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Identifying pathways for beta cell generation is essential for cell therapy in diabetes. Here we investigate the potential of 17β-estradiol (E2) and estrogen receptor (ER) signaling for stimulating beta cell generation during embryonic development and in severely injured adult pancreas. Estradiol concentration, ER activity and number of ERα transcripts were enhanced in pancreas injured by partial duct ligation (PDL), along with nuclear localization of ERα in beta cells. PDL-induced proliferation of beta cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and beta cell growth in PDL pancreas were impaired when ERα was turned off chemically or genetically (ERα(-/-)), while in situ delivery of E2 promoted beta cell formation. In embryonic pancreas, beta cell replication, number of Ngn3(+) progenitor cells and expression of key transcription factors of the endocrine lineage were decreased by ERα inactivation. The present study reveals that E2 and ERα signaling can drive betacell replication and formation in mouse pancreas.

OBJECTIVE Generating functional β-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G1- to S-phase transition during the cycling of many cell types and is required for pancreatic β-cell growth and function. However, the consequences of overexpression of E2F1 in β-cells are unknown. RESEARCH DESIGN AND METHODS The effects of E2F1 overexpression on β-cell proliferation and function were analyzed in isolated rat β-cells and in transgenic mice. RESULTS Adenovirus AdE2F1-mediated overexpression of E2F1 increased the proliferation of isolated primary rat β-cells 20-fold but also enhanced β-cell death. Coinfection with adenovirus AdAkt expressing a constitutively active form of Akt (protein kinase B) suppressed β-cell death to control levels. At 48 h after infection, the total β-cell number and insulin content were, respectively, 46 and 79% higher in AdE2F1+AdAkt-infected cultures compared with untreated. Conditional overexpression of E2F1 in mice resulted in a twofold increase of β-cell proliferation and a 70% increase of pancreatic insulin content, but did not increase β-cell mass. Glucose-challenged insulin release was increased, and the mice showed protection against toxin-induced diabetes. CONCLUSIONS Overexpression of E2F1, either in vitro or in vivo, can stimulate β-cell proliferation activity. In vivo E2F1 expression significantly increases the insulin content and function of adult β-cells, making it a strategic target for therapeutic manipulation of β-cell function.

Experimentally induced injury is an established strategy for studying mechanisms of tissue remodeling with the final goal of developing new regenerative therapies. Under normal physiological conditions, proliferation and differentiation of progenitor cells, including even canonical stem cell−like activity, can be stimulated in tissues, such as brain and liver, that have a low cellular turnover rate (1,2). The presence of stem/progenitor cells in the pancreas could be relevant to normal homeostatic maintenance of various cell types in this organ, such as endocrine hormone−expressing cells, enzyme-secreting acinar cells, and the less secretory exocrine duct cells. Further, pancreatic stem/progenitor cells may be a possible source for replenishing cells destroyed by autoimmune disease or other stressors. We speculate that proliferative progenitors might be isolated, expanded, and differentiated in vitro to alleviate the donor scarcity in human islet transplantation and may therefore be developed as a therapy for diabetes. However, the existence and exact location of adult stem- or progenitor-like cells that can give rise to functional β-cells is highly controversial. This Perspective focuses on findings from a severe insult model (partial duct ligation [PDL]) with a long history (3). PDL received renewed attention when a 2008 study combined it with genetic reporter strategies now possible in mice to try to identify and isolate cells acting as β-cell progenitors (4). In vivo β-cell neogenesis under PDL was recently substantiated (5,6). Because the outcomes from this technique appear to vary across laboratories, we summarize and discuss some of the reported discrepancies to help identify current limitations and pitfalls of this model as well as opportunities for forward progress. The mechanisms leading to replacement of the endogenous β-cell pool have been studied under several cell ablation paradigms to test for the existence and type of cells in the adult mouse pancreas that are …

Nutrient homeostasis is known to be regulated by pancreatic islet tissue. The function of islet β-cells is controlled by a glucose sensor that operates at physiological glucose concentrations and acts in synergy with signals that integrate messages originating from hypothalamic neurons and endocrine cells in gut and pancreas. Evidence exists that the extrapancreatic cells producing and secreting these (neuro)endocrine signals also exhibit a glucose sensor and an ability to integrate nutrient and (neuro)hormonal messages. Similarities in these cellular and molecular pathways provide a basis for a network of coordinated functions between distant cell groups, which is necessary for an appropriate control of nutrient homeostasis. The glucose sensor seems to be a fundamental component of these control mechanisms. Its molecular characterization is most advanced in pancreatic β-cells, with important roles for glucokinase and mitochondrial oxidative fluxes in the regulation of ATP-sensitive K+ channels. Other glucose-sensitive cells in the endocrine pancreas, hypothalamus, and gut were found to share some of these molecular characteristics. We propose that similar metabolic signaling pathways influence the function of pancreatic α-cells, hypothalamic neurons, and gastrointestinal endocrine and neural cells.

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

The polymorphic variable number of tandem repeats in the 5′ upstream region of the human insulin gene is a well-known non-human leukocyte antigen locus contributing to genetic susceptibility to IDDM. Controversy exists about the question as to whether INS susceptibility haplotypes are or are not preferentially inherited together with HLA-DR4 haplotypes. We investigated whether genetic interaction between INS and the HLA complex can be better defined using DQ genotypic and phenotypic markers in addition to DR serology. The 5′ INS 1/1 genotype was positively associated with IDDM both in non-DR4 subjects (relative risk = 4.3; 95% confidence interval, 1.6–11.5) and DR4 subjects (relative risk = 4.2; 95% confidence interval, 1.9–9.0). Further subdivision of IDDM patients and matched control subjects according to HLA-DQA1 and HLA-DQB1 genotype or phenotype also failed to show any association between 5′ INS and HLA class II genes in diabetic patients. The 5′ INS and HLA class II polymorphisms therefore provide independent risk markers, which may both contribute to the genetic screening of a high-risk population among nondiabetic individuals.

We previously reported that interleukin-1beta (IL-1beta) alone does not cause apoptosis of beta-cells, whereas when combined with gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), it exerts a distinct apoptotic effect. Studies in beta-cell lines indicated that IL-1beta reduced expression of islet brain (IB)-1/JNK interacting protein (JIP)-1, a JNK scaffold protein with antiapoptotic action. We examined whether variations in IB1/JIP-1 expression in purified primary beta-cells affect their susceptibility to cytokine-induced apoptosis. Exposure to IL-1beta for 24 It decreased cellular IB1/JIP-1 content by 66 +/- 17%; this IL-1beta effect was maintained in the presence of TNF-alpha + IFN-gamma, which did not influence IB1/JIP-1 levels by themselves. Addition of IL-1beta to TNF-alpha + IFN-gamma increased apoptosis from 20 +/- 2% to 59 +/- 5%. A similar increase in TNF-alpha + IFN-gamma-induced apoptosis was produced by adenoviral expression of antisense IB1/JIP-1 and was not further enhanced by addition of IL-1beta, indicating that IL-1beta-mediated suppression of IB1/JIP-1 in beta-cells increases their susceptibility to cytokine-induced apoptosis. However, adenovirally mediated overexpression of IB1/JIP-1 also potentiated TNF-alpha + IFN-gamma-induced apoptosis, suggesting that the antiapoptotic effect of IB1/JIP-1 depends on well-defined cellular levels. We conclude that the IB1/JIP-1 level in beta-cells can control their susceptibility to apoptosis independent of JNK signaling.

Cytokine-induced beta -cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1 beta (IL-1 beta), and its activity promotes the expression of several beta -cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1 beta + gamma -interferon [IFN-gamma])-induced expression of NF-kappaB in beta -cell apoptosis, rat beta -cells were infected with the recombinant adenovirus AdI kappaB((SA)2), which contained a nondegradable mutant form of inhibitory KB(SA)2, a I kappaB((SA)2) with S32A and S36A that locks NF-kappaB in, cytosolic protein complex, preventing its nuclear action. Expression of I kappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdI kappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta -cell survival after IL-1 beta + IFN-gamma treatment was significantly improved by I kappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta -cells.