Elsevier

Brain Research

Volume 945, Issue 1, 26 July 2002, Pages 50-59
Brain Research

Research report
Intracerebroventricular administration of α-melanocyte stimulating hormone increases phosphorylation of CREB in TRH- and CRH-producing neurons of the hypothalamic paraventricular nucleus

https://doi.org/10.1016/S0006-8993(02)02619-7Get rights and content

Abstract

Changes in circulating leptin levels, as determined by nutritional status, are important for the central regulation of neuroendocrine axes. Among these effects, fasting reduces TRH gene expression selectively in the hypothalamic paraventricular nucleus (PVN), which can be reversed by leptin administration. Intracerebroventricular (i.c.v.) infusion of α-MSH recapitulates the effects of leptin on hypophysiotropic TRH neurons, completely restoring proTRH mRNA to levels in fed animals despite continuation of the fast, making α-MSH a candidate for mediating the central effects of leptin. As α-MSH binds to a G-protein coupled receptor that activates cAMP and α-MSH stimulates the TRH promoter through the phosphorylation of the transcription factor CREB in vitro, we determined whether i.c.v. injection of α-MSH to rats regulates phosphorylation of CREB, specifically in hypophysiotropic TRH neurons of PVN. As α-MSH also induces the activation of CRH gene expression in the PVN, we further determined whether i.c.v. injection of α-MSH regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. In vehicle-treated animals, only rare neurons contained nuclear phospho-CREB (PCREB) immunoreactivity in the parvocellular PVN. I.c.v. injection of 10 μg α-MSH dramatically increased the number of PCREB-immunolabeled cell nuclei in the PVN in fasted groups at 10 min postinjection, particularly in the medial, periventricular, anterior and ventral parvocellular subdivisions, whereas a moderate increase of PCREB immunoreactivity was observed at 30 min and PCREB immunoreactivity was lowest at 1 h postinfusion. Double immunolabeling with proTRH antiserum revealed that following i.c.v. α-MSH infusion at 10 min, the majority of TRH neurons contained PCREB in the anterior (71%), medial (83%) and periventricular (63%) parvocellular subdivisions. The percentage of double-labeled TRH neurons declined at 30 min and 1 h post α-MSH infusion. Similarly, only 16% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle treated animals, whereas 10 min following α-MSH infusion the percentage of CRH neurons colocalizing with the PCREB rose to 54%, then fell to 37 and 17% at 30 and 60 min postinfusion, respectively. These data demonstrate that i.c.v. α-MSH administration increases the phosphorylation of CREB in several subdivisions of the PVN including TRH and CRH neurons in the medial and periventricular parvocellular subdivisions, suggesting that phosphorylation of CREB may be necessary for α-MSH-induced activation of the TRH and CRH genes. The increase in PCREB in the anterior and ventral parvocellular subdivisions of the PVN, regions linked to nonhypophysiotropic functions such as autonomic regulation, would also imply a role for these neurons in anorectic and energy wasting responses of melanocortin signaling.

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.

References (50)

  • T.A. Roeling et al.

    Efferent connections of the hypothalamic ‘grooming area’ in the rat

    Neuroscience

    (1993)
  • P.E. Sawchenko et al.

    Circuits and mechanisms governing hypothalamic responses to stress: a tale of two paradigms

    Prog. Brain Res.

    (2000)
  • B.M. Spiegelman et al.

    Obesity and the regulation of energy balance

    Cell

    (2001)
  • J.B. Tatro

    Melanotropin receptors in the brain are differentially distributed and recognize both corticotropin and α-melanocyte stimulating hormone

    Brain Res.

    (1990)
  • R.H. Thompson et al.

    Organization of inputs to the dorsomedial nucleus of the hypothalamus: a reexamination with Fluorogold and PHAL in the rat

    Brain Res. Brain Res. Rev.

    (1998)
  • J.E. Wikberg et al.

    New aspects on the melanocortins and their receptors

    Pharmacol. Res.

    (2000)
  • M.M. Wirth et al.

    Paraventricular hypothalamic α-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects

    Peptides

    (2001)
  • R.A. Adan et al.

    Differential effects of melanocortin peptides on neural melanocortin receptors

    Mol. Pharmacol.

    (1994)
  • R.S. Ahima et al.

    Role of leptin in the neuroendocrine response to fasting

    Nature

    (1996)
  • M. Bamshad et al.

    Central nervous system origins of the sympathetic nervous system outflow to white adipose tissue

    Am. J. Physiol.

    (1998)
  • N.G. Blake et al.

    Effect of food deprivation and altered thyroid status on the hypothalamic–pituitary–thyroid axis in the rat

    J. Endocrinol.

    (1992)
  • J.K. Elmquist et al.

    Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei

    Proc. Natl. Acad. Sci. USA

    (1998)
  • C. Fekete et al.

    α-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression

    J. Neurosci.

    (2000)
  • C. Fekete et al.

    Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic–pituitary–thyroid axis during fasting

    J. Neurosci.

    (2000)
  • M. Hagiwara et al.

    Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A

    Mol. Cell. Biol.

    (1993)
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