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Glucagon-like peptide 1 (GLP-1) suppresses ghrelin levels in humans via increased insulin secretion
Regulatory Peptides 143 (2007) 64 – 68 www.elsevier.com/locate/regpep
Glucagon-like peptide 1 (GLP-1) suppresses ghrelin levels in humans via increased insulin secretion Dirk Hagemann a , Jens J. Holst b , Arnica Gethmann a , Matthias Banasch a , Wolfgang E. Schmidt a , Juris J. Meier a,⁎ b
a Department of Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Germany Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark
Received 24 January 2007; received in revised form 10 March 2007; accepted 10 March 2007 Available online 20 March 2007
Abstract Introduction: Ghrelin is an orexigenic peptide predominantly secreted by the stomach. Ghrelin plasma levels rise before meal ingestion and sharply decline afterwards, but the mechanisms controlling ghrelin secretion are largely unknown. Since meal ingestion also elicits the secretion of the incretin hormone glucagon-like peptide 1 (GLP-1), we examined whether exogenous GLP-1 administration reduces ghrelin secretion in humans. Patients and methods: 14 healthy male volunteers were given intravenous infusions of GLP-1(1.2 pmol·kg− 1 min− 1) or placebo over 390 min. After 30 min, a solid test meal was served. Venous blood was drawn frequently for the determination of glucose, insulin, C-peptide, GLP-1 and ghrelin. Results: During the infusion of exogenous GLP-1 and placebo, GLP-1 plasma concentrations reached steady-state levels of 139 ± 15 pmol/l and 12 ± 2 pmol/l, respectively (p b 0.0001). During placebo infusion, ghrelin levels were significantly reduced in the immediate postprandial period (p b 0.001), and rose again afterwards. GLP-1 administration prevented the initial postprandial decline in ghrelin levels, possibly as a result of delayed gastric emptying, and significantly reduced ghrelin levels 150 and 360 min after meal ingestion (p b 0.05). The patterns of ghrelin concentrations in the experiments with GLP-1 and placebo administration were inversely related to the respective plasma levels of insulin and Cpeptide. Conclusions: GLP-1 reduces the rise in ghrelin levels in the late postprandial period at supraphysiological plasma levels. Most likely, these effects are indirectly mediated through its insulinotropic action. The GLP-1-induced suppression of ghrelin secretion might be involved in its anorexic effects. © 2007 Elsevier B.V. All rights reserved. Keywords: GLP-1; Glucagon-like peptide 1; Ghrelin; Appetite; Energy homeostasis
1. Introduction Energy homeostasis is tightly controlled by the interplay of afferent neural signals, circulating fuels and the secretion of gastrointestinal hormones [1,2]. Among the peptide hormones from the gut known to regulate appetite and food intake, glucagon-like peptide 1 (GLP-1) and ghrelin seem to act as key regulators [3,4]. Thus, GLP-1 induces satiety and inhibits food consumption through a combination of direct central nervous
⁎ Corresponding author. Tel.: +49 234 509 1; fax: +49 234 509 2309. E-mail address: [email protected] (J.J. Meier). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.03.002
effects as well as an inhibition of gastric emptying [3,5–7]. As a consequence, chronic administration of GLP-1 or its longacting analogues leads to a progressive and sustained reduction in energy intake and body weight [8–11]. In contrast, ghrelin has been shown to promote appetite and increase caloric intake both in rodents and in humans [12,13]. Endogenous ghrelin levels rise prior to meal ingestion and sharply decline afterwards, thereby suggesting a physiological role as an endogenous appetite factor [14,15]. The mechanisms subserving the postprandial decline in ghrelin levels are yet unclear, and different mechanisms have been proposed. One possible explanation for this phenomenon is a direct neural suppression of ghrelin release induced e.g. by gastric distention and
D. Hagemann et al. / Regulatory Peptides 143 (2007) 64–68
subsequent vagal nervous stimulation [16]. Alternatively, it seems possible that fuel substrates, especially glucose, act to suppress the secretion of ghrelin [17]. In addition, it has been suggested that the postprandial rise in insulin secretion or the secretion of incretin hormones, namely glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), mediates the postprandial drop in ghrelin levels [18–22]. In support of the latter hypothesis, an inverse association between ghrelin and GLP-1 plasma concentrations after meal intake has been described [20,23]. However, since any such association might as well be secondary, e.g. mediated by the interplay between GLP-1, insulin secretion and gastric emptying, it seemed of interest to directly examine the effects of exogenous GLP-1 on ghrelin secretion. We have previously studied the effects of exogenous GLP-1 on postprandial lipid concentrations in healthy human volunteers [24]. This enabled us to examine the changes in ghrelin plasma levels in response to GLP-1 administration as well. Therefore, in the present studies, we sought to address, whether GLP-1 suppresses ghrelin secretion in humans and, if so, whether this is likely to be a direct or indirect effect. 2. Patients and methods 2.1. Study protocol The study protocol was approved by the ethics committee of the Ruhr-University of Bochum on 04/02/2003 (registrationnumber: 2074) prior to the study. Written informed consent was obtained from all participants. Parts of this study related to the effects of GLP-1 on insulin secretion and lipid metabolism have been published previously [24,25]. The present report focuses on the effects of GLP-1 on ghrelin secretion in the same experiments.
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laboratory parameters and a clinical examination was performed. If subjects met the inclusion criteria, they were recruited for the following tests. On separate occasions, either GLP-1 (7–36 amide) (1.2 pmol kg− 1 min− 1) or placebo was administered intravenously over 390 min (− 30 to 360 min). At 0 min, a mixed test meal (250 kcal) was ingested. Capillary and venous blood samples were collected frequently throughout the experiments for the determination of glucose, GLP-1, ghrelin, insulin and Cpeptide. The tests were carried out in randomised order. An interval of at least 2 days was kept between the tests in order to avoid carry-over effects. 2.4. Peptide Synthetic synthetic GLP-1 (7–36)amide was a kind gift from Restoragen, Inc., Nebrasca, USA. The peptide was sterile filtered and processed for infusion as described [7]. 2.5. Experimental procedures The tests were performed in the morning after an overnight fast with the subjects in a supine position throughout the experiments with the upper body lifted by 30°. Two forearm veins were punctured with a teflon cannula (Moskito 123, 18 gauge, Vygon, Aachen, Germany), and kept patent using 0.9% NaCl (for blood sampling and for GLP-1/placebo administration, respectively). The experiments were started after drawing basal blood samples (− 45 and − 30 min) with the infusion of GLP-1 (7–36) amide or placebo at − 30 min. After 0 min, a standard test meal (one egg, two slices of white bread, 5 g of margarine, 150 ml of water; 55% carbohydrates, 15% protein, 30% lipids; total caloric content: 250 kcal) was served. Capillary and venous blood samples were collected at 30-min intervals.
2.2. Participants 2.6. Blood specimen 14 healthy male volunteers participated in the study. The age was 24.2 ± 2.0 years (means ± SD), and the body mass index was 24.7 ± 2.2 kg/m2. HbA1c was 5.4 ± 0.2% (normal range: 4.8– 6.0%). None of the participants had a history of gastrointestinal disorders, had previously undergone abdominal surgery, or was taking any medication with a known modulating effect on gastrointestinal motility or glucose and lipid metabolism. All participants were advised to maintain their usual dietary habits and to avoid strenuous exercise prior to the experiments. From all participants, blood was drawn in the fasting state for measurements of standard hematological and clinical chemistry parameters. None of the participants had any abnormalities in terms of anemia (hemoglobin b 120 g/l), an elevation in liver enzymes (ALAT, ASAT, AP, γ-GT) to higher activities than double the respective normal value, or elevated creatinine concentrations (N1.5 mg/dl [114 μmol/l]). 2.3. Study design All participants were studied on three occasions: At a screening visit blood was drawn in the fasting state for
Venous blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol®; 20000 KIU/ml, 200 μl/10 ml blood; Bayer AG, Leverkusen, Germany and kept on ice. After centrifugation at 4 °C, plasma for hormone analyses was kept frozen at − 28 °C. Capillary blood samples (approximately 100 μl) were added to NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. 2.7. Laboratory determinations Glucose was measured as described [7] using a Glucose Analyser 2 (Beckman Instruments, Munich, Germany). Insulin and C-peptide levels were measured by ELISA as described [24,25]. GLP-1 immunoreactivity was determined using a radioimmunoassays specific for the C-terminus of the peptide [7,26]. This assay measures the sum of the intact peptide plus the primary metabolite, GLP-1 (9–36 amide) using the antiserum 89390 and synthetic GLP-1 (7–36 amide) as standard. The assay cross-reacts less than 0.01% with C-terminally truncated
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ation below 10%. Quality controls were always within acceptable limits. 2.8. Statistical analysis
Fig. 1. Plasma concentrations of GLP-1 (total concentrations) during the intravenous administration of GLP-1 (1.2 pmol·kg− 1·min− 1) or placebo in 14 healthy male subjects. At t = 0 min, a mixed test (250 kcal) meal was served (arrow). Data are presented as means ± SEM. p-values were calculated using paired repeated-measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant differences (p b 0.05) at individual time points (one-way ANOVA).
fragments, and 83% with GLP-1 (9–36 amide). The detection limit was 3 pmol/l. Plasma ghrelin concentrations were determined as described [27] using a radioimmunoassay kit from Linco Research (St. Charles, Missouri) (Cat. #GHRT-89k) which measures total (intact as well as desoctanoylated ghrelin). In our hands the sensitivity was 100 pg/ml, and intra-assay coefficients of vari-
Results are reported as mean ± SEM. All statistical calculations were carried out using paired repeated-measures analysis of variance (ANOVA) using Statistica Version 5.0 (Statsoft Europe, Hamburg, Germany). This analysis provides p-values for the overall differences between the experiments (A), differences over time (B), and for the interaction of experiment with time (AB). If a significant interaction of treatment and time (AB) was documented (p b 0.05), individual values were compared by paired one-way ANOVA. A two-sided p-value b 0.05 was taken to indicate significant differences. 3. Results During the infusion of exogenous GLP-1, plasma concentrations were raised from 10 ± 3 pmol/l to steady-state levels of 139 ± 15 pmol/l (p b 0.0001; Fig. 1). In contrast, GLP-1 levels were rather unchanged during placebo administration. Fasting ghrelin concentrations were not significantly different between the experiments with GLP-1 and placebo administration (Fig. 2). During placebo infusion, ghrelin levels were lowered between t = 60 min and t = 90 min after meal ingestion (p b 0.001), and rose again afterwards. GLP-1 administration tended to reduce ghrelin concentrations already prior to
Fig. 2. Plasma concentrations of glucose (A), ghrelin (B), insulin (C), and C-peptide (D) during the intravenous administration of GLP-1 (1.2 pmol·kg− 1·min− 1) or placebo in 14 healthy male subjects. At t = 0 min, a mixed test (250 kcal) meal was served (arrows). Data are presented as means ± SEM. p-values were calculated using paired repeated-measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant differences (p b 0.05) at individual time points (one-way ANOVA).
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meal ingestion (Fig. 2). The immediate postprandial decline in plasma ghrelin levels was absent during GLP-1 infusion. However, ghrelin levels were 9.4 ± 3.9% lower during GLP-1 administration between t = 150 min and t = 360 min compared to placebo (p b 0.05; Fig. 2). Interestingly, the patterns of ghrelin plasma concentrations observed during the experiments with GLP-1 and placebo administration seemed to be inversely related to the respective plasma levels of insulin and C-peptide ([24]; Fig. 2). 4. Discussion In the present studies, we examined the effects of exogenous GLP-1 on ghrelin secretion in humans. We report that the typical postprandial decline in ghrelin levels was absent during GLP-1 infusion. However, in the late postprandial period (t = 150 to t = 360 min), ghrelin plasma concentrations were significantly lower during GLP-1 administration than with placebo. These results directly lead to the question, whether the observed GLP-1 effects on ghrelin secretion were based on a direct interaction between both hormones, or rather indirectly mediated by other factors. In support of the former possibility, the GLP-1 receptor is widely expressed on the stomach [4,28], even though a direct localisation of the GLP-1 receptor on gastric X/A-like cells has not yet been shown. Nevertheless, a couple of points argue against such reasoning. In fact, while GLP-1 inhibited ghrelin secretion in the late postprandial period, there was even a slight increase in ghrelin levels from t = 60 to t = 120 min after the test meal (Fig. 2). Furthermore, the patterns of ghrelin concentrations determined during the experiments with placebo and GLP-1 administration appeared inversely related to the respective insulin and C-peptide levels, thereby suggesting that the changes in ghrelin levels were mediated by the GLP-1 effects on insulin secretion. Along these lines, the observed lack of ghrelin suppression in the early postprandial period might be due to the GLP-1 induced reduction of insulin secretion, which was caused by its marked deceleration of gastric emptying [7,24]. In contrast, in the late postprandial period (120–360 min), when gastric emptying was almost complete and, consequently, insulin secretion was augmented [24], ghrelin levels were lower in the experiments with GLP-1 administration. Taken together, these data suggest that ghrelin release is primarily controlled by changes in insulin secretion, in line with previous reports from our and other groups [18,19,27]. Other factors, such as GLP-2 [27], glucose [17], or somatostatin [29] might further contribute to the postprandial decline in ghrelin levels. It should also be noted that despite GLP-1 plasma concentrations exceeding the physiological range by ∼ 5-fold, the overall suppression of ghrelin secretion was rather modest (∼10% in the late postprandial period). Therefore, while based on these experiments a minor direct effect of GLP-1 on ghrelin secretion cannot be completely ruled out, the incretin hormone is unlikely to play a major role as a physiological regulator of ghrelin secretion. Recently, Perez-Tilve and colleagues reported a reduction in ghrelin levels after both central and intraperitoneal administra-
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tion of the long-acting GLP-1 analogue exendin-4 as well as of the GLP-1 receptor antagonist Exendin (9–39) in fasted rats [21]. In contrast, administration of a DPP-4 inhibitor and the GLP-1 analogue Ser8-GLP-1(7–36)-NH2 was without any effect on ghrelin secretion. The authors therefore suggested that the observed reduction in ghrelin levels might be mediated by an exendin-specific receptor distinct from the known GLP-1 receptor [21]. The present data in humans do not lend support to the idea of a direct GLP-1 effect on ghrelin secretion. Nevertheless, the prolonged suppression of ghrelin secretion in the late postprandial period might contribute to the anorexic actions of GLP-1 observed in rodents as well as in humans [5,30]. In conclusion, the present studies have shown that GLP-1 at supraphysiological levels reduces the rise in ghrelin levels in the late postprandial period in healthy humans. Most likely, these effects are mediated through the insulinotropic action of GLP-1. The GLP-1-induced suppression of ghrelin secretion might contribute to its anorexic effects. Acknowledgements The technical assistance of Birgit Baller and Lone Bagger is gratefully acknowledged. This study was supported by grants from the Deutsche Forschungsgemeinschaft (grant Me 2096/3-1 to JJM), the Danish Medical Research Council (to JJH) and the Ruhr-University Bochum (grant F-515-06 to JJM). References [1] Horvath TL. The hardship of obesity: a soft-wired hypothalamus. Nat Neurosci 2005;8:561–5. [2] Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–71. [3] Meier JJ, Gallwitz B, Schmidt WE, Nauck MA. Glucagon-like peptide 1 (GLP-1) as a regulator of food intake and body weight: therapeutic perspectives. Eur J Pharmacol 2002;440:269–79. [4] van der Lely AJ, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–57. [5] Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide-1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998;101: 515–20. [6] Gutzwiller J-P, Göke B, Drewe J, et al. Glucagon-like peptide-1: a potent regulator of food intake in humans. Gut 1999;44:81–6. [7] Meier JJ, Gallwitz B, Salmen S, et al. Normalization of glucose concentrations and deceleration of gastric emptying after solid meals during intravenous glucagon-like peptide 1 in patients with type 2 diabetes. J Clin Endocrinol Metab 2003;88:2719–25. [8] Zander M, Madsbad S, Madsen JL, Holst JJ. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and betacell function in type 2 diabetes: a parallel-group study. Lancet 2002;359: 824–30. [9] Defronzo RA, Ratner RE, Han J, et al. Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes. Diabetes Care 2005;28:1092–100. [10] Kendall DM, Riddle MC, Rosenstock J, et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in patients with type 2 diabetes treated with metformin and a sulfonylurea. Diabetes Care 2005;28: 1083–91. [11] Buse JB, Henry RR, Han J, et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care 2004;27:2628–35.
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[12] Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908–13. [13] Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding. Nature 2001;409:194–8. [14] Cummings DE, Purnell JQ, Frayo RS, et al. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50: 1714–9. [15] Cummings DE, Weigle DS, Frayo RS, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002;346: 1623–30. [16] Sugino T, Yamaura J, Yamagishi M, et al. Involvement of cholinergic neurons in the regulation of the ghrelin secretory response to feeding in sheep. Biochem Biophys Res Commun 2003;304:308–12. [17] Nakagawa E, Nagaya N, Okumura H, et al. Hyperglycaemia suppresses the secretion of ghrelin, a novel growth-hormone-releasing peptide: responses to the intravenous and oral administration of glucose. Clin Sci (Lond) 2002;103:325–8. [18] Saad MF, Bernaba B, Hwu CM, et al. Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 2002;87:3997–4000. [19] Mohlig M, Spranger J, Otto B, et al. Euglycemic hyperinsulinemia, but not lipid infusion, decreases circulating ghrelin levels in humans. J Endocrinol Investig 2002;25:RC36–8. [20] Djurhuus CB, Hansen TK, Gravholt C, et al. Circulating levels of ghrelin and GLP-1 are inversely related during glucose ingestion. Horm Metab Res 2002;34:411–3. [21] Perez-Tilve D, Gonzalez-Matias L, Alvarez-Crespo M, et al. Exendin-4 potently decreases ghrelin levels in fasting rats. Diabetes 2007;56:143–51.
[22] Rudovich NN, Dick D, Moehlig M, et al. Ghrelin is not suppressed in hyperglycemic clamps by gastric inhibitory polypeptide and arginine. Regul Pept 2005;127:95–9. [23] Djurhuus CB, Hansen TK, Holst JJ, Schmitz O. Feast or famine — are GLP-1 and ghrelin secretion intertwined. Horm Metab Res 2002;34:353–4. [24] Meier JJ, Gethmann A, Goetze O, et al. Glucagon-like peptide 1 (GLP-1) abolishes the postprandial rise in triglyceride concentrations and lowers free fatty acid levels in humans. Diabetologia 2006;49:452–8. [25] Meier JJ, Gethmann A, Nauck MA, et al. The glucagon-like peptide 1 metabolite GLP-1 (9–36)amide reduces postprandial glycemia independently of gastric emptying and insulin secretion in humans. Am J Physiol Endocrinol Metabol Gastrointest Physiol 2006;290:E1118–23. [26] Deacon CF, Nauck MA, Toft-Nielsen M, et al. Both subcutaneously and intravenously administered glucagon-like peptide 1 are rapidly degraded from the NH2-terminus in type 2-diabetic patients and in healthy subjects. Diabetes 1995;44:1126–31. [27] Banasch M, Bulut K, Hagemann D, et al. Glucagon-like peptide 2 inhibits ghrelin secretion in humans. Regul Pept 2007;137:173–8. [28] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656–60. [29] Barkan AL, Dimaraki EV, Jessup SK, et al. Ghrelin secretion in humans is sexually dimorphic, suppressed by somatostatin, and not affected by the ambient growth hormone levels. J Clin Endocrinol Metab 2003;88:2180–4. [30] Turton MD, D OS, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379:69–72.
Glucagon-like peptide 1 (GLP-1) suppresses ghrelin levels in humans via increased insulin secretion Dirk Hagemann a , Jens J. Holst b , Arnica Gethmann a , Matthias Banasch a , Wolfgang E. Schmidt a , Juris J. Meier a,⁎ b
a Department of Medicine I, St. Josef-Hospital, Ruhr-University Bochum, Germany Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark
Received 24 January 2007; received in revised form 10 March 2007; accepted 10 March 2007 Available online 20 March 2007
Abstract Introduction: Ghrelin is an orexigenic peptide predominantly secreted by the stomach. Ghrelin plasma levels rise before meal ingestion and sharply decline afterwards, but the mechanisms controlling ghrelin secretion are largely unknown. Since meal ingestion also elicits the secretion of the incretin hormone glucagon-like peptide 1 (GLP-1), we examined whether exogenous GLP-1 administration reduces ghrelin secretion in humans. Patients and methods: 14 healthy male volunteers were given intravenous infusions of GLP-1(1.2 pmol·kg− 1 min− 1) or placebo over 390 min. After 30 min, a solid test meal was served. Venous blood was drawn frequently for the determination of glucose, insulin, C-peptide, GLP-1 and ghrelin. Results: During the infusion of exogenous GLP-1 and placebo, GLP-1 plasma concentrations reached steady-state levels of 139 ± 15 pmol/l and 12 ± 2 pmol/l, respectively (p b 0.0001). During placebo infusion, ghrelin levels were significantly reduced in the immediate postprandial period (p b 0.001), and rose again afterwards. GLP-1 administration prevented the initial postprandial decline in ghrelin levels, possibly as a result of delayed gastric emptying, and significantly reduced ghrelin levels 150 and 360 min after meal ingestion (p b 0.05). The patterns of ghrelin concentrations in the experiments with GLP-1 and placebo administration were inversely related to the respective plasma levels of insulin and Cpeptide. Conclusions: GLP-1 reduces the rise in ghrelin levels in the late postprandial period at supraphysiological plasma levels. Most likely, these effects are indirectly mediated through its insulinotropic action. The GLP-1-induced suppression of ghrelin secretion might be involved in its anorexic effects. © 2007 Elsevier B.V. All rights reserved. Keywords: GLP-1; Glucagon-like peptide 1; Ghrelin; Appetite; Energy homeostasis
1. Introduction Energy homeostasis is tightly controlled by the interplay of afferent neural signals, circulating fuels and the secretion of gastrointestinal hormones [1,2]. Among the peptide hormones from the gut known to regulate appetite and food intake, glucagon-like peptide 1 (GLP-1) and ghrelin seem to act as key regulators [3,4]. Thus, GLP-1 induces satiety and inhibits food consumption through a combination of direct central nervous
⁎ Corresponding author. Tel.: +49 234 509 1; fax: +49 234 509 2309. E-mail address: [email protected] (J.J. Meier). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.03.002
effects as well as an inhibition of gastric emptying [3,5–7]. As a consequence, chronic administration of GLP-1 or its longacting analogues leads to a progressive and sustained reduction in energy intake and body weight [8–11]. In contrast, ghrelin has been shown to promote appetite and increase caloric intake both in rodents and in humans [12,13]. Endogenous ghrelin levels rise prior to meal ingestion and sharply decline afterwards, thereby suggesting a physiological role as an endogenous appetite factor [14,15]. The mechanisms subserving the postprandial decline in ghrelin levels are yet unclear, and different mechanisms have been proposed. One possible explanation for this phenomenon is a direct neural suppression of ghrelin release induced e.g. by gastric distention and
D. Hagemann et al. / Regulatory Peptides 143 (2007) 64–68
subsequent vagal nervous stimulation [16]. Alternatively, it seems possible that fuel substrates, especially glucose, act to suppress the secretion of ghrelin [17]. In addition, it has been suggested that the postprandial rise in insulin secretion or the secretion of incretin hormones, namely glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), mediates the postprandial drop in ghrelin levels [18–22]. In support of the latter hypothesis, an inverse association between ghrelin and GLP-1 plasma concentrations after meal intake has been described [20,23]. However, since any such association might as well be secondary, e.g. mediated by the interplay between GLP-1, insulin secretion and gastric emptying, it seemed of interest to directly examine the effects of exogenous GLP-1 on ghrelin secretion. We have previously studied the effects of exogenous GLP-1 on postprandial lipid concentrations in healthy human volunteers [24]. This enabled us to examine the changes in ghrelin plasma levels in response to GLP-1 administration as well. Therefore, in the present studies, we sought to address, whether GLP-1 suppresses ghrelin secretion in humans and, if so, whether this is likely to be a direct or indirect effect. 2. Patients and methods 2.1. Study protocol The study protocol was approved by the ethics committee of the Ruhr-University of Bochum on 04/02/2003 (registrationnumber: 2074) prior to the study. Written informed consent was obtained from all participants. Parts of this study related to the effects of GLP-1 on insulin secretion and lipid metabolism have been published previously [24,25]. The present report focuses on the effects of GLP-1 on ghrelin secretion in the same experiments.
65
laboratory parameters and a clinical examination was performed. If subjects met the inclusion criteria, they were recruited for the following tests. On separate occasions, either GLP-1 (7–36 amide) (1.2 pmol kg− 1 min− 1) or placebo was administered intravenously over 390 min (− 30 to 360 min). At 0 min, a mixed test meal (250 kcal) was ingested. Capillary and venous blood samples were collected frequently throughout the experiments for the determination of glucose, GLP-1, ghrelin, insulin and Cpeptide. The tests were carried out in randomised order. An interval of at least 2 days was kept between the tests in order to avoid carry-over effects. 2.4. Peptide Synthetic synthetic GLP-1 (7–36)amide was a kind gift from Restoragen, Inc., Nebrasca, USA. The peptide was sterile filtered and processed for infusion as described [7]. 2.5. Experimental procedures The tests were performed in the morning after an overnight fast with the subjects in a supine position throughout the experiments with the upper body lifted by 30°. Two forearm veins were punctured with a teflon cannula (Moskito 123, 18 gauge, Vygon, Aachen, Germany), and kept patent using 0.9% NaCl (for blood sampling and for GLP-1/placebo administration, respectively). The experiments were started after drawing basal blood samples (− 45 and − 30 min) with the infusion of GLP-1 (7–36) amide or placebo at − 30 min. After 0 min, a standard test meal (one egg, two slices of white bread, 5 g of margarine, 150 ml of water; 55% carbohydrates, 15% protein, 30% lipids; total caloric content: 250 kcal) was served. Capillary and venous blood samples were collected at 30-min intervals.
2.2. Participants 2.6. Blood specimen 14 healthy male volunteers participated in the study. The age was 24.2 ± 2.0 years (means ± SD), and the body mass index was 24.7 ± 2.2 kg/m2. HbA1c was 5.4 ± 0.2% (normal range: 4.8– 6.0%). None of the participants had a history of gastrointestinal disorders, had previously undergone abdominal surgery, or was taking any medication with a known modulating effect on gastrointestinal motility or glucose and lipid metabolism. All participants were advised to maintain their usual dietary habits and to avoid strenuous exercise prior to the experiments. From all participants, blood was drawn in the fasting state for measurements of standard hematological and clinical chemistry parameters. None of the participants had any abnormalities in terms of anemia (hemoglobin b 120 g/l), an elevation in liver enzymes (ALAT, ASAT, AP, γ-GT) to higher activities than double the respective normal value, or elevated creatinine concentrations (N1.5 mg/dl [114 μmol/l]). 2.3. Study design All participants were studied on three occasions: At a screening visit blood was drawn in the fasting state for
Venous blood was drawn into chilled tubes containing EDTA and aprotinin (Trasylol®; 20000 KIU/ml, 200 μl/10 ml blood; Bayer AG, Leverkusen, Germany and kept on ice. After centrifugation at 4 °C, plasma for hormone analyses was kept frozen at − 28 °C. Capillary blood samples (approximately 100 μl) were added to NaF (Microvette CB 300; Sarstedt, Nümbrecht, Germany) for the immediate measurement of glucose. 2.7. Laboratory determinations Glucose was measured as described [7] using a Glucose Analyser 2 (Beckman Instruments, Munich, Germany). Insulin and C-peptide levels were measured by ELISA as described [24,25]. GLP-1 immunoreactivity was determined using a radioimmunoassays specific for the C-terminus of the peptide [7,26]. This assay measures the sum of the intact peptide plus the primary metabolite, GLP-1 (9–36 amide) using the antiserum 89390 and synthetic GLP-1 (7–36 amide) as standard. The assay cross-reacts less than 0.01% with C-terminally truncated
66
D. Hagemann et al. / Regulatory Peptides 143 (2007) 64–68
ation below 10%. Quality controls were always within acceptable limits. 2.8. Statistical analysis
Fig. 1. Plasma concentrations of GLP-1 (total concentrations) during the intravenous administration of GLP-1 (1.2 pmol·kg− 1·min− 1) or placebo in 14 healthy male subjects. At t = 0 min, a mixed test (250 kcal) meal was served (arrow). Data are presented as means ± SEM. p-values were calculated using paired repeated-measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant differences (p b 0.05) at individual time points (one-way ANOVA).
fragments, and 83% with GLP-1 (9–36 amide). The detection limit was 3 pmol/l. Plasma ghrelin concentrations were determined as described [27] using a radioimmunoassay kit from Linco Research (St. Charles, Missouri) (Cat. #GHRT-89k) which measures total (intact as well as desoctanoylated ghrelin). In our hands the sensitivity was 100 pg/ml, and intra-assay coefficients of vari-
Results are reported as mean ± SEM. All statistical calculations were carried out using paired repeated-measures analysis of variance (ANOVA) using Statistica Version 5.0 (Statsoft Europe, Hamburg, Germany). This analysis provides p-values for the overall differences between the experiments (A), differences over time (B), and for the interaction of experiment with time (AB). If a significant interaction of treatment and time (AB) was documented (p b 0.05), individual values were compared by paired one-way ANOVA. A two-sided p-value b 0.05 was taken to indicate significant differences. 3. Results During the infusion of exogenous GLP-1, plasma concentrations were raised from 10 ± 3 pmol/l to steady-state levels of 139 ± 15 pmol/l (p b 0.0001; Fig. 1). In contrast, GLP-1 levels were rather unchanged during placebo administration. Fasting ghrelin concentrations were not significantly different between the experiments with GLP-1 and placebo administration (Fig. 2). During placebo infusion, ghrelin levels were lowered between t = 60 min and t = 90 min after meal ingestion (p b 0.001), and rose again afterwards. GLP-1 administration tended to reduce ghrelin concentrations already prior to
Fig. 2. Plasma concentrations of glucose (A), ghrelin (B), insulin (C), and C-peptide (D) during the intravenous administration of GLP-1 (1.2 pmol·kg− 1·min− 1) or placebo in 14 healthy male subjects. At t = 0 min, a mixed test (250 kcal) meal was served (arrows). Data are presented as means ± SEM. p-values were calculated using paired repeated-measures ANOVA and denote A: differences between the experiments, B: differences over time and AB: differences due to the interaction of experiment and time. Asterisks indicate significant differences (p b 0.05) at individual time points (one-way ANOVA).
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meal ingestion (Fig. 2). The immediate postprandial decline in plasma ghrelin levels was absent during GLP-1 infusion. However, ghrelin levels were 9.4 ± 3.9% lower during GLP-1 administration between t = 150 min and t = 360 min compared to placebo (p b 0.05; Fig. 2). Interestingly, the patterns of ghrelin plasma concentrations observed during the experiments with GLP-1 and placebo administration seemed to be inversely related to the respective plasma levels of insulin and C-peptide ([24]; Fig. 2). 4. Discussion In the present studies, we examined the effects of exogenous GLP-1 on ghrelin secretion in humans. We report that the typical postprandial decline in ghrelin levels was absent during GLP-1 infusion. However, in the late postprandial period (t = 150 to t = 360 min), ghrelin plasma concentrations were significantly lower during GLP-1 administration than with placebo. These results directly lead to the question, whether the observed GLP-1 effects on ghrelin secretion were based on a direct interaction between both hormones, or rather indirectly mediated by other factors. In support of the former possibility, the GLP-1 receptor is widely expressed on the stomach [4,28], even though a direct localisation of the GLP-1 receptor on gastric X/A-like cells has not yet been shown. Nevertheless, a couple of points argue against such reasoning. In fact, while GLP-1 inhibited ghrelin secretion in the late postprandial period, there was even a slight increase in ghrelin levels from t = 60 to t = 120 min after the test meal (Fig. 2). Furthermore, the patterns of ghrelin concentrations determined during the experiments with placebo and GLP-1 administration appeared inversely related to the respective insulin and C-peptide levels, thereby suggesting that the changes in ghrelin levels were mediated by the GLP-1 effects on insulin secretion. Along these lines, the observed lack of ghrelin suppression in the early postprandial period might be due to the GLP-1 induced reduction of insulin secretion, which was caused by its marked deceleration of gastric emptying [7,24]. In contrast, in the late postprandial period (120–360 min), when gastric emptying was almost complete and, consequently, insulin secretion was augmented [24], ghrelin levels were lower in the experiments with GLP-1 administration. Taken together, these data suggest that ghrelin release is primarily controlled by changes in insulin secretion, in line with previous reports from our and other groups [18,19,27]. Other factors, such as GLP-2 [27], glucose [17], or somatostatin [29] might further contribute to the postprandial decline in ghrelin levels. It should also be noted that despite GLP-1 plasma concentrations exceeding the physiological range by ∼ 5-fold, the overall suppression of ghrelin secretion was rather modest (∼10% in the late postprandial period). Therefore, while based on these experiments a minor direct effect of GLP-1 on ghrelin secretion cannot be completely ruled out, the incretin hormone is unlikely to play a major role as a physiological regulator of ghrelin secretion. Recently, Perez-Tilve and colleagues reported a reduction in ghrelin levels after both central and intraperitoneal administra-
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tion of the long-acting GLP-1 analogue exendin-4 as well as of the GLP-1 receptor antagonist Exendin (9–39) in fasted rats [21]. In contrast, administration of a DPP-4 inhibitor and the GLP-1 analogue Ser8-GLP-1(7–36)-NH2 was without any effect on ghrelin secretion. The authors therefore suggested that the observed reduction in ghrelin levels might be mediated by an exendin-specific receptor distinct from the known GLP-1 receptor [21]. The present data in humans do not lend support to the idea of a direct GLP-1 effect on ghrelin secretion. Nevertheless, the prolonged suppression of ghrelin secretion in the late postprandial period might contribute to the anorexic actions of GLP-1 observed in rodents as well as in humans [5,30]. In conclusion, the present studies have shown that GLP-1 at supraphysiological levels reduces the rise in ghrelin levels in the late postprandial period in healthy humans. Most likely, these effects are mediated through the insulinotropic action of GLP-1. The GLP-1-induced suppression of ghrelin secretion might contribute to its anorexic effects. Acknowledgements The technical assistance of Birgit Baller and Lone Bagger is gratefully acknowledged. This study was supported by grants from the Deutsche Forschungsgemeinschaft (grant Me 2096/3-1 to JJM), the Danish Medical Research Council (to JJH) and the Ruhr-University Bochum (grant F-515-06 to JJM). References [1] Horvath TL. The hardship of obesity: a soft-wired hypothalamus. Nat Neurosci 2005;8:561–5. [2] Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–71. [3] Meier JJ, Gallwitz B, Schmidt WE, Nauck MA. Glucagon-like peptide 1 (GLP-1) as a regulator of food intake and body weight: therapeutic perspectives. Eur J Pharmacol 2002;440:269–79. [4] van der Lely AJ, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–57. [5] Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide-1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998;101: 515–20. [6] Gutzwiller J-P, Göke B, Drewe J, et al. Glucagon-like peptide-1: a potent regulator of food intake in humans. Gut 1999;44:81–6. [7] Meier JJ, Gallwitz B, Salmen S, et al. Normalization of glucose concentrations and deceleration of gastric emptying after solid meals during intravenous glucagon-like peptide 1 in patients with type 2 diabetes. J Clin Endocrinol Metab 2003;88:2719–25. [8] Zander M, Madsbad S, Madsen JL, Holst JJ. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and betacell function in type 2 diabetes: a parallel-group study. Lancet 2002;359: 824–30. [9] Defronzo RA, Ratner RE, Han J, et al. Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes. Diabetes Care 2005;28:1092–100. [10] Kendall DM, Riddle MC, Rosenstock J, et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in patients with type 2 diabetes treated with metformin and a sulfonylurea. Diabetes Care 2005;28: 1083–91. [11] Buse JB, Henry RR, Han J, et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care 2004;27:2628–35.
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