolism, but also result in profound functional reorganization of several neuroendocrine axes [2], [9]. For example, while the biosynthesis and secretion of thyrotropin-releasing hormone (TRH) in hypophysiotropic neurons of the paraventricular nucleus (PVN) are regulated by a thyroid hormone-dependent negative feedback control mechanism [16], [39], these neurons are also powerfully affected by nutritional status. During fasting, a significant reduction of TRH gene expression occurs selectively in the PVN [4], the seat of hypophysiotropic TRH neurons, an effect that is completely reversed by systemic administration of the adipostatic hormone, leptin [21]. The arcuate nucleus of the mediobasal hypothalamus has been recognized as a primary target site for leptin’s actions [9] which may be transmitted by extensive monosynaptic and parallel multisynaptic pathways to hypophysiotropic neurons of the PVN [5], [27], [46].
An intact arcuate nucleus appears to be a prerequisite for the ability of leptin to regulate the hypothalamic–pituitary–thyroid axis [22]. Moreover, intracerebroventricular (i.c.v.) infusion of α-MSH recapitulates the effect of leptin on hypophysiotropic TRH neurons, completely restoring proTRH mRNA levels, despite continuation of the fast [6], making this arcuate nucleus-derived peptide an important candidate to mediate the effects of leptin on hypophysiotropic TRH neurons. I.c.v. administration of α-MSH also reactivates corticotropin-releasing hormone (CRH) gene expression in the PVN of fasting animals [7], indicating that α-MSH is capable of acting simultaneously on diverse neuroendocrine axes.
Characterization of melanocortin receptors has revealed that all couple in a stimulatory fashion to cAMP and induce the transcription of genes by activation of protein kinase A (PKA) [30]. The TRH as well as CRH promoters contain a consensus cAMP response element (CRE) [20], [38], suggesting that these genes are regulated by binding of the cAMP response element binding protein or CREB. CREB is a constitutively expressed transcription factor whose phosphorylation by PKA at serine-133 activates a number of well-characterized neuropeptide genes [28], [38]. Indeed, recent in vitro evidence in a heterologous cell system indicates that phosphorylated CREB (PCREB) can bind to the CRE in the TRH promoter and activate the gene [13].
To determine whether a similar mechanism of cell signaling by α-MSH is observed in vivo, we examined the presence of PCREB in the nucleus TRH and CRH producing neurons in the PVN following the i.c.v. administration of α-MSH. Further, we hypothesized that if α-MSH induces the phosphorylation of CREB in target neurons in the PVN, then the immunocytochemical delineation of PCREB would identify other neuronal populations in the PVN affected by melanocortin signaling including the locus of those neurons involved in anorexia and energy disposal.
Section snippets
Animals
These experiments were performed on adult male Sprague–Dawley rats (Taconic Farms, Germantown, NY, USA) weighing 210–230 g. The animals were housed individually in cages under standard environmental conditions (light between 0600 and 1800 h; temperature, 22±1 °C; rat chow and water available ad libitum). All experimental protocols were reviewed and approved by the Animal Research Committee at the New England Medical Center and Tufts University School of Medicine.
Animal preparation and α-MSH infusion
Ten days prior to
Effect of fasting on PCREB immunoreactivity in the PVN
In the fed state, the PCREB immunolabeling was confined to the nuclei of magnocellular neurosecretory neurons, with limited immunostaining in the parvocellular subdivision (Fig. 1A). In the fasting state, PCREB immunolabeling was even further reduced with fewer nuclei in the magnocellular subdivision and nearly complete absence of immunoreactivity in the parvocellular subdivision (Fig. 1B). Thus, presence of PCREB in TRH and CRH neurons of the PVN in response to α-MSH was further investigated
Discussion
The mechanisms by which animals adapt to inadequate nutrient availability has been vastly expanded by the discovery of the white fat-derived hormone, leptin [50]. During fasting, circulating levels of leptin are decreased, orchestrating a series of central responses that result in energy conservation [2]. Included among these is suppression of the hypothalamic–pituitary–thyroid axis, largely due to inhibition of proTRH gene expression selectively in hypophysiotropic neurons of the hypothalamic
Acknowledgements
This work was supported by NIH Grant DK37021.
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