Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms

Highlights

• Choroid plexus cells internalize less Fr-ghrelin in GHSR-deficient mice.

• Hypothalamic tanycytes internalize Fr-ghrelin in a GHSR-independent manner.

• Systemically-injected ghrelin shows lower transport to CSF in mice lacking GHSR.

• Ghsr mRNA is detected in choroid plexus cells, but undetectable in tanycytes.

Abstract

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

Introduction

Ghrelin is an acylated peptide hormone secreted from enteroendocrine cells of the gastrointestinal tract (Kojima et al., 1999). Ghrelin acts via a G-protein coupled receptor, named the growth hormone secretagogue receptor (GHSR), which is highly expressed in the pituitary and the brain (Howard et al., 1996). A main target of ghrelin is the central nervous system, where ghrelin regulates growth hormone secretion, food intake, blood glucose homeostasis and stress responses, among other functions (Yanagi et al., 2018). Since no physiologically relevant levels of ghrelin are synthesized in the brain (Cabral et al., 2017b), plasma ghrelin needs to be transported into the brain in order to gain access to its neuronal targets. Ghrelin-responsive neurons are widely distributed in a number of well-characterized brain nuclei; however, the accessibility of plasma ghrelin to most of these targets is strikingly low (Cabral et al, 2013, 2015; Perello et al., 2019; Schaeffer et al., 2013).

In order to gain access to the brain, ghrelin could either cross the blood-brain barrier, diffuse through the fenestrated capillaries at circumventricular organs (CVOs) or cross the blood-cerebrospinal fluid (CSF) barrier (Perello et al., 2019). The transport of mouse ghrelin across the blood-brain barrier in a blood-to-brain direction appears to be very low under normal conditions (Banks, 2002). Plasma ghrelin passively extravasates from the fenestrated capillaries of the CVOs, such as the hypothalamic median eminence (ME) and the area postrema (AP) (Cabral et al, 2014, 2017a; 2014; Schaeffer et al., 2013). However, this entry pathway for circulating ghrelin limits its action to GHSR-expressing neurons located nearby fenestrated capillaries, such as the ventromedial region of the arcuate nucleus (ARH), which contains fenestrated capillaries that branch from the hypophyseal system, or the AP itself (Cabral et al, 2014, 2017a; 2014; Ciofi, 2011; Schaeffer et al., 2013). Notably, recent evidence showed that plasma ghrelin is also selectively transported across the blood-CSF barrier and reaches the CSF (Uriarte et al., 2018). Since the monolayer of ependymal cells that line the brain ventricles displays a variable level of permeability (Mullier et al., 2010; Smith et al., 2004), CSF ghrelin can diffuse to the periventricular brain parenchyma and reach some populations of GHSR-expressing neurons that are not reached by plasma ghrelin. Thus, the blood-CSF barrier appears to play a key role for the central actions of ghrelin.

The blood-CSF barrier is formed by the choroid plexus epithelial (CPE) cells and the hypothalamic β-type tanycytes (Banks, 2019; Prevot et al., 2018). The CPE cells form a layer of cuboidal cells that surrounds a core of fenestrated capillaries in the brain ventricles and produce CSF. The β-type tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle and bridge the CSF with the fenestrated capillaries of the ME. Little is known about the mechanisms by which cells of the blood-CSF barrier transport circulating ghrelin into the CSF (Perello et al., 2019). The blood-CSF barrier does not allow the free diffusion of plasma ghrelin into the CSF, as indicated by the fact that CSF ghrelin levels are significantly smaller than plasma ghrelin levels. In sheep, CSF ghrelin levels show a pulsatile profile, with around half of the peaks in the CSF preceded by peaks in plasma, and systemically-injected ghrelin increases CSF ghrelin levels around 40–50 min after treatment (Grouselle et al., 2008). In mice, CSF ghrelin levels also increase after systemic administration of the hormone, and such elevation seems to be important for the full orexigenic action of the systemically-injected hormone since ghrelin-induced food intake is partially reduced by immuno-neutralization of ghrelin in the CSF (Uriarte et al., 2018). At cellular level, systemically-injected fluorescent ghrelin is internalized by CPE cells and by hypothalamic tanycytes in mice (Cabral et al., 2014; Schaeffer et al., 2013; Uriarte et al., 2018). Fluorescent ghrelin is also rapidly internalized by rat tanycytes in vitro, and this tanycytic ghrelin shuttle likely plays a role in the postnatal ARH development (Collden et al., 2015). Currently, the mechanisms transporting ghrelin through the blood-CSF barrier are uncertain. Given the potent effects of ghrelin treatment in several species, including humans, it seems very likely that this hormone (or ghrelin-mimetic compounds) would be used to treat patients suffering a variety of disorders, such as loss of appetite or weight loss. Thus, gaining insights into the mechanisms that transport ghrelin into the brain is a major aspect of the biology of ghrelin that may have clinical implications. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier in adult mice occurs in a GHSR-dependent manner.