Glucagon-Like Peptide-2 Stimulates S-Phase Entry of Intestinal Lgr5+ Stem Cells.

BACKGROUND & AIMS: Leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5)+/olfactomedin-4 (Olfm4)+ intestinal stem cells (ISCs) in the crypt base are crucial for homeostatic maintenance of the epithelium. The gut hormone, glucagon-like peptide-21-33 (GLP-2), stimulates intestinal proliferation and growth; however, the actions of GLP-2 on the Lgr5+ ISCs remain unclear. The aim of this study was to determine whether and how GLP-2 regulates Lgr5+ ISC cell-cycle dynamics and numbers. METHODS: Lgr5-Enhanced green-fluorescent protein - internal ribosome entry site - Cre recombinase - estrogen receptor T2 (eGFP-IRES-creERT2) mice were acutely administered human Glycine2 (Gly2)-GLP-2, or the GLP-2-receptor antagonist, GLP-23-33. Intestinal epithelial insulin-like growth factor-1-receptor knockout and control mice were treated chronically with human Gly2 (hGly2)-GLP-2. Cell-cycle parameters were determined by 5-Ethynyl-2'-deoxyuridine (EdU), bromodeoxyuridine, antibody #Ki67, and phospho-histone 3 labeling and cell-cycle gene expression. RESULTS: Acute hGly2-GLP-2 treatment increased the proportion of eGFP+EdU+/OLFM4+EdU+ cells by 11% to 22% (P < .05), without affecting other cell-cycle markers. hGly2-GLP-2 treatment also increased the ratio of eGFP+ cells in early to late S-phase by 97% (P < .001), and increased the proportion of eGFP+ cells entering S-phase by 218% (P < .001). hGly2-GLP-2 treatment induced jejunal expression of genes involved in cell-cycle regulation (P < .05), and increased expression of Mcm3 in the Lgr5-expressing cells by 122% (P < .05). Conversely, GLP-23-33 reduced the proportion of eGFP+EdU+ cells by 27% (P < .05), as well as the expression of jejunal cell-cycle genes (P < .05). Finally, chronic hGly2-GLP-2 treatment increased the number of OLFM4+ cells/crypt (P < .05), in an intestinal epithelial insulin-like growth factor-1-receptor-dependent manner. CONCLUSIONS: These findings expand the actions of GLP-2 to encompass acute stimulation of Lgr5+ ISC S-phase entry through the GLP-2R, and chronic induction of Lgr5+ ISC expansion through downstream intestinal insulin-like growth factor-1 signaling.

GLP-2, EGF, and the Intestinal Epithelial IGF-1 Receptor Interactions in the Regulation of Crypt Cell Proliferation.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal growth and proliferation through downstream mediators, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1). EGF synergistically enhances the proliferative actions of IGF-1 in intestinal cell lines, and both of these factors are known to be essential for the trophic effects of GLP-2 in vivo. However, whether EGF and IGF-1 interact to mediate the proliferative actions of GLP-2 in vivo remains unknown. Normal and knockout (KO) mice lacking the intestinal epithelial IGF-1 receptor (IE-IGF-1R) were therefore treated chronically with EGF and/or long-acting human hGly2GLP-2, followed by determination of intestinal growth parameters. Intestines from control and IE-IGF-1R KO mice were also used to generate organoids (which lack the GLP-2 receptor) and were treated with EGF and/or IGF-1. Combination treatment with EGF and hGly2GLP-2 increased small intestinal weight and crypt-villus height in C57Bl/6 mice in an additive manner, whereas only hGly2GLP-2 treatment increased crypt cell proliferation. However, although combination treatment also increased small intestinal weight and crypt-villus height in IE-IGF-1R KO mice, the proliferative responses to hGly2GLP-2 alone or with EGF were diminished in these animals. Finally, IGF-1 treatment of organoids undergoing EGF withdrawal was not additive to the effect of EGF replacement on proliferation, but could restore normal proliferation in the absence of EGF. Together, these findings demonstrate that the intestinal proliferative effects of hGly2GLP-2 are augmented by exogenous EGF in a manner that is partially dependent upon IE-IGF-1R signaling.