BACKGROUND & AIMS: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. METHODS: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. RESULTS: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline. CONCLUSIONS: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.[on SciFinder (R)]