Abstract Body (Do not enter title and authors here): Background:Dysregulated lipid handling in the intestine contributes to dyslipidemia and atherosclerotic cardiovascular disease. The gut hormone glucagon-like peptide-2 (GLP-2) triggers the release of “pre-formed” chylomicrons stored in the intestine during post-absorptive state. Previous studies have implicated the involvement of neural pathways in enhanced dietary lipid absorption by GLP-2. Our group showed for the first time that GLP-2 acts through gut-brain neural pathway in mobilizing stored lipids from the intestine in post-absorptive state. The key players in this pathway remain unknown. Central GLP-2 receptor (GLP-2R) and melanocortin 4 receptor (MC4R) have been shown to mediate GLP-2’s effects on feeding behaviour and liver glucose metabolism, but their roles in intestinal lipid handling have not been examined. Hypothesis:Peripheral GLP-2 mobilizes intestinal lipid storage during post-absorptive state acting through central GLP-2R and MC4R. Methods:Sprague-Dawley rats were implanted with an intracerebroventricular (i.c.v.) cannula and a mesenteric lymph duct cannula. Following recovery, they received a lipid load (Intralipid 20%) into the duodenum. Four hours later, they were infused i.c.v. with a GLP-2R antagonist (GLP-2(11-33)), an MC4R antagonist (SHU9119), or placebo. One hour following infusion, they were treated with intraperitoneal GLP-2 and post-absorptive lipid output from the intestine was assessed. Spatial transcriptomics analysis was conducted to detect the mRNA expression changes in three different cell types (endothelial, smooth muscle, and neuronal cells) in jejunum among the three i.c.v. treatment groups. Results:Blockade of central GLP-2R or MC4R significantly attenuated the effects of GLP-2 in stimulating lymph triglyceride output. Specifically, central GLP-2R blockade had significantly lower triglyceride output (p<0.05) at 30 mins while central MC4R blockade had a significantly lower triglyceride output from 15 to 30 mins (p<0.001) compared to central PBS treatment. Spatial transcriptomics identified differentially affected genes in jejunal cells following peripheral GLP-2 treatment with or without central receptor blockade. Conclusion:These results support the critical roles of central GLP-2R and MC4R in a neural pathway for peripheral GLP-2 in mobilizing lipids stored in the intestine during post-absorptive state, potentially through modulating differential gene expression in specific cell types in the intestine.