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Endocrinology, doi:10.1210/en.2007-0226
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Endocrinology Vol. 148, No. 8 3998-4006
Copyright © 2007 by The Endocrine Society

Sensory Stimuli Directly Acting at the Central Nervous System Regulate Gastric Ghrelin Secretion. An ex Vivo Organ Culture Study

Luisa M. Seoane, Omar Al-Massadi, J. Eduardo Caminos, Sulay A. Tovar, Carlos Dieguez and Felipe F. Casanueva

Endocrinología Molecular (L.M.S., O.A.-M., F.F.C.), Area de Investigacion, Complejo Hospitalario Universitario de Santiago (CHUS), E-15780, Santiago de Compostela, Spain; Departaments of Medicine (O.A.-M., F.F.C.) and Physiology (J.E.C., S.A.T., C.D.), Santiago de Compostela University, 15782 Santiago de Compostela, Spain; Centro de Investigaciones Biomédicas en Red (CIBER): Fisiopatología de la obesidad y nutrición (CB06/03) (L.M.S., C.D., F.F.C.), Instituto de Salud Carlos III, Spain; and Department of Physiology (J.E.C.), School of Medicine, National University of Colombia, 1101 Bogotá, Colombia

Address all correspondence and requests for reprints to: Luisa M. Seoane, Area de Investigacion, Complejo Hospitalario Universitario de Santiago de Compostela, P.O. Box 563, E-15780, Santiago de Compostela, Spain. E-mail: fssisisc{at}usc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin, a novel gastrointestinal hormone involved in GH regulation, has been postulated as a relevant orexigenic peptide released by splanchnic tissues. Descriptive studies have shown that plasma ghrelin levels increase in states of negative energy balance or fasting, while decreasing in obesity and after feeding. In the present study, a novel organ-culture model of gastric tissue explants obtained from rat donors has been validated for ex vivo experiments. Fasting induced gastric ghrelin release as well as ghrelin mRNA expression that were reflected in plasma. Interestingly, those changes were fully reverted by 15 min of refeeding before stomach extraction. Unexpectedly, when animals were allowed 15 min before explant extraction to see or smell, but not eat, the food (tease feeding), ghrelin secretion was suppressed just like in gastric explants from refed animals. This effect was blocked when the animals were subjected to surgical vagotomy or treated with atropine sulphate. In conclusion, gastric explants were a suitable model for testing ghrelin mechanism of secretion in vitro, and they were found to maintain memory of the previously received signals. Similar to feeding, tease feeding resulted in suppression of ghrelin discharge by explants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, THE ENDOGENOUS ligand for the GH secretagogue receptor, is a 28-amino-acid peptide with a serine three residue n-octanoylated strongly involved in the regulation of GH secretion and energy homeostasis (1). Although ghrelin is expressed in a large number of tissues such as pituitary, hypothalamus, thyroid, and placenta (2, 3, 4). It is found in greatest quantity in the gastric fundus (5). Ghrelin mRNA expression, as well as the peptide itself, have been localized in the X/A-like cells within the acid-producing oxyntic glands of rat stomachs. However, ghrelin immunoreactive cells are not strictly confined to oxyntic mucosa because ghrelin is also synthesized and secreted from the duodenum, ileum, cecum, and colon (6). Ghrelin is considered an incretin, i.e. a gastrointestinal hormone regulated by and regulating nutritional status.

Several studies have shown that intracerebroventricular and/or ip ghrelin administration stimulates food intake as well as adiposity (7, 8). Ghrelin activates the expression of orexigenic neuropeptides such as neuropeptide Y and agouti-related peptide, in the hypothalamic arcuate nucleus. This leads to an increase in food intake and body weight (9).

Gastric ghrelin expression is conditioned by nutritional status. Thus, hypoglycemia, leptin, and fasting up-regulate ghrelin (7, 10). On the other hand, in obesity, ghrelin plasma levels are low and increase after hypocaloric diet treatment (11). Exceptions to this are patients with Prader-Willi syndrome, who present elevated ghrelin levels that may contribute to their voracious appetite, hyperphagia, and obesity (12, 13). In patients with anorexia nervosa, plasma ghrelin levels are elevated and return to normal levels after partial weight recovery (14). Human studies have reported a preprandial increase and a postprandial decline in plasma ghrelin levels, suggesting that it may play a physiological role in hunger and meal initiation (7, 11). However, there is still much controversy regarding the mechanisms regulating these changes (15).

The cephalic phase of gastrointestinal responses to food intake interacts with the gastric and intestinal phases to promote the absorption and use of incoming substrates. The cephalic response is activated by the thought, sight, smell, and taste of food, which produce an appetizing effect (16). When subjects are exposed to food-related sensory stimuli, vagal efferent fibers from the solitary tract nucleus are activated, and some gastrointestinal hormones are released; these hormones are considered cephalic phase reflexes (17). As one of these, ghrelin has been cited for its potential role in the anticipatory meal response (18). This is reminiscent of insulin, which presents a preprandial surge of secretion to minimize the prandial increases in blood glucose (19). The mechanisms controlling ghrelin secretion after food exposure have not yet been described (18).

The objectives of the present paper were three. First, the aim was to validate an ex vivo model suitable for assessing direct ghrelin secretion from the stomach and the ghrelin regulation mechanism by nutrients. Second, it was to gain insight into the control of ghrelin secretion directly by the stomach and, third, to observe whether ghrelin is secreted in anticipation of actual food intake.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
For all experiments, adult Sprague-Dawley rats were used. Animal research was conducted according to protocols approved by the Animal Care Committee of Santiago de Compostela University. Rats were housed in 12-h light/12-h dark cycles with free access to food and water. The animals were assigned to one of four weight-matched experimental groups (n = 10): 1) ad libitum-fed group with 24-h access to food and water; 2) a fasting group in which rats were deprived of food for 36 h before euthanasia; 3) the refeeding group in which the animals were deprived of food for 36 h but were allowed to have free access to food 15 min before euthanasia, and finally, 4) a tease feeding group, in which after 36 h of deprivation, the animals were allowed to smell and see food, but not to eat it, for 15 min before euthanasia.

Additional experimental groups were used to assess the effects of surgical vagotomy and cholinergic blockade on ghrelin levels. Two different groups of vagotomized animals were studied: one of them under fasting conditions (b) and the other under tease feeding conditions (d). Sham operated rats were used as controls. To test the effect of cholinergic blockade, two experimental groups were treated with atropine sulfate (0.5 mg/kg ip, dissolved in sterile saline; Sigma-Aldrich, St. Louis, MO) 30 min before the beginning of fasting conditions or tease feeding conditions. Experiments were performed during the first 2 h of the light cycle. After euthanasia, a blood sample for ghrelin analysis was obtained, and stomachs were excised.

Tissue explants culture
To obtain ex vivo tissue, the stomachs were immediately excised and transported to the incubator in sterile Krebs-Ringer-HEPES buffer [NaCl, 125 nmol/liter; KCl, 5 nmol/liter; MgSO4, 1.2 nmol/liter; KH2PO4, 1.3 nmol/liter; CaCl2, 2 nmol/liter; glucose, 6 nmol/liter; and HEPES 25 nmol/liter (pH = 7.4)]. After blood vessels and connective tissue were removed, stomach tissue was washed with sterile Krebs-Ringer-HEPES. Tissue explants, mostly gastric fundus, with an approximate weight of 2 g, were placed in six-well dishes containing 2.5 ml DMEM supplemented with penicillin (100 U/ml) and streptomycin sulfate (100 µg/ml), and incubated at 37 C under a humidified atmosphere of 95% air-5% CO2. After a preincubation period of 1 h, the medium was discarded, and 2.5-ml fresh medium was dispensed into each well. Culture medium was then collected at 1, 2, or 3 h, and tissue was weighted with a precision scale. Media and plasma were stored at –20 C until ghrelin assay.

Surgical vagotomy
The surgical procedure was performed aseptically, and all surgical instruments were sterilized before use. Animals were operated under ketamine-xylazine anesthesia. Rats were placed on their backs, and a midline abdominal incision was made. The liver was carefully moved to the right to expose the esophagus. Dorsal and ventral branches of the vagus nerve were then exposed and dissected from the esophagus. Each branch of the nerve was ligated with surgical suture at two points, as distally as possible to prevent bleeding, and cauterized between the sutures. The abdominal muscles and the skin were then sutured with surgical silk. Sham surgeries were also performed, in which each trunk of the nerve was exposed, but not tied or cauterized. One week after vagotomy, the animals were euthanized, and the stomachs were excised as described previously. The effectiveness of the vagotomy was assessed by postmortem stomach observation. Only the rats that showed an increase in stomach size after vagotomy were included.

RT-PCR
RT-PCR protocol conditions and quantitative real-time PCR amplification and detection for gastric mucosa ghrelin were performed on a Roche Light Cycler system (Roche Molecular Biochemicals, Mannheim, Germany), as previously described (20). RNA extraction was performed on the samples according to the manufacturer’s protocol (Invitrogen, Barcelona, Spain). RNA was resuspended in diethylpyrocarbonate water, and the integrity of total RNA was checked by agarose gel electrophoresis and 28S and 18S rRNAs visualized after ethidium bromide staining (data not shown). Two micrograms of total RNA were reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) into cDNA, as previously described (21).

PCR was performed using 3-µl cDNA template with specific primers for ghrelin. Rat hypoxanthine-guanine phosphoribosyltransferase (HPRT), designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), was used as an internal control gene. The fluorescence spectra were recorded during the elongation phase of each PCR cycle. Contamination with genomic DNA was excluded by using total RNA samples as template in which no amplification product was detected. Furthermore, intron-spanning primers for ghrelin and HPRT housekeeping were used to support further the absence of genomic DNA (3). After the final cycle, the melting curve was determined to check that only one product had been produced, and the PCR product was electrophoresed on a 1.5% agarose/0.5x Tris-borate EDTA gel containing ethidium bromide to confirm that the product was the expected size. Relative quantification of PCR products was then based on value differences using the comparative cycle threshold method (20). Ghrelin mRNA levels were normalized with respect to the HPRT level in each sample. This experiment was performed on eight animals per group.

Time-course studies
To perform plasmatic ghrelin and insulin time-course studies, intracardiac cannulas were implanted under ketamine-xylazine anesthesia, as previously described (9). After surgery, the animals were placed directly in isolation test chambers for 5 d and given free access to food and water. The day of the experiment, the animals were assigned to one of the four experimental groups described previously, and blood samples (0.3 ml) were withdrawn at the appropriate times: 15, 30, 45, and 60 min. Serum was kept at –20 C until RIA analysis.

Biochemical analysis
Total ghrelin levels were determined by a double antibody RIA using reagents kits and methods provided by Phoenix Pharmaceuticals Inc. (Belmont, CA). Samples for tissue explants were obtained directly from culture medium. For testing plasma ghrelin levels, the samples were obtained from trunk blood by decapitation, and were collected in tubes containing EDTA (1 mg/ml blood) and aprotinin (500 U/ml blood; Sigma-Aldrich). Samples were immediately centrifuged and then subjected to RIA, as previously described (22). The limit of assay sensitivity was 1 pg/ml. Results were expressed as picograms per milliliter of ghrelin per gram of tissue in culture media or as picograms per milliliter in plasma.

Plasma insulin levels were determined by a double antibody RIA using insulin RIA kits provided by Phoenix Pharmaceuticals Inc.

Plasma glucose concentrations were determined by a glucose autoanalyzer (Accu-Chek sensor, Roche Diagnostics GmbH, Mannheim, Germany). The rats were weighed on two occasions: at the start of fasting and just before euthanasia. Data were expressed as mean ± SE and assessed by the Mann-Whitney U test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the adequate parameters for the organ culture model used here, two variables were analyzed: fasting period and incubation time. Stomach explants from animals with ad libitum food intake (control group) were compared with tissue from rats deprived of food for 12, 36, and 60 h. All groups were compared after 1 h of incubation, 2 h, and 3 h. No meaningful changes were detected in any of the groups at 1 h. However, differences were found at 2 h of incubation, but only in donors that had fasted for 36 h or more (Fig. 1BGo). Thus, the model was set at 2-h incubation of explants from donors deprived of food for 36 h. The secretion from ad libitum animals explants was 2366 ± 217-pg/ml/g tissue, and after 36 h of fasting was of 3501 ± 338-pg/ml/g tissue. This represents 148% of the control group (P < 0.01).


Figure 1
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  		FIG. 1. Gastric ghrelin secretion directly from tissue explants at 1 (A), 2 (B), and 3 h (C) of incubation presented as percent over control (mean ± SE). Samples were measured in duplicate (n = 10–15). *, P < 0.05; **, P < 0.01 vs. control.

 
Having established a proper model, the focus became the determination of whether ghrelin changes in plasma under different metabolic situations were due to direct variations in gastric release. Groups of experimental animals with either ad libitum food intake or 36-h fasting were studied (Fig. 2AGo). As expected, fasting significantly increased plasmatic ghrelin values to 300% over controls (620 ± 66 pg/ml fasting animals vs. 225 ± 45 pg/ml in controls; P < 0.01). In the groups that had fasted for 36 h, 15 min of refeeding previous to explants extraction induced a nonsignificant (NS) reduction in ghrelin levels (529 ± 96 pg/ml) vs. fasting group.


Figure 2
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  		FIG. 2. A, Plasmatic ghrelin concentration (n = 10–15) in the different experimental conditions. B, Gastric ghrelin secretion from tissue explants to the incubation medium (**, P < 0.01 vs. control group; ++, P < 0.01 vs. fasting group). C, Ghrelin mRNA expression in gastric mucosa by real-time RT-PCR, standardized by HPRT mRNA levels (**, P < 0.01 vs. control group).

 
It is noteworthy that the plasmatic ghrelin changes were a direct reflection of the stomach secretion of ghrelin into the incubation media (Fig. 2BGo). Fasting for 36 h enhanced stomach release of ghrelin (3549 ± 343 pg/ml/g of tissue), which is 150% vs. control (P < 0.01), and that increase was significantly and strikingly reduced by just 15 min of refeeding (2058.44 ± 205.6 pg/ml/g), which is 87% over control (P < 0.01). The model was considered validated because this refeeding was able to counteract the fasting induced increase in ghrelin secretion. The changes in ghrelin release were supported by changes in gastric ghrelin mRNA (Fig. 2CGo) (fasting 36 h 19 ± 0.7 arbitrary units vs. control 14 ± 0.8 arbitrary units; P < 0.01), but refeeding did not induce significant alteration in mRNA (refeeding 17 ± 0.9 arbitrary units, NS vs. fasting). The NS change was most probably due to the fact that 15 min of refeeding was too short a time to modify the previously enhanced mRNA expression induced by fasting.

The most unexpected finding was the effect of tease feeding on gastric ghrelin secretion (Fig. 2AGo). In the tease feeding group, which was allowed to watch and smell, but not to eat food, for 15 min before euthanasia, a reduction was found in ghrelin release after incubation similar to that obtained for the ad libitum feeding group (tease feeding: 2484 ± 505 pg/ml/g of tissue; P < 0.01 vs. fasting). This change, unrelated to food intake, was most probably due to an inhibition of the gastric release, seeing as ghrelin mRNA levels were not modified with respect to the fasting group (Fig. 2CGo). On the other hand, animals subjected to surgical vagotomy did not show any significant difference between tease feeding and fasting, in either plasmatic ghrelin levels or gastric ghrelin secretion. In the tease feeding group, gastric ghrelin levels were 83.1% of values obtained for fasting animals, and plasmatic ghrelin levels were 100% (Fig. 3Go). Identical results were obtained in animals treated with atropine sulfate 30 min before exposure to the tease feeding stimulus: the gastric ghrelin values of secretion were 100.94%, and the plasmatic ghrelin levels were 115.7% of the fasting group.


Figure 3
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  		FIG. 3. A, Plasmatic ghrelin concentration (n = 10) and gastric ghrelin secretion (B) from tissue explants to the incubation medium in rats fasted 36 h without (white bars) or with tease feeding (black bars) in animals subjected to either vagotomy or atropine administration. **, P < 0.01 vs. fast group.

 
As proof that animals complied with the experimental model, plasma glucose was found to lower significantly in both the fasting and tease feeding rats but recovered normal values in the refeeding group (Fig. 4AGo). Insulin levels in plasma were slightly increased in the tease feeding rats with respect to fasting, but in the same way than glucose, increased with refeed to normal values (Fig. 4BGo). Body weight also showed compliance with the fasting protocol (Fig. 4CGo). To control the short-term time dynamic of the plasmatic changes observed, plasma ghrelin was measured at shorter intervals in freely moving cannulated rats (Fig. 5AGo). In the fasting group, plasma ghrelin remained high throughout, while refeeding induced a reduction in ghrelin levels that was evident at 15 and 30 min [620 ± 66 pg/ml fast vs. 222 ± 45 pg/ml control and 529 ± 96 pg/ml refeed at 15 min (**, P < 0.01 vs. control); 573 ± 76 pg/ml at 36 h fasting vs. 250 ± 34 pg/ml control and 441 ± 82 pg/ml in refeed at 30 min (**, P < 0.01 vs. control)]. On the other hand, tease feeding induced a dramatic reduction in ghrelin levels evident at 15 min (468 ± 37 pg/ml tease feeding, NS vs. refeed) and 30 min (450 ± 27 pg/ml tease feeding, NS vs. refeed), but the reduction disappeared at 60 min (45 min: 296 ± 62 pg/ml control, 487 ± 87 pg/ml fast, 317.5 ± 73 pg/ml refeed, and 426 ± 36 pg/ml tease feeding; and 60 min: 312 ± 62 pg/ml control, 481 ± 69 pg/ml fast, 400 ± 60 pg/ml refeed, and 585 ± 103 tease feeding).


Figure 4
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  		FIG. 4. A, Plasma glucose levels in: ad libitum animals (control), fast 36 h, 15 min refeed after fasting, and tease feeding for 15 min after fasting expressed as mean ± SE (n = 10) (**, P < 0.01; *, P < 0.05). B, Plasma insulin levels in: ad libitum animals (control), fast 36 h, 15 min refeed after fasting, and tease feeding for 15 min after fasting expressed as mean ± SE (n = 10) (**, P < 0.01; *, P < 0.05). C, Body weight of experimental animals before and after fasting for 36 h.

 

Figure 5
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  		FIG. 5. A, Plasmatic ghrelin levels (pg/ml) at 15, 30, 45, and 60 min [**, P < 0.01; *, P < 0.05 (n = 10)]. B, Plasma glucose levels (pg/ml) at 15, 30, 45, and 60 min [*, P < 0.05 (n = 10)]. C, Plasma insulin levels (pg/ml) at 15, 30, 45, and 60 min [*, P < 0.05 vs. control groups (n = 10)].

 
It was measured insulin and glucose plasma levels in parallel to ghrelin to assess that the changes observed in plasma ghrelin were not mediated by changes in insulin. It was found that insulin (Fig. 5BGo) and glucose concentration were not affected by tease feeding because the values obtained were similar to that obtained in the fasting group.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To assess the direct regulation of ghrelin secretion by the stomach, an organ culture model of gastric tissue has been standardized in the present study. With this model, it has been proved that the food-mediated changes in plasma ghrelin levels are due to variations in ghrelin release by the stomach. However, there were two findings that were more surprising. First, excised tissues incubated for a total of 3 h, despite no longer being under central nervous system (CNS) control, still maintained the previous CNS conditioning. In fact, 15 min of feeding or even tease feeding, led to a reduction in gastric ghrelin secretion that was maintained 1–3 h after excision of the explant. Second, tease feeding (smell and sight of food, but not intake) was able to reduce gastric secretion of ghrelin in a way similar to true feeding. These findings strongly suggest that the influence of food on ghrelin secretion is partly mediated by nondigestive sensory signals, perhaps involving the vagus nerve. The findings also indicate that the neuronal network of the myenteric plexus is endowed with medium-term memory.

As the first known orexigenic peptide coming from splanchnic tissues, ghrelin has raised considerable interest in the scientific community. A large number of communications have described the variations in ghrelin plasma levels in diverse clinical and experimental situations (1, 7, 12). It is well known that plasma ghrelin increases in states of negative energy balance, like fasting, and that it normalizes after refeeding (10). However, the mechanism of regulation has not been described, leading to the unproved assumption that any changes in plasma ghrelin are a reflection of changes in gastric release (23). A model that allows direct assessment of gastric ghrelin secretion would improve knowledge of ghrelin regulation. In the present study, a model for assessing gastric ghrelin secretion directly from gastric tissue explants was validated, and the optimal incubation period for the gastric explants was found to be 2 h. In states of fasting and refeeding, the observed ghrelin changes reported here in plasma, incubation media, and mRNA unambiguously validate this model for use in further studies.

Fasting induced an increase in both gastric ghrelin secretion and mRNA content, generating an increase in ghrelin plasma levels. The fact that refeeding rapidly reversed those changes demonstrates, for first time, that the often-cited ghrelin changes in plasma under such conditions (24) are a direct reflection of changes in the gastric release of ghrelin. However, the effect of food intake on circulating ghrelin levels was found to involve a more complex mechanism of action than was previously thought. The time-course study of plasmatic ghrelin levels in freely moving rats showed that refeeding produced a strong and time-dependent reversion of the fasting-induced increase in plasma ghrelin levels; at 45 min of refeeding, ghrelin values were identical to those obtained in fed animals. Refeeding for just 15 min was enough to induce a quick blockade of ghrelin secretion from the stomach, but the time of refeeding was too short to affect enhanced ghrelin synthesis. These data suggest that ghrelin secretion directly from the stomach is the first target of ingested food and that inhibitory signals stop the release of previously synthesized ghrelin.

The cephalic phase reflexes are anticipatory changes in the digestion process that allow a more efficient use of food. These complex changes are mostly mediated by enteric hormones whose release is dependent on CNS-generated neural stimuli, more than on nutrient-induced stimulation (25). It has been reported in many species that the cephalic response to sensory stimulations of food produces an increase in insulin, glucagon, and other incretins mediated by vagus nerve action on the pancreas (26). The present study aimed to analyze whether something similar could be controlling gastric ghrelin secretion. Human studies on this kind of anticipatory responses are limited, and some were not able to show clear results because psychological, cognitive, and social attitudes toward food influence individual responses (27). Similarly, studies have been performed in animals to assess the cephalic phase reflexes, but their mechanisms were not elucidated (25). The tease feeding used in this study is a model to study whether ghrelin is secreted as an anticipatory response to an imminent feeding or is activated by food stimuli; the model has been validated in the present work by the measure of insulin levels in which it was found a anticipatory response of insulin to sensory stimulus of food in the tease feeding group (Fig. 4BGo), although this change is not relevant to mediate the effect of this stimulus on plasma ghrelin as it was observed in insulin time-course study (Fig. 5BGo). Surprisingly, it was found that tease feeding and true refeeding produced similar effects on gastric ghrelin secretion and ghrelin plasma levels. The great difference between the two models was that in refeeding, the restored circulating ghrelin levels were maintained through the time, whereas in tease feeding, there was just a transient effect. These changes affected gastric ghrelin secretion but did not alter expression because enhanced ghrelin mRNA was unaltered.

Interestingly, previous studies in sheep with pseudofeeding, in which the animals were provided with food wrapped in a nylon bag that could be swallowed but not digested, reported that the preprandial pulse of circulating ghrelin was reverted (25). This suggests that the regulation of circulating ghrelin is not merely due to absorption of nutrients, although in that model, the mechanical stimuli to esophagus and gastric wall were fully operative. In the tease feeding model presented here, there was no direct mechanical contact between food and esophagus or stomach; the only operative factor was sensorial stimuli acting at the CNS level because the animals could smell and see the food but not swallow, taste, or chew it. Nevertheless, a clear effect of blockade in ghrelin secretion was found. The present study suggests that ghrelin secretion directly from the stomach is not only due to direct mechanical contact with the gastric wall, digestion, or absorption of nutrients. Another relevant factor involved in this process is the CNS sensorial stimuli, which induce the cephalic responses. The implication of vagus nerve mediating the cephalic response of ghrelin to food-related stimuli is supported in the present study by the fact that both vagotomy and cholinergic blockade with atropine sulfate prevented the tease feeding-mediated ghrelin reduction because the values of gastric ghrelin secretion and plasmatic ghrelin levels in this group did not differ from those observed in fasting animals. We propose that ghrelin could be a neurally mediated integrative factor, constituting a link among the sensory qualities of food, neural activation, and nutrient metabolism. Moreover, ghrelin could be considered an incretin hormone.

Gastric tissue excised from the organism maintained the previous secretory status for a period of 3 h after excision, suggesting that the neurons from the enteric system of the gastric tissue have medium-term memory. Previous studies showed that there exists a molecular memory of synaptic activity at the enteric nervous system (27). Electrophysiological studies provide evidence that the prolonged activation of myenteric plexus neurons of the guinea pig contribute to a slowly developing, sustained increase in excitability of the neurons associated with depolarization and that this increased excitability lasted for up to 3.5 h after stimulation (28).

In conclusion, the present study presents a suitable model for studying ghrelin secretion directly from the stomach, which eliminates interference produced by other organs. This new model demonstrated that food intake regulation of plasmatic ghrelin is mediated by gastric tissue because changes in nutritional status first affect ghrelin secretion before expression or circulating levels. For the first time, it has been shown that sensorial stimuli related to food, but without food ingestion, are able to modify gastric ghrelin secretion and circulating ghrelin levels in the same way that true food ingestion does, demonstrating that factors other than those caused by nutrient digestion or absorption are involved in ghrelin regulation. This regulation of ghrelin secretion from the stomach by food-related stimuli is mediated by a medium-term memory mechanism from the system of sensory neurons integrated in the enteric nervous system.


   Acknowledgments
 
We thank Ms. Mary Lage and Dr. Francisco Barreiro for their expert technical assistance.


   Footnotes
 
This research was supported by grants from the FIS (Fondo de Investigación Sanitaria) and the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (CP04/00158, PI 060935, PI 060705, and PI042251), and Xunta de Galicia (PGIDIT05BTF20802PR and PGIDIT06PXIB918360PR) and European Union (LSHM-CT-2003-503041). L.M.S. currently holds research contracts from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 10, 2007

Abbreviations: CNS, Central nervous system; HPRT, hypoxanthine-guanine phosphoribosyltransferase; NS, nonsignificant.

Received February 15, 2007.

Accepted for publication April 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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