Phenotype of entero-endocrine L cells becomes restricted during development.

BACKGROUND: Glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP) are hormones secreted by L and K cells, respectively, and by LK cells. To characterize L and K cells during development, we examined ileum from embryonic (e)- 12 to e-17. RESULTS: GLP-1 cells were first seen at e-15 and their number increased at e-17. At e-17, most GLP-1 cells co-expressed GIP. The transcription factors Pax6 and Pdx-1 are required for GIP expression, while Pax6 activates the expression of GLP-1. At e-17, the mucosa has GIP+ Pax6+, GIP+ Pdx-1+, GLP-1+ Pax6+, and GLP-1+ Pdx-1+ cells. Unlike ileal L cells of postnatal and adult mice, a subset of ileal L cells of e-17 embryos co-expressed GLP-1 and glucagon (Glu). Glu-positive cells contain proprotein-convertase 2 (PC2) and PC3/1, the enzymes responsible for Glu and GLP-1 synthesis, respectively. CONCLUSIONS: Our findings indicate that most GLP-1+ cells of ileum of e-17 embryos co-express GIP and, therefore, are LK cells. In addition, a subset of GLP-1+ cells of embryos but not of neonates co-express glucagon, indicating that the expression of Glu in GLP-1+ cells disappears after birth.

Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy.

PURPOSE: Connective tissue growth factor (CTGF) is a profibrotic factor that induces extracellular matrix (ECM) production and angiogenesis, two processes involved in diabetic retinopathy (DR). In this study, we examined whether insulin therapy or a CTGF-specific small interfering RNA (siRNA) administered to diabetic rats decreased the levels of CTGF and of selected putative downstream genes in the retina. METHODS: Rats with streptozotocin-induced diabetes were used. Animals received either no treatment for 12 weeks or were administered constant insulin therapy. MRNA and protein levels of CTGF and select ECM genes were determined using real-time PCR and western blotting of the retina. Localization of CTGF in the retina was visualized using immunohistochemistry. A group of diabetic rats received intravitreal injection of CTGF siRNA, and the retinas were examined three days later. RESULTS: CTGF mRNA and protein significantly increased in the retinas of diabetic rats. Immunohistochemistry indicated that retinal Müller cells of diabetic rats expressed CTGF. Hyperglycemia upregulated mRNA levels of fibronectin, laminin ß1, collagen IVα3, and vascular endothelial growth factor (VEGF), and this increase was prevented by insulin therapy. Treatment of diabetic rats with CTGF siRNA decreased laminin ß1, collagen IVα3 mRNA, and CTGF mRNA and protein but did not affect fibronectin or vascular endothelial growth factor mRNA levels. CONCLUSIONS: These results indicate that CTGF and ECM genes can be regulated using insulin. Importantly, these results also suggest that CTGF regulates changes in ECM molecules in DR.

Ablation of the glucagon receptor gene increases fetal lethality and produces alterations in islet development and maturation.

Although glucagon (GLU) plays a pivotal role in glucose homeostasis, its role in the regulation of fetal growth and maturation is poorly understood. These issues were examined in a line of mice with a global deletion of the GLU receptor (Gcgr-/-), which are characterized by lower blood glucose levels and by alpha- and delta-cell hyperplasia in adults. Ablation of Gcgr was deleterious to fetal survival; it delayed beta-cell differentiation and perturbed the proportion of beta- to alpha-cells in embryonic islets. In adults, the mutation inhibited the progression of alpha-cells to maturity, affected the expression of several beta-cell-specific genes, and resulted in an augmentation of the alpha-, beta-, and delta-cell mass. This increase was due to an augmentation in both islet number and in the rate of proliferation of cells expressing GLU or insulin. These findings suggest that GLU participates in a feedback loop that regulates the proportion of the different endocrine cell types in islets, the number of islets per pancreas, and development of the mature alpha-cell phenotype.

Functional activity of murine intestinal mucosal cells is regulated by the glucagon-like peptide-1 receptor.

To determine whether the glucagon-like peptide-1 receptor (GLP-1r) plays a role in the regulation of intestinal functional activity, we analyzed the distribution of the GLP-1r in mouse tissues and tested if tissues expressing the receptor respond to exendin-4 and exendin (9-39) amide, a GLP-1r agonist and antagonist respectively. In ileum, Glp1r mRNA level was two fold higher in extracts from epithelial cells than non-epithelial tissues. By immunohistochemistry, the receptor was localized to the mucosal cell layer of villi of ileum and colon, to the myenteric and submucosal plexus and to Paneth cells. Intravenous administration of exendin-4 to CD-1 mice induced expression of the immediate early gene c-fos in mucosal cells but not in cells of the enteric plexuses or in L cells of ileum. The induction of c-fos was inhibited by the voltage-gated sodium channel blocker tetrodotoxin. Exendin-4 also increased c-fos expression in ileal segments in vitro, suggesting that this action of the analog was independent of an extrinsic input. The induction of c-fos expression by exendin-4 was inhibited by exendin (9-39) amide, indicating that the action of exendin-4 was mediated by activation of the receptor. Our findings indicate that the GLP-1r is involved in ileal enterocyte and Paneth cell function, that the GLP-1 analog activates c-fos expression in the absence of an extrinsic input and that some of the actions of the receptor is/are mediated by voltage-gated Na channels.

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

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.

Differential expression of glucagon and glucagon-like peptide 1 receptors in mouse pancreatic alpha and beta cells in two models of alpha cell hyperplasia.

Glucose homeostasis is determined by a balance between insulin and glucagon, produced by beta and alpha cells of the pancreas respectively. The levels of circulating hormones is partly determined by the mass of these two endocrine cell types. However, in contrast to beta cells, the identity of the signals regulating alpha cell number is not known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) and mice with ablation of prohormone convertase 2 (PC2), the enzyme involved in the conversion of proglucagon into mature glucagon, develop alpha cell hyperplasia. These observations and the fact that Gcgr-/- mice exhibit high levels of circulating glucagon-like peptide-1 (GLP-1) suggested that members of the glucagon family of peptides could be directly involved in the regulation of alpha cell number. In this study we sought to determine whether alpha cells express receptors for Glucagon (Gcgr) and/or the glucagon-like peptide-1 (GLP1r). We examined the expression of these receptors in islets of Gcgr-/-, PC2-/- mice and control littermates, in an alpha (alphaTC1/9) and in a beta (betaTC3) cell line. Gcgr was expressed exclusively by islet beta cells, but not by alpha cells, of the two lines of mice lacking glucagon signaling. Similarly, betaTC but not alphaTC cells, expressed Gcgr. The expression of GLP1r by alpha cells was determined by the genotype and age of the mice. In embryos, GLU+ cells of Gcgr+/+ mice cells express GLP1r during early development, but not in adults. In contrast, alpha cells of Gcgr-/- mice were GLP1r+ throughout life, reflecting the immature state of GLU+ cells when Gcgr is deleted. Unlike alpha cells, beta cells of all mice lines examined initiate GLP1r expression after birth. These results suggest that GLP-1 may affect the maturation of postnatal but not prenatal beta cells. In addition, they also suggest that the incretin could mediate alpha cell proliferation, inducing the development of alpha cell hyperplasia in Gcgr-/- mice.

Nestin expression in pancreatic endocrine and exocrine cells of mice lacking glucagon signaling.

Nestin, a marker of neural stem cells, is also expressed by cells located in the epithelium of the pancreatic primordium and by a subpopulation of exocrine cells but not by endocrine cells. These findings raised the possibility that the pancreatic epithelium is heterogeneous and comprised of subpopulations of exocrine/nestin-positive and endocrine/nestin-negative precursor cells. We examined this issue in two mutant mouse models characterized by protracted expression of several embryonal properties in islet cells. One mutant line comprises mice lacking mature glucagon due to abrogation of proprotein convertase-2 (PC2(-/-)), responsible for the conversion of proglucagon into glucagon, while the second line consists of mice with a global deletion of the glucagon receptor (Gcgr(-/-)). We demonstrate that nestin is transiently expressed by acinar cells and by insulin and glucagon cells of islets of both lines of mice. In addition, the lack of glucagon signaling increased nestin mRNA levels in pancreas of mutant embryos and adult mice. We conclude that nestin+ cells located in the pancreatic primordium generate the cells of the endocrine and exocrine lineages. Furthermore, our results suggest that nestin expression is regulated by glucagon signaling.