Establishment of a pancreatic β cell proliferation model in vitro and a platform for diabetes drug screening

Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.     

A Brief History of Pancreatic Reg: Implications as to its Clinical Importance

Although the pancreatic regenerating (reg) gene, was first isolated from a rat regenerating islets in 1988, its protein product was originally described in the 1970s. Reg proteins arise from a multigene family with three subtypes, and have a protein structure similar to calcium dependent lectins. Reg I and II have been implicated in control of pancreatic development and may play a role in maintenance of the beta-cell mass in the mature pancreas. Administration of reg I protein has been used in experimental animals as a therapy for surgically-induced diabetes mellitus. Reg I protein is also an inhibitor of calcium carbonate crystalization, important in maintaining the fluidity of pancreatic juice. The reg III gene, whose protein product is pancreatitis associated protein, is induced during pancreatic inflammation. Serum levels of reg III protein are a sensitive marker of severity of pancreatitis. It is an endogenous pancreatic factor that prevents the bacteria infection and scavenges oxygen-derived free radicals. Reg mRNA has been detected in non-pancreatic tissue such as the enterochromaffin-like cells of the stomach, neoplastic tissues of the colon, the small intestine, nervous system, liver tumors, and pituitary. Reg proteins are mitogens to intestinal epithelial cells, pancreatic ductal, beta cells, and Schwann cells, and are likely important to the overall integrity of the pancreas and gastrointestinal tract.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Identification of a novel Reg family gene, Reg IIIdelta, and mapping of all three types of Reg family gene in a 75 kilobase mouse genomic region

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of three subtypes (types I, II, III) based on the primary structures of the encoded proteins of the genes. We have isolated three types of mouse Reg family gene (Reg I, Reg II, Reg IIIalpha, Reg IIIbeta and Reg IIIgamma) [Unno et al. (1993) J. Biol. Chem. 268, 15974-15982; Narushima et al. (1997) Gene 185, 159-168]. In the present study, by Southern blot analysis of a mouse bacterial artificial chromosome clone containing the five Reg family genes in combination with PCR cloning of every interspace fragment between adjacent genes, the Reg family genes were mapped to a contiguous 75kb region of the mouse genome according to the following order: 5'-Reg IIIbeta-Reg IIIalpha-Reg II-Reg I-Reg IIIgamma-3'. In the process of ordering the genes, we sequenced the 6.8kb interspace fragment between Reg IIIbeta and Reg IIIalpha and encountered a novel type III Reg gene, Reg IIIdelta. This gene is divided into six exons spanning about 3kb, and encodes a 175 amino acid protein with 40-52% identity with the other five mouse Reg (regenerating gene product) proteins. Reg IIIdelta was expressed predominantly in exocrine pancreas, but not in normal islets, hyperplastic islets, intestine or colon, whereas both Reg I and Reg II were expressed in hyperplastic islets and Reg IIIalpha, Reg IIIbeta and Reg IIIgamma were expressed strongly in the intestinal tract. Possible roles of Reg IIIdelta and the widespread occurrence of the Reg IIIdelta gene in mammalian genomes are discussed.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.     

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.     

Reg3α Overexpression Protects Pancreatic β Cells from Cytokine-Induced Damage and Improves Islet Transplant Outcome

The process of islet transplantation for treating type 1 diabetes has been limited by the high level of graft failure. This may be overcome by locally delivering trophic factors to enhance engraftment. Regenerating islet-derived protein 3α (Reg3α) is a pancreatic secretory protein which functions as an antimicrobial peptide in control of inflammation and cell proliferation. In this study, to investigate whether Reg3α could improve islet engraftment, a marginal mass of syngeneic islets pretransduced with adenoviruses expressing Reg3α or control EGFP were transplanted under the renal capsule of streptozotocin-induced diabetic mice. Mice receiving islets with elevated Reg3α production exhibited significantly lower blood glucose levels (9.057 ± 0.59 mmol/L versus 13.48 ± 0.35 mmol/L, P < 0.05) and improved glucose-stimulated insulin secretion (1.80 ± 0.17 ng/mL versus 1.16 ± 0.16 ng/mL, P < 0.05) compared with the control group. The decline of apoptotic events (0.57% ± 0.15% versus 1.06% ± 0.07%, P < 0.05) and increased β-cell proliferation (0.70% ± 0.10% versus 0.36% ± 0.14%, P < 0.05) were confirmed in islet grafts overexpressing Reg3α by morphometric analysis. Further experiments showed that Reg3α production dramatically protected cultured islets and pancreatic β cells from cytokine-induced apoptosis and the impairment of glucose-stimulated insulin secretion. Moreover, exposure to cytokines led to the activation of MAPKs in pancreatic β cells, which was reversed by Reg3α overexpression in contrast to control group. These results strongly suggest that Reg3α could enhance islet engraftments through its cytoprotective effect and advance the therapeutic efficacy of islet transplantation.     

Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.01) and the difference persisted even at 7-11 days of age. Analysis of embryonic pancreata revealed impaired branching morphogenesis and delayed islet cell differentiation in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.01), indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) were markedly higher, while no apparent differences were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.     

PED/PEA-15 Inhibits Hydrogen Peroxide-Induced Apoptosis in Ins-1E Pancreatic Beta-Cells via PLD-1

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg_PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E_PED/PEA-15). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E_PED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E_PED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.     

Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus

Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. The expression of Reg3β (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed β-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3β was achieved in the pancreatic islets of RIP-I/Reg3β mice, which exhibited normal islet histology, β-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3β mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3β overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3β is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.     

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.     

Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.     

Cyclin D1 activation through ATF-2 in Reg-induced pancreatic beta-cell regeneration

Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.     

Imaging pancreatic beta-cells in the intact pancreas

We have developed a method to visualize fluorescent protein-labeled beta-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the beta-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, beta-cell clusters, and single beta-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary beta-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red beta-cells. The imaging technique that we have developed, coupled with the transgenic mice in which beta-cells and beta-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.     

Differentiation of CD24- pancreatic ductal cell-derived cells into insulin-secreting cells

Pancreatic progenitor cells represent both a potential source of transplantable islets for the treatment of diabetes and a valuable instrument for the investigation of the tumorigenesis of pancreatic carcinoma. It has been reported that pancreatic ductal cells of adults have the characteristics of pancreatic progenitors, but whether these cells can generate endocrine cells requires verification. Here, the differentiation of daughter cells of CD24(-) pancreatic ductal cells into insulin-secreting cells in vitro is reported. Crude pancreatic ductal cells were first obtained from adult mice by gradient centrifugation, and then the CD24(-) cells were isolated with a fluorescence-activated cell sorter. The isolated cells were cultured in serum-containing medium at clonal density to form epithelial colonies (ECs). The ECs were then stimulated with basic fibroblast growth factor (bFGF). After 72 h, insulin-secreting cells were observed in the ECs. These results indicate that the daughter cells of CD24(-) pancreatic ductal cells can differentiate into insulin-secreting cells in vitro when stimulated with exogenous bFGF. Therefore, CD24(-) pancreatic ductal cells have the potential to be pancreatic progenitor cells.     

Islet Formation during the Neonatal Development in Mice

The islet of Langerhans is a unique micro-organ within the exocrine pancreas, which is composed of insulin-secreting beta-cells, glucagon-secreting alpha-cells, somatostatin-secreting delta-cells, pancreatic polypeptide-secreting PP cells and ghrelin-secreting epsilon-cells. Islets also contain non-endocrine cell types such as endothelial cells. However, the mechanism(s) of islet formation is poorly understood due to technical difficulties in capturing this dynamic event in situ. We have developed a method to monitor beta-cell proliferation and islet formation in the intact pancreas using transgenic mice in which the beta-cells are specifically tagged with a fluorescent protein. Endocrine cells proliferate contiguously, forming branched cord-like structures in both embryos and neonates. Our study has revealed long stretches of interconnected islets located along large blood vessels in the neonatal pancreas. Alpha-cells span the elongated islet-like structures, which we hypothesize represent sites of fission and facilitate the eventual formation of discrete islets. We propose that islet formation occurs by a process of fission following contiguous endocrine cell proliferation, rather than by local aggregation or fusion of isolated beta-cells and islets. Mathematical modeling of the fission process in the neonatal islet formation is also presented.