Abnormal Expression of an Insulin Synthesizing Enzyme in Islets of Adult Autoantibody Positive Donors.

The observation that the two active forms of proprotein convertase 1/3 (PC1/3) were differentially expressed in beta cells of normal islets raised the possibility that this heterogeneity is lost during type 1 diabetes (T1D) progression. To test this hypothesis, the expression of the convertase was evaluated by confocal microscopy in sections of human pancreas of autoantibody positive (AA+) and T1D donors and compared with that of control. Islets of T1D pancreas were comprised of beta cells expressing either low or high PC1/3 levels and all islets of a pancreatic section contained only one beta cell type. Pancreata of AA+ donors contained either of these two classes of islets intermixed with normal islets comprised of beta cells with heterogeneous PC1/3 expression. This alteration affected the expression of proinsulin and insulin, which in most AA+ and T1D donors were lower than in controls. The present results indicate that the heterogeneity of PC1/3 expression is lost in all beta cells in a subset islets of AA+ donors and in all islets of T1D donors. These findings suggest that the heterogeneity of PC1/3 expression is a biomarker of human beta cell health and that its loss coincides with the initial stages of T1D.

Human Islets Contain a Beta Cell Type That Expresses Proinsulin But Not the Enzyme That Converts the Precursor to Insulin.

In pancreatic beta cells, proinsulin (ProIN) undergoes folding in endoplasmic reticulum/Golgi system and is translocated to secretory vesicles for processing into insulin and C-peptide by the proprotein convertases (PC)1/3 and PC2, and carboxypeptidase E. Human beta cells show significant variation in the level of expression of PC1/3, the critical proconvertase involved in proinsulin processing. To ascertain whether this heterogeneity is correlated with the level of expression of the prohormone and mature hormone, the expression of proinsulin, insulin, and PC1/3 in human beta cells was examined. This analysis identified a human beta cell type that expressed proinsulin but lacked PC1/3 (ProIN+PC1/3-). This beta cell type is absent in rodent islets and is abundant in human islets of adults but scarce in islets from postnatal donors. Human islets also contained a beta cell type that expressed both proinsulin and variable levels of PC1/3 (ProIN+PC1/3+) and a less abundant cell type that lacked proinsulin but expressed the convertase (ProIN-PC1/3+). These cell phenotypes were altered by type 2 diabetes. These data suggest that these three cell types represent different stages of a dynamic process with proinsulin folding in ProIN+PC1/3- cells, proinsulin conversion into insulin in ProIN+PC1/3+cells, and replenishment of the proinsulin content in ProIN-PC1/3+ cells.

Heterogeneous Expression of Proinsulin Processing Enzymes in Beta Cells of Non-diabetic and Type 2 Diabetic Humans.

Although there is evidence indicating transcriptional and functional heterogeneity in human beta cells, it is unclear whether this heterogeneity extends to the expression level of the enzymes that process proinsulin to insulin in beta cells. To address this question, the expression levels of prohormone convertases (PC) 1/3, proprotein convertase 2 (PC2), and carboxypeptidase E (CPE) were determined in immune-stained sections of human pancreas. In non-diabetic donors, the level of proprotein convertase 1/3 (PC1/3) expression varied among beta cells of each islet but the average per islet was similar for all islets of each donor. Although the average PC1/3 expression of all islets examined per sample was unique for each pancreas, donors had similar levels of proinsulin/insulin expression. PC2 expression in beta cells showed less pronounced inter- and intraislet variation while CPE levels were fairly constant. The relationship between PC1/3 and PC2 expression levels was variable among different donors. Type 2 diabetes had an uneven effect on the expression levels of all three enzymes as they decrease only in some islets in a section. These findings suggest the presence of intraislet, but not interislet, variation in the expression of the proinsulin processing enzymes in non-diabetic subjects and a heterogeneous effect of type 2 diabetes on enzyme expression in islets.

Mouse insulin cells expressing an inducible RIPCre transgene are functionally impaired.

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.

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.

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.

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.

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.

Reducing plasma membrane sphingomyelin increases insulin sensitivity.

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.

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.

Adenoviral-mediated inhibition of connective tissue growth factor in Rat-2 cells.

Connective tissue growth factor (CTGF) is a profibrotic factor shown to induce extracellular matrix production and angiogenesis, two processes involved in the development of diabetic retinopathy (DR). In this study we tested the effect of a recombinant adenovirus encoding for a CTGF antisense oligonucleotide (rAdASO) on the levels of transforming growth factor-beta (TGF-beta) induced expression of CTGF in Rat-2 fibroblasts. Using semi-quantitative RT-PCR, there was a 2-fold increase in CTGF message induced by TGF-beta. Western blot and immunocytochemical analyses revealed a significant increase in CTGF protein level. This upregulation of CTGF by TGF-beta was inhibited by infection with rAdASO. These findings indicate that infection of the Rat-2 cells with rAdASO was effective in decreasing TGF-beta-induced CTGF expression. These results indicate that this viral vector might have therapeutic potential to control elevated CTGF levels that occur in DR.

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.

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.

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.

Islet-derived multipotential cells/progenitor cells.

The pancreatic beta-cell has a pivotal role in the regulation of glucose homeostasis; its death leads to type I diabetes. Neogenesis of beta-cells, the differentiation of beta-cells from non-beta-cells, could be an important mechanism of islet cell repopulation. To examine the ability of the adult pancreas to generate new beta-cells, we characterized the phenotype of beta precursor cells in embryos and then determined that cells expressing embryonic traits appeared in islets of adult mouse pancreas following deletion of preexisting insulin cells by streptozotocin, a specific beta-cell toxin. These precursor cells generated new beta-cells (NBCs) that repopulated the islets. The number of NBCs increased dramatically after restoration of normoglycemia by insulin therapy. Future studies will seek to identify the source of the NBCs and to examine the mechanisms that lead to their differentiation.