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

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.

Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain.

The stomach-derived hormone ghrelin mainly acts in the brain. Studies in mice have shown that the accessibility of ghrelin into the brain is limited and that it mainly takes place in some circumventricular organs, such as the median eminence. Notably, some known brain targets of ghrelin are distantly located from the circumventricular organs. Thus, we hypothesized that ghrelin could also access the brain via the blood-cerebrospinal fluid (CSF) barrier, which consists of the choroid plexus and the hypothalamic tanycytes. Using systemic injection of ghrelin or fluorescent-ghrelin in mice, we found that cells of the blood-CSF barrier internalize these molecules. In time-response studies, we found that peripherally injected fluorescent-ghrelin quickly reaches hypothalamic regions located in apposition to the median eminence and more slowly reaches the periventricular hypothalamic parenchyma, adjacent to the dorsal part of the third ventricle. Additionally, we found that CSF ghrelin levels increase after the systemic administration of ghrelin, and that central infusions of either an anti-ghrelin antibody, which immuno-neutralizes CSF ghrelin, or a scrambled version of ghrelin, which is also internalized by cells of the blood-CSF barrier, partially impair the orexigenic effect of peripherally injected ghrelin. Thus, current evidence suggests that the blood-CSF barrier can transport circulating ghrelin into the brain, and that the access of ghrelin into the CSF is required for its full orexigenic effect.

Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid.

Ghrelin is a stomach-derived peptide hormone that acts in the brain to regulate many important physiological functions. Ghrelin receptor, named the growth hormone secretagogue receptor (GHSR), is present in many brain areas with or without obvious direct access to ghrelin circulating in the bloodstream. Ghrelin is also present in the cerebrospinal fluid (CSF) but the brain targets of CSF ghrelin are unclear. Here, we studied which brain areas are accessible to ghrelin present in the CSF. For this purpose, we centrally injected mice with fluorescein-labeled ghrelin (F-ghrelin) peptide tracer and then systematically mapped the distribution of F-ghrelin signal through the brain. Our results indicated that centrally injected F-ghrelin labels neurons in most of the brain areas where GHSR is present. Also, we detected F-ghrelin uptake in the ependymal cells of both wild-type and GHSR-null mice. We conclude that CSF ghrelin is able to reach most of brain areas expressing GHSR. Also, we propose that the accessibility of CSF ghrelin to the brain parenchyma occurs through the ependymal cells in a GHSR-independent manner.

Cerebrospinal fluid concentrations of ghrelin in patients with multiple sclerosis.

Ghrelin, an orexigenic peptide, exerts immunomodulatory and other effects in the CNS and has neuroprotective properties. Ghrelin is predominantly produced by X/A-like cells in the gastric mucosa. Hence, ghrelin's main source of production lies outside the CNS while important functions of ghrelin are operated by specific receptors in the CNS. We analyzed 82 samples of our repository (45 samples form patients with multiple sclerosis and 37 control samples) for cerebrospinal fluid (CSF) and corresponding serum concentrations of ghrelin. Desacyl ghrelin concentrations were measured with a commercially available enzyme immunometric assay. We validated the assay for CSF samples. The test-retest reproducibility for ghrelin in CSF samples was excellent. Ghrelin CSF concentrations were higher in patients with multiple sclerosis (p<0.02) compared with controls. CSF concentrations correlated with serum concentrations in patients with multiple sclerosis (p<0.01). No such correlation was found in controls. Our findings endorse existing hypotheses that ghrelin affects the central inflammatory process in MS. The correlation between serum and CSF concentrations in MS, but not in controls, suggests a differential regulation of blood-to-brain transport mechanisms for ghrelin in MS and indicates that central effects of ghrelin in MS might be amenable to pharmacological manipulation of the systemic ghrelin secretion.

Concentrations of hormones and metabolites in cerebrospinal fluid and plasma of dairy cows during the periparturient period.

During early lactation, high-yielding dairy cows often show insufficient feed intake (FI) and, as a consequence, they enter into a negative energy balance associated with an altered pattern of plasma metabolites and hormones. These act as short- and long-term hunger or satiety signals in the brain and play an important role in the control of FI. Metabolites and hormones also occur in cerebrospinal fluid (CSF), which surrounds the hypothalamus and brainstem, 2 major centers of FI regulation. The CSF hormone and metabolite concentrations are mainly under control of the blood-brain barrier. Consequently, CSF hormone and metabolite concentrations differ from those in blood. However, the contribution of putative orexigenic and anorexigenic CSF signals possibly leading to insufficient FI of high-yielding dairy cows during early lactation has not been studied so far. Therefore, the aim of this study was to elucidate associations existing between both plasma and CSF hormones and metabolites during the periparturient period. Ten multiparous German Holstein dairy cows were fed ad libitum and samples of CSF from the spinal cord and blood from the jugular vein were withdrawn before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. Feed intake started to decrease from d 5 before calving and increased thereafter. Glucose, ß-hydroxybutyrate (BHBA), cholesterol, nonesterified fatty acids, urea (all enzymatic), lactate (colorimetric), amino acids (HPLC), osmolality (osmometer), ghrelin (RIA), leptin (ELISA), and resistin (Western immunoblot) were measured in both CSF and plasma, whereas free fatty acids (gas chromatography-mass spectrometry) and volatile fatty acids (gas chromatography-flame-ionization detector) were determined in plasma only. Whereas leptin concentrations decreased after calving in both plasma and CSF, ghrelin concentrations were not altered, and abundances of total resistin and its hexamers decreased only in plasma. Although plasma concentrations of cholesterol and nonesterified fatty acids changed during the periparturient period, their concentrations were not affected in CSF. In contrast, CSF Gln concentration tended to increase until calving, whereas CSF concentrations of BHBA, α-aminobutyric acid, Cit, Gly, Ile, Val, and Leu were increased in early lactation compared with the preparturient period. Because Gln is known to serve as neuronal substrate generating ATP, Gln is suggested to act as a central anorexigenic signal shortly before parturition. Moreover, due to their known anorexic effect, BHBA and Leu may potentially act as central signals and thereby suppress a sufficient increase in FI during early lactation.

Pulsatile cerebrospinal fluid and plasma ghrelin in relation to growth hormone secretion and food intake in the sheep.

As in other species, exogenous administration of ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptors can stimulates feeding behaviour and GH secretion in the sheep. However, the importance of endogenous ghrelin for these two functions as well as its central or peripheral origin remained to be established. In the present study, cerebrospinal fluid (CSF) ghrelin concentrations were measured in five anoestrous ewes and found to be more than 1000-fold lower than circulating plasma levels, in keeping with the even lower concentration in hypothalamic compared to abomasum tissue extracts. Cluster analysis indicated that CSF ghrelin levels were markedly pulsatile, with a greater number of peaks than plasma ghrelin. Pulsatility parameters were closer for GH and CSF ghrelin than between GH and plasma ghrelin. Plasma ghrelin and GH levels were significantly correlated in three out of five ewes but CSF ghrelin and GH in one ewe only. Half of the CSF ghrelin episodes were preceded by a ghrelin peak in plasma with a 22-min delay. Cross-correlations between plasma GH and plasma or CSF ghrelin did not reach significance but a trend towards cross-correlation was observed from 20 to 0 min between plasma and CSF ghrelin. At 09.00 h, when food was returned to ewes, voluntary food intake did not elicit a consistent change in plasma or CSF ghrelin levels. By contrast, a peripheral ghrelin injection (1 mg, i.v.) immediately stimulated feeding behaviour and GH secretion. These effects were concomitant with a more than ten-fold increase in plasma ghrelin levels, whereas CSF ghrelin values only doubled 40-50 min after the injection. This suggests that peripherally-injected ghrelin crosses the blood-brain barrier, but only in low amount and with relatively slow kinetics compared to its effects on GH release and food intake. Taken together, the results obtained in the present study support the notion that, in the ovariectomised-oestradiol implanted sheep model, peripheral ghrelin injection rapidly induces GH secretion, and feeding behaviour, probably by acting on growth hormone secretagogue receptor subtype 1 located in brain regions in which the blood-brain barrier is not complete (e.g. the arcuate nucleus).