Leading article

M. KAPICA1, I. PUZIO1, I. KATO2, A. KUWAHARA3, R. ZABIELSKI4


ROLE OF FEED-REGULATING PEPTIDES
ON PANCREATIC EXOCRINE SECRETION



1Department of Biochemistry and Animal Physiology, University of Life Sciences in Lublin, Poland, 2Department of Bioorganic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, 3Ho, Kanagawa-machi Kanazawa, Japan, 3Laboratory of Physiology, University of Shizuoka, Shizuoka, Japan, 4Department of Physiological Sciences, Warsaw University of Life Sciences, Warsaw, Poland


  In recent two decades a group of feed intake-regulating peptides (i.e., leptin, apelin, ghrelin, obestatin and orexins) have been discovered. Besides the central nervous system these regulatory peptides are produced and released by the gastrointestinal (GI) endocrine cells and neurons, and functional receptors were found in the GI tract and the pancreas. High expression of feed intake-regulating peptides was found in the stomach; however, they may be expressed in other GI tissues too. The peptides control gastrointestinal functions, modulate orexigenic drive and energy metabolism via different mechanisms. Basal leptin, apelin, ghrelin and obestatin plasma concentrations correlated with BMI, and we observed significant reduction of ghrelin and leptin concentrations following fundectomy in rats. We have shown previously that exogenous leptin and ghrelin (a peptide derived from the same preprohormone as obestatin) inhibit the secretion of rat pancreatic juice through a neurohormonal mechanism. Intravenous obestatin was found to stimulate pancreatic protein output in anaesthetized rat via a CCK-vagal-dependent mechanism, whilst a direct action of obestatin on rat pancreatic acini in vitro resulted in opposite effect. Intravenous boluses of apelin reduced the juice volume, protein and trypsin outputs in a dose-dependent manner. However, apelin administered into the duodenal lumen significantly increased pancreatic protein and trypsin outputs through a vagal mechanism. Orexin A and B were found to stimulate insulin release, though on the rat exocrine pancreas orexin A had no effect, and the effect of orexin B was weak. Concluding, feed intake-regulating peptides participate in controlling the exocrine pancreas.

Key words: apelin, ghrelin, leptin, obestatin, orexins



INTRODUCTION

The regulation of food intake is complex. It requires discrete nuclei within the central nervous system (CNS) to detect signals from the periphery regarding metabolic status, process and integrate this information in a coordinated manner and to provide appropriate responses to ensure that the organism does not enter a state of positive or negative energy balance. Factors controlling appetite (initiation, frequency and termination of food ingestion) and the energy intake influence also the overall digestive function of the gastrointestinal (GI) system involving the pancreas. This seems to be a reasonable way to integrate the overall human and animal activity with food intake and the effectiveness of enzymatic degradation by pancreatic juice. Moreover, the energy costs of pancreatic juice enzyme synthesis and electrolyte secretion are high. A number of regulatory peptides and proteins is released from the GI tract in response to changes in the nutritional state to influence the central mechanisms involved in regulation of appetite, satiety and energy balance through a range of blood, local paracrine, gastrointestinal luminal, and neural pathways. In this paper we will briefly review some of them (leptin, ghrelin, obestatin, apelin and orexin-A and -B) which have been discovered in the past two decades as central and peripheral factors controlling food intake, energy homeostasis and overall body activity, and more recently also for controlling the exocrine pancreas.


LEPTIN

Leptin was discovered by the team of Friedmann as a 167 amino acid hormone secreted by adipose tissue into the blood thereby controlling (inhibiting) food intake and energy expenditure (1). A few years later, leptin was considered also as an gastrointestinal hormone, since leptin and leptin receptors were found in the gastrointestinal system. Important production of leptin in the stomach, estimated by us to be a 25% of total leptin in rats (2), was localized to chief cells in the fundic region (3). In humans, plasma leptin peaks 1-2 after food intake, and significant reduction below the control level is observed after 24-fasting (4). Fully functional leptin receptors (Ob-Rb) were demonstrated in the stomach (5, 6), small intestine (7, 8) as well as in the pancreas tissues. In the pancreas, leptin receptors were found on the b-cells (9) and on the acinar cells (10, 11). First available data on leptin effect on the exocrine pancreas in vivo were confusing showing either inhibition (12) or stimulation (13). More recently we have found an explanation for these conflicting results. Matyjek and co-workers (14) demonstrated that in anaesthetized rats intravenous leptin boluses reduce pancreatic secretion (mostly protein output and pancreatic enzyme activities) by inhibiting neurohormonal cholecystokinin (CCK)-vagal-dependent mechanism which controls the exocrine pancreas. On the other hand, leptin boluses administered into the duodenal lumen significantly stimulated pancreatic protein secretion presumably through the release of endogenous CCK (2). This result is in agreement with earlier observations by Brzozowski et al. (15) who demonstrated that exogenous leptin may increase the concentration of circulating CCK in the blood plasma and gastrointestinal lumen, and Yuan et al. (16) who found that leptin might modulate the potency of CCK signals which are responsible for stimulation of vagal afferent fibers. In acinar pancreatic cell in vitro preparation, Matyjek et al. (14) observed a tendency towards a slight reduction of amylase release following leptin administration thought it did not reach statistical significance. This suggests marginal, if any, direct influence of leptin on the exocrine pancreas. Earlier studies demonstrated also significant inhibition of insulin production and release by leptin (17, 18, 19, 20). Therefore inhibition of insulo-acinar axis function may be another explanation of leptin mechanisms in controlling the exocrine pancreas. Concluding, it seems that leptin was the first appetite regulator with established role in controlling the exocrine pancreas function. However, the direct physiological role on the exocrine pancreas is rather doubtful.


GHRELIN

Circulating ghrelin is produced predominantly by the gastric mucosa (21). Accordingly, Matyjek et al. (2) demonstrated that in rats two months after fundectomy, the concentration of plasma ghrelin was as low as 0.25 ng/ml, while in non-operated rats it was ca. 7.7 ng/ml. Substantially lower than in the stomach amounts of ghrelin were detected in the intestine and pancreas, and behind the GI tract in the kidney, immune system, placenta, testis, pituitary, lung, and hypothalamus (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Ghrelin was shown to stimulate feed intake when injected either centrally (35, 36, 37, 38, ) or peripherally (36, 39). In humans, significant elevations in plasma ghrelin were observed just before meals, suggesting its role as a hunger signal (40,41).

Ghrelin and ghrelin receptors (GHS-R) were found in pancreatic islets (28, 29, 33, 42, 43, 44, 45, 46). Low plasma ghrelin levels were associated with elevated fasting insulin levels and insulin resistance in humans (47; 48). These findings suggest an important role for ghrelin in the regulation of insulin release. In addition, plasma ghrelin level correlated inversely with obesity (49; 50, 51). Hence, ghrelin could be involved in energy and glucose metabolism, in which insulin plays a crucial role. It has been found that endogenous ghrelin inhibits glucose-induced insulin release via GHS-R1a, as demonstrated by the marked increase in the insulin response to glucose after blockade of endogenous ghrelin (43). Moreover, administration of exogenous ghrelin suppresses further insulin secretion in glucose-stimulated insulin secretion (52, 53). Release of ghrelin from pancreatic islets was assessed by comparing ghrelin level in the pancreatic vein (splenic vein) with that in the pancreatic artery (celiac artery) in anaesthetized rats. The concentrations of both acylated-ghrelin and desacyl-ghrelin in the pancreatic vein were significantly higher than those in the pancreatic artery indicating that ghrelin is produced by the pancreas (54). Depending on dose and experimental conditions, ghrelin has been shown to either inhibit or stimulate insulin secretion in experimented animals (55, 56, 57, 28). Ghrelin has been reported to stimulate insulin secretion from isolated rat pancreatic islets (55, 28) and also in rats in vivo (57). Conversely, ghrelin has been reported able to blunt insulin secretion from isolated rat pancreas after stimulation with glucose, arginine, and carbachol (55). Systemic action of exogenous ghrelin to elevate blood glucose levels has been well documented in humans and rodents (58, 59, 54). In mice fasted overnight, intraperitoneal administration of ghrelin at concentrations of 1 and 10 nmol/kg significantly elevated blood glucose levels at 30 min after administration (54). Contribution of ghrelin from the stomach and other sources was assessed using gastrectomized (GX) rats lacking stomach-derived ghrelin. In GX rats, plasma concentrations of acylated-ghrelin were markedly reduced to 16% of control (5.2±0.7 fmol/ml in GX rats vs. 32.5±9.7 fmol/ml in normal rats), indicative of lack of stomach derived ghrelin. The remaining levels of acylated-ghrelin may be derived substantially from the intestine, the second largest source of ghrelin (60, 29). Although the circulating acylated-ghrelin was dramatically reduced in GX rats, i.p. injection of GHS-R antagonist [D-Lys3]-GHRP-6 increased plasma insulin concentrations at 30 min in GX rats to a similar extent to that in normal rats (61). These results suggest that the effect of GHS-R antagonist is not due to antagonism of circulating stomach-derived ghrelin, but primarily to blockade of local ghrelin including that in the islets. This finding suggests that the ghrelin produced by pancreas serves as a local regulator of insulin release, although it might not substantially contribute to the pool of circulating ghrelin.

Zhang et al. (62) reported that ghrelin-28 is a potent inhibitor of rat pancreatic exocrine secretion in vivo and in vitro. In our studies intravenous bolus infusions of pentaghrelin reduced in a dose-dependent manner the secretion of pancreatic juice and protein output, both in the non-stimulated and CCK-8-stimulated conditions (63). The effect on the juice volume, however, was weak as compared to that on protein output (Fig. 1). Reduction in trypsin activity output reflected the protein output pattern. Administration of pentaghrelin failed to affect the non-stimulated pancreatic secretion in rats subjected to vagotomy, capsaicin pre-treatment (neurotoxin that inactivates vagal afferent pathways) and tarazepide, a benzodiazepine CCK1 receptor antagonist (63). Data taken together suggest that elevation of plasma ghrelin which occurs during fasting may contribute to a reduction of pancreatic exocrine secretion through multiple mechanisms involving those originating in the gut mucosa and enteric nerves, in particular the insulo-acinar axis and duodenal neurohormonal CCK-vagal-dependent mechanism.

Fig. 1. Effect of intravenous bolus infusions of pentaghrelin (0, 1.2, 12 and 50 nmol kg-1 body weight, b. wt.) on pancreatico-biliary juice (PBJ) protein output: basal (vehicle), CCK-8-stimulated (12 and 120 pmol kg-1 b. wt. h-1), and basal secretion following vagotomy, capsaicin and tarazepide treatment in anaesthetized rats. Each bar represents a 15 min PBJ sample after infusion of vehicle alone or pentaghrelin. Mean and S.E.M. (n=6). The mean value was significantly different from respective vehicle infusion (one-way ANOVA for repeated measurements followed by Tukey's test, * P<0.05, ** P<0.01, *** P<0.001). Adapted from ref. 63.


OBESTATIN

Obestatin is a recently identified 23-amino acid peptide, which is derived from the preproghrelin gene (64). Obestatin appears to have opposite actions to ghrelin on the regulation of food intake, emptying of the stomach, and body weight in rodents, and could be a part of dual system connecting the gut and brain to regulate energy homeostasis (64). Obestatin also reduces fluid intake by acting on the thirst center in the brain (64). The G-protein coupled receptor GPR39 (65) was proposed to be the receptor for obestatin (64), but this has not been confirmed by other studies (66; 67). This receptor is present in many regions of the brain and peripheral tissues including pancreas (65; 64, 68, 69). Obestatin immunoreactivity positively correlated with insulin concentrations (68), but Green et al. (70) failed to find any effects of this peptide on the glucose homeostasis. Plasma obestatin displays an ultradian rhythmicity, similar to the secretion patterns of ghrelin and growth hormone, but circulating ghrelin and obestatin levels are not strictly correlated. For example, after gastric bypass, ghrelin levels are almost unchanged, but obestatin levels are reduced (71), and after a 24-h fast plasma ghrelin is increased whilst plasma obestatin is decreased (72). Surgical removal of the stomach by gastrectomy reduced the levels of circulating obestatin and ghrelin by 50%-80% in rats (73, 74). Significant levels of obestatin immunoreactivity were recently reported in perinatal rat pancreas (68). Obestatin receptor, GPR39, expression was found in the rat pancreas (64, 68). Interestingly, there is a significant correlation between insulin and obestatin concentrations in the postnatal pancreas, raising the possibility that obestatin could play a role in the control of insulin secretion (68). Little is known, however, on the effects on the exocrine pancreas. In recent preliminary studies in rats we have reported that exogenous obestatin may stimulate the pancreatic protein output and trypsin activity following intravenous and intraduodenal administration (Fig. 2). The effect was dose-dependent, but it required intact vagal supply (75). More studies are necessary, however, to understand a physiological role of obestatin-ghrelin team on the exocrine pancreas.

Fig. 2. Effect of intravenous obestatin boluses (30, 100 and 300 nmol/kg body weight, b. wt.) on pancreatic protein output in anaesthetized rats in control and following vagomy. The secretion of pancreato-billiary juice was sampled in 15 min intervals (samples numbered from 1 to 10). Mean and S.E.M. Arrows indicate bolus infusions. The mean value was significantly different from respective vehicle infusion (one-way ANOVA for repeated measurements followed by Tukey's test, * P<0.05). Adapted from ref. 75.


APELIN

Apelin was first discovered in bovine gastric mucosa by Tatemoto et al. (76). The peptide is an endogenous ligand for a G protein-coupled orphan receptor, APJ. Real time RT-PCR assays were used to show localization of apelin, which is produced in several tissues of the body, including the heart, brain, lung, pregnant and lactating breast, and GI tract (77, 78). Biologically active peptides are apelin-36, and its fragments 13 and 12 (79). Apelin was shown to modulate appetite by feedback signals such as leptin (80) or ghrelin (81, 82). Moreover, apelin is increased in hyperinsulinemia-associated obesity disorders in which feeding behavior and energy balance are altered (79, 83). These findings have led to the suggestion that apelin could have a role modulating feeding behavior and energy homeostasis. In the gastric mucosa, apelin is involved in gastric cell proliferation and controlling exocrine and endocrine functions (84, 85). Apelin is secreted by adipose tissue and its production is increased in obesity (79). Intracerebroventricular injection of apelin-13 decreased food intake in fed but not in fasted rats (86), while daytime administration of apelin-12 stimulated feeding (80). Low APJ mRNA expression has previously been shown in human pancreas but it was not detected in rat pancreas (87). More recently, Sörhede Winzell et al. (88) showed that apelin-36 inhibits glucose-stimulated insulin secretion both in vivo and in vitro in mice. in vivo, the peptide reduced the increase in circulating insulin after an intravenous glucose challenge. Apelin inhibited the insulin response to intravenous glucose in high-fat fed mice. These mice are obese and insulin resistant and the high-fat fed mouse has been shown to be a good model for studies of islet perturbations due to insulin resistance (88). The inhibition of glucose-stimulated insulin secretion by apelin would fit to the activation of the sympathetic nervous system, since such an activation is associated with impaired insulin secretion (89). The high abundance of apelin receptor (APJ) mRNA in hypothalamus (78, 90) with high expression in the paraventricular and supraoptic nuclei (78) would support a role of apelin in the central regulation of hormone release. Sörhede Winzell et al. (88) showed the expression of apelin receptor mRNA in isolated islets, which would suggest a direct effect of apelin on the ß-cells. Indeed, administration of apelin-36 inhibited glucose-stimulated insulin secretion from isolated mouse islets (88). Until recently, the role of apelin in controlling the exocrine pancreas became unknown. Just recently we made preliminary studies in in vivo and in vitro in rats. Intravenous apelin boluses decreased juice volume, protein and trypsin outputs in a dose-dependent manner, an effect corroborating with the mentioned above reduction of insulin release. In contrast, intraduodenal administration of apelin stimulated the pancreatic secretion. Both pharmacological blockade (atropine) and vagotomy could abolish all apelin effects. The direct effect of apelin on the pancreatic acini needs to be considered rather as pharmacological one, since the elevation of amylase release in vitro was significant only with 10-6 M apelin (91).


OREXINS

Orexin-A and -B (also named hypocretin-1 and 2) have been discovered by two independent research teams in 1998 by subtractive cDNA cloning and orphan receptor technologies (92, 93). Prepro-orexin is enzymatically matured into two peptides, orexin-A and orexin-B which are 33- and 28-amino-acid peptides, respectively. Orexins are neuropeptides present in hypothalamic neurons that project throughout the central nervous system to nuclei involved in the control of feeding, sleep-wakefulness, stereotype behaviors, addictions, neuroendocrine homeostasis and autonomic regulation (94). Direct injection of orexin-A into brain has been shown to increase short term feeding in rats (95, 96) and mice (97), while orexin-B had only a minor effect (98). Correspondingly, systemic injection of anti-orexin antibody resulted in reduced food intake in rats (99, 100). One of the first gastrointestinal effects reported was that of intracisternal injection of orexin-A to stimulate gastric acid secretion in conscious rats (101). Orexin-B injections had no effect. Orexin-A likely acts in the brain to stimulate acid secretion by modulating the dorsal vagal complex nuclei. Indeed, intraperitoneal administration of orexin in rats was ineffective and vagotomy or atropine abolished the action of central orexin (101). Miyasaka et al. (102) obtained similar effects concerning pancreas exocrine secretion: stimulation with orexin-A, and no effect with orexin-B. Pretreatment with cholinergic receptor antagonists, hexamethonium and atropine, abolished the stimulatory effect of orexin-A, which indicates mediation of cholinergic in pancreatic response.

Majority of orexin-positive neurons in the dorsal motor nuclei of the dorsal vagal complex were found to project into the gastric fundus or corpus, whereas only a few supplied the antrum, pylorus, duodenum or caecum (103). These neurons may contribute to GI motility regulation. Besides central effects, a number of local actions in the gastrointestinal tract and endocrine system was reported as reviewed elsewhere (94, 104). Orexin-A immunoreactivity was found in guinea pig stomach, in the endocrine cells of the pyloric glands, and a subset of these cells contained gastrin (105). The following extensive immunohistochemistry study by Kirchgessner and Liu (106) demonstrated orexins A and B and their receptors (OX1 and OX2) in a number of gastrointestinal tissues and enteric nerves including pancreas; abundant orexins-A and OX1 expression was found in the b-cells. Kirchgessner and Liu (106) speculated that GI orexins could prime the gut for the gastric and intestinal phase of secretion.

Orexins were found throughout the small intestine of rodents (rats, mice and guinea pigs) and humans and in guinea pig distal colon (105). Prepro-orexin mRNA was detected by PCR in longitudinal muscle of rat intestine (105). Orexin-containing neurons were immunodetected in both myenteric and submucosal plexuses (105). Orexins were present in nerve terminals making synaptic contacts with other neurons, especially with vasoactive intestinal peptide (VIP)-containing submucosal neurons. Orexin-immunoreactive neurons were also observed in the circular muscle layer, surrounding submucosal blood vessels and mucosa (105). The extensive network of orexin-containing nerve fibers in the mucosa encircles the crypts and travels within the lamina propria to the tips of the villi. This large distribution of intestinal orexin fibers suggests the possibility of various biological effects of endogenous orexins, such as enteric neuronal excitation, gut motility, blood flow and processes of epithelial absorption and/or secretion. This hypothesis is in agreement with the presence of orexin receptors in the intestinal wall (105). Orexin-A excites secretomotor neurons in the guinea pig submocosal plexus and controls gut motility by increasing the velocity of propulsion in isolated guinea pig colon (107). Kirchgessner i Liu (106) showed that prolonged fasting stimulates expression of orexins-A in the submucosal neurons in the duodenum.

Subcutaneous injection of orexin-A in rats resulted in an increase of plasma insulin level (108, 109). A direct effect of orexins on endocrine pancreas is likely since orexin-A stimulates insulin secretion in perfused rat pancreas in vitro (108) and immunoreactivity from orexin-A and orexin receptor has been detected in rodent insulin-positive endocrine cells (105). Intravenous injections of orexin-A and B did not affect pancreatic secretion in anaesthetized rats. The negative results were obtained in both, fed and 20 h starved rats (104). In contrast, intraduodenal infusions of orexin-B in rats fasted for 20 h resulted in significant stimulation of juice outflow, and a tendency toward increase in protein output. These data suggest that luminal orexins may control the juice flow in long-starved rats through as yet unknown mechanism. One of probable pathways is stimulation through insulo-acinar axis.


SUMMARY

A limited number of data accumulated so far suggest that the above mentioned regulatory peptides may synchronize the regulation of appetite with production and secretion of pancreatic juice. Circulating leptin after meal inhibits food intake simultaneously with inhibition of pancreatic enzyme secretion leading to reduced efficacy of digestion in the gut lumen. Local, luminal stimulation by leptin after meal is presumably less important due to dilution effect in the full stomach and duodenum. In contrast, hunger hormone - ghrelin stimulates appetite and when released locally before a meal it may participate in the stimulation of pancreatic enzymes via CCK and neurohormonal CCK1-vagal dependent mechanism. The rationale for circulating ghrelin in terms of the exocrine pancreas function remains unclear but it helps to control insulin release. The physiological role of obestatin-ghrelin team as well as that of apelin remains further elucidation. Orexins seem to play a minor role in controlling food intake in comparison to their contribution to maintenance of wakefulness, stereotypic behaviors, reward seeking and drug addiction (104). In regard to the exocrine pancreas, orexin-B may help to maintain basal secretion of juice during long periods of fasting.

Acknowledgments: Grant no N303 043 32/1447, Ministry for Research and Higher Education, Poland.

Conflict of interest statement: None declared.



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R e c e i v e d : June 10, 2008
A c c e p t e d : July 30, 2008

Author’s address: Malgorzata Kapica, D.V.M., Ph.D., Department of Biochemistry and Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, ul. Akademicka 12, 20-950 Lublin, Poland. Tel:. +48(81)4456768; Fax: +48(81) 4456973;
e-mail: malgorzata.kapica@ar.lublin.pl