Ghrelin and NUCB2/nesfatin-1 are expressed in the same gastric cell and differentially correlated with body mass index in obese subjects

The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55–65 kg/m²), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40–50 kg/m², 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.     

Effects of CP 55,940--agonist of CB1 cannabinoid receptors on ghrelin and somatostatin producing cells in the rat pancreas

Cannabinoids participate in the modulation of numerous functions in the human organism, increasing the sense of hunger, affecting carbohydrate and lipid metabolism, and controlling systemic energy balance mechanisms. Moreover, they influence the endocrine system functions, acting via two types of receptors, CB1 and CB2. The aim of the present study was to examine the number, distribution and activity of ghrelin and somatostatin producing endocrine cells in the pancreas of rats after a single administration of selective CP 55,940 agonist of CB1 receptor. The study was performed on 20 rats. Neuroendocrine cells were identified by immunohistochemical reactions, involving specific antibodies against ghrelin and somatostatin. The distribution and number of ghrelin- and somatostatin-immunoreactive cells were separately studied in five pancreas islets of each section. A performed analysis showed a decreased number of somatostatin-immunoreactive cells and a weak immunoreactivity of ghrelin and somatostatin containing neuroendocrine cells in the pancreatic islets of experimental rats, compared to control animals. The obtained results suggest that a single administration of a selective CP 55,940 agonist of CB1 receptor influences the immunoreactivity of endocrine cells with ghrelin and somatostatin expression in the pancreas islets.     

Immunoreactivity of gastric ECL and A-like cells in fasted and fed rats and mice.

The oxyntic mucosa of rat and mouse stomach harbors histamine-producing ECL cells and ghrelin-producing A-like cells. The ECL cells are known to be active when the circulating gastrin levels are elevated in response to food intake. The A-like cells are the main source of circulating ghrelin. In response to starvation, the circulating ghrelin is elevated as a hunger signal. The aim of the present work was to study the correlation between the immunoreactivities and cellular activities of the ECL cells and A-like cells. Rats were either fed or fasted for 48 h and mice for 24 h. Immunohistochemical examination with antiserum against chromogranin A-derived fragment pancreastatin revealed both the ECL cells and the A-like cells without a difference between fasted and fed animals. Histamine was limited to the ECL cells with no significant difference between fasted and fed animals. Histidine decarboxylase (HDC) immunoreactivity occurred predominately in the ECL cells of the fed, but not fasted, animals in which the HDC enzymatic activity in the oxyntic mucosa was higher than in fasted animals. Ghrelin immunoreactivity was increased in terms of intensity, but not cell density in fasted animals. Thus, the immunoreactivities of ECL cells and A-like cells might be affected by starvation.     

Immunoreactivity of gastric ECL and A-like cells in fasted and fed rats and mice

The oxyntic mucosa of rat and mouse stomach harbors histamine-producing ECL cells and ghrelin-producing A-like cells. The ECL cells are known to be active when the circulating gastrin levels are elevated in response to food intake. The A-like cells are the main source of circulating ghrelin. In response to starvation, the circulating ghrelin is elevated as a hunger signal. The aim of the present work was to study the correlation between the immunoreactivities and cellular activities of the ECL cells and A-like cells. Rats were either fed or fasted for 48 h and mice for 24 h. Immunohistochemical examination with antiserum against chromogranin A-derived fragment pancreastatin revealed both the ECL cells and the A-like cells without a difference between fasted and fed animals. Histamine was limited to the ECL cells with no significant difference between fasted and fed animals. Histidine decarboxylase (HDC) immunoreactivity occurred predominately in the ECL cells of the fed, but not fasted, animals in which the HDC enzymatic activity in the oxyntic mucosa was higher than in fasted animals. Ghrelin immunoreactivity was increased in terms of intensity, but not cell density in fasted animals. Thus, the immunoreactivities of ECL cells and A-like cells might be affected by starvation.     

Somatostatin suppresses ghrelin secretion from the rat stomach

Ghrelin is an acylated peptide that stimulates food intake and the secretion of growth hormone. While ghrelin is predominantly synthesized in a subset of endocrine cells in the oxyntic gland of the human and rat stomach, the mechanism regulating ghrelin secretion remains unknown. Somatostatin, a peptide produced in the gastric oxyntic mucosa, is known to suppress secretion of several gastrointestinal peptides in a paracrine fashion. By double immunohistochemistry, we demonstrated that somatostatin-immunoreactive cells contact ghrelin-immunoreactive cells. A single intravenous injection of somatostatin reduced the systemic plasma concentration of ghrelin in rats. Continuous infusion of somatostatin into the gastric artery of the vascularly perfused rat stomach suppressed ghrelin secretion in both dose- and time-dependent manner. These findings indicate that ghrelin secretion from the stomach is regulated by gastric somatostatin.     

Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues

Ghrelin is a new gastric peptide involved in food intake control and growth hormone release. We aimed to assess its cell localisation in man during adult and fetal life and to clarify present interspecies inconsistencies of gastric endocrine cell types. A specific serum generated against amino acids 13-28 of ghrelin was tested on fetal and adult gastric mucosa and compared with ghrelin in situ hybridisation. Immunogold electron microscopy was performed on normal human, rat and dog adult stomach. Ghrelin cells were detected in developing gut, pancreas and lung from gestational week 10 and in adult human, rat and dog gastric mucosa. By immunogold electron microscopy, gastric ghrelin cells showed distinctive morphology and hormone reactivity in respect to histamine enterochromaffin-like, somatostatin D, glucagon A or serotonin enterochromaffin cells. Ghrelin cells were characterised by round, compact, electron-dense secretory granules of P/D(1) type in man (mean diameter 147+/-30 nm), A-like type in the rat (183+/-37 nm) and X type in the dog (273+/-49 nm). It is concluded that, ghrelin is produced by well-defined cell types, which in the past had been labelled differently in various mammals mostly because of the different size of their secretory granule. In man ghrelin cells develop during early fetal life.     

Synergistic action of gastrin and ghrelin on gastric acid secretion in rats

Gastrin and ghrelin are secreted from G cells and X/A-like cells in the stomach, respectively, and respective hormones stimulate gastric acid secretion by acting through histamine and the vagus nerve. In this study, we examined the relationship between gastrin, ghrelin and gastric acid secretion in rats. Intravenous (iv) administration of 3 and 10 nmol of gastrin induced transient increases of ghrelin levels within 10 min in a dose-dependent manner. Double immunostaining for ghrelin and gastrin receptor revealed that a proportion of ghrelin cells possess gastrin receptors. Although (iv) administration of gastrin or ghrelin induced significant gastric acid secretion, simultaneous treatment with both hormones resulted in a synergistic, rather than additive, increase of gastric acid secretion. This synergistic increase was not observed in vagotomized rats.These results suggest that gastrin may directly stimulate ghrelin release from the stomach, and that both hormones may increase gastric acid secretion synergistically.     

Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. We have morphologically characterized the cells that produce obestatin and ghrelin in new-born and adult Sprague-Dawley rats that were freely fed, fasted, or subjected to gastric bypass surgery or reserpine treatment. Tissue samples collected from the gastrointestinal tract and pancreas were examined by double-immunofluorescence staining, immunoelectron microscopy, and conventional electron microscopy. Obestatin was present in the stomach, duodenum, jejunum, colon, and pancreas. In the stomach, differences were noted in the development of obestatin- and preproghrelin-immunreactive (IR) cells on the one hand and ghrelin-IR cells on the other, particularly 2 weeks after birth. Preproghrelin- and obestatin-IR cells were more numerous than ghrelin-IR cells in the stomach, suggesting the lack of ghrelin in some A-like cells. Most obestatin-producing cells in the stomach were distributed in the basal part of the oxyntic mucosa; these cells co-localized with chromogranin A (pancreastatin) and vesicle monoamine transporters type 1 and 2, but not with serotonin or histidine decarboxylase. Immunoelectron microscopy revealed the obestatin- and ghrelin-producing cells to be A-like cells, characterized by numerous highly electron-dense granules containing ghrelin and obestatin. Some granules exhibited an even electron density with thin electron-lucent halos, suggestive of monoamines. Feeding status, gastric bypass surgery, and reserpine treatment had no obvious effect on the A-like cells. In the pancreas, obestatin was present in the peripheral part of the islets, with a distribution distinct from that of glucagon-producing A cells, insulin-producing beta cells, and cells producing pancreatic polypeptide Y. Thus, obestatin and ghrelin co-localize with an anticipated monoamine in A-like cells in the stomach, and obestatin is found in pancreatic islets. This study was supported by a grant from the Cancer Foundation of St. Olav’s Hospital, Trondheim, Norway.     

Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2

Clinical studies are evaluating the efficacy of synthetic ghrelin agonists in postoperative ileus management. However, the control of ghrelin secretion under conditions of postoperative gastric ileus is largely unknown. Peripheral somatostatin inhibits ghrelin secretion in animals and humans. We investigated the time course of ghrelin changes postsurgery in fasted rats and whether somatostatin receptor subtype 2 (sst(2)) signaling is involved. Abdominal surgery (laparotomy and 1-min cecal palpation) induced a rapid and long-lasting decrease in plasma acyl ghrelin levels as shown by the 64, 67, and 59% reduction at 0.5, 2, and 5 h postsurgery, respectively, compared with sham (anesthesia alone for 10 min, P < 0.05). Levels were partly recovered at 7 h and fully restored at 24 h. The percentage of acyl ghrelin reduction was significantly higher than that of desacyl ghrelin at 2 h postsurgery and not at any other time point. This was associated with a 48 and 23% decrease in gastric and plasma ghrelin-O-acyltransferase protein concentrations, respectively (P < 0.001). Ghrelin-positive cells in the oxyntic mucosa expressed sst(2a) receptor and the sst(2) agonist S-346-011 inhibited fasting acyl ghrelin levels by 64 and 77% at 0.5 and 2 h, respectively. The sst(2) antagonist S-406-028 prevented the abdominal surgery-induced decreased circulating acyl ghrelin but not the delayed gastric emptying assessed 0.5 h postinjection. These data show that activation of sst(2) receptor located on gastric X/A-like cells plays a key role in the rapid inhibition of circulating acyl ghrelin induced by abdominal surgery while not being primarily involved in the early phase of postoperative gastric ileus.     

The Gastric CB1 Receptor Modulates Ghrelin Production through the mTOR Pathway to Regulate Food Intake

Over the years, the knowledge regarding the relevance of the cannabinoid system to the regulation of metabolism has grown steadily. A central interaction between the cannabinoid system and ghrelin has been suggested to regulate food intake. Although the stomach is the main source of ghrelin and CB1 receptor expression in the stomach has been described, little information is available regarding the possible interaction between the gastric cannabinoid and ghrelin systems in the integrated control of energy homeostasis. The main objective of the present work was to assess the functional interaction between these two systems in terms of food intake using a combination of in vivo and in vitro approaches. The present work demonstrates that the peripheral blockade of the CB1 receptor by rimonabant treatment decreased food intake but only in food-deprived animals. This anorexigenic effect is likely a consequence of decreases in gastric ghrelin secretion induced by the activation of the mTOR/S6K1 intracellular pathway in the stomach following treatment with rimonabant. In support of this supposition, animals in which the mTOR/S6K1 intracellular pathway was blocked by chronic rapamycin treatment, rimonabant had no effect on ghrelin secretion. Vagal communication may also be involved because rimonabant treatment was no longer effective when administered to animals that had undergone surgical vagotomy. In conclusion, to the best of our knowledge, the present work is the first to describe a CB1 receptor-mediated mechanism that influences gastric ghrelin secretion and food intake through the mTOR pathway.     

A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control

Ghrelin is a 28 a.a. gastric peptide, recently identified as a natural ligand of the growth hormone secretagogue receptor (orphan receptor distinct from the receptor for growth hormone releasing hormone). In the present study, radioimmunoassay demonstrated ghrelin-like material in the rat oxyntic mucosa with moderate amounts also in antrum and duodenum. Small amounts were found in the distal intestines and pancreas. Northern blot analysis revealed abundant ghrelin mRNA in the oxyntic mucosa. Immunocytochemistry demonstrated ghrelin-immunoreactivity in endocrine-like cells in the oxyntic mucosa. Such cells occurred in low numbers also in the antrum and duodenum. The rat oxyntic mucosa is rich in endocrine (chromogranin A/pancreastatin-immunoreactive) cells, such as the histamine-rich ECL cells (65-75% of the endocrine cells), the A-like cells (20-25%) and the D cells (somatostatin cells) (10%). The ghrelin-immunoreactive (IR) cells contained pancreastatin but differed from ECL cells and D cells by being devoid of histamine-forming enzyme (ECL cell constituent) and somatostatin (D cell constituent). Hence, ghrelin seems to occur in the A-like cells. The ghrelin-IR cells in the antrum were distinct from the gastrin cells, the serotonin-containing enterochromaffin cells and the D cells. Conceivably, ghrelin cells in the antrum and distally in the intestines also belong to the A-like cell population. The concentration of ghrelin in the circulation was lowered by about 80% following the surgical removal of the acid-producing part of the stomach in line with the view that the oxyntic mucosa is the major source of ghrelin. The serum ghrelin concentration was higher in fasted rats than in fed rats; it was reduced upon re-feeding and seemed unaffected by 1-week treatment with the proton pump inhibitor omeprazole, resulting in elevated serum gastrin concentration. Infusion of gastrin-17 for 2 days failed to raise the serum ghrelin concentration. Omeprazole treatment for 10 weeks raised the level of HDC mRNA but not that of ghrelin mRNA or somatostatin mRNA in the oxyntic mucosa. Hence, unlike the ECL cells, ghrelin-containing A-like cells do not seem to operate under gastrin control.     

Role of gastrin in the development of gastric mucosa,ECL cells and A-like cells in newborn and young rats

Histamine-producing ECL cells and ghrelin-producing A-like cells are endocrine/paracrine cell populations in the acid-producing part of the rat stomach. While the A-like cells operate independently of gastrin, the ECL cells respond to gastrin with mobilization of histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. Gastrin is often assumed to be the driving force behind the postnatal development of the gastric mucosa in general and the ECL cells in particular. We tested this assumption by examining the oxyntic mucosa (with ECL cells and A-like cells) in developing rats under the influence of YF476, a cholecystokinin-2 (CCK(2)) receptor antagonist. The drug was administered by weekly subcutaneous injections starting at birth. The body weight gain was not affected. Weaning occurred at days 15-22 in both YF476-treated and age-matched control rats. Circulating gastrin was low at birth and reached adult levels 2 weeks after birth. During and after weaning (but not before), YF476 greatly raised the serum gastrin concentration (because of abolished acid feedback inhibition of gastrin release). The weight of the stomach was unaffected by YF476 during the first 2-3 weeks after birth. From 4 to 5 weeks of age, the weight and thickness of the gastric mucosa were lower in YF476-treated rats than in controls. Pancreastatin-immunoreactive cells (i.e. all endocrine cells in the stomach) and ghrelin-immunoreactive cells (A-like cells) were few at birth and increased gradually in number until 6-8 weeks of age (control rats). At first, YF476 did not affect the development of the pancreastatin-immunoreactive cells, but a few weeks after weaning, the cells were fewer in the YF476 rats. The ECL-cell parameters (oxyntic mucosal histamine and pancreastatin concentrations, the histidine decarboxylase (HDC) activity, the HDC mRNA levels and serum pancreastatin concentration) increased slowly until weaning in both YF476-treated and control rats. From then on, there was a further increase in the ECL-cell parameters in control rats but not in YF476 rats. The postnatal development of the ghrelin cells (i.e. the A-like cells) and of the A-like cell parameters (the oxyntic mucosal ghrelin concentration and the serum ghrelin concentrations) was not affected by YF476 at any point.We conclude that gastrin affects neither the oxyntic mucosa nor the endocrine cells before weaning. After weaning, CCK(2) receptor blockade is associated with a somewhat impaired development of the oxyntic mucosa and the ECL cells. While gastrin stimulation is of crucial importance for the onset of acid secretion during weaning and for the activation of ECL-cell histamine formation and secretion, the mucosal and ECL-cell growth at this stage is only partly gastrin-dependent. In contrast, the development of the A-like cells is independent of gastrin at all stages.     

Cannabinoids stimulate prostaglandin production by human gestational tissues through a tissue- and CB1-receptor-specific mechanism

Endocannabinoids have been implicated in the mechanisms of implantation, maintenance of pregnancy, and parturition in women. Intrauterine prostaglandin production and actions are also critical in each of these mechanisms. Hence, we have evaluated the effects of cannabinoids on prostaglandin biosynthesis by human gestational membranes. Explants of term amnion and choriodecidua were established and treated with the endogenous endocannabinoids 2-arachidonoyl glycerol and anandamide, as well as the synthetic cannabinoid CP55,940, to determine their ability to modulate PGE(2) production. The explants were also treated with CP55,940 in the presence of either SR141716A (a potent and selective antagonist of the cannabinoid receptor CB1) or NS398 [a cyclooxygenase (COX)-2 inhibitor] to determine whether any observed stimulation of PGE(2) production was mediated through the CB1-receptor and/or COX-2 activity. All three cannabinoids caused a significant increase in PGE(2) production in the amnion but not in the choriodecidua. However, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. The enhanced PGE(2) production caused by CP55,940 was abrogated by cotreatment with either SR141716A or NS398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by CB1 agonism and COX-2. Data from Western blotting show that cannabinoid treatment results in the upregulation of COX-2 expression. This study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labor and membrane rupture.     

Expression in Escherichia coli and characterisation of the human central CB1 and peripheral CB2 cannabinoid receptors

The human central CB1 and peripheral CB2 cannabinoid receptors were expressed in Escherichia coli as fusion proteins with the maltose-binding protein at their amino-termini and a hexa-histidine/Flag tag at their carboxyl-termini. Western blot analysis of the expressed proteins revealed considerable degradation of the CB1 fusion, which failed to bind either the cannabinoid agonist CP 55,940 or the CB1-specific antagonist SR 141716A. In contrast, the CB2 fusion was well-expressed and bound several cannabinoids with affinities comparable to those observed in mammalian expression systems. This revised version was published online in November 2006 with corrections to the Cover Date.     

Enhanced ghrelin secretion in rats with cysteamine-induced duodenal ulcers

Ghrelin, produced and secreted by the A-like cells of the stomach, stimulates growth hormone secretion, gastric motility, and food intake. Cysteamine inhibits the release of somatostatin and induces the formation of duodenal ulcers in rats. The present study was conducted to investigate the dynamics of ghrelin secretion in rats treated with cysteamine. Male Wistar rats (7 wk old) were administered three doses of cysteamine (400 mg/kg) orally; at 50 h after the first dose, duodenal ulcers were induced, and the plasma level of somatostatin and gastric density of somatostatin-immunoreactive cells were significantly reduced. The plasma total and active ghrelin levels were significantly higher in the cysteamine-treated rats than in the control rats, whereas the gastric ghrelin levels, number of gastric ghrelin-immunoreactive cells, and preproghrelin mRNA expression levels were significantly lower. Even at the time points of 2 and 10 h after the first dose of cysteamine, at which time no significant ulcer formation or antral neutrophil accumulation was yet noted, a significant increase in the plasma ghrelin level and decrease in the gastric ghrelin level were observed. Furthermore, although lansoprazole treatment attenuated the duodenal ulceration induced by cysteamine, the increase in the plasma level of ghrelin could still be demonstrated. Because an inverse correlation was found between the plasma ghrelin and somatostatin levels, the inhibition of somatostatin secretion may be associated with the increased ghrelin secretion. In conclusion, an increase in the plasma ghrelin level precedes the formation of duodenal ulcers in rats treated with cysteamine.     

Ghrelin expression in gut endocrine growths

We aimed to assess the occurrence of ghrelin, a new gut hormone, in endocrine growths of the stomach. In addition, since ghrelin has been detected in other gut derivatives during adult and/or fetal life, we also studied endocrine tumours of the pancreas, intestine and lung. A specific serum generated against amino acids 13-28 of ghrelin was tested on 16 specimens of gastric mucosa with endocrine cell hyperplasia and on 75 endocrine tumours. Ghrelin-immunoreactive cells were moderately represented in normal, atrophic or hypertrophic gastric mucosa, as a rule with no obvious hyperplastic changes even in the presence of concurrent, prominent enterochromaffin-like cell hyperplasia associated with hypergastrinemia. Ghrelin cells were also found in tumour cell fractions of well-differentiated gastric (25 of 33, 76%), pancreatic (6 of 15, 40%) and pulmonary (4 of 8) endocrine tumours. No ghrelin immunoreactivity was detected in 14 intestinal tumours and in five poorly differentiated endocrine carcinomas of the stomach or pancreas. We conclude that ghrelin cells may take part in gut endocrine growths, with special reference to well-differentiated endocrine tumours of the stomach, independently from associated signs of endocrine hyperfunction.     

The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system

Introduction

Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction.

Methods

The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors.

Results and Conclusions

Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.     

A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.     

Topically administered CB(2)-receptor agonist,JWH-133, does not decrease intraocular pressure (IOP) in normotensive rabbits

Recent IOP and receptor localization studies suggest that the IOP effects of cannabinoids are mediated via ocular CB(1) receptors. However, it is not yet known whether CB(2) receptor agonists decrease IOP. In this study, the IOP-lowering effects of the CB(2) receptor agonist JWH-133 were studied in normotensive rabbits, and compared with CP55,940. JWH-133 and CP55,940 were dissolved in aqueous hydroxypropyl-beta-cyclodextrin (HP-beta-CD) solutions and propylene glycol. The eye drops (25 microl) were administered unilaterally to the rabbit eye, and IOPs were measured at fixed time intervals. JWH-133, dissolved in either HP-beta-CD (doses = 10 microg and 25 microg) or propylene glycol (dose = 62.5 microg), did not have any effect on IOP when compared to vehicle treatments. In contrast, CP55,940 formulated in HP-beta-CD (doses = 25 microg and 62.5 microg) or propylene glycol (dose = 62.5 microg) reduced IOP significantly compared to vehicle treatments. The results suggest that topically administered CB(2) receptor agonist, JWH-133, does not decrease IOP in normotensive rabbits at the doses and formulations used, and thus, CB(2) receptor agonists may not be useful as cannabinoid-based IOP-lowering therapeutics.     

Dual Neuroprotective and Neurotoxic Effects of Cannabinoid Drugs in Vitro

Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.