Elsevier

Peptides

Volume 31, Issue 1, January 2010, Pages 145-149
Peptides

Glucocorticoids increase NPY gene expression in the arcuate nucleus by inhibiting mTOR signaling in rat hypothalamic organotypic cultures

https://doi.org/10.1016/j.peptides.2009.09.036Get rights and content

Abstract

The mammalian target of rapamycin (mTOR) has been implicated in the regulation of physiological functions such as cell growth and proliferation, and glucocorticoids reportedly inhibit mTOR signaling in peripheral tissues. Recent studies suggest that the mTOR signaling in the hypothalamus plays a critical role in maintaining energy homeostasis. In this study, we examined whether the mTOR signaling in the hypothalamus is involved in the regulation of neuropeptide Y (Npy) gene expression in the arcuate nucleus by glucocorticoids. In the hypothalamic organotypic cultures, the incubation with rapamycin significantly inhibited the mTOR signaling which was shown by decreases in the levels of phosphorylated p70S6K1 and S6. Similar to the action of the mTOR inhibitor rapamycin, dexamethasone (DEX), a synthetic glucocorticoid, also inhibited the mTOR signaling in the hypothalamic explants. Analyses of the explants with in situ hybridization demonstrated that the DEX or rapamycin alone significantly increased Npy gene expression in the arcuate nucleus, but that there were no additive effects of DEX and rapamycin on the expression. These data suggest that glucocorticoids upregulate the Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling, at least in part.

Introduction

The mammalian target of rapamycin (mTOR) is a highly conserved serine–threonine kinase whose activity affects several physiological functions such as cell growth and proliferation [17], [22], [31]. In vitro, cellular levels of ATP increase mTOR signaling, and mTOR itself is thought to serve as an ATP sensor [8]. The mTOR is known to induce the phosphorylation of ribosomal S6 kinase 1 (p70S6K1) and translation inhibitor factor 4E-binding protein, and these proteins in turn control downstream targets such as S6 [10]. Recent studies suggest that mTOR signaling pathways in the hypothalamus are involved in the regulation of energy balance [6], [7], [14], [21], [29]. The hypothalamic mTOR signaling is reportedly inhibited under fasting [7] and activated by the administration of leptin, ciliary neurotrophic factor (CNTF) and fatty acid synthase inhibitor, all of which are known as anorexigenic agents [5], [7], [18]. Moreover, constitutive-active p70S6K1 and dominant-negative p70S6K1 expressed in the hypothalamus decreased and increased food intake as well as body weight, respectively [3]. These data suggest that the hypothalamic mTOR acts as the master regulator of energy balance that integrates nutritional and hormonal signals. It is also demonstrated that the majority of neuropeptide Y (NPY) neurons in the arcuate nucleus express mTOR [7]. Thus, it is possible that the effects of the hypothalamic mTOR on energy balance are mediated, at least in part, via the NPY neurons.

Glucocorticoids, synthesized in and released from the adrenal glands [27], are one of the orexigenic peripheral signals. An excess of glucocorticoids causes obesity, whereas their depletion leads to marked anorexia in humans [27]. Previously, we demonstrated that glucocorticoids stimulated Npy gene expression in the arcuate nucleus in rat hypothalamic organotypic cultures, and that ghrelin and insulin, representative orexigenic and anorexigenic hormones, respectively, affected Npy gene expression only in the presence of glucocorticoids [9], [23]. These data suggest that glucocorticoids not only stimulate food intake but also play a permissive role in the regulation of energy balance, and that NPY neurons in the arcuate nucleus are the possible site of action for glucocorticoids.

While it is reported that glucocorticoids inhibit mTOR signaling in peripheral tissues [16], [24], [25], [28], it is not clear whether glucocorticoids affect the mTOR signaling pathway in the central nervous system. In the present study, we examined whether or not glucocorticoids affect mTOR signaling in the hypothalamus to regulate Npy gene expression in the arcuate nucleus.

Section snippets

Hypothalamic organotypic cultures

Rat hypothalamic slice-explant cultures were performed as described previously [1], [2], [11]. Sprague–Dawley (SD) pups, 7–9 days old (Chubu Science Materials, Nagoya, Japan; lights on from 9:00 to 21:00), were killed by decapitation, and hypothalamic tissues were sectioned at 350 μm thickness on a Mcllwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). Three coronal slices containing arcuate nucleus were separated and placed in HBSS (Invitrogen, Grand Island, NY) enriched with

Effects of rapamycin on mTOR signaling in hypothalamic cultures

Incubation with 10−6 M rapamycin for 24 h significantly decreased the levels of p-p70S6K1 as well as p-S6 in hypothalamic organotypic cultures (Fig. 1A and B). On the other hand, rapamycin did not significantly affect p70S6K1 or S6 levels at 24 h (Fig. 1A and B).

Effects of DEX on mTOR signaling in hypothalamic cultures

The levels of p-p70S6K1 as well as p-S6 were significantly decreased with the incubation of 10−8 M DEX for 24 h in hypothalamic organotypic cultures (Fig. 1C and D). The incubation with 10−8 M DEX did not significantly affect p70S6K1 and S6

Discussion

In the present study, we demonstrated that glucocorticoids inhibited the hypothalamic mTOR signaling and that the inhibition of mTOR signaling increased Npy mRNA expression in the arcuate nucleus in the hypothalamic organotypic cultures. These data suggest that glucocorticoids stimulate Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling in the hypothalamus, at least in part.

The hypothalamic organotypic cultures employed in this study have been shown to maintain the

Disclosure statement

All authors have nothing to declare.

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