We previously reported that new beta cells differentiated in pancreatic islets of mice in which diabetes was produced by injection of a high dose of the beta cell toxin streptozotocin (SZ), which produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing beta cells, a population of insulin containing cells reappeared in islets. However, the number of new beta cells was small, and the animals remained severely hyperglycemic. In the present study, we tested whether restoration of normoglycemia by exogenous administered insulin would enhance beta cell differentiation and maturation. We found that beta cell regeneration improved in SZ-treated mice animals that rapidly attained normoglycemia following insulin administration because the number of beta cells per islet reached near 40% of control values during the first week after restoration of normoglycemia. Two presumptive precursor cell types appeared in regenerating islets. One expressed the glucose transporter-2 (Glut-2), and the other cell type coexpressed insulin and somatostatin. These cells probably generated the monospecific cells containing insulin that repopulated the islets. We conclude that beta cell neogenesis occurred in adult islets and that the outcome of this process was regulated by the insulin-mediated normalization of circulating blood glucose levels.

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.

To date, the role of pancreatic hormones in pancreatic islet growth and differentiation is poorly understood. To address this issue, we examined mice with a disruption in the gene encoding prohormone convertase 2 (PC2). These mice are unable to process proglucagon, prosomatostatin, and other neuroendocrine precursors into mature hormones. Initiation of insulin (IN) expression during development was delayed in PC2 mutant mice. Cells containing IN were first detected in knockout embryos on d 15 of development, 5 d later than in wild-type littermates. However, the IN(+) cells of d 15 PC2 mutant mice coexpressed glucagon, as did the first appearing beta-cells of controls. In addition, lack of PC2 perturbed the pattern of expression of transcription factors presumed to be involved in the determination of the mature alpha-cell phenotype. Thus, in contrast to controls, alpha-cells of mutant mice had protracted expression of Nkx 6.1 and Pdx-1, but did not express Brn-4. Islets of adult mutant mice also contained cells coexpressing insulin and somatostatin, an immature cell type found only in islets of the wild-type strain during development. In addition to the effects on islet cell differentiation, the absence of PC2 activity resulted in a 3-fold increase in the rate of proliferation of proglucagon cells during the perinatal period. This increase contributed to the development of alpha-cell hyperplasia during postnatal life. Furthermore, the total beta-cell volume was increased 2-fold in adult mutants compared with controls. This increase was due to islet neogenesis, as the number of islets per section was significantly higher in knockout mice compared with wild-type mice, whereas both strains had similar rates of IN cell proliferation. These results indicate that hormones processed by PC2 affected processes that regulate islet cell differentiation and maturation in embryos and adults.

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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.

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 ß 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 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 (αTC1/9) and in a beta (βTC3) 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, ßTC but not αTC cells, expressed Gcgr. The expression of GLP-1r by alpha cells was determined by the genotype and age of the mice. In embryos, GLU+ cells of Gcgr+/+ mice cells express GLP-1r during early development, but not in adults. In contrast, alpha cells of Gcgr−/− mice were GLP-1r+ throughout life, reflecting the immature state of GLU+ cells when Gcgr is deleted. Unlike alpha cells, beta cells of all mice lines examined initiate GLP-1r 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.

We previously reported that new beta cells differentiated in pancreatic islets of mice in which diabetes was produced by injection of a high dose of the beta cell toxin streptozotocin (SZ), which produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing beta cells, a population of insulin containing cells reappeared in islets. However, the number of new beta cells was small, and the animals remained severely hyperglycemic. In the present study, we tested whether restoration of normoglycemia by exogenous administered insulin would enhance beta cell differentiation and maturation. We found that beta cell regeneration improved in SZ-treated mice animals that rapidly attained normoglycemia following insulin administration because the number of beta cells per islet reached near 40% of control values during the first week after restoration of normoglycemia. Two presumptive precursor cell types appeared in regenerating islets. One expressed the glucose transporter-2 (Glut-2), and the other cell type coexpressed insulin and somatostatin. These cells probably generated the monospecific cells containing insulin that repopulated the islets. We conclude that beta cell neogenesis occurred in adult islets and that the outcome of this process was regulated by the insulin-mediated normalization of circulating blood glucose levels.