Unacylated ghrelin is not a functional antagonist but a full agonist of the type 1a growth hormone secretagogue receptor (GHS-R)

Abstract

Recent findings demonstrate that the effects of ghrelin can be abrogated by co-administered unacylated ghrelin (UAG). Since the general consensus is that UAG does not interact with the type 1a growth hormone secretagogue receptor (GHS-R), a possible mechanism of action for this antagonistic effect is via another receptor. However, functional antagonism of the GHS-R by UAG has not been explored extensively. In this study we used human GHS-R and aequorin expressing CHO-K1 cells to measure [Ca²⁺]_i following treatment with UAG. UAG at up to 10⁻⁵ M did not antagonize ghrelin induced [Ca²⁺]_i. However, UAG was found to be a full agonist of the GHS-R with an EC₅₀ of between 1.6 and 2 μM using this in vitro system. Correspondingly, UAG displaced radio-labeled ghrelin from the GHS-R with an IC₅₀ of 13 μM. In addition, GHS-R antagonists were found to block UAG induced [Ca²⁺]_i with approximately similar potency to their effect on ghrelin activation of the GHS-R, suggesting a similar mode of action. These findings demonstrate in a defined system that UAG does not antagonize activation of the GHS-R by ghrelin. But our findings also emphasize the importance of assessing the concentration of UAG used in both in vitro and in vivo experimental systems that are aimed at examining GHS-R independent effects. Where local concentrations of UAG may reach the high nanomolar to micromolar range, assignment of GHS-R independent effects should be made with caution.

Introduction

Ghrelin was discovered through its ability to activate the type 1a growth hormone secretagogue receptor (GHS-R) and stimulate growth hormone release in vivo (Smith et al., 2005, van der Lely et al., 2004). An evolutionarily conserved feature of ghrelin is the acylation of its third residue, usually with n-octanoic and, less commonly, with n-decanoic acid (Hosoda et al., 2003). Kojima et al. (1999) were the first to describe the requirement that ghrelin be acylated on its third serine residue for activation of the GHS-R in the nanomolar range, with an EC₅₀ for increased [Ca²⁺]_i of 2.5 × 10⁻⁹ M. In the circulation, ghrelin also occurs as a unacylated isoform (UAG) at 10–50 times the concentration of acylated ghrelin (Kojima and Kangawa, 2005, and our unpublished observations).

Of great interest to us has been the finding that in humans co-administration of UAG can antagonize the metabolic effects of ghrelin in vivo. Ghrelin administration causes hyperglycemia, hypoinsulinemia, increased circulating free fatty acids and worsening insulin sensitivity, but these effects are reversed or prevented by co-administration with UAG (Broglio et al., 2004, Gauna et al., 2004). These effects seem to be specific to ghrelin's metabolic activity since UAG has no impact on GH, PRL or ACTH secretion (Broglio et al., 2004). This suggested a direct action on the endocrine pancreas, and perhaps on hepatic glucose production. In relation to these in vivo findings, we have shown that UAG not only suppresses glucose output, but also blocks ghrelin induced glucose release by primary hepatocytes (Gauna et al., 2005). In support of these findings, a recent report demonstrated the antagonistic effect in fish where ghrelin's orexigenic effects were blocked by administration of UAG. Furthermore, this effect appears to occur both centrally and peripherally (Matsuda et al., 2006). There are now many reports of direct biological activity of UAG in vitro that suggest a receptor mediated cellular response, perhaps via a specific receptor that is not GHS-R (Baldanzi et al., 2002, Cassoni et al., 2004, Chen et al., 2005, Gauna et al., 2006, Muccioli et al., 2004, Nanzer et al., 2004, Thompson et al., 2003, Toshinai et al., 2006). Despite these findings, the current consensus appears to be that UAG is inactive as an agonist of the GHS-R. However, the possibility remains that UAG is somehow able to block the ghrelin response by antagonizing the GHS-R. Therefore, we have explored in more detail, in a defined in vitro system, the ability of UAG to antagonize activation of the GHS-R by ghrelin, the potency of UAG at the GHS-R, and the effects of GHS-R antagonists.