Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk.

BACKGROUND AND PURPOSE: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.

Feeding behavior during long-term hexarelin administration in young and old rats.

Ghrelin, a 28-amino-acid peptide isolated from the stomach, is the natural ligand of the GH-secretagogues receptor-1a (GHS-R1a) and, so far, the only discovered circulating appetite-stimulating hormone. Similarly to ghrelin, many synthetic compounds belonging to the GHS family stimulate both GH secretion and feeding, whereas some stimulate GH secretion only. In the past years, studies have focused on the potential of the GHS to stimulate GH release during long-term treatment in humans and experimental animals. Few data are available about the extraendocrine effects of the GHS during several weeks of treatment, particularly in old rats. The aim of the present study was first to identify the lowest dose of hexarelin giving maximal stimulation of food intake both in young (3-month-old) and old rats (24-month-old). A dose-response study (80-320 microg/kg, s.c.) revealed that hexarelin at the dose of 80 microg/kg gave reproducibly maximal stimulation of food consumption in young as well as in old rats. Second, we evaluated the effect of 8-week daily sc treatment with hexarelin in young and old male rats. The outcome of the chronic study was that hexarelin (80 microg/kg, s.c., once daily) maintained a persistent significant orexigenic action throughout the treatment period, both in young and old rats. Interestingly, hexarelin treatment did not affect body weight gain either in young or old rats. We conclude that hexarelin is endowed with long-lasting orexigenic activity and might represent a potential therapeutic approach for pathological conditions characterized by a decline in food intake.

Ghrelin control of GH secretion and feeding behaviour: the role of the GHS-R1a receptor studied in vivo and in vitro using novel non-peptide ligands.

Energy homeostasis is controlled by a complex regulatory system of molecules that affect food intake and that are critical for maintaining a stable body weight during life. Ghrelin is a peptide of 28 amino acid synthesized predominantly by the stomach and the gut, which activate the type 1a growth hormone (GH) secretagogue receptor (GHS-R1a), a G-protein coupled receptor. The acylated form of ghrelin potently stimulates GH secretion both in vitro and in vivo in several animal species, including humans. Beside the endocrine effect, ghrelin shows also extraendocrine activities, including stimulation of feeding behaviour. Several classes of small synthetic peptide and non-peptide ligands of the GHS-R1a have been described and are able to release GH and stimulate food intake. However, in time, it appeared that the stimulating effects on GH secretion could be divorced from those on food intake, suggesting that more than a single receptor might be involved. Several experimental data have even questioned the physiological role of ghrelin in the control of GH secretion and energy metabolism. By using novel agonists, partial agonists, and antagonists for the GHS-R1a receptor, we have studied whether the stimulation of this receptor could account for the purported physiological role of ghrelin. Our results demonstrate that the ability to bind in vitro the GHS-R1a is not predictive of the in vivo biological activity of the compounds and that the endocrine and extraendocrine effects could be mediated also by receptors different from the GHS-R1a.

Chronic intracerebroventricular TLQP-21 delivery does not modulate the GH/IGF-1-axis and muscle strength in mice.

OBJECTIVE: Biallelic ablation of VGF determines a dwarf phenotype. VGF precursor protein encodes for different biologically active peptides none of which has been related to growth or muscular abnormalities. Here we present the first attempt to fill this gap. We tested the hypothesis that a recently identified VGF-derived peptide, TLQP-21, shown to centrally modulate metabolic functions, could also modulate growth hormone (GH)-axis and muscle strength. DESIGN: Adult male mice were chronically icv injected with TLQP-21 (15 microg/day for 14 days). Physiological, molecular and behavioral parameters related to the GH/IGF-1-axis were investigated. RESULTS: Except for a reduction in the soleus weight, TLQP-21 did not affect GH/IGF-1-axis mediators, muscle strength and muscle weight. CONCLUSIONS: Results collected exclude a role for TLQP-21 in modulating the GH/IGF1-axis and muscle functions. VGF-derived peptides involved in the dwarf phenotype of VGF-/- mice have to be identified yet.

Central dysregulations in the control of energy homeostasis and endocrine alterations in anorexia and bulimia nervosa.

In the last decades we have come to understand that the hypothalamus is a key region in controlling energy homeostasis. A number of control models have been proposed to explain the regulation of feeding behavior in physiological and pathological conditions, but all those based on imbalances of single factors fail to explain the disrupted regulation of energy supply in eating disorders such as anorexia nervosa and bulimia nervosa, as well as other psychiatric disorders. A growing amount of evidence demonstrates that many signaling molecules originated within the brain or coming from the adipose tissue or the gastro-enteric tract are involved in the highly complex process controlling food intake and energy expenditure. The recent discovery of leptin, ghrelin, and other factors have made it possible to penetrate in the still undefined pathophysiology of eating disorders with the hope of finding effective treatments for such diseases.

Obestatin inhibits feeding but does not modulate GH and corticosterone secretion in the rat.

Obestatin is a recently discovered 23 amino acids peptide derived from the ghrelin gene. As opposed to ghrelin, obestatin was shown to inhibit food intake in mice. The aims of this research were to study the effects of acute obestatin treatment on feeding behavior in the rat and its effects on GH and corticosterone secretion. Our results demonstrate that in young-adult male rats, obestatin effectively blunts the hunger caused by short-term starvation. Obestatin did not modify GH secretion in 10-day-old rats and did not antagonize the GH-releasing effects of hexarelin. Moreover, obestatin administration had no effects on spontaneous corticosterone secretion. In conclusion, these data demonstrate that in young-adult male rats the newly discovered obestatin can inhibit feeding but does not modify GH and corticosterone release in infant rats.

Effects of hexarelin against acid-independent and acid-dependent ulcerogens in the rat.

The effects of intracerebroventricular (icv) or subcutaneous (sc) hexarelin (Hexa) administration, against gastric ulcers induced by ethanol (50%, 1 ml/rat/os) or Indomethacin (20 mg/kg/os) were examined in conscious rats. Hexa at 1 nmol/rat, icv or 10 nmol/kg, sc reduced ethanol-induced ulcers by 47% and 32% respectively. Hexa, but not ghrelin significantly worsened (+40%) Indomethacin-induced ulcers when injected sc. Hexa-gastroprotection against ethanol-induced ulcers was removed by the GHS-R antagonist (D-Lys3)-GRPR-6 and by the inhibitor of NO-synthase (NOS) Nomega-nitro-L-arginine methyl ester. Semiquantitative RT-PCR assay of gastric NOS mRNA isoforms revealed that the reduction in iNOS-derived NO and the increase of constitutive-derived NO are relevant for the gastroprotection of Hexa against ethanol-induced gastric damage.

In vitro and in vivo pharmacological role of TLQP-21, a VGF-derived peptide, in the regulation of rat gastric motor functions.

BACKGROUND AND PURPOSE: Vgf gene expression has been detected in various endocrine and neuronal cells in the gastrointestinal tract. In this study we investigated the pharmacological activity of different VGF-derived peptides. Among these, TLQP-21, corresponding to the 556-576 fragment of the protein was the unique active peptide, and its pharmacological profile was further studied. EXPERIMENTAL APPROACH: The effects of TLQP-21 were examined in vitro by smooth muscle contraction in isolated preparations from the rat gastrointestinal tract and, in vivo, by assessing gastric emptying in rats. Rat stomach tissues were also processed for immunohistochemical and biochemical characterization. KEY RESULTS: In rat longitudinal forestomach strips, TLQP-21 (100 nmol x L(-1)-10 micromol x L(-1)) concentration-dependently induced muscle contraction (in female rats, EC(50) = 0.47 micromol.L(-1), E(max): 85.7 +/- 7.9 and in male rats, 0.87 micromol x L(-1), E(max): 33.4 +/- 5.3; n = 8), by release of prostaglandin (PG)E(2) and PGF(2a) from the mucosal layer. This effect was significantly antagonized by indomethacin and selective inhibitors of either cyclooxygenase-1 (S560) or cyclooxygenase-2 (NS398). Immunostaining and biochemical studies confirmed the presence of VGF in the gastric neuronal cells. TLQP-21, injected i.c.v. (2-32 nmol per rat), significantly decreased gastric emptying by about 40%. This effect was significantly (P < 0.05) blocked by i.c.v. injection of indomethacin, suggesting that, also in vivo, this peptide acts in the brain stimulating PG release. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that this VGF-derived peptide plays a central and local role in the regulation of rat gastric motor functions.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity.

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography-tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 mug/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue beta2-AR (beta2 adrenergic receptor) and white adipose tissue (WAT) PPAR-delta (peroxisome proliferator-activated receptor delta), beta3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Ghrelin in gastroenteric pathophysiology.

Ghrelin, an acylated peptide produced predominantly by the stomach, has been discovered to be a natural ligand of the growth hormone secretagogue receptor 1a (GHS-R1a). It is localized in distinct cells of the gastric mucosa, mainly distributed in the mid portion of the oxyntic gland characterized by P/D1 granules in man and X/A-like granules in rodents. The ghrelin cell represents the second most frequent endocrine cell type after the enterochromaffin-like cells in gastric oxyntic mucosa, pointing to a potentially relevant role in the physiology of the stomach. Ghrelin has no relevant homology with any known gastrointestinal peptide and displays strong GH-releasing activity both in animals and in humans. However, in addition to stimulating GH secretion, ghrelin possesses several other endocrine and extraendocrine biological activities that are explained by the widespread distribution of ghrelin and GHS-R1a expression. In the rat, ghrelin exerts a control in gastric acid secretion and motility: the gastric acid secretion is stimulated by peripheral administration of high doses of ghrelin, but inhibited by very low doses of ghrelin delivered into the central nervous system. Moreover, ghrelin provides a potent and dose-related gastroprotective action against ethanol- and stress-induced gastric ulcers. The integrity of both nitric oxide (NO) system and capsaicin afferent nerves are required for the gastroprotective effect of ghrelin, whereas the vagus nerve might be involved in conveying ghrelinergic signal from periphery to the brain. In addition, prostaglandins derived by the constitutive cyclooxygenase (COX) activity are essential for the protective activity of ghrelin in ethanol and stress-induced gastric lesions. Given its prevailing role in physiological and pathophysiological gastric function, the discovery of ghrelin will open new perspectives and potential clinical implications in the gastroenteric field.