Gladys Teitelman

Precursor Cells in Mouse Islets Generate New β-Cells in Vivo during Aging and after Islet Injury

Whereas it is believed that the pancreatic duct contains endocrine precursors, the presence of insulin progenitor cells residing in islets remain controversial. We tested whether pancreatic islets of adult mice contain precursor beta-cells that initiate insulin synthesis during aging and after islet injury. We used bigenic mice in which the activation of an inducible form of Cre recombinase by a one-time pulse of tamoxifen results in the permanent expression of a floxed human placental alkaline phosphatase (PLAP) gene in 30% of pancreatic beta-cells. If islets contain PLAP(-) precursor cells that differentiate into beta-cells (PLAP(-)IN(+)), a decrease in the percentage of PLAP(+)IN(+) cells per total number of IN(+) cells would occur. Conversely, if islets contain PLAP(+)IN(-) precursors that initiate synthesis of insulin, the percentage of PLAP(+)IN(+) cells would increase. Confocal microscope analysis revealed that the percentage of PLAP(+)IN(+) cells in islets increased from 30 to 45% at 6 months and to 60% at 12 months. The augmentation in the level of PLAP in islets with time was confirmed by real-time PCR. Our studies also demonstrate that the percentage of PLAP(+)IN(+) cells in islets increased after islet injury and identified putative precursors in islets. We postulate that PLAP(+)IN(-) precursors differentiate into insulin-positive cells that participate in a slow renewal of the beta-cell mass during aging and replenish beta-cells eliminated by injury.

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.

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. Developmental Dynamics 236:1126–1133, 2007.

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.

The role of pregnancy hormones in the regulation of Pdx-1 expression.

During pregnancy, pancreatic beta cells undergo changes that are probably due to an increase in the lactogenic hormones prolactin (PRL) and placental lactogen (PL). Since the transcription factor PDX-1 is involved in the regulation of the beta cell function and phenotype, we tested the possibility that the effect of PRL on beta cells was mediated by PDX-1. Exposure of islet cells to PRL in vitro resulted in increased levels of PDX-1 protein and mRNA and a stimulation of pdx-1 transcription. However, PDX-1 levels in islets exposed in vivo to high concentration of prolactin was similar to controls. In vitro studies suggested that the up-regulation of PDX-1 by PRL was opposed by glucocorticoids (GC) at concentrations similar to those present in pregnant and control female mice. We conclude that, although pdx-1 is a key regulator of beta cell specific genes, it does not appear to play a central role in the up-regulation of islet cell function during pregnancy.

Mouse Insulin Cells Expressing an Inducible RIPCre Transgene Are Functionally Impaired

Background: We tested whether insulin cells expressing an inducible RIP-Cre transgene display a normal phenotype. Results: RipCre insulin cells die when the stimulation of insulin synthesis is protracted. Conclusion: Beta cells expressing an inducible RIP-Cre transgene are functionally deficient. Significance: The use of an inducible RIPCre system to examine beta cell gene function/development could distort experimental results. We used cre-lox technology to test whether the inducible expression of Cre minimize the deleterious effect of the enzyme on beta cell function. We studied mice in which Cre is linked to a modified estrogen receptor (ER), and its expression is controlled by the rat insulin promoter (RIP). Following the injection of tamoxifen (TM), CreER- migrates to the nucleus and promotes the appearance of a reporter protein, enhanced yellow fluorescent protein (EYFP), in cells. Immunocytochemical analysis indicated that 46.6 ± 2.1% insulin cells of adult RIPCreER- EYFP expressed EYFP. RIPCreER-EYFP (+TM) mice were normoglycemic throughout the study, and their glucose tolerance test results were similar to control CD-1 mice. However, an extended exposure to reagents that stimulate insulin synthesis was detrimental to the survival of IN+EYFP+cells. The administration of an inhibitor of the enzyme dipeptidyl-peptidase (DPP4i), which prevents the cleavage of glucagon-like peptide (GLP-1), to adult RIPCreER-EYFP mice lead to a decrease in the percentage of IN+EYFP+ to 17.5 ± 1.73 and a significant increase in apoptotic cells in islets. Similarly, a 2-week administration of the GLP-1 analog exendin 4 (ex-4) induced an almost complete ablation of IN+ expressing a different reporter protein and a significant decrease in the beta cell mass and rate of beta cell proliferation. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient. Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique.

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. Developmental Dynamics, 2012.

Islet Precursor Cells in Adult Pancreatic Islets

The search for new sources of β cells is driven by the shortage of islets of Langerhans suitable for replacement therapy for type I diabetes. Recent advances in stem cell research, described in other sections of this book, have led to promising sources of insulin-producing cells (1, 2, 3, 4, 5). In addition, significant progress in the elucidation of the molecular program that guides the differentiation of islet cells during development (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) will certainly provide tools to expand the population of embryonic β-precursor cells and promote their differentiation into mature, insulin-producing cells. The adult pancreas is another potential source of β-precursor cells. The existence of islet progenitor cells in mature pancreas can be inferred from the fact that new islets are normally formed during postnatal life (23,24) and that a dramatic increase in islet number occurs in response to various stimuli (25, 26, 27). The presence of “endocrine stem/progenitor cells” could not only provide a source of cells suitable for transplantation, but also could replenish the β-cell population that has been depleted by injury or disease.