Regulation of mouse intestinal L cell progenitors proliferation by the glucagon family of peptides

Abstract

Glucagon like peptide-1 (GLP-1) and GLP-2 are hormones secreted by intestinal L cells that stimulate glucose-dependent insulin secretion and regulate intestinal growth, respectively. Mice with deletion of the glucagon receptor (Gcgr) have high levels of circulating GLP-1 and GLP-2. We sought to determine whether the increased level of the glucagon-like peptides is due to L cell hyperplasia. We found, first, that high levels of the glucagon-like peptides increase L cell number but does not affect the number of other intestinal epithelial cell types. Second, a large proportion of ileal L cells of Gcgr(-/-) mice coexpressed glucose-dependent insulinotropic peptide (GIP). Cells coexpressing GIP and GLP-1 are termed LK cells. Third, the augmentation in L cell number was due to a higher rate of proliferation of L cell progenitors rather than to the entrance of mature L cells into the cell cycle. Fourth, a high concentration of the glucagon-like peptides in the circulation augmented the mRNA levels of transcription factors expressed by late but not early enteroendocrine progenitors. Fifth, the administration of exendin 9-39, a GLP-1 receptor antagonist, resulted in a decrease in the rate of L cell precursor proliferation. Finally, we determined that L cells do not express the GLP-1 receptor, suggesting that the effect of GLP-1 is mediated by paracrine and/or neuronal signals. Our results suggest that GLP-1 plays an important role in the regulation of L cell number.

Fig. 1.

Increase in L cell number in ileum. A and B, Photomicrographs illustrate cells immunostained for GLP-1 in ileum of Gcgr^+/+ (A) and Gcgr^−/− (B) mice. Scale bar, 20 μm. C, Histogram documents that the number of L cells per villus and crypt is higher in Gcgr^−/− than in Gcgr^+/+ mice; n = 6 mice per line, at least 300 villi, and crypts per mouse were scored. **, P < 0.005. D, Immunohistochemical localization of GLP-1 (red) and GIP (green) in a LK cell (yellow) located in the ileum of Gcgr^−/− mice. Scale bar, 15 μm. E, The number of cells coexpressing GLP-1 and GIP (LK cells) per villus plus crypt of ileum is higher in Gcgr^−/− than in Gcgr^+/+ mice; n = 4 mice per line. *, P < 0.05. F and G, Representative sections document that the thickness of the intestinal wall and villi length were similar in Gcgr^+/+ (F) and Gcgr^−/− (G) mice. H, Villi in ileum of Gcgr^+/+ and Gcgr^−/− have similar surface area; n = 4 mice per line, and at least 100 villi were scored per animal. I, Graph illustrates the specific increase in the ratio of LK cells over total number of L cells scored in ileum of Gcgr^−/− and Gcgr^+/+ mice. *, P < 0.05; n = 4 per line. J, Duodenum of Gcgr^−/− and Gcgr^+/+ mice have similar number of cells coexpressing GLP-1 and GIP (LK cells); n = 4 mice per line. K, Photomicrograph illustrates two GLP-1⁺ (cytoplasm, red) and Pdx-1⁺ (nucleus, green) cells in the ileum of Gcgr^−/− mice. Scale bar, 15 μm. For all measurement of cell numbers, at least 300 villi plus crypt were scored.

Fig. 2.

The number of other intestinal epithelial cells is not affected. The number of goblet (A–C), serotonin (D–F), and CCK (G–I) cells in the ileum is similar in Gcgr^+/+ and Gcgr^−/− mice. At least 200 villi per mouse for each cell type, three mice per line, were examined. The three cell types are illustrated in the respective photomicrographs. Photomicrographs A, D, and G correspond to Gcgr^+/+ mice; photomicrographs B, E, and H correspond to Gcgr^−/− mice. Scale bars, 50 μm (A and B) and 20 μm (D, E, G, and H). Goblet cells were identified using the PAS technique, whereas serotonin and CCK cells were identified by immunocytochemistry.

Fig. 3.

A, Increased gut length in Gcgr^−/− compared with Gcgr^+/+. B, Plasma level of GLP-2 was higher in Gcgr^−/− than in Gcgr^+/+ mice. C, Effect of DPP4 inhibition in CD-1 mice. Sections of ileum of CD-1 mice were stained for visualization of GLP-1, and the number of L cells per surface area of villi was determined; n = 4 mice per treatment, at least 300 villi, and crypts per mouse were scored per antibody staining. CD, Control diet; ED, experimental diet. D–F, Apoptosis; D and E, caspase3⁺ cells in ileum (D) and colon (E) of Gcgr^−/− mice. Note cluster of stained cells in villus of ileum. Scale bar, 30 μm. F, Deletion of Gcgr inhibited apoptosis of intestinal epithelial cells. The number of apoptotic cells (caspase 3⁺), was lower in Gcgr^−/− than in Gcgr^+/+ mice (200 crypts scored per mouse and three mice examined per strain). G, Mature L cells are quiescent. Photomicrograph illustrates a section of ileal crypts of Gcgr^−/− mice processed for visualization of GLP-1 (red) and Ki67 (green). Note that the GLP-1⁺ cell (arrowhead) does not contain Ki67. Scale bar, 20 μm. This cell is shown in the inset at higher magnification. **, P < 0.005 and ***, P < 0.001.

Fig. 4.

Increased L cell precursor proliferation. A and B, Predictions from analysis of rate of precursor cell proliferation after a long-term exposure to BrdU. Precursors that differentiated before the BrdU administration lack BrdU (BrdU⁻GLP-1⁺, old L cells). In model A, a precursor cell (BrdU⁻GLP-1⁻) divides only once during the exposure to BrdU, and the two daughter cells (BrdU⁺GLP-1⁻) become quiescent and initiate GLP-1 expression (BrdU⁺GLP-1⁺). In B, precursor cells undergo two cycles of cell division, generating four daughter cells before becoming quiescent and differentiating into L cells. The ratio of (new) BrdU⁺GLP-1⁺ cells generated during the period of exposure to BrdU to the total number of L cells scored (GLP1⁺ BrdU⁻ plus GLP-1⁺ BrdU⁺) is higher in B than in A. C, Photomicrograph illustrates a new L cell [GLP-1⁺ (red) BrdU⁺ (green)] in Gcgr^−/− mice generated from proliferating progenitors. This cell is shown in higher magnification in the inset. D, The number of GLP-1⁺BrdU⁺ cells per total number of GLP-1⁺ cells was determined in ileum of Gcgr^+/+, Gcgr^−/−, and Gcgr^−/− mice injected with Ex 9–39; n = 3 mice per group. Results from Gcgr^−/− mice were normalized to the value obtained for the Gcgr^+/+ line. Note the increase in the number of new L cells in Gcgr^−/− mice and its decrease in Gcgr^−/− mice that received Ex 9–39; n = 3 mice per group. **, P < 0.01; *, P < 0.05.

Fig. 5.

Increased length of TA region. A–C, Comparison of the length of the TA region in colonic crypts of Gcgr^+/+ (A), Gcgr^−/− (B), and Gcgr^−/− mice plus Ex 9–39 (C) immunostained for Ki67. Note that proliferating cells occupy the lower half of the crypt in A and the majority of the crypt in B. The length of the TA region decreased in Gcgr^−/− mice injected with Ex 9–39 (C). Scale bar, 30 μm. D, Morphometric analysis confirms the augmentation in the size of the TA region in Gcgr^−/− mice and the inhibitory effect of the GLP-1r antagonist on the length of the TA region; n = 3 mice per line, and at least 25 correctly oriented colonic crypts were measured. *, P < 0.05; **, P < 0.005. E and F, Cell size was determined in cytospin preparations of isolated mucosa from Gcgr^+/+ (E) and Gcgr^−/− (F) immunostained for Ki67 (green, nuclei) and β-catenin (red, cell membrane). Scale bar, 25 μm. G, Real-time PCR analysis indicates an increase in Ki67 mRNA level in Gcgr^−/− when compared with Gcgr^+/+ mice and a decrease in Gcgr^−/− mice that received Ex 9–39. Results are expressed as fold increase over levels found in Gcgr^+/+ mice; n = 4. ***, P < 0.001.

Fig. 6.

L cells do not express the GLP-1r. Photomicrograph illustrates a section of ileum of Gcgr^−/− mice processed for visualization of GLP-1 (A) and GLP-1r (B). Each color is illustrated in a separate panel. Note that the GLP-1⁺ cell in A does not express the GLP-1r in B; the position of the GLP-1⁺ cells is illustrated in B with an arrowhead. Scale bar, 20 μm. C, Photomicrograph illustrates a pancreatic islet of wild-type mouse immunostained for GLP-1r as a positive control. D, Pancreatic islet incubated with GLP-1r antibody immunoabsorbed with a specific GLP-1r blocking peptide lacks immunostaining. Scale bars, 20 μm. The circle of bars indicates the location of an islet.

Fig. 7.

Effect of ablation of Gcgr on expression of transcription factors. A, Real-time PCR analysis of TF expression in isolated intestinal cells of ileum of Gcgr^+/+, Gcgr^−/−, and Gcgr^−/− mice that received Ex 9–39. Fold change in Gcgr^−/− and Gcgr^−/− plus Ex 9–39 was normalized to values of Gcgr^+/+. Note that the level of expression of Ngn3 mRNA was similar in Gcgr^+/+ and Gcgr^−/− mice, whereas mRNA coding for NeuroD, Nkx2.2, Pdx-1, and Pax6 increased in the mutant strain and diminished after Ex 9–39 treatment; n = 3 mice per line. *, P < 0.05. B, Real-time PCR analysis indicates that the level of preproglucagon (PPG), PC3/1, and DPP4 mRNA was similar in Gcgr^+/+ and Gcgr^−/− mice. Results are the average from three independent experiments. C and D, The level of GIP mRNA measured by real-time PCR (C) and of GIP level in the circulation (D) was similar in Gcgr^+/+ and Gcgr^−/− mice. Three mice per line were examined.

Fig. 8.

A, Proposed model for enteroendocrine cell differentiation in mouse ileum. Stem cells generate endocrine and nonendocrine lineages. Ngn3 induces endocrine differentiation in progenitors. A subset of progenitors are activated by GLP-1 to increase expression of TF, such as NeuroD, Nkx2.2, Pdx-1, and Pax6, that are involved in differentiation of these progenitors to the LK endocrine fate. Our results indicate that Ex 9–39 inhibits the involvement of GLP-1 in this process. Progenitors that will differentiate into L cells express Pax6, which activates the glucagon gene. Precursors that will generate LK and K cells activate Pax6 plus Pdx-1 and Pdx-1 expression, respectively. B, Extension of Pdx-1 regional expression results in increased LK cell number. This model illustrates that in Gcgr^+/+, the caudal boundary of Pdx-1 expression terminates in the anterior gastrointestinal tract. In Gcgr^−/−, high levels of the glucagon-like peptides enhance Pdx-1 expression and extend its caudal boundary up to the ileum, leading to an increased number of LK cells in the caudal gastrointestinal tract. D, Duodenum; I, ileum; J, jejunum. Bar indicates the rostrocaudal gradient of Pdx-1 expression; E, endocrine; EE, enteroendocrine.