LEAP2 reduces postprandial glucose excursions and ad libitum food intake in healthy men

Summary 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.

In brief LEAP2 was recently discovered as a hormone in rodents, which modulates ghrelin actions on plasma glucose and appetite. In a randomized, double-blind, placebo-controlled trial, Hagemann et al. demonstrate that exogenous LEAP2 reduces postprandial glucose excursions and food intake in healthy men.

INTRODUCTION
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. 1 The gastric hormone ghrelin, an endogenous GHSR agonist, stimulates food intake and gastrointestinal motility and increases plasma glucose. 1,2 Ghrelin, a peptide hormone, requires an acylation to obtain full activity, and its expression is regulated according to energy status. 1,2 Thus, plasma ghrelin concentrations rise during conditions with energy deficit, such as fasting and calorie restriction, and fall after food intake or with obesity. 3,4 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. 5 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. 4,6,7 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 a-melanocyte-stimulating hormone and inhibited by agouti-related peptide. 8 LEAP2 is synthesized as a 77-amino-acid prepropeptide and is processed into several truncated forms, including the mature 40-amino-acid residue peptide (LEAP2  ; here called LEAP2), which is identical in mice and humans. 5 Plasma LEAP2 concentrations are regulated inversely compared with plasma ghrelin in several metabolic settings. 4 Hence, LEAP2 concentrations have been reported to decrease during weight loss and increase with obesity. 4 Whether plasma concentrations of LEAP2 increase in response to food intake in humans as seen in rodents remains to be clarified. 4,7 Mani et al. 4 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. 7 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, LEAP2 [38][39][40][41][42][43][44][45][46][47] , however without glucoregulatory effect in a clinical proof-of-concept trial. 7 Interestingly, LEAP2 [38][39][40][41][42][43][44][45][46][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. 6,7 Thus, the pharmacological potential of LEAP2 family peptides deserves to be further investigated.
To date, bariatric surgery is the most effective treatment of obesity. 9 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, 10,11 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 ghrelin 4 and elevated expression levels following RYGB. 7 The administration of ghrelin in rodents and humans stimulates food intake and increases plasma glucose. 5,12,13 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; 5 however, this finding could not be confirmed in succeeding studies. 6,7,14 Nevertheless, LEAP2 treatment has consistently been shown to impair ghrelininduced food intake and hyperglycemia in rodents. 5,6,[14][15][16] 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 mice 17 and reduced binge-like eating after central administration of a truncated LEAP2 form in mice. 18 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.

RESULTS
Intravenous infusion of LEAP2 results in supraphysiological steady-state plasma concentrations in lean men 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, placebocontrolled 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.
Exogenous LEAP2 alters the postprandial plasma glucose response 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 (AUC 0-255 min ) and the postprandial AUC (AUC 60-255 min ) were lower during LEAP2 infusion compared with placebo (Table 2), demonstrating a reduced postprandial glucose response during LEAP2 infusion.
Insulinotropic and anti-lipolytic effects of exogenous LEAP2 during fasting 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 AUC 0-60 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  (20)(21)(22)(23)(24)(25) fasting state, circulating glycerol (AUC 0-60 min ) concentrations were lower during LEAP2 infusion compared with placebo infusion ( Figure 2H; Table 2), suggesting a decreased lipolytic activity.
No differences in AUC 0-60 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).

Exogenous LEAP2 does not alter gastric emptying
Because ghrelin is known to increase the gastric emptying rate, 19 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. 20 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.
Exogenous LEAP2 decreases plasma concentrations of GH, but not ghrelin GH release reflects activity of the GHSR and is stimulated by acute administration of ghrelin. 21 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 (AUC 0-60 min ) and postprandial (AUC 60-255 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 rodents 5,14 and suggest that LEAP2 may reduce GH release independently of plasma concentrations of ghrelin in humans. The study was designed as a randomized, doubleblind, placebo-controlled, crossover study including 20 healthy, young men. IC, indirect calorimetry; LEAP2, liver-expressed antimicrobial peptide 2; TC, thermal imaging camera; VAS, visual analog scale.
No effect of LEAP2 on sensations of hunger, satiety, prospective food consumption, fullness, nausea, comfort, and thirst As ghrelin infusion in humans has been reported to increase appetite sensations, 13 we investigated the effect of LEAP2 on several appetite-related sensations using visual analog scale (VAS) ratings. We found no differences in VAS ratings of hunger, satiety, prospective food consumption, fullness, nausea, comfort, or thirst from experimental days with LEAP2 and placebo infusions (Figures 3A-3G). Moreover, we found no differences between LEAP2 and placebo infusions when comparing the VAS ratings given just before (time = 255 min) as well as just after (time $272 min) the ad libitum meal. Thus, changes in VAS ratings of hunger, prospective food consumption, or nausea could not explain the reduced food intake observed during LEAP2 infusion. Importantly, VAS ratings indicated a low level of nausea and a high level of comfort throughout both study days ( Figures 3E and 3F).
Exogenous LEAP2 does not affect resting energy expenditure, utilization of metabolic substrates, or brown adipose tissue thermogenesis Since ghrelin has been proposed as a regulator of systemic energy expenditure, 1 we investigated the effect of exogenous LEAP2 on resting energy expenditure, respiratory quotient, and protein turnover in healthy individuals assessed by indirect calorimetry and renal urea excretion, respectively. Using this approach, we found no differences in resting energy expenditure or respiratory quotient between LEAP2 and placebo infusions ( Figure 3H; respiratory quotient data not shown). Moreover, we found no difference in renal urea excretion at the two consecutive time points or total urea excretion between study days (data not shown). As ghrelin has been suggested to suppress brown adipose tissue (BAT) activity in rodents, 1 we also investigated BAT thermogenesis using a thermal imaging camera measuring temperatures of the skin above the two supraclavicular BAT depots and over the manubrium of the sternum; the latter as a control reflecting overall cutaneous circulation. We found no differences in BAT thermogenesis between LEAP2 and placebo infusions calculated as the average of the supraclavicular skin temperatures subtracting the manubrium skin temperature ( Figure 3I), indicating that LEAP2 does not affect BAT thermogenesis in humans.
Exogenous LEAP2 reduces food intake during an ad libitum meal Next, food intake was assessed by an ad libitum meal test at the end of the infusion period. Here, we found an $12% reduction in energy intake in the healthy, young men during LEAP2 infusion compared with placebo demonstrated by reduced total caloric intake and caloric intake per kg body weight ( Figures 4A and  4B). In addition to reduced food intake, a shorter meal duration during LEAP2 infusion was also observed ( Figure 4C).

GHSR-dependent effect of LEAP2 dosing on plasma glucose and food intake in mice
We evaluated the effects of a single subcutaneous dose of LEAP2 on glucose metabolism and food intake in GHSR-null mice and wild-type littermates to tease out GHSR-dependent effects. In line with the observed effects in humans, LEAP2 dosing significantly diminished increases in plasma glucose during ad libitum food intake 1 h after dosing in wild-type mice as compared with vehicle ( Figure 5A). In contrast, a similar dose of LEAP2 did not affect plasma glucose in GHSR-null mice ( Figure 5A), indicating that the glucose-lowering effect of LEAP2 in the postprandial state may be mediated through the GHSR. Of note, the glucose-lowering effect of LEAP2 was only observed after 1 h and not at the later time points ( Figure 4A). In line with the human   Figure 5B), but no effect of LEAP2 was found in GHSR-null mice ( Figure 5B). Together, these results suggest that the GHSR is a prerequisite for the observed metabolic effects of LEAP2 in mice and provide in vivo insights into LEAP2 as a GHSR ligand with opposing actions to the orexigenic hormone ghrelin. In a separate cohort of fasted female wild-type mice, we observed no effect of exogenous LEAP2 on fasting insulin concentrations following LEAP2 dosing ( Figure 5C), contrasting our human data. As in humans, LEAP2 did not affect fasting plasma glucose concentrations in wildtype mice either ( Figure 5D).

Pharmacokinetic parameters of exogenous LEAP2 in healthy men
In order to describe the kinetics of exogenous LEAP2, 8 of the 20 study participants were subjected to a separate intravenous infusion of LEAP2 reaching supraphysiological steadystate plasma concentrations followed by a 150-min washout period ( Figure S1A). Based on a pharmacokinetic model taking baseline plasma concentrations of LEAP2 into account, 22 we estimated the volume of distribution of LEAP2 to 14 ± 5.3 L, metabolic clearance rate of LEAP2 to 0.86 ± 0.17 L/min, half-life of LEAP2 to 9.1 ± 2.5 min, and endogenous production rate of LEAP2 to 1.9 ± 0.14 nmol/min. Taken

DISCUSSION
LEAP2 was recently discovered as an endogenous circulating hormone in rodents with antagonistic effects on ghrelin actions and inverse effects on GHSR constitutive activity. 5,6 Here, we demonstrate that exogenous LEAP2 reduces postprandial glucose excursions and suppresses food intake in healthy men. In addition, we report a lower glucose peak and food intake in wild-type mice, but not in GHSR-null mice, after LEAP2 administration, suggesting that the plasma glucose-and appetitelowering effects of LEAP2 are mediated by the ghrelin receptor, GHSR, in mice. The observed effects on both glucose homeostasis and appetite regulation may revitalize the GHSR as therapeutic target in metabolic diseases in the future.
A pioneering study characterizing the pharmacological effects of LEAP2 in rodents 5 demonstrated a lower food intake in mice treated with LEAP2 compared with vehicle-treated mice. Using a randomized, double-blind, placebo-controlled crossover design, we now demonstrate that exogenous LEAP2 reduces food intake during an ad libitum meal test (of pasta Bolognese) in healthy men. On average, participants consumed 432 kJ less during LEAP2 infusion compared with placebo (corresponding to an $12% reduction) and spent less time eating before feeling comfortably full. For comparison, 432 kJ corresponds approximately to the energy content in a 25 cL soft drink. Importantly, this anorectic effect was observed without a concomitant increase in nausea. A sustained reduction of 432 kJ per meal for longer periods would translate into robust weight loss, but it remains to be determined whether the effect is reproducible in overweight individuals and whether it is sustained with prolonged dosing of LEAP2 and whether the response can be optimized by altering the LEAP2 dosage. For perspective, acute GLP-1 infusion (inducing an $8-fold increase in plasma concentration of GLP-1) in healthy, normal-weight, young men resulted in a similar reduction in energy intake of $12% during a comparable ad libitum meal test. 23 Interestingly, the appetite-suppressing effect of LEAP2 was reproduced in wild-type mice, but not in GHSR-null mice, showing that the GHSR is required for the reduced food intake observed in LEAP2-treated mice. Our data confirm the finding by Ge et al. 5 demonstrating a lower food intake in LEAP2-treated compared with vehicle-treated mice, in contrast to a number of other studies. 6,7,14 The inconsistent finding on LEAP2-induced modulation of food intake may be related to the level of stress in the studied animals, as stress hormones strongly induce ghrelin secretion. In our study designed to measure plasma glucose, we expect high level of stress and accordingly high plasma ghrelin concentrations. However, the fact that LEAP2 administration consistently has been shown to reverse ghrelin-induced food intake 5,6,14,16 makes it conceivable that LEAP2 mainly acts as a competitive antagonist impairing ghrelin-induced activation of GHSR in rodents. This is further supported by a recent study indicating that LEAP2 deletion in mice enhances the actions of ghrelin. 15 Of note, it remains to be explored whether exogenous LEAP2 reduces food intake in animal models of impaired metabolic control, such as diet-induced obesity.
The insulinotropic properties of the LEAP2 family of peptide hormones in vitro 6,7 prompted us to investigate the effect of exogenous LEAP2 on postprandial glucose metabolism in healthy humans. Here, we report reduced postprandial glucose excursions following a standardized liquid mixed meal during LEAP2 infusion. Plasma profiles of insulin and C-peptide revealed an insulinotropic effect of LEAP2 during fasting, but not postprandially. A concomitant increase in the C-peptide/glucose ratio during fasting indicates that LEAP2 may increase the b cell sensitivity to glucose in the fasted state without enhancing glucose-stimulated insulin secretion after meal ingestion, which is in agreement with our recent findings for the endogenous LEAP2 fragment, LEAP2 [38][39][40][41][42][43][44][45][46][47] . 7 The mechanisms underlying the reduced postprandial plasma glucose excursions observed in the present study remain uncertain, but we speculate that LEAP2 may act through brain-mediated regulation of hepatic glucose production, as seen with insulin, 24 or increase peripheral insulin sensitivity, the latter possibly as an indirect effect to decreased plasma concentrations of GH. 25 Moreover, we cannot rule out that the increased insulin concentrations observed during LEAP2 infusion in the fasting state may prime insulin-sensitive tissues before meal ingestion. Gastric emptying was not altered by exogenous LEAP2, as previously reported for ghrelin, 19 suggesting that the lower postprandial plasma glucose peak observed during LEAP2 infusion is not a result of delayed gastric emptying of the meal.
We observed a small increment in plasma glucagon concentrations during LEAP2 infusion. The increase in plasma glucagon concentration may counteract the glucose-lowering effect of exogenous LEAP2. The GHSR has been localized to both a and b cells as well as somatostatin-producing d cells and pancreatic polypeptide cells in the human and mouse pancreas. 17,[26][27][28] It may be of interest, therefore, to investigate whether a GHSRdependent glucoregulatory effect of LEAP2 is affected by the intra-islet communication that is thought to exist between the different GHSR-expressing pancreatic cell types. 26,29 The LEAP2-induced reduction of postprandial plasma glucose excursions was reproducible in wild-type mice, but not in GHSRnull mice, when measuring plasma glucose 1 h after dosing. This suggests that the glucose-lowering effect of LEAP2 in mice is dependent on the GHSR. It should be noted that three studies have already investigated the effect of exogenous LEAP2 on glucose metabolism in rodents: (1) in a recent study in rats, 14 it was demonstrated that centrally injected LEAP2 suppresses the hyperglycemic effect of centrally injected ghrelin; (2) a decrease in plasma glucose following intraperitoneally administered LEAP2 in mice was recently reported; 17 and (3) in male NMRI mice, LEAP2 did not affect the blood glucose during an oral glucose tolerance test. 7 Taken together, these results suggest that any glucose-lowering effect of LEAP2 might depend on several factors, including way of LEAP2 administration, nature of the glycemia, and the phenotype of the organism investigated.
During fasting, GH promotes lipid mobilization and oxidation as a means of fat utilization. 25 Exogenous ghrelin has been reported to induce lipolysis in humans independently of GH, 30 mirroring the physiological effect of endogenous ghrelin and GH during fasting. 1 This prompted us to investigate the effect of exogenous LEAP2 on plasma concentrations of triglycerides, free fatty acids, and glycerol-the substrate and products, respectively, of lipolysis-in fasting and postprandial states. Compared with placebo, LEAP2 infusion decreased glycerol in the fasted state (AUC 0-60 min ) and plasma concentrations of both glycerol and free fatty acids were lower preprandially (at time point 60 min), suggesting inhibition of lipolysis during LEAP2 infusion. It remains to be established whether LEAP2 has any anti-lipolytic effect or whether lipolysis may be indirectly mediated via effects on insulin secretion and/or GH secretion. Because ghrelin has been proposed to modulate systemic energy expenditure, 1 we tested whether exogenous LEAP2 could have the opposite effect on resting energy expenditure, substrate utilization, and BAT thermogenesis. We observed no effect on resting energy expenditure or utilization of carbohydrates, fat, or protein-the latter assessed by respiratory quotient and renal urea excretion. Moreover, no effect on BAT thermogenesis was found. Limitations of these measurements should be considered. Indirect calorimetry depends on the participant's ability to rest, and in 23 tests out of 160, the readings did not reach steady state, probably due to the other study procedures and therefore limited test time. Thermal imaging of skin temperatures depends on other factors than BAT thermogenesis, such as the surrounding temperature. To correct for the overall cutaneous heat production in the air-conditioned room, we assessed BAT thermogenesis by manubrium-corrected supraclavicular skin temperatures. Of note, this study was powered to detect differences in food intake, and thus, regarding the secondary endpoints, our study might be underpowered.
We measured plasma concentrations of GH as a sign of GHSR activation, since this is the most sensitive physiological response to GHSR activation. 5 Plasma concentration of GH was lower during LEAP2 infusion compared with placebo, indicating reduced GHSR activity. Interestingly, plasma profiles of ghrelin did not differ between LEAP2 and placebo infusions, pointing to absence of feedback regulation of ghrelin secretion, known to occur for many other hormonal systems. Taken together, our data confirm previous in vitro findings that LEAP2 downregulates GHSR activity independently of ghrelin concentrations, 4,6,7 thus suggesting that the metabolic effects of exogenous LEAP2 observed in humans are mediated-at least partly-through GHSR. Whether the decrease in plasma concentration of GH observed postprandially in this study is involved in the reduction of the postprandial glucose excursions remains to be explored. As previously speculated, exogenous LEAP2 does not seem to inhibit the production or secretion of ghrelin. 5 The plasma profile of GH during placebo infusion followed a pattern similar to that of ghrelin, which is consistent with the known physiological regulation according to energy status, 31 i.e., increased concentrations during fasting, decreased postprandial concentrations, and returning to fasting concentrations at the end of the postprandial period. The pulsatile pattern of GH secretion may explain the large variation observed in the dataset. 32 Importantly, both GH and ghrelin plasma concentrations were within physiological ranges reported previously. 31 Until now, the effects of LEAP2 exposure have not been investigated in humans. Importantly, no adverse reactions were observed during the infusions. LEAP2 infusion resulted in a 2.6-fold steady-state concentration as compared with placebo infusion, reaching supraphysiological concentrations as compared with previous plasma measurements in lean individuals. 4 Plasma concentrations of LEAP2 during placebo infusion and baseline were within concentration ranges reported previously, 4,7 suggesting that LEAP2 is an endogenous peptide measured in the nanomolar range. Moreover, our data confirm that endogenous LEAP2 assessed during placebo infusion was not affected by a liquid mixed meal test in lean individuals in agreement with previous findings in lean individuals. 4 Whether endogenous LEAP2 concentrations change with food intake in overweight individuals is presently uncertain due to contrasting findings. 4,7 Of notice, we cannot rule out that the observed effects of exogenous LEAP2 would have been more pronounced employing a higher infusion rate of LEAP2, and future studies will, therefore, be required to investigate a possible doseresponse relationship. The pharmacokinetic model of exogenous LEAP2 shows a half-life of LEAP2 amounting to $9 min; thus, an extension of the half-life is necessary-for example, through lipidation-if LEAP2 should have a pharmacological potential in the future. Limitations of the study Plasma concentrations of insulin-like growth factor 1 were not measured. Also, the study was designed to investigate the effects of exogenous LEAP2 in healthy men and was not designed to answer mechanistic effects of exogenous LEAP2 in humans. Since only one dose of LEAP2 was used, the dose-response relationship remains to be investigated.
In conclusion, we demonstrate that exogenous LEAP2 resulting in supraphysiological plasma concentrations reduces postprandial glucose excursions and ad libitum food intake in young, healthy men. These effects were also observed in wild-type mice, but not in GHSR-null mice, suggesting that the glucoselowering and appetite-suppressing effects of LEAP2-at least partly-depend on the GHSR in mice. Together, these findings raise the possibility that LEAP2 might constitute a clinically relevant pharmacological target for the treatment of obesity.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  Exclusion criteria included anemia, serum creatinine above the normal range and/or albuminuria, history of hepatobiliary and/or gastrointestinal disorder and/or plasma liver enzymes (alanine or aspartate aminotransferases) above two times the normal range, allergy or intolerance to ingredients included in the standardized meals, first-degree relatives with diabetes and/or hemoglobin A 1c >48 mmol/mol [6.5%], regular tobacco smoking and/or use of other nicotine-containing products and any physical or psychological condition or ongoing medication that the investigators evaluated would interfere with trial participation.

Ethics approval
The study was approved by the Scientific-Ethical Committee of the Capital Region of Denmark (registration number H-19038628) and the Danish Data Protection Agency (approval number P-2020-125). The study was conducted in accordance with the Declaration of Helsinki (7 th revision, 2013) and registered at ClinicalTrials.gov (registration numbers NCT04621409 and NCT04897984). Informed consent was obtained from all participants before inclusion.

Mouse models
All animals had ad libitum access to water and a chow diet (Altromin, 1310, Brogaarden, Denmark) and were housed in a 22 C temperature-controlled environment under a 12:12 h light-dark cycle (lights on from 6 AM to 6 PM) unless otherwise stated. All animal studies were approved by the Danish Animal Experiments Inspectorate and performed according to institutional guidelines. Animal licenses: 2017-15-0202-00119 and 2019-15-0201-00289. Wild C57/BL6 mice were purchased from Janvier labs (Le Genest-Saint-Isle, France). GHSR-null mice were kindly donated by J. Zigman and have been used in a previous publication. 33 All mice had two weeks to habituate in their corresponding cages before start of studies.

METHOD DETAILS
Peptides and preparation of infusions Human LEAP2 was synthesized by Vivitide (Louisville, Kentucky, USA). The peptide was confirmed to be identical to the natural peptide by high-performance liquid chromatography and mass spectrometry, amino acid sequencing and elemental analysis by the manufacturer. The final purity was >98%. The LEAP2 was dissolved in saline (9 mg/mL) with 0.5% human albumin, sterile filtrated and tested for endotoxins and sterility by the Capital Region Pharmacy (Herlev, Denmark) before dispensed into vials stored at À20 C until use. To prevent oxidation of methionine in the LEAP2 amino acid sequence, this was performed under a flow of nitrogen, and vials were blanked with nitrogen before filling. On experimental days, infusions were prepared by an unblinded staff member diluting LEAP2 to a total volume of 500 mL in saline (9 mg/mL, Fresenius Kabi, Uppsala, Sweden) containing 0.5% human albumin (5% solution, CSL Behring, Lyngby, Denmark). Placebo infusions consisted of 500 mL saline with 0.5% human albumin. Apart from preparing the infusion solutions, the unblinded staff member was not involved in the study. The infusion rate of LEAP2 in humans was estimated from a previous study of mice subjected to a single intravenous dose of LEAP2. 5 Synthetic LEAP2 used for animal studies was synthesized by Novo Nordisk (Bagsvaerd, Denmark), confirmed identical to the natural peptide by high-performance liquid chromatography and dissolved in phosphate buffer (50 mmol/L; pH 7.4) and saline (70 mmol/L).

Clinical study design and experimental procedures
The study was designed as a randomized, double-blind, placebo-controlled, crossover study and included two experimental days with intravenous infusion of LEAP2 ($25 pmol/kg/min = 115 ng/kg/min) or placebo (saline) with minimum one week between visits. A prespecified randomization list was generated from www.sealedenvelope.com with a block size of four. The study was conducted from October 2020 to February 2021 at Center for Clinical Metabolic Research, Copenhagen University Hospital -Herlev and Gentofte, Copenhagen, Denmark. See Figure 1 for an overview of the clinical study design. Each experimental day was preceded by 12 h of fasting including any liquids and 48 h of abstinence from alcohol, strenuous physical activity, medicine and intermittent fasting and/ or excessive eating. Participants were instructed to keep a food diary for 48 h prior to experimental days. No experimental days were planned on Mondays since weekend activities often differ from activities during weekdays. In the evening before the experimental days (between 6 and 8 PM), the participants consumed a standardized meal (558 g pasta Bolognese; same energy content per 100 g as the ad libitum meal) and were asked to remain fasting until the experimental day. At the beginning of each experimental day, a cannula was inserted in a cubital vein in each arm, one for blood sampling and one for peptide infusion. The forearm for blood sampling was wrapped in a heating pad (45 C) to arterialize the venous blood. LEAP2 or placebo infusion was started at timepoint 0 min. At time 65-70 min, the participants were given a liquid mixed meal (2.97 mL/kg body weight; 1,010 kJ, 29.7 g carbohydrate, 9.6 g protein and 9.3 g fat per 100 mL; Nutricia Nutridrink, Allerød, Denmark) mixed with 1.5 g acetaminophen plus 50 mL of water. An ad libitum meal was served 260 min after start of the infusion and consisted of pasta Bolognese (565 kJ, 15.0 g carbohydrate, 5.3 g protein and 5.6 g fat per 100 mL) together with 400 mL of water. The ad libitum meal was served in undisturbed surroundings, and participants were instructed to eat until they felt comfortably full. The infusion was terminated when participants had finished eating.

Indirect calorimetry
Resting energy expenditure and respiratory quotient were measured by indirect calorimetry (Vyntus CPX Canopy; Vyaire Medical, Hö chberg, Germany) for 15 min at timepoints À25 (baseline), 45, 120 and 240 min, with the participants awake and in a supine position from 5 min prior to each test. Participants rested in a hospital bed for 30 min before the first test and throughout the experimental day. The gas and flow analyzers were calibrated before each test. The first 5 min and the last minute of data collection were subsequently discarded, and the mean of a 5-min steady state period defined as a coefficient of variation <10% for VO 2 and CO 2 was used to estimate resting energy expenditure and respiratory quotient for each test. The 5-min steady state period with the lowest resting energy expenditure and with a respiratory quotient between 0.7 and 1.0 was considered. In a few cases without a 5-min steady state period, a 4-min steady state period was considered. 35,36 In the present study, a steady state period of 5 min was successfully reached in 135 out of 160 tests. One participant was excluded from the subsequent data analysis due to no steady state period in any of the four tests during an experimental day. A 4-min steady state period was considered in six cases reaching a total of 23 excluded tests from the data analysis.

Thermal imaging
To assess BAT thermogenesis, temperature of the upper chest was measured from À10 to 0 min, 0 to 15 min, 90 to 105 min and 180 to 195 min using a thermal imaging camera (FLIR A655sc; FLIR Systems, Wilsonville, Oregon, USA). Participants were positioned approximately one meter from the camera in a hospital bed with slight elevation of the head and wearing long-sleeved shirts that exposed the upper chest. The emissivity for measurement of human skin temperatures was set to 0.98 as detailed by the manufacturer. Thermal images were collected at 30-s intervals and analyzed using the FLIR tools program (FLIR Systems, Wilsonville, Oregon, USA). Post hoc, the two supraclavicular regions and a region corresponding to the manubrium of the sternum were marked as regions of interest and the maximum temperature per image was recorded. An average of records from the two supraclavicular regions was used. The manubrium region was used as a negative control reflecting the overall cutaneous heat production. [37][38][39] For analysis of BAT thermogenesis, manubrium-corrected temperature was calculated as the average of the supraclavicular skin temperatures minus the manubrium skin temperature. The first and last minutes of each measurement period were omitted from analysis. Average room temperature was 22.5 ± 0.06 C adjusted by two air conditioners (Electrolux EXP35U538CW; AB Electrolux, Stockholm, Sweden) and recorded four times during each experimental day. Average humidity was 31.2 ± 0.39%. Due to technical issues with the camera, data from 3 of 40 experimental days are missing.

Urine sampling
To assess the amount of protein breakdown after the liquid mixed meal, urine samples were collected at timepoints 135 and 255 min for measurement of renal urea excretion and total urine production. At timepoint 60 min, 5 min prior to ingestion of the liquid mixed meal, participants were asked to empty their urinary bladder. Urine samples were stored at À80 C until analysis, and urine urea was measured by spectrophotometric methods following enzymatic hydrolysis and oxidation (Siemens Atellica CH 930; Siemens Healthcare, Ballerup, Denmark).

Human plasma analyses
For analysis of plasma glucose, blood was distributed in fluoride heparin-coated tubes, centrifuged immediately for 30 s (12,100 g, room temperature) and measured bedside by the glucose oxidase method (YSI 2900 Biochemistry Analyzer; YSI Incorporated, Yellow Springs, Ohio, USA). For analysis of LEAP2, blood was collected in chilled K 3 EDTA tubes each containing 250 KIU of aprotinin. For analysis of acyl ghrelin, blood was collected in chilled K 2 EDTA tubes added 20 mL pefabloc (100 mg/mL; Merck Millipore, Burlington, Massachusetts, USA). Subsequently, pefabloc-treated plasma was acidified with HCl (12.5 mL 2M HCl per 500 mL plasma; Merck Millipore, Burlington, Massachusetts, USA). For analysis of glucagon, blood was collected in K 2 EDTA tubes. For analysis of GH, insulin, C-peptide, free fatty acids, glycerol, triglycerides and acetaminophen (paracetamol), blood was collected in tubes with lithium and heparin. All tubes were immediately cooled on ice, centrifuged for 15 min (2,000 g, 4 C) and stored at À80 C until analysis. LEAP2 concentrations were determined by an in-house radioimmunoassay directed against the N-terminal part of LEAP2. LEAP-2 was used for standards, and the tracer was a peptide composed of the 12 N-terminal amino acids of LEAP2, with a 125 I-labelled tyrosine added to the C-terminal end. The precision and accuracy of the assay were evaluated based on European Medicines Agency's guidelines for bioanalytical assay validation. The detection range of the assay was 5-500 pmol/L. Intra-and inter-assay variation was below 12%. Acyl ghrelin was measured by use of a radioimmunoassay kit (GHRA-88HK, Merck Millipore, Burlington, Massachusetts, Student's paired t test was used to test for differences in baseline, peak, time to peak, AUC and mean values and ad libitum food intake in men. Indirect calorimetry and thermal imaging data were fitted to a repeated-measures mixed effects model with the intervention and timepoints as independent variables with interaction and Geisser-Greenhouse correction. Sidá k's post hoc test was used to compare LEAP2 to placebo for each timepoint. For analysis of endogenous LEAP2 concentrations in response to a liquid mixed meal test, postprandial data during placebo infusion were fitted to a repeated-measures mixed effects model with time as a fixed effect and the individual participant as a random effect with Geisser-Greenhouse correction. Dunnett's post hoc test was used to compare endogenous LEAP2 concentrations at each timepoint (75 to 255 min) to the postprandial baseline. Mouse plasma glucose, food intake and insulin data were analyzed by a repeated-measures two-way ANOVA with Sidá k's post hoc test. Statistical analyses were carried out using GraphPad Prism v9.0.0 and v9.1.1 (GraphPad Software, San Diego, California). A twosided p value < 0.05 was considered statistically significant. The population size calculation was based on data from a previous study investigating the reproducibility of an ad libitum meal in healthy men. 41 Eighteen participants were needed to detect a difference of minimum 500 kJ in ad libitum food intake with a with-in subject difference of 739 kJ, a power of 80% and the above-mentioned significance level. To ensure sufficient power, 20 participants were included.
We constructed and applied a pharmacokinetic model to the data using the software Monolix (Monolix version 2019R1; Lixoft SAS, Antony, France). The parameters were estimated using the Stochastic Approximation EM algorithm. 22 The one-compartment model assumed input from both endogenous LEAP2 secretion and the exogenous LEAP2 infusion assuming that LEAP2 kinetics followed a single-exponential elimination. We assumed that the endogenous production of LEAP2 was constant i.e. not suppressed during infusion of exogenous LEAP2. This model probably represents a simplification of the actual physiological LEAP2 kinetics, but the data fitted well to model predictions.