Investigating the Effects of 24 Hour LEAP2 Infusion in Individuals With Overweight and Obesity

Ghrelin is the only known circulating gut hormone with an appetite-stimulating (orexigenic) effect, acting through the growth hormone secretagogue receptor to increase food intake, promote fat storage, influence glucose metabolism, stimulate growth hormone release, and accelerate gastric emptying. Because this receptor retains some activity independent of ghrelin binding, blocking it may reveal effects beyond simple hormone suppression. LEAP2 (liver-expressed antimicrobial peptide 2) is a naturally occurring hormone that acts as a competitive antagonist and inverse agonist at the ghrelin receptor, offering a specific tool to transiently block ghrelin signaling in humans. The investigators' group has previously shown that short-term LEAP2 infusion reduces ad libitum food intake and improves postprandial glucose excursions in both lean individuals and individuals with obesity. Whether these effects persist, or how they evolve, over a full 24-hour period of ghrelin receptor blockade has not been established, and this study is designed to address that gap.

Eligible participants will attend a screening visit including medical history, biometric measurements, bioelectrical impedance analysis of body composition, blood pressure and heart rate, a screening blood panel, and (for female participants) a urine pregnancy test. Food-related addictive behavior will be characterized using the Yale Food Addiction Scale 2.0 to describe the study population.

Each participant will complete two experimental days at the Center for Clinical Metabolic Research, Gentofte/Herlev Hospital, separated by a washout of at least 7 days; for female participants, both visits will be scheduled in the same phase of the menstrual cycle. Before each visit, participants follow a standardized pre-visit protocol: replicating the same self-selected evening meal, an overnight fast, and avoidance of alcohol, strenuous exercise, prolonged fasting, and excessive caloric intake in the preceding 48 hours. A continuous glucose monitor is applied at least 24 hours before each visit.

On each 24-hour visit day, two intravenous cannulas are placed (one for infusion, one for arterialized venous blood sampling). Following a supine baseline assessment, participants receive a continuous intravenous infusion of either LEAP2 (40 pmol/kg/min) or matching saline placebo, prepared and labeled by an unblinded staff member not otherwise involved in study procedures. At 45 minutes, a standardized liquid mixed meal containing paracetamol (used to track gastric emptying) is consumed. Standardized ad libitum lunch, dinner, and breakfast meals are offered at approximately 5, 11, and 23 hours after infusion start, with continuous access to a standardized snack buffet between meals; amounts consumed are determined by weighing food before and after each meal. A structured light-activity protocol (short walks after lunch and dinner) is included to standardize energy expenditure conditions.

Throughout each visit, the study collects blood and urine samples at predefined time points; hourly visual analogue scale (VAS) assessments of hunger, fullness, satiety, prospective food intake, nausea, thirst, and comfort (except during the nocturnal period); indirect calorimetry at baseline and before each meal to measure resting energy expenditure and substrate oxidation; and the Leeds Food Preference Questionnaire at baseline and before the lunch and breakfast meals to assess explicit and implicit food preferences. Blood pressure, heart rate, and continuous glucose values are monitored throughout. After the breakfast meal, a subcutaneous adipose tissue biopsy is obtained under local anesthesia for transcriptomic, proteomic, and histological analysis.

Statistical analyses will use linear mixed models with treatment and period as fixed effects and an unstructured covariance structure to account for repeated measurements and potential variance heterogeneity between interventions, consistent with ICH E9 statistical guidance for crossover trials. The primary analysis will compare 24-hour ad libitum caloric intake between LEAP2 and placebo; secondary endpoints will be adjusted for multiple testing using the Benjamini-Hochberg method; exploratory endpoints will be reported descriptively with confidence intervals without formal hypothesis testing. The sample size of 18 completers was calculated based on a within-subject standard deviation for 24-hour energy intake derived from the investigators' prior LEAP2 studies, targeting detection of a clinically relevant 800 kJ reduction with 80% power at a two-sided alpha of 0.05.