The ups and downs of growth hormone secretagogue receptor signaling
TL;DR: The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control as mentioned in this paper, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions.
Abstract: The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.
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TL;DR: The first known LEAP2-KO mouse line was generated in this paper, where the metabolic effects of genetic leaper-expressed antimicrobial peptide-2 (LEAP2) deletion were determined.
Abstract: Objective The hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. Methods We generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. Results LEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2× higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10× higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2–119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. Conclusions LEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.
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TL;DR: The authors investigated the effects of exogenous LEAP2 on post-prandial glucose metabolism and ad libitum food intake in a randomized, double-blind, placebo-controlled, crossover trial of 20 healthy men.
Abstract: •Exogenous LEAP2 lowers postprandial plasma glucose excursions•Exogenous LEAP2 suppresses ad libitum food intake•During fasting, exogenous LEAP2 increases insulin secretion and suppresses lipolysis•The GHSR is required for eliciting LEAP2 effects in mice The gastric hormone ghrelin stimulates food intake and increases plasma glucose through activation of the growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) has been proposed to inhibit actions of ghrelin through inverse effects on GHSR activity. Here, we investigate the effects of exogenous LEAP2 on postprandial glucose metabolism and ad libitum food intake in a randomized, double-blind, placebo-controlled, crossover trial of 20 healthy men. We report that LEAP2 infusion lowers postprandial plasma glucose and growth hormone concentrations and decreases food intake during an ad libitum meal test. In wild-type mice, plasma glucose and food intake are reduced by LEAP2 dosing, but not in GHSR-null mice, pointing to GHSR as a potential mediator of LEAP2’s glucoregulatory and appetite-suppressing effects in mice. The gastric hormone ghrelin stimulates food intake and increases plasma glucose through activation of the growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) has been proposed to inhibit actions of ghrelin through inverse effects on GHSR activity. Here, we investigate the effects of exogenous LEAP2 on postprandial glucose metabolism and ad libitum food intake in a randomized, double-blind, placebo-controlled, crossover trial of 20 healthy men. We report that LEAP2 infusion lowers postprandial plasma glucose and growth hormone concentrations and decreases food intake during an ad libitum meal test. In wild-type mice, plasma glucose and food intake are reduced by LEAP2 dosing, but not in GHSR-null mice, pointing to GHSR as a potential mediator of LEAP2’s glucoregulatory and appetite-suppressing effects in mice. The growth hormone (GH) secretagogue receptor (GHSR) modulates fundamental physiological functions, including regulation of food intake, glucose homeostasis, and GH release from the anterior pituitary gland.1Müller T.D. Nogueiras R. Andermann M.L. Andrews Z.B. Anker S.D. Argente J. Batterham R.L. Benoit S.C. Bowers C.Y. Broglio F. et al.Ghrelin.Mol. Metab. 2015; 4: 437-460Google Scholar The gastric hormone ghrelin, an endogenous GHSR agonist, stimulates food intake and gastrointestinal motility and increases plasma glucose.1Müller T.D. Nogueiras R. Andermann M.L. Andrews Z.B. Anker S.D. Argente J. Batterham R.L. Benoit S.C. Bowers C.Y. Broglio F. et al.Ghrelin.Mol. Metab. 2015; 4: 437-460Google Scholar,2Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach.Nature. 1999; 402: 656-660Google Scholar Ghrelin, a peptide hormone, requires an acylation to obtain full activity, and its expression is regulated according to energy status.1Müller T.D. Nogueiras R. Andermann M.L. Andrews Z.B. Anker S.D. Argente J. Batterham R.L. Benoit S.C. Bowers C.Y. Broglio F. et al.Ghrelin.Mol. Metab. 2015; 4: 437-460Google Scholar,2Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach.Nature. 1999; 402: 656-660Google Scholar Thus, plasma ghrelin concentrations rise during conditions with energy deficit, such as fasting and calorie restriction, and fall after food intake or with obesity.3Ariyasu H. Takaya K. Tagami T. Ogawa Y. Hosoda K. Akamizu T. Suda M. Koh T. Natsui K. Toyooka S. 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: 4753-4758Google Scholar,4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar Recently, liver-expressed antimicrobial peptide 2 (LEAP2), a 40-amino-acid peptide expressed in the liver and the small intestine, was identified as another GHSR ligand.5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar LEAP2 is both an inverse agonist of GHSR that downregulates the constitutive activity of GHSR and a competitive antagonist that impairs ghrelin-induced activation of GHSR.4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar,6M’Kadmi C. Cabral A. Barrile F. Giribaldi J. Cantel S. Damian M. Mary S. Denoyelle S. Dutertre S. Péraldi-Roux S. et al.N-terminal liver-expressed antimicrobial peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor.J. Med. Chem. 2019; 62: 965-973Google Scholar,7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar Thus, the activity of GHSR is controlled at least in part by two circulating, gut-derived ligands with opposing actions. This type of dual regulation of receptor signaling properties is unusual in human physiology, but it has previously been described for the melanocortin receptors. For example, the melanocortin-4 receptor—an important receptor for appetite regulation—also displays constitutive activity, which is further activated by α-melanocyte-stimulating hormone and inhibited by agouti-related peptide.8Baldini G. Phelan K.D. The melanocortin pathway and control of appetite-progress and therapeutic implications.J. Endocrinol. 2019; 241: R1-R33Google Scholar LEAP2 is synthesized as a 77-amino-acid prepropeptide and is processed into several truncated forms, including the mature 40-amino-acid residue peptide (LEAP238–77; here called LEAP2), which is identical in mice and humans.5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar Plasma LEAP2 concentrations are regulated inversely compared with plasma ghrelin in several metabolic settings.4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar Hence, LEAP2 concentrations have been reported to decrease during weight loss and increase with obesity.4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar Whether plasma concentrations of LEAP2 increase in response to food intake in humans as seen in rodents remains to be clarified.4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar,7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar Mani et al.4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar observed a postprandial rise in LEAP2 plasma concentration in obese individuals eligible for bariatric surgery, but not in lean individuals; however, we could not confirm a postprandial increase in LEAP2 plasma levels in a similar group of obese individuals.7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar Recently, we reported an increased expression level of LEAP2 following the bariatric surgical procedure Roux-en-Y gastric bypass (RYGB) and robust insulinotropic properties in vitro of another endogenous LEAP2 fragment, LEAP238–47, however without glucoregulatory effect in a clinical proof-of-concept trial.7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar Interestingly, LEAP238–47 retains the inverse agonistic properties on GHSR, suggesting that the insulinotropic characteristics of the fragment may be directly linked to downregulation of constitutive GHSR activity.6M’Kadmi C. Cabral A. Barrile F. Giribaldi J. Cantel S. Damian M. Mary S. Denoyelle S. Dutertre S. Péraldi-Roux S. et al.N-terminal liver-expressed antimicrobial peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor.J. Med. Chem. 2019; 62: 965-973Google Scholar,7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar Thus, the pharmacological potential of LEAP2 family peptides deserves to be further investigated. To date, bariatric surgery is the most effective treatment of obesity.9Maciejewski M.L. Arterburn D.E. Van Scoyoc L. Smith V.A. Yancy W.S. Weidenbacher H.J. Livingston E.H. Olsen M.K. Bariatric surgery and long-term durability of weight loss.JAMA Surg. 2016; 151: 1046Google Scholar Despite documented beneficial effects of individual gut hormones, such as glucagon-like peptide 1 (GLP-1) and peptide YY, on glycemic control and appetite regulation following RYGB surgery,10Moffett R.C. Docherty N.G. le Roux C.W. The altered enteroendocrine reportoire following roux-en-Y-gastric bypass as an effector of weight loss and improved glycaemic control.Appetite. 2021; 156: 104807Google Scholar,11Svane M.S. Jørgensen N.B. Bojsen-Møller K.N. Dirksen C. Nielsen S. Kristiansen V.B. Toräng S. Wewer Albrechtsen N.J. Rehfeld J.F. Hartmann B. et al.Peptide YY and glucagon-like peptide-1 contribute to decreased food intake after Roux-en-Y gastric bypass surgery.Int. J. Obes. 2016; 40: 1699-1706Google Scholar the exact mechanisms that regulate changes in gut-derived signals and link them with metabolic control are not well understood. GHSR and ghrelin regulate a wealth of metabolic functions connecting gut and brain and have been considered as possible neuroendocrine therapeutic targets. However, despite two decades of intensive research in the field, no viable clinical candidate has been developed. Consequently, the discovery of the endogenous inverse agonist LEAP2 may reveal a potential therapeutic target for ghrelin-related diseases, including type 2 diabetes and obesity, due to its reciprocal relationship with ghrelin4Mani B.K. Puzziferri N. He Z. Rodriguez J.A. Osborne-Lawrence S. Metzger N.P. Chhina N. Gaylinn B. Thorner M.O. Thomas E.L. et al.LEAP2 changes with body mass and food intake in humans and mice.J. Clin. Invest. 2019; 129: 3909-3923Google Scholar and elevated expression levels following RYGB.7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar The administration of ghrelin in rodents and humans stimulates food intake and increases plasma glucose.5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar,12Cornejo M.P. Mustafá E.R. Cassano D. Banères J.L. Raingo J. Perello M. The ups and downs of growth hormone secretagogue receptor signaling.FEBS J. 2021; 288: 7213-7229Google Scholar,13Druce M.R. Wren A.M. Park A.J. Milton J.E. Patterson M. Frost G. Ghatei M.A. Small C. Bloom S.R. Ghrelin increases food intake in obese as well as lean subjects.Int. J. Obes. 2005; 29: 1130-1136Google Scholar Several groups have investigated the effect of exogenous LEAP2 on food intake and plasma glucose in rodents. A pioneering study demonstrated lower food intake in LEAP2-treated compared with vehicle-treated mice;5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar however, this finding could not be confirmed in succeeding studies.6M’Kadmi C. Cabral A. Barrile F. Giribaldi J. Cantel S. Damian M. Mary S. Denoyelle S. Dutertre S. Péraldi-Roux S. et al.N-terminal liver-expressed antimicrobial peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor.J. Med. Chem. 2019; 62: 965-973Google Scholar,7Hagemann C.A. Zhang C. Hansen H.H. Jorsal T. Rigbolt K.T.G. Madsen M.R. Bergmann N.C. Heimbürger S.M.N. Falkenhahn M. Theis S. et al.Identification and metabolic profiling of a novel human gut-derived LEAP2 fragment.J. Clin. Endocrinol. Metab. 2021; 106: e966-e981Google Scholar,14Islam M.N. Mita Y. Maruyama K. Tanida R. Zhang W. Sakoda H. Nakazato M. Liver-expressed antimicrobial peptide 2 antagonizes the effect of ghrelin in rodents.J. Endocrinol. 2020; 244: 13-23Google Scholar Nevertheless, LEAP2 treatment has consistently been shown to impair ghrelin-induced food intake and hyperglycemia in rodents.5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar,6M’Kadmi C. Cabral A. Barrile F. Giribaldi J. Cantel S. Damian M. Mary S. Denoyelle S. Dutertre S. Péraldi-Roux S. et al.N-terminal liver-expressed antimicrobial peptide 2 (LEAP2) region exhibits inverse agonist activity toward the ghrelin receptor.J. Med. Chem. 2019; 62: 965-973Google Scholar,14Islam M.N. Mita Y. Maruyama K. Tanida R. Zhang W. Sakoda H. Nakazato M. Liver-expressed antimicrobial peptide 2 antagonizes the effect of ghrelin in rodents.J. Endocrinol. 2020; 244: 13-23Google Scholar, 15Shankar K. Metzger N.P. Singh O. Mani B.K. Osborne-Lawrence S. Varshney S. Gupta D. Ogden S.B. Takemi S. Richard C.P. et al.LEAP2 deletion in mice enhances ghrelin’s actions as an orexigen and growth hormone secretagogue.Mol. Metab. 2021; 53: 101327Google Scholar, 16Barrile F. M’Kadmi C. De Francesco P.N. Cabral A. García Romero G. Mustafá E.R. Cantel S. Damian M. Mary S. Denoyelle S. et al.Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region.Mol. Cell. Endocrinol. 2019; 498: 110573Google Scholar Notably, only two studies besides the study of Ge et al. have demonstrated effects of LEAP2 administration alone, i.e., a decrease in plasma glucose after intraperitoneal LEAP2 administration in mice17Gupta D. Dowsett G.K.C. Mani B.K. Shankar K. Osborne-Lawrence S. Metzger N.P. Lam B.Y.H. Yeo G.S.H. Zigman J.M. High coexpression of the ghrelin and LEAP2 receptor GHSR with pancreatic polypeptide in mouse and human islets.Endocrinology. 2021; 162: bqab148Google Scholar and reduced binge-like eating after central administration of a truncated LEAP2 form in mice.18Cornejo M.P. Castrogiovanni D. Schiöth H.B. Reynaldo M. Marie J. Fehrentz J.A. Perello M. Growth hormone secretagogue receptor signalling affects high-fat intake independently of plasma levels of ghrelin and LEAP2, in a 4-day binge eating model.J. Neuroendocrinol. 2019; 31: e12785Google Scholar Whether exogenous LEAP2 affects food intake and glucose metabolism in humans has not been investigated. In the present study, we studied the effects of exogenous LEAP2 in healthy men and show that it lowers postprandial glucose excursions and suppresses food intake (effects that may be mediated via the GHSR as informed by experiments in GHSR-null mice). Whether the striking effects of exogenous LEAP2 in humans will revitalize the GHSR as a therapeutic target in metabolic diseases awaits further studies. To investigate the metabolic effects of LEAP2 in humans, we carried out an intravenous infusion of LEAP2 (∼25 pmol/kg/min), resulting in supraphysiological plasma concentrations in 20 lean, young men (see Table 1 for baseline characteristics and Figure 1 for an overview of the randomized, double-blind, placebo-controlled study design). Using an in-house radioimmunoassay directed against the N-terminal part of LEAP2, we determined LEAP2 plasma concentrations during LEAP2 and placebo infusions. During LEAP2 and placebo infusions, the baseline concentrations of LEAP2 plasma concentrations were similar (Figure 2A ). During LEAP2 infusion, plasma concentrations of LEAP2 reached steady state after 45 min of infusion with a mean concentration of 41.2 ± 1.1 ng/mL (Figure 2A), ∼2.6-fold higher than the mean plasma LEAP2 concentration during placebo infusion (Figure 2A). During placebo infusion, no change in LEAP2 concentration was found in response to the liquid mixed meal (Figure 2A), suggesting that endogenous LEAP2 concentrations are not acutely affected by food intake in lean, young men. The participants reported no adverse events during the infusions.Table 1Baseline characteristics of study participantsMale/female (n/n)20/0Age (years)23 (20–25)Weight (kg)80.3 (74.9–88.2)Height (m)1.87 (1.81–1.92)BMI (kg/m2)23.1 (22.3–25.0)Fasting plasma glucose (mmol/L)5.2 (5.0–5.3)HbA1c (mmol/mol)32 (29–33)Data are presented as median (interquartile range [IQR]). BMI, body mass index; HbA1c, glycated hemoglobin A1c. Open table in a new tab Figure 2LEAP2 alters postprandial plasma concentrations of glucose, glucagon, and growth hormone during a liquid mixed meal test and fasting plasma concentrations of insulin, C-peptide, C-peptide/glucose ratio, glucagon, and glycerol in healthy, young menShow full captionPlasma concentrations of LEAP2 (A), glucose (B), insulin (C), C-peptide (D), C-peptide/glucose ratio (E), glucagon (F), free fatty acids (G), glycerol (H), triglycerides (I), acetaminophen (J), growth hormone (K), and acyl ghrelin (L). Bold dotted line, infusion start (0 min); thin dotted line, liquid mixed meal test (65 min); blue square symbols, LEAP2 infusion; gray round symbols, placebo infusion (n = 20). Data are presented as mean ± SEM. Student’s paired t test of AUC60–255 min: p = 0.017 (B), p < 0.001 (F), and p < 0.001 (K). Student’s paired t test of AUC0–60 min: p < 0.001 (C), p = 0.018 (D), p = 0.003 (E), p = 0.038 (F), and p = 0.039 (H). AUC, area under the curve.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data are presented as median (interquartile range [IQR]). BMI, body mass index; HbA1c, glycated hemoglobin A1c. Plasma concentrations of LEAP2 (A), glucose (B), insulin (C), C-peptide (D), C-peptide/glucose ratio (E), glucagon (F), free fatty acids (G), glycerol (H), triglycerides (I), acetaminophen (J), growth hormone (K), and acyl ghrelin (L). Bold dotted line, infusion start (0 min); thin dotted line, liquid mixed meal test (65 min); blue square symbols, LEAP2 infusion; gray round symbols, placebo infusion (n = 20). Data are presented as mean ± SEM. Student’s paired t test of AUC60–255 min: p = 0.017 (B), p < 0.001 (F), and p < 0.001 (K). Student’s paired t test of AUC0–60 min: p < 0.001 (C), p = 0.018 (D), p = 0.003 (E), p = 0.038 (F), and p = 0.039 (H). AUC, area under the curve. First, we assessed the effect of exogenous LEAP2 on postprandial glucose metabolism in healthy men. For this purpose, participants were given a standardized liquid mixed meal (65 min after infusion start). During LEAP2 infusion, the postprandial plasma glucose peaks were lower than during placebo infusion (Figure 2B; Table 2). Furthermore, the area under the curve (AUC) for the entire infusion period (AUC0–255 min) and the postprandial AUC (AUC60–255 min) were lower during LEAP2 infusion compared with placebo (Table 2), demonstrating a reduced postprandial glucose response during LEAP2 infusion.Table 2Overview of plasma measurements in the clinical studyLEAP2Placebo (saline)p value (paired t test)Glucose Baseline (mmol/L)5.02 ± 0.065.01 ± 0.070.900 Peak (mmol/L)6.21 ± 0.126.58 ± 0.130.005 Time to peak (min)140 ± 12.7137 ± 10.70.808 Postprandial baseline (mmol/L)4.96 ± 0.055.10 ± 0.080.077 Postprandial end (mmol/L)4.48 ± 0.114.75 ± 0.120.056 AUC (mmol/L × min)1,288 ± 13.41,336 ± 18.50.013 –60 min (mmol/L × min)295 ± 3.02300 ± 3.670.198 AUC60–255 min (mmol/L × min)992 ± 12.41,037 ± 16.90.017Insulin Baseline (pmol/L)36.4 ± 1.7036.3 ± 2.080.984 Peak (pmol/L)347 ± 38.6356 ± 35.40.734 Time to peak (min)110 ± 6.28120 ± 9.230.433 Postprandial baseline (pmol/L)38.6 ± 2.0931.1 ± 2.080.017 AUC (nmol/L × min)33.8 ± 2.6333.3 ± 2.340.775 AUC0–60 min (nmol/L × min)2.39 ± 0.091.92 ± 0.13<0.001 AUC60–255 min (nmol/L × min)31.5 ± 2.6031.4 ± 2.280.985C-peptide Baseline (pmol/L)360 ± 21.4350 ± 15.80.673 Peak (pmol/L)1,524 ± 1061,647 ± 1250.317 Time to peak (min)136 ± 9.01160 ± 11.20.028 Postprandial baseline (pmol/L)365 ± 20.2313 ± 15.90.010 AUC (nmol/L × min)218 ± 11.7222 ± 11.50.697 AUC0–60 min (nmol/L × min)22.5 ± 1.1819.7 ± 1.000.018 AUC60–255 min (nmol/L × min)196 ± 11.2202 ± 10.90.490C-peptide/glucose ratio Baseline (pmol/mmol)71.4 ± 3.9369.8 ± 2.820.675 Peak (pmol/mmol)269 ± 17.9268 ± 17.60.959 Time to peak (min)140 ± 9.24153 ± 10.30.179 Postprandial baseline (pmol/mmol)73.6 ± 4.0361.4 ± 3.030.002 AUC (nmol/mmol × min)41.4 ± 2.3440.8 ± 1.890.705 AUC0–60 min (nmol/mmol × min)4.57 ± 0.233.92 ± 0.180.003 AUC60–255 min (nmol/mmol × min)36.8 ± 2.2336.8 ± 1.781.000Glucagon Baseline (pmol/L)6.57 ± 0.767.23 ± 0.680.237 Peak (pmol/L)13.0 ± 0.9411.2 ± 0.720.003 Time to peak (min)116 ± 12.5117 ± 17.10.926 Postprandial baseline (pmol/L)7.35 ± 0.825.35 ± 0.670.005 AUC (nmol/L × min)2.26 ± 0.201.74 ± 0.14<0.001 AUC0–60 min (nmol/L × min)0.446 ± 0.0440.375 ± 0.0350.038 AUC60–255 min (nmol/L × min)1.82 ± 0.171.36 ± 0.13<0.001Free fatty acids Baseline (μmol/L)339 ± 18.4361 ± 20.90.419 Nadir (μmol/L)58.4 ± 3.9457.7 ± 3.700.860 Time to nadir (min)155 ± 7.63168 ± 11.10.317 Postprandial baseline (μmol/L)272 ± 16.4360 ± 28.00.013 AUC (mmol/L × min)43.2 ± 2.4948.1 ± 2.740.171 AUC0–60 min (mmol/L × min)18.9 ± 1.1820.9 ± 1.430.258 AUC60–255 min (mmol/L × min)24.4 ± 1.5127.2 ± 1.540.150Glycerol Baseline (μmol/L)26.6 ± 2.4528.4 ± 3.120.379 Nadir (μmol/L)11.7 ± 2.1212.1 ± 2.440.761 Time to nadir (min)144 ± 10.2164 ± 11.80.201 Postprandial baseline (μmol/L)22.9 ± 2.1231.4 ± 3.650.007 AUC (mmol/L × min)4.87 ± 0.575.49 ± 0.700.080 AUC0–60 min (mmol/L × min)1.65 ± 0.151.95 ± 0.190.039 AUC60–255 min (mmol/L × min)3.22 ± 0.443.54 ± 0.540.211Triglycerides Baseline (μmol/L)920 ± 62.2953 ± 70.40.539 Peak (μmol/L)1,275 ± 84.81,353 ± 1130.279 Time to peak (min)195 ± 5.33192 ± 6.850.725 Postprandial baseline (μmol/L)847 ± 48.3916 ± 73.20.159 AUC (mmol/L × min)263 ± 16.7279 ± 22.10.251 AUC0–60 min (mmol/L × min)53.5 ± 3.1456.8 ± 4.410.284 AUC60–255 min (mmol/L × min)210 ± 13.8222 ± 17.90.258Growth hormone Baseline (ng/mL)0.594 ± 0.3000.299 ± 0.1060.372 Nadir (ng/mL)0.042 ± 0.0040.084 ± 0.0120.001 Time to nadir (min)116 ± 17.2128 ± 13.90.592 Postprandial baseline (ng/mL)0.121 ± 0.0480.432 ± 0.1330.014 AUC (ng/mL × min)33.8 ± 11.8106 ± 18.30.001 AUC0–60 min (ng/mL × min)20.2 ± 9.7632.0 ± 9.570.312 AUC60–255 min (ng/mL × min)13.6 ± 2.3273.7 ± 14.5<0.001Acyl ghrelin Baseline (ng/mL)0.155 ± 0.0110.158 ± 0.0100.730 Nadir (ng/mL)0.088 ± 0.0060.084 ± 0.0040.343 Postprandial baseline (ng/mL)0.161 ± 0.0120.172 ± 0.0130.264 AUC (ng/mL × min)31.2 ± 1.9431.7 ± 1.960.634 AUC0–60 min (ng/mL × min)9.50 ± 0.679.90 ± 0.650.387 AUC60–255 min (ng/mL × min)21.7 ± 1.3321.8 ± 1.340.875Acetaminophen Peak (μmol/L)65.1 ± 2.5266.3 ± 2.750.548 Time to peak (min)209 ± 6.34215 ± 7.930.297Data are presented as mean ± SEM. AUC, area under the curve; LEAP2, liver-expressed antimicrobial peptide 2. Open table in a new tab Data are presented as mean ± SEM. AUC, area under the curve; LEAP2, liver-expressed antimicrobial peptide 2. We measured plasma concentrations of insulin, C-peptide, glucagon, and calculated C-peptide/glucose ratio—the latter as a measure of pancreatic beta cell secretion—before and after the liquid mixed meal test (i.e., during fasting and postprandial conditions) in the healthy, young men. In the fasting state, the circulating concentrations of insulin and C-peptide and C-peptide/glucose ratio were higher during LEAP2 infusion compared with placebo, as assessed by AUC0–60 min (Figures 2C–2E; Table 2). Accordingly, postprandial baseline values (i.e., at the end of the fasting period, time = 60 min) were higher for both insulin, C-peptide, and C-peptide/glucose ratio (Figures 2C–2E; Table 2). The insulinotropic effect of LEAP2 during fasting was supported by a shorter time to peak for C-peptide during LEAP2 infusion (Table 2). An increase of ∼2 pmol/L in glucagon concentrations was observed both in the fasting state and postprandially during LEAP2 infusion (Figure 2F; Table 2). Plasma concentrations of free fatty acids, glycerol, and triglycerides were measured during fasting and postprandial conditions (Figures 2G–2I; Table 2). In the fasting state, circulating glycerol (AUC0–60 min) concentrations were lower during LEAP2 infusion compared with placebo infusion (Figure 2H; Table 2), suggesting a decreased lipolytic activity. No differences in AUC0–60 min values were found for free fatty acids or triglycerides, but postprandial baseline concentrations (time = 60 min) of free fatty acids and glycerol were lower during LEAP2 infusion (Table 2). Because ghrelin is known to increase the gastric emptying rate,19Levin F. Edholm T. Schmidt P.T. Grybäck P. Jacobsson H. Degerblad M. Höybye C. Holst J.J. Rehfeld J.F. Hellström P.M. et al.Ghrelin stimulates gastric emptying and hunger in normal-weight humans.J. Clin. Endocrinol. Metab. 2006; 91: 3296-3302Google Scholar we admixed acetaminophen to the liquid mixed meal in order to evaluate postprandial plasma excursions of acetaminophen as an indirect marker of gastric emptying, as previously validated.20Willems M. Otto Quartero A. Numans M.E. How useful is paracetamol absorption as a marker of gastric emptying? A systematic literature study.Dig. Dis. Sci. 2001; 46: 2256-2262Google Scholar Peak plasma acetaminophen concentrations and time to peak plasma acetaminophen concentrations revealed no difference between LEAP2 and placebo infusions (Figure 2J; Table 2), suggesting that exogenous LEAP2 does not alter gastric emptying rate of a liquid mixed meal. GH release reflects activity of the GHSR and is stimulated by acute administration of ghrelin.21Takaya K. Ariyasu H. Kanamoto N. Iwakura H. Yoshimoto A. Harada M. Mori K. Komatsu Y. Usui T. Shimatsu A. et al.Ghrelin strongly stimulates growth hormone (GH) release in humans.J. Clin. Endocrinol. Metab. 2000; 85: 4908-4911Google Scholar To test whether exogenous LEAP2 reduces the production or secretion of GH and ghrelin, we measured plasma concentrations of both hormones (Figures 2K and 2L; Table 2). Notably, GH concentrations were suppressed during LEAP2 infusion compared with placebo, as assessed by AUC (Table 2). When assessing the fasting (AUC0–60 min) and postprandial (AUC60–255 min) periods separately, only the postprandial period was lower during LEAP2 infusion compared with placebo (Table 2), suggesting that LEAP2 mainly suppresses GH postprandially. The postprandial GH suppression was supported by a lower nadir during LEAP2 infusion compared with placebo infusion (Table 2). Plasma concentrations of ghrelin (measured as active acylated ghrelin) were unaffected by LEAP2 infusion (Table 2); hence, the metabolic effects of exogenous LEAP2 in healthy men do not seem to be a result of altered plasma concentrations of ghrelin. Together, these results support previous findings on suppression of ghrelin-induced GH release in a dose-dependent manner in rodents5Ge X. Yang H. Bednarek M.A. Galon-Tilleman H. Chen P. Chen M. Lichtman J.S. Wang Y. Dalmas O. Yin Y. et al.LEAP2 is an endogenous antagonist of the ghrelin receptor.Cell Metab. 2018; 27: 461-469.e6Google Scholar,14Islam M.N. Mita Y. Maruyama K. Tanida R. Zhang W. Sakoda H. Nakazato M. Liver-expressed antimicrobial peptide 2 antagonizes the effect of ghrelin in rodents.J. Endocrinol. 2020; 244: 13-23Google Scholar and suggest that LEAP2 may reduce GH release independently of plasma concentrations of ghrelin in humans. As ghrelin infusion in humans has been reported to increase appetite sensations,13Druce M.R. Wren A.M. Park A.J. Milton J.E. Patterson M. Frost G. Ghatei M.A.
18 citations
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TL;DR: In this paper, a variety of in vivo and in vitro studies were performed to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner.
12 citations
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TL;DR: In this article, the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on voltage-gated calcium channels type 2.2 (CaV2.2) was investigated.
Abstract: The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.
9 citations
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TL;DR: In this paper , the authors discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin, and conclude that studies using rats have provided the most congruent evidence indicating that the VN mediates some actions of Ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations.
Abstract: Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.
7 citations
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TL;DR: The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelIn, a peptide specifically releases GH both in vivo and in vitro.
Abstract: Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivo and in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide 'ghrelin' (ghre is the Proto-Indo-European root of the word 'grow'). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.
8,073 citations
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TL;DR: It is proposed that ghrelin, in addition to its role in regulating GH secretion, signals the hypothalamus when an increase in metabolic efficiency is necessary, suggesting an involvement in regulation of energy balance.
Abstract: The discovery of the peptide hormone ghrelin, an endogenous ligand for the growth hormone secretagogue (GHS) receptor, yielded the surprising result that the principal site of ghrelin synthesis is the stomach and not the hypothalamus Although ghrelin is likely to regulate pituitary growth hormone (GH) secretion along with GH-releasing hormone and somatostatin, GHS receptors have also been identified on hypothalamic neurons and in the brainstem Apart from potential paracrine effects, ghrelin may thus offer an endocrine link between stomach, hypothalamus and pituitary, suggesting an involvement in regulation of energy balance Here we show that peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats Intracerebroventricular administration of ghrelin generated a dose-dependent increase in food intake and body weight Rat serum ghrelin concentrations were increased by fasting and were reduced by re-feeding or oral glucose administration, but not by water ingestion We propose that ghrelin, in addition to its role in regulating GH secretion, signals the hypothalamus when an increase in metabolic efficiency is necessary
3,894 citations
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TL;DR: The hypothesis that ghrelin plays a physiological role in meal initiation in humans is supported by the clear preprandials rise and postprandial fall in plasma ghrelIn levels.
Abstract: The recently discovered orexigenic peptide ghrelin is produced primarily by the stomach and circulates in blood at levels that increase during prolonged fasting in rats. When administered to rodents at supraphysiological doses, ghrelin activates hypothalamic neuropeptide Y/agouti gene-related protein neurons and increases food intake and body weight. These findings suggest that ghrelin may participate in meal initiation. As a first step to investigate this hypothesis, we sought to determine whether circulating ghrelin levels are elevated before the consumption of individual meals in humans. Ghrelin, insulin, and leptin were measured by radioimmunoassay in plasma samples drawn 38 times throughout a 24-h period in 10 healthy subjects provided meals on a fixed schedule. Plasma ghrelin levels increased nearly twofold immediately before each meal and fell to trough levels within 1 h after eating, a pattern reciprocal to that of insulin. Intermeal ghrelin levels displayed a diurnal rhythm that was exactly in phase with that of leptin, with both hormones rising throughout the day to a zenith at 0100, then falling overnight to a nadir at 0900. Ghrelin levels sampled during the troughs before and after breakfast correlated strongly with 24-h integrated area under the curve values (r = 0.873 and 0.954, respectively), suggesting that these convenient, single measurements might serve as surrogates for 24-h profiles to estimate overall ghrelin levels. Circulating ghrelin also correlated positively with age (r = 0.701). The clear preprandial rise and postprandial fall in plasma ghrelin levels support the hypothesis that ghrelin plays a physiological role in meal initiation in humans.
2,869 citations
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TL;DR: The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by gh Relin derived from the stomach, which plays important roles for maintaining GH release and energy homeostasis in vertebrates.
Abstract: Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through the GHS-R, a G protein-coupled receptor whose ligand has only been discovered recently. Using a reverse pharmacology paradigm with a stable cell line expressing GHS-R, we purified an endogenous ligand for GHS-R from rat stomach and named it "ghrelin," after a word root ("ghre") in Proto-Indo-European languages meaning "grow." Ghrelin is a peptide hormone in which the third amino acid, usually a serine but in some species a threonine, is modified by a fatty acid; this modification is essential for ghrelin's activity. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. In addition, ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus, a region known to control food intake. Ghrelin is orexigenic; it is secreted from the stomach and circulates in the bloodstream under fasting conditions, indicating that it transmits a hunger signal from the periphery to the central nervous system. Taking into account all these activities, ghrelin plays important roles for maintaining GH release and energy homeostasis in vertebrates.
2,740 citations
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TL;DR: Ghrelin is the first circulating hormone demonstrated to stimulate food intake in man and is a potentially important new regulator of the complex systems controlling food intake and body weight.
Abstract: Ghrelin is a recently identified endogenous ligand for the growth hormone secretagogue receptor. It is synthesized predominantly in the stomach and found in the circulation of healthy humans. Ghrelin has been shown to promote increased food intake, weight gain and adiposity in rodents. The effect of ghrelin on appetite and food intake in man has not been determined. We investigated the effects of intravenous ghrelin (5.0 pmol/kg/min) or saline infusion on appetite and food intake in a randomised double-blind cross-over study in nine healthy volunteers. There was a clear-cut increase in energy consumed by every individual from a free-choice buffet (mean increase 28 ± 3.9%, p<0.001) during ghrelin compared with saline infusion. Visual analogue scores for appetite were greater during ghrelin compared to saline infusion. Ghrelin had no effect on gastric emptying as assessed by the paracetamol absorption test. Ghrelin is the first circulating hormone demonstrated to stimulate food intake in man. Endogenous ghr...
2,476 citations