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Effects of insulin, leptin, and glucagon on ghrelin secretion from isolated perfused rat stomach
Regulatory Peptides 119 (2004) 77 – 81 www.elsevier.com/locate/regpep
Effects of insulin, leptin, and glucagon on ghrelin secretion $ from isolated perfused rat stomach Jun Kamegai *, Hideki Tamura, Takako Shimizu, Shinya Ishii, Hitoshi Sugihara, Shinichi Oikawa Department of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8603, Japan Received 7 November 2003; received in revised form 7 January 2004; accepted 14 January 2004
Abstract Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally purified from the rat stomach. Although ghrelin has been recognized as an important regulator of energy metabolism, the regulation of the ghrelin secretion is largely unknown. Here, we examined the direct effects of insulin, leptin, and glucagon on the release of ghrelin from the isolated rat stomach. The isolated pancreas – spleen – duodenum deprived preparation of rat stomach was used. After a baseline control infusion into the left gastric artery, insulin, leptin, or glucagon were infused for 15 min at concentrations of 0.1, 1, and 10 nM. The levels of immunoreactive ghrelin in the venous effluents were measured with a radioimmunoassay. Insulin and leptin inhibited ghrelin secretion dose-dependently (total amount of ghrelin release: insulin at 1 nM, 73.5 F 7.3% of the control infusion; leptin at 1 nM, 81.8 F 2.5% of the control infusion; n = 5, P < 0.05), while glucagon increased it dose-dependently (total amount of ghrelin released at 10 nM was 143.9 F 19.3% of the control infusion; n = 5, P < 0.01). These results indicate that the ghrelin responses observed in vivo could be due to direct effects of multiple hormonal signals on the stomach. D 2004 Elsevier B.V. All rights reserved. Keywords: Ghrelin; Stomach; Insulin; Leptin; Glucagon
1. Introduction Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor, was originally purified from the rat stomach [1]. Ghrelin has been recognized as an important regulator of energy metabolism and GH secretion. Ghrelin induces adiposity by stimulating food intake, as well as a reduction in fat utilization [2 –6]. On the other hand, we and others have reported that treatment with ghrelin stimulates GH secretion from the pituitary in vivo and in vitro [1,7,8]. Ghrelin is synthesized primarily in endocrine cells in the stomach and is present in the circulation [9]. However, the regulation of the ghrelin secretion from the stomach is still largely unknown.
Abbreviations: AGRP, agouti-related protein; DKRBG, dextran Krebs – Ringer bicarbonate buffer containing glucose; GH, growth hormone; NPY, neuropeptide Y; STZ, streptozotocin. $ This work was supported by a Grant-in-Aid for scientific research (C) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to J.K.) (KAKENHI 13671165) and a Grant from Hakujikai, Institute of Gerontology (to J.K.). * Corresponding author. Tel.: +81-3-3822-2131; fax: +81-3-3943-4313. E-mail address: [email protected] (J. Kamegai). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.01.012
We have previously reported that streptozotocin (STZ)induced hypoinsulinemic diabetic rats had increased plasma ghrelin levels, and that insulin treatment decreased plasma ghrelin levels [10]. Therefore, we hypothesized that insulin directly decreases the secretion of ghrelin from the stomach. However, to our best knowledge, the reports on the effects of insulin on ghrelin synthesis and release are conflicting. Insulin treatment increased [11], or did not affect [12], the expression of the ghrelin gene in the stomach of rodents, whereas the administration of insulin increased plasma concentrations of ghrelin in rodents [12] but did not affect it in humans [13]. Recently, it has been reported that hyperinsulinemia reduced [14,15] or did not change [16] ghrelin levels in human subjects using the euglycemic hyperinsulinemic clamp method. Thus, the effect of insulin on ghrelin secretion is uncertain. On the other hand, leptin, an adipocyte-derived anorexic hormone, helps to regulate food intake and energy expenditure through specific receptors located in the hypothalamus. Leptin treatment suppresses food intake by decreasing the neuronal activity of neuropeptide Y (NPY)/agouti-related protein (AGRP)-containing neurons [17,18]. Recently, we reported that plasma concentrations of ghrelin in STZtreated diabetic rats were regulated in the opposite direction
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to plasma concentrations of leptin through STZ with and without insulin treatments [10]. These data are consistent with the concept that the regulation of ghrelin secretion, as well as its biological effects, is reciprocal to those of leptin [19]. However, the direct interaction between the two hormones have not been elucidated. We previously reported that GH-releasing hormone, a member of a structurally related protein family that includes vasoactive intestinal peptide, glucagon, and secretin, and whose cognate receptors use cAMP as a second messenger, increases the level of ghrelin mRNA in the pituitary [20]. Since glucagon and cAMP also enhanced the activity of the ghrelin promoter [21], we hypothesized that glucagon increases ghrelin release from the stomach. Because functional receptors for insulin, leptin, and glucagon occur in the stomach [22 – 25], we examined in this report the effects of insulin, leptin, and glucagon on ghrelin release from the stomach using perfusion of the isolated stomach.
2. Materials and methods
gastric artery by means of a peristaltic pump at a flow rate of 1.5 ml/min. 2.3. Experimental design and hormone assay After an equilibration period of 30 min and a basal period of 10 min of perfusion with DKRBG alone, the solution with insulin (Humalin-RR; Eli Lilly, Indianapolis, IN), leptin (Sigma, Tokyo, Japan), or glucagon (Peptide Institute, Osaka, Japan) was infused for 15 min at concentrations of 10 10, 10 9, and 10 8 M, followed by a 10-min washout period. The control experiment was performed by the infusion of the solution alone. Only one perfusion was done in each stomach, and five perfusions were performed for each dose of each hormone. All the venous effluents were collected at the times indicated in the figures into tubes which contained EDTA/2 Na (1.5 mg/ml), aprotinin (500 U/ ml), and 1N HCl (26.5 Al/ml), and stored at 70 C until assayed. The levels of immunoreactive ghrelin were measured with a radioimmunoassay kit (Phoenix Pharmaceuticals, Mountain View, CA), as previously described [10]. All data are presented as the mean F SEM. The data were
2.1. Animals Male Sprague – Dawley rats (250 – 280 g; Saitama Experimental Animal Supply Saitama, Japan) were housed in airconditioned animal quarters, with lights on between 08:00 and 20:00 h, and given food and water ad libitum. Experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Nippon Medical School Animal Care Research Committee. 2.2. Preparation of the isolated perfused rat stomach The perfusion of the rat stomach was performed by using the isolated pancreas – spleen – duodenum deprived preparation as described previously [25]. After a 48-h fast, the animals were anesthetized with 50 mg/kg of pentobarbital intraperitoneally. After a midline laparotomy, polyethylene cannulae were inserted into the left gastric artery and gastric vein. All other blood vessels and the pancreas were carefully excluded by ligation, and then the spleen was removed. After the esophagocardial junction was ligated, the esophagus and vagus nerve were removed at approximately 90 min before the hormone infusion, and then a catheter was inserted into the stomach through the pyloric ring to drain the gastric juice, and finally the duodenum was removed. All perfusions were performed with 4.6% dextran 40 (Wako Chemical, Tokyo, Japan) Krebs – Ringer bicarbonate buffer containing 5.5 mM glucose (DKRBG). The perfusate was continuously gassed with 95% O2 and 5% CO2 and maintained at pH 7.4 and PaO2 350 mm Hg. Both the perfusate and the stomach preparation were kept at 37 jC throughout the experiment. The perfusate was infused into the left
Fig. 1. Effect of insulin infusion on ghrelin release from the isolated rat stomach. After a 48-h fast, the adult male rat stomach was used for the experiment. After a basal period of 10 min perfusion with DKRBG alone into the left gastric artery, the solution with insulin was infused for 15 min, followed by a 10 min washout period. All the venous effluents were collected, and the levels of immunoreactive ghrelin were measured with a RIA. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, compared to the control group.
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analyzed with an ANOVA using Duncan’s New Multiple Range test. P < 0.05 was considered significant.
3. Results In the control experiments with perfusion of only the solution, the ghrelin levels in the effluents at the beginning of the experiments (at 10 min) were 297.5 F 4.2 pg/ml, and at the end of the experiments (at 25 min) the levels were 322.6 F 16.8 pg/ml, which did not change significantly throughout the perfusion period (Figs. 1– 3). The secretion of ghrelin in response to insulin is shown in Fig. 1. The ghrelin secretion was decreased during the insulin infusion period, and significantly decreased compared to that with the control solution (10 nM; 5– 15 min). After the termination of the insulin infusion, the ghrelin levels tended to increase. The secretion of ghrelin in response to leptin is shown in Fig. 2. Leptin infusion decreased ghrelin secretion, and after a 10-min (10 nM) or a 15-min (1 nM) infusion of leptin, the ghrelin secretion was significantly decreased compared to that with the control solution. After the beginning of the leptin infusion at 10 nM, the secretion of ghrelin decreased gradually throughout the observation period, even continued after the end of the leptin infusion.
Fig. 3. Effect of glucagon infusion on ghrelin release from the isolated rat stomach. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, **P < 0.01, compared to the control group.
The secretion of ghrelin in response to glucagon is shown in Fig. 3. The ghrelin secretion was increased during the glucagon infusion period, and significantly increased compared to that with the control solution (10 nM; 3 and 5 min).
Table 1 Total amount of ghrelin secretion during the infusion of insulin, leptin, and glucagon
Control Insulin
Leptin
Glucagon
a
Fig. 2. Effect of leptin infusion on ghrelin release from the isolated rat stomach. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, **P < 0.01, compared to the control group.
Dose of peptide (M)
Total amounts of ghrelin releasea (pg/15 min)
Relative amounts of ghrelin releaseb (%)
0 10 10 10 10 10 10 10 10 10
4689 F 133 4289 F 443 3444 F 278* 3008 F 500** 4209 F 301 3839 F 97* 3544 F 207* 5194 F 179 5895 F 568 6749 F 739**
100.0 F 3.5 91.4 F 11.6 73.5 F 7.3* 64.2 F 13.0** 89.7 F 7.8 81.8 F 2.5* 75.6 F 5.4* 110.7 F 4.6 125.7 F 14.8 143.9 F 19.3**
10 9 8 10 9 8 10 9 8
Total amounts of immunoreactive ghrelin released during a 15-min period of hormone infusion. b The amounts of immunoreactive ghrelin released by hormones are expressed as a percentage of the amount released by the control solution. The data represent the mean F SEM (n = 5 animals/group). * P < 0.05. ** P < 0.01, compared to the control group.
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After the termination of the glucagon infusion, the ghrelin levels tended to decrease. Total amounts of ghrelin secretion during 15 min of the infusion of insulin, leptin, and glucagon are listed in Table 1. Leptin and insulin evoked a significant and dose-dependent decrease of ghrelin secretion, whereas the glucagon infusion evoked a significant and dose-dependent increase of ghrelin secretion.
In conclusion, the present study demonstrated for the first time that insulin and leptin at physiological concentrations directly inhibit ghrelin secretion from the isolated perfused rat stomach, while glucagon increased it. These results indicated that some of the changes in the secretion of ghrelin observed in vivo could be due to the direct effects of multiple hormonal signals on the stomach.
Acknowledgements 4. Discussion We thank Ms. Masayo Asizawa for technical assistance. The present study demonstrated that insulin and leptin at concentrations from 10 10 to 10 8 M directly inhibit the secretion of ghrelin from the isolated perfused rat stomach in a dose-dependent manner, while glucagon, in contrast, increases it. It has been reported that the administration of insulin in vivo decreases [14,15], increases [12], or does not change [13,16] the secretion of ghrelin. Insulin treatment in vivo has, directly or indirectly, multiple influences on the secretions of several hormones, e.g., glucagon, GH, cortisol/ cortisone, and catecholamines, and on plasma glucose. Therefore, the direct decreasing effect of insulin on ghrelin release could have been masked by the influence of the other hormonal conditions on ghrelin release in the in vivo experiments. Under physiological conditions, negative correlations have been reported between plasma insulin and ghrelin concentrations [26,27]. The results of the present study suggest that postprandial insulin surges could be at least in part responsible for the reduction of the plasma ghrelin levels after food intake [26]. Leptin is the most important signal which reflects peripheral energy balance [28,29]. Leptin decreases food intake by decreasing the neuronal activity of NPY/AGRP-containing neurons [17,18], whereas ghrelin activates NPY/AGRP neuronal activity, resulting in an increase of food intake [3– 5]. Thus, the biological effects of the two peripheral hormones on the hypothalamus are reciprocal. However, peripheral interactions of the two hormones have not been defined. In this study, we demonstrated that leptin directly decreases the secretion of ghrelin. These results are consistent with the recent report that moderate hyperleptinemia prevents an increase of plasma ghrelin during moderate short-term caloric restriction [30]. These results indicated that the anorexic effect of leptin may occur through an action on the stomach by decreasing ghrelin secretion as well as through a hypothalamic action. Alternatively, since ghrelin receptors are expressed in adipose tissue, the principal site of leptin synthesis [1], it is conceivable that ghrelin might affect leptin secretion. On the other hand, in this study using an isolated stomach perfusion system, glucagon infusion enhanced the release of ghrelin dose-dependently. However, since a relatively high dose (10 nM) of glucagon was required to increase the secretion of ghrelin significantly, further studies will be necessary to determine the physiological role of glucagon in ghrelin secretion.
References [1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656 – 60. [2] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908 – 13. [3] Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I. Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression. Endocrinology 2000;141:4797 – 800. [4] Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and agouti-related protein mRNA levels and body weight in rats. Diabetes 2001;50:2438 – 43. [5] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, et al. A role for ghrelin in the central regulation of feeding. Nature 2001;409:194 – 8. [6] Shuto Y, Shibasaki T, Otagiri A, Kuriyama H, Ohata H, Tamura H, et al. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding and adiposity. J Clin Invest 2002;109:1429 – 36. [7] Tamura H, Kamegai J, Shimizu T, Ishii S, Sugihara H, Oikawa S. Ghrelin stimulates GH but not food intake in arcuate nucleus ablated rats. Endocrinology 2002;143:3268 – 75. [8] Tannenbaum GS, Epelbaum J, Bowers CY. Interrelationship between the novel peptide ghrelin and somatostatin/growth hormone-releasing hormone in regulation of pulsatile growth hormone secretion. Endocrinology 2003;144:967 – 74. [9] Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, et al. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 2001;86:4753 – 8. [10] Ishii S, Kamegai J, Tamura H, Shimizu T, Sugihara H, Oikawa S. Role of ghrelin in streptozotocin-induced diabetic hyperphagia. Endocrinology 2002;143:4934 – 7. [11] Toshinai K, Mondal MS, Nakazato M, Date Y, Murakami N, Kojima M, et al. Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration. Biochem Biophys Res Commun 2001;281:1220 – 1. [12] Lee HM, Wang G, Englander EW, Kojima M, Greeley Jr. GH. Ghrelin, a new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations. Endocrinology 2002;143:185 – 90. [13] Caixa´s A, Bashore C, Nash W, Pi-Sunyer FX, Laferre`re B. Insulin, unlike food intake, does not suppress ghrelin in human subjects. J Clin Endocrinol Metab 2002;87:1902 – 6. [14] Saad MF, Bernaba B, Hwu CM, Jinagouda S, Fahmi S, Kogosov E, et al. Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 2002;87:3997 – 4000.
J. Kamegai et al. / Regulatory Peptides 119 (2004) 77–81 [15] Riis AL, Hansen TK, Moller N, Weeke J, Jorgensen JO. Hyperthyroidism is associated with suppressed circulating ghrelin levels. J Clin Endocrinol Metab 2003;88:853 – 7. [16] Schaller G, Schmidt A, Pleiner J, Woloszczuk W, Wolzt M, Luger A. Plasma ghrelin concentrations are not regulated by glucose or insulin: a double-blind, placebo-controlled crossover clamp study. Diabetes 2003;52:16 – 20. [17] Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, et al. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995;377:530 – 2. [18] Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of target of leptin action in rat hypothalamus. J Clin Invest 1996;98:1101 – 6. [19] Horvath TL, Diano S, Sotonyi P, Heiman M, Tschop M. Ghrelin and the regulation of energy balance—a hypothalamic perspective. Endocrinology 2001;142:4163 – 9. [20] Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Oikawa S. Regulation of the ghrelin gene: growth hormone-releasing hormone upregulates ghrelin mRNA in the pituitary. Endocrinology 2001;142: 4154 – 7. [21] Kishimoto M, Okimura Y, Nakata H, Kudo T, Iguchi G, Takahashi Y, et al. Cloning and characterization of the 5( V)-flanking region of the human ghrelin gene. Biochem Biophys Res Commun 2003;305: 186 – 92. [22] Chiba T, Taminato T, Kadowaki S, Abe H, Chihara K, Matsukura S, et al. Effects of insulin and pancreatic polypeptide on gastric somatostatin release. Diabetes 1980;29:292 – 5.
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[23] Madaus S, Schusdziarra V, Dummer W, Classen M. The effect of glucose and insulin on vagally induced gastrin, bombesin-like immunoreactivity and somatostatin secretion from the perfused rat stomach. Neuropeptides 1991;18:215 – 22. [24] Goiot H, Attoub S, Kermorgant S, Laigneau JP, Lardeux B, Lehy T, et al. Antral mucosa expresses functional leptin receptors coupled to STAT-3 signaling, which is involved in the control of gastric secretions in the rat. Gastroenterology 2001;121:1417 – 27. [25] Chiba T, Taminato T, Kadowaki S, Abe H, Chihara K, Seino Y, et al. Effects of glucagon, secretin, and vasoactive intestinal polypeptide on gastric somatostatin and gastrin release from isolated perfused rat stomach. Gastroenterology 1980;79:67 – 71. [26] Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50:1714 – 9. [27] Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50:707 – 9. [28] Cummings DE, Schwartz MW. Genetics and pathophysiology of human obesity. Annu Rev Med 2003;54:453 – 71. [29] Wilding JP. Neuropeptides and appetite control. Diabet Med 2002;19: 619 – 27. [30] Barazzoni R, Zanetti M, Stebel M, Biolo G, Cattin L, Guarnieri G. Hyperleptinemia prevents increased plasma ghrelin concentration during short-term moderate caloric restriction in rats. Gastroenterology 2003;124:1188 – 92.
Effects of insulin, leptin, and glucagon on ghrelin secretion $ from isolated perfused rat stomach Jun Kamegai *, Hideki Tamura, Takako Shimizu, Shinya Ishii, Hitoshi Sugihara, Shinichi Oikawa Department of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8603, Japan Received 7 November 2003; received in revised form 7 January 2004; accepted 14 January 2004
Abstract Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, was originally purified from the rat stomach. Although ghrelin has been recognized as an important regulator of energy metabolism, the regulation of the ghrelin secretion is largely unknown. Here, we examined the direct effects of insulin, leptin, and glucagon on the release of ghrelin from the isolated rat stomach. The isolated pancreas – spleen – duodenum deprived preparation of rat stomach was used. After a baseline control infusion into the left gastric artery, insulin, leptin, or glucagon were infused for 15 min at concentrations of 0.1, 1, and 10 nM. The levels of immunoreactive ghrelin in the venous effluents were measured with a radioimmunoassay. Insulin and leptin inhibited ghrelin secretion dose-dependently (total amount of ghrelin release: insulin at 1 nM, 73.5 F 7.3% of the control infusion; leptin at 1 nM, 81.8 F 2.5% of the control infusion; n = 5, P < 0.05), while glucagon increased it dose-dependently (total amount of ghrelin released at 10 nM was 143.9 F 19.3% of the control infusion; n = 5, P < 0.01). These results indicate that the ghrelin responses observed in vivo could be due to direct effects of multiple hormonal signals on the stomach. D 2004 Elsevier B.V. All rights reserved. Keywords: Ghrelin; Stomach; Insulin; Leptin; Glucagon
1. Introduction Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor, was originally purified from the rat stomach [1]. Ghrelin has been recognized as an important regulator of energy metabolism and GH secretion. Ghrelin induces adiposity by stimulating food intake, as well as a reduction in fat utilization [2 –6]. On the other hand, we and others have reported that treatment with ghrelin stimulates GH secretion from the pituitary in vivo and in vitro [1,7,8]. Ghrelin is synthesized primarily in endocrine cells in the stomach and is present in the circulation [9]. However, the regulation of the ghrelin secretion from the stomach is still largely unknown.
Abbreviations: AGRP, agouti-related protein; DKRBG, dextran Krebs – Ringer bicarbonate buffer containing glucose; GH, growth hormone; NPY, neuropeptide Y; STZ, streptozotocin. $ This work was supported by a Grant-in-Aid for scientific research (C) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to J.K.) (KAKENHI 13671165) and a Grant from Hakujikai, Institute of Gerontology (to J.K.). * Corresponding author. Tel.: +81-3-3822-2131; fax: +81-3-3943-4313. E-mail address: [email protected] (J. Kamegai). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.01.012
We have previously reported that streptozotocin (STZ)induced hypoinsulinemic diabetic rats had increased plasma ghrelin levels, and that insulin treatment decreased plasma ghrelin levels [10]. Therefore, we hypothesized that insulin directly decreases the secretion of ghrelin from the stomach. However, to our best knowledge, the reports on the effects of insulin on ghrelin synthesis and release are conflicting. Insulin treatment increased [11], or did not affect [12], the expression of the ghrelin gene in the stomach of rodents, whereas the administration of insulin increased plasma concentrations of ghrelin in rodents [12] but did not affect it in humans [13]. Recently, it has been reported that hyperinsulinemia reduced [14,15] or did not change [16] ghrelin levels in human subjects using the euglycemic hyperinsulinemic clamp method. Thus, the effect of insulin on ghrelin secretion is uncertain. On the other hand, leptin, an adipocyte-derived anorexic hormone, helps to regulate food intake and energy expenditure through specific receptors located in the hypothalamus. Leptin treatment suppresses food intake by decreasing the neuronal activity of neuropeptide Y (NPY)/agouti-related protein (AGRP)-containing neurons [17,18]. Recently, we reported that plasma concentrations of ghrelin in STZtreated diabetic rats were regulated in the opposite direction
78
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to plasma concentrations of leptin through STZ with and without insulin treatments [10]. These data are consistent with the concept that the regulation of ghrelin secretion, as well as its biological effects, is reciprocal to those of leptin [19]. However, the direct interaction between the two hormones have not been elucidated. We previously reported that GH-releasing hormone, a member of a structurally related protein family that includes vasoactive intestinal peptide, glucagon, and secretin, and whose cognate receptors use cAMP as a second messenger, increases the level of ghrelin mRNA in the pituitary [20]. Since glucagon and cAMP also enhanced the activity of the ghrelin promoter [21], we hypothesized that glucagon increases ghrelin release from the stomach. Because functional receptors for insulin, leptin, and glucagon occur in the stomach [22 – 25], we examined in this report the effects of insulin, leptin, and glucagon on ghrelin release from the stomach using perfusion of the isolated stomach.
2. Materials and methods
gastric artery by means of a peristaltic pump at a flow rate of 1.5 ml/min. 2.3. Experimental design and hormone assay After an equilibration period of 30 min and a basal period of 10 min of perfusion with DKRBG alone, the solution with insulin (Humalin-RR; Eli Lilly, Indianapolis, IN), leptin (Sigma, Tokyo, Japan), or glucagon (Peptide Institute, Osaka, Japan) was infused for 15 min at concentrations of 10 10, 10 9, and 10 8 M, followed by a 10-min washout period. The control experiment was performed by the infusion of the solution alone. Only one perfusion was done in each stomach, and five perfusions were performed for each dose of each hormone. All the venous effluents were collected at the times indicated in the figures into tubes which contained EDTA/2 Na (1.5 mg/ml), aprotinin (500 U/ ml), and 1N HCl (26.5 Al/ml), and stored at 70 C until assayed. The levels of immunoreactive ghrelin were measured with a radioimmunoassay kit (Phoenix Pharmaceuticals, Mountain View, CA), as previously described [10]. All data are presented as the mean F SEM. The data were
2.1. Animals Male Sprague – Dawley rats (250 – 280 g; Saitama Experimental Animal Supply Saitama, Japan) were housed in airconditioned animal quarters, with lights on between 08:00 and 20:00 h, and given food and water ad libitum. Experiments were conducted according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Nippon Medical School Animal Care Research Committee. 2.2. Preparation of the isolated perfused rat stomach The perfusion of the rat stomach was performed by using the isolated pancreas – spleen – duodenum deprived preparation as described previously [25]. After a 48-h fast, the animals were anesthetized with 50 mg/kg of pentobarbital intraperitoneally. After a midline laparotomy, polyethylene cannulae were inserted into the left gastric artery and gastric vein. All other blood vessels and the pancreas were carefully excluded by ligation, and then the spleen was removed. After the esophagocardial junction was ligated, the esophagus and vagus nerve were removed at approximately 90 min before the hormone infusion, and then a catheter was inserted into the stomach through the pyloric ring to drain the gastric juice, and finally the duodenum was removed. All perfusions were performed with 4.6% dextran 40 (Wako Chemical, Tokyo, Japan) Krebs – Ringer bicarbonate buffer containing 5.5 mM glucose (DKRBG). The perfusate was continuously gassed with 95% O2 and 5% CO2 and maintained at pH 7.4 and PaO2 350 mm Hg. Both the perfusate and the stomach preparation were kept at 37 jC throughout the experiment. The perfusate was infused into the left
Fig. 1. Effect of insulin infusion on ghrelin release from the isolated rat stomach. After a 48-h fast, the adult male rat stomach was used for the experiment. After a basal period of 10 min perfusion with DKRBG alone into the left gastric artery, the solution with insulin was infused for 15 min, followed by a 10 min washout period. All the venous effluents were collected, and the levels of immunoreactive ghrelin were measured with a RIA. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, compared to the control group.
J. Kamegai et al. / Regulatory Peptides 119 (2004) 77–81
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analyzed with an ANOVA using Duncan’s New Multiple Range test. P < 0.05 was considered significant.
3. Results In the control experiments with perfusion of only the solution, the ghrelin levels in the effluents at the beginning of the experiments (at 10 min) were 297.5 F 4.2 pg/ml, and at the end of the experiments (at 25 min) the levels were 322.6 F 16.8 pg/ml, which did not change significantly throughout the perfusion period (Figs. 1– 3). The secretion of ghrelin in response to insulin is shown in Fig. 1. The ghrelin secretion was decreased during the insulin infusion period, and significantly decreased compared to that with the control solution (10 nM; 5– 15 min). After the termination of the insulin infusion, the ghrelin levels tended to increase. The secretion of ghrelin in response to leptin is shown in Fig. 2. Leptin infusion decreased ghrelin secretion, and after a 10-min (10 nM) or a 15-min (1 nM) infusion of leptin, the ghrelin secretion was significantly decreased compared to that with the control solution. After the beginning of the leptin infusion at 10 nM, the secretion of ghrelin decreased gradually throughout the observation period, even continued after the end of the leptin infusion.
Fig. 3. Effect of glucagon infusion on ghrelin release from the isolated rat stomach. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, **P < 0.01, compared to the control group.
The secretion of ghrelin in response to glucagon is shown in Fig. 3. The ghrelin secretion was increased during the glucagon infusion period, and significantly increased compared to that with the control solution (10 nM; 3 and 5 min).
Table 1 Total amount of ghrelin secretion during the infusion of insulin, leptin, and glucagon
Control Insulin
Leptin
Glucagon
a
Fig. 2. Effect of leptin infusion on ghrelin release from the isolated rat stomach. The data represent the mean F SEM (n = 5 animals/group). *P < 0.05, **P < 0.01, compared to the control group.
Dose of peptide (M)
Total amounts of ghrelin releasea (pg/15 min)
Relative amounts of ghrelin releaseb (%)
0 10 10 10 10 10 10 10 10 10
4689 F 133 4289 F 443 3444 F 278* 3008 F 500** 4209 F 301 3839 F 97* 3544 F 207* 5194 F 179 5895 F 568 6749 F 739**
100.0 F 3.5 91.4 F 11.6 73.5 F 7.3* 64.2 F 13.0** 89.7 F 7.8 81.8 F 2.5* 75.6 F 5.4* 110.7 F 4.6 125.7 F 14.8 143.9 F 19.3**
10 9 8 10 9 8 10 9 8
Total amounts of immunoreactive ghrelin released during a 15-min period of hormone infusion. b The amounts of immunoreactive ghrelin released by hormones are expressed as a percentage of the amount released by the control solution. The data represent the mean F SEM (n = 5 animals/group). * P < 0.05. ** P < 0.01, compared to the control group.
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After the termination of the glucagon infusion, the ghrelin levels tended to decrease. Total amounts of ghrelin secretion during 15 min of the infusion of insulin, leptin, and glucagon are listed in Table 1. Leptin and insulin evoked a significant and dose-dependent decrease of ghrelin secretion, whereas the glucagon infusion evoked a significant and dose-dependent increase of ghrelin secretion.
In conclusion, the present study demonstrated for the first time that insulin and leptin at physiological concentrations directly inhibit ghrelin secretion from the isolated perfused rat stomach, while glucagon increased it. These results indicated that some of the changes in the secretion of ghrelin observed in vivo could be due to the direct effects of multiple hormonal signals on the stomach.
Acknowledgements 4. Discussion We thank Ms. Masayo Asizawa for technical assistance. The present study demonstrated that insulin and leptin at concentrations from 10 10 to 10 8 M directly inhibit the secretion of ghrelin from the isolated perfused rat stomach in a dose-dependent manner, while glucagon, in contrast, increases it. It has been reported that the administration of insulin in vivo decreases [14,15], increases [12], or does not change [13,16] the secretion of ghrelin. Insulin treatment in vivo has, directly or indirectly, multiple influences on the secretions of several hormones, e.g., glucagon, GH, cortisol/ cortisone, and catecholamines, and on plasma glucose. Therefore, the direct decreasing effect of insulin on ghrelin release could have been masked by the influence of the other hormonal conditions on ghrelin release in the in vivo experiments. Under physiological conditions, negative correlations have been reported between plasma insulin and ghrelin concentrations [26,27]. The results of the present study suggest that postprandial insulin surges could be at least in part responsible for the reduction of the plasma ghrelin levels after food intake [26]. Leptin is the most important signal which reflects peripheral energy balance [28,29]. Leptin decreases food intake by decreasing the neuronal activity of NPY/AGRP-containing neurons [17,18], whereas ghrelin activates NPY/AGRP neuronal activity, resulting in an increase of food intake [3– 5]. Thus, the biological effects of the two peripheral hormones on the hypothalamus are reciprocal. However, peripheral interactions of the two hormones have not been defined. In this study, we demonstrated that leptin directly decreases the secretion of ghrelin. These results are consistent with the recent report that moderate hyperleptinemia prevents an increase of plasma ghrelin during moderate short-term caloric restriction [30]. These results indicated that the anorexic effect of leptin may occur through an action on the stomach by decreasing ghrelin secretion as well as through a hypothalamic action. Alternatively, since ghrelin receptors are expressed in adipose tissue, the principal site of leptin synthesis [1], it is conceivable that ghrelin might affect leptin secretion. On the other hand, in this study using an isolated stomach perfusion system, glucagon infusion enhanced the release of ghrelin dose-dependently. However, since a relatively high dose (10 nM) of glucagon was required to increase the secretion of ghrelin significantly, further studies will be necessary to determine the physiological role of glucagon in ghrelin secretion.
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