Highlights d Enteroendocrine differentiation and labeling in human small intestinal organoids d RNA-seq and peptide LC-MS profiling of purified human GLP-1-secreting L-cells d Human ileal L-cells are electrically active and respond to nutritional stimuli

Aims/hypothesis Insulin-like peptide-5 (INSL5) is found only in distal colonic L cells, which co-express glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). GLP-1 is a well-known insulin secretagogue, and GLP-1 and PYY are anorexigenic, whereas INSL5 is considered orexigenic. We aimed to clarify the metabolic impact of selective stimulation of distal colonic L cells in mice. Methods Insl5 promoter-driven expression of Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) was employed to activate distal colonic L cells (L distalDq). IPGTT and food intake were assessed with and without DREADD activation. Results L distalDq cell stimulation with clozapine N-oxide (CNO; 0.3 mg/kg i.p.) increased plasma GLP-1 and PYY (2.67-and 3.31-fold, respectively); INSL5 was not measurable in plasma but was co-secreted with GLP-1 and PYY in vitro. IPGTT (2 g/kg body weight) revealed significantly improved glucose tolerance following CNO injection. CNO-treated mice also exhibited reduced food intake and body weight after 24 h, and increased defecation, the latter being sensitive to 5-hydroxytryptamine (5-HT) receptor 3 inhibition. Pre-treatment with a GLP1 receptor-blocking antibody neutralised the CNO-dependent improvement in glucose tolerance but did not affect the reduction in food intake, and an independent group of animals pair-fed to the CNOtreatment group demonstrated attenuated weight loss. Pre-treatment with JNJ-31020028, a neuropeptide Y receptor type 2 antagonist, abolished the CNO-dependent effect on food intake. Assessment of whole body physiology in metabolic cages revealed L distalDq cell stimulation increased energy expenditure and increased activity. Acute CNO-induced food intake and glucose homeostasis outcomes were maintained after 2 weeks on a high-fat diet. Conclusions/interpretation This proof-of-concept study demonstrates that selective distal colonic L cell stimulation has beneficial metabolic outcomes.