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