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The discovery that long noncoding RNA TUNAR regulates β-cell proliferation may be important in designing new treatments for diabetes.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within b-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in b-cells selectively activated protein kinase R–like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion. Fil: Ghiasi, Seyed Mojtaba. Universidad de Copenhagen; Dinamarca Fil: Dahlby, Tina. Universidad de Copenhagen; Dinamarca Fil: Andersen, Caroline Hede. Universidad de Copenhagen; Dinamarca Fil: Haataja, Leena. University of Michigan; Estados Unidos Fil: Petersen, Sólrun. Universidad de Copenhagen; Dinamarca Fil: Omar-Hmeadi, Muhmmad. Uppsala Universitet; Suecia Fil: Yang, Mingyu. Uppsala Universitet; Suecia Fil: Pihl, Celina. Universidad de Copenhagen; Dinamarca Fil: Bresson, Sophie Emilie. Universidad de Copenhagen; Dinamarca Fil: Khilji, Muhammad Saad. Universidad de Copenhagen; Dinamarca Fil: Klindt, Kristian. Universidad de Copenhagen; Dinamarca Fil: Cheta, Oana. Universidad de Copenhagen; Dinamarca Fil: Perone, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Copenhagen; Dinamarca Fil: Tyrberg, Björn. Astrazeneca. IMED Biotech Unit; Suecia Fil: Prats, Clara. Universidad de Copenhagen; Dinamarca Fil: Barg, Sebastian. Uppsala Universitet; Suecia Fil: Tengholm, Anders. Uppsala Universitet; Suecia Fil: Arvan, Peter. University of Michigan; Estados Unidos Fil: Mandrup-Poulsen, Thomas. Universidad de Copenhagen; Dinamarca Fil: Marzec, Michal Tomasz. Universidad de Copenhagen; Dinamarca

Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when β-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible β5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of β5i-containing intermediate proteasomes was significantly increased in these cells, as was β5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon β5i small interfering RNA-mediated knockdown. Finally, the fraction of β-cells expressing the β5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that β5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.

International audience; A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the β5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1β stimulating proinsulin biosynthesis. These findings suggest that the β cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic β cells, as this might facilitate autoantigen presentation by β cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in β cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes.We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the β5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by non-toxic concentrations of IL-1β stimulating proinsulin biosynthesis. These findings suggest that the β cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

Stress-related changes in β-cell mRNA levels result from a balance between gene transcription and mRNA decay. The regulation of RNA decay pathways has not been investigated in pancreatic β-cells. We found that no-go and nonsense-mediated RNA decay pathway components (RDPCs) and exoribonuclease complexes were expressed in INS-1 cells and human islets. Pelo, Dcp2, Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by inflammatory cytokines in INS-1 cells under conditions where central β-cell mRNAs were downregulated. These changes in RDPC mRNA or corresponding protein levels were largely confirmed in INS-1 cells and rat/human islets. Cytokine-induced upregulation of Pelo, Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by inducible nitric oxide synthase inhibition, as were endoplasmic reticulum (ER) stress, inhibition of Ins1/2 mRNA, and accumulated insulin secretion. Reactive oxygen species inhibition or iron chelation did not affect RDPC expression. Pelo or Xrn1 knockdown (KD) aggravated, whereas Smg6 KD ameliorated, cytokine-induced INS-1 cell death without affecting ER stress; both increased insulin biosynthesis and medium accumulation but not glucose-stimulated insulin secretion in cytokine-exposed INS-1 cells. In conclusion, RDPCs are regulated by inflammatory stress in β-cells. RDPC KD improved insulin biosynthesis, likely by preventing Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but Smg6 KD ameliorated, cytokine-mediated β-cell death, possibly through prevention of proapoptotic and antiapoptotic mRNA degradation, respectively.

Pregnancy is associated with increased β-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. Aims/hypothesis This study tests the hypothesis that lncRNAs regulate β-cell proliferation in response to prolactin in the context of β-cell mass compensation in pregnancy. Methods The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 β-cell line by EdU incorporation and cell count. Results We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary β-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates β-cell growth. Conclusions/interpretation During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse β-cell proliferation and may be a crucial component of β-cell proliferation in β-cell mass adaptation in both health and disease.

Control of  -cell function and mass is tightly linked to glucose homeostasis. Failing  -cells inevitably lead to diabetes. Recently, several contradictory studies have been published arguing against or in favor of various mechanisms controlling  -cell mass regulation. Here we review the literature on control of adult  -cell mass and aim to reconcile thereby the contradictions. We discuss the role of � -cell proliferation and neogenesis, both in mice and man. We also discuss the influence of genetic predisposition on  -cell mass control. We conclude that  -cell generation in the adult human and mouse likely depends on many paths to assure sufficient numbers of  -cells at any given time, thereby balancing mechanisms for negative regulation of  -cell numbers. A simple model with only one pathway does not fit the current literature.

The basic helix-loop-helix (bHLH) gene Hes6 is known to promote neural differentiation in vitro. Here, we report the expression and functional studies of Hes6 in the inner ear. The expression of Hes6 appears to be parallel to that of Math1 (also known as Atoh1), a bHLH gene necessary and sufficient for hair cell differentiation. Hes6 is expressed initially in the presumptive hair cell precursors in the cochlea. Subsequently, the expression of Hes6 is restricted to morphologically differentiated hair cells. Similarly, the expression of Hes6 in the vestibule is in the hair cell lineage. Hes6 is dispensable for hair cell differentiation, and its expression in inner ear hair cells is abolished in the Math1-null animals. Furthermore, the introduction of Hes6 into the cochlea in vitro is not sufficient to promote sensory or neuronal differentiation. Therefore, Hes6 is downstream of Math1 and its expression in the inner ear delineates the sensory lineage. However, the role of Hes6 in the inner ear remains elusive.

Engraftment (i.e., the adaptation of transplanted pancreatic islets to their new surroundings with regard to revascularization, reinnervation, and reorganization of other stromal compartments) is of crucial importance for the survival and function of the endocrine cells. Previous studies suggest that transplantation induces both vascular and stromal dysfunctions in the implanted islets when compared with endogenous islets. Thus the vascular density and the blood perfusion of islet grafts is decreased and accompanied with a capillary hypertension. This leads to hypoxic conditions, with an associated shift toward anaerobic metabolism in grafted islets. An improved engraftment will prevent or compensate for the vascular/stromal dysfunction seen in transplanted islets and thereby augment survival of the islet implant. By such means the number of islets needed to cure the recipient will be lessened. This will increase the number of patients that can be transplanted with the limited material available.

Achieving normoglycemia is the goal of diabetes therapy. Potentially, there are many ways to achieve this goal, including transplantation of cells exhibiting glucose-responsive insulin secretion. However, to be applicable to the large number of people who might benefit from beta cell replacement, an unlimited supply of beta cells must be found. To address this problem, we have been developing cell lines from the human endocrine pancreas. In one case, a cell line, betalox5, has been developed from human islets that can be induced under some circumstances to differentiate into functional beta cells exhibiting appropriate glucose-responsive insulin secretion. Inducing differentiation is complex, requiring the activation of multiple signaling pathways, including those downstream of those involved in cell-cell contact and the glucagon-like peptide-1 receptor. In addition, transfer of the PDX-1 gene is also necessary to render the cells competent for differentiation. However, it is clear that many other genes are involved in maintaining the commitment of betalox5 cells towards the beta cell lineage. Understanding the complement of genes required to establish and maintain a beta cell lineage commitment would be enormously helpful in efforts to develop a cell line that can be used for beta cell replacement therapies. Here, we provide further information on the characteristics of cell lines that we have developed from the human pancreas that are relevant to the development of a beta cell replacement therapy for diabetes.

Using immortalized human pancreatic endocrine cell lines, we have shown previously that differentiation into hormoneexpressing cells requires cell-cell contact acting in synergy with the homeodomain transcription factor pancreatic duodenal homeobox-1 (PDX-1). Although differentiation is associated with a decrease in cell proliferation, the mechanisms behind this relationship are not known. Using TRM-6, a cell line, andlox5, a-cell line, we show here that cell-cell contact and subsequent endocrine differentiation lead to a down-regulation of the c-myc protooncogene. Overexpression of c-Myc obtained with an inducible c-Myc-estrogen receptor fusion protein results in an increase in cell proliferation and the ablation of hormone expression. Moreover, we show that although c-Myc is expressed in a subset of cells from the human fetal and adult pancreas, it is absent in differentiated endocrine cells. The mechanism by which c-Myc interferes with hormone expression may be through effects on the homeodomain transcription factor PDX-1, as immunostaining for PDX-1 in cells with activated c-Myc revealed a redistribution of PDX-1 from the nucleus to the cytoplasm. These results suggest that c-Myc plays a central role in a cell-cell contactmediated switch mechanism by which cell division vs. differentiation in endocrine cells is determined. (J Clin Endocrinol Metab 87: 3475–3485, 2002)

β-Cells rapidly secrete insulin in response to acute increases in plasma glucose but, upon further continuous exposure to glucose, insulin secretion progressively decreases. Although the mechanisms are unclear, this mode of regulation suggests the presence of a time-dependent glucosensory system that temporarily attenuates insulin secretion. Interestingly, early-stage β-cell dysfunction is often characterized by basal (ie, fasting) insulin hypersecretion, suggesting a disruption of these related mechanisms. Because sweet taste receptors (STRs) on β-cells are implicated in the regulation of insulin secretion and glucose is a bona fide STR ligand, we tested whether STRs mediate this sensory mechanism and participate in the regulation of basal insulin secretion. We used mice lacking STR signaling (T1R2−/− knockout) and pharmacologic inhibition of STRs in human islets. Mouse and human islets deprived of STR signaling hypersecrete insulin at short-term fasting glucose concentrations. Accordingly, 5-hour fasted T1R2−/− mice have increased plasma insulin and lower glucose. Exposure of isolated wild-type islets to elevated glucose levels reduced STR expression, whereas islets from diabetic (db/db) or diet-induced obese mouse models show similar down-regulation. This transcriptional reprogramming in response to hyperglycemia correlates with reduced STR function in these mouse models, leading to insulin hypersecretion. These findings reveal a novel mechanism by which insulin secretion is physiologically regulated by STRs and also suggest that, during the development of diabetes, STR function is compromised by hyperglycemia leading to hyperinsulinemia. These observations further suggest that STRs might be a promising therapeutic target to prevent and treat type 2 diabetes.

The beta-cell toxin alloxan, which produces oxygen radicals, is a model substance in studies of type 1 diabetes. Recently, human beta-cells have been found to be relatively resistant to this toxin. To clarify species differences in alloxan diabetogenicity, and oxygen radical toxicity, mouse, rat, rabbit, dog, pig, human and guinea pig islets have been studied after alloxan exposure. Using a standardized in vivo model, where islets were transplanted to nude mice, the different islets were compared. The results demonstrated that mouse and rat islet grafts were morphologically disturbed by alloxan and ROS. Rabbit and dog islet graft morphology was reasonably intact; and human, porcine, and guinea pig islet grafts were all well preserved. Furthermore, ultrastructural signs of apoptosis and necrosis, disturbances in the insulin secretory pattern during and after an alloxan perifusion, and islet lysosomal enzyme activities were studied in vitro in islets from some species. Guinea pig beta-cells were affected by alloxan, but a regeneration process compensated for the observed apoptotic and necrotic cell death. Human islets did not show any signs of alloxan-induced damage in the different models studied. Finally, no correlation between high alloxan sensitivity and high lysosomal enzyme activity was found. Thus, the beta-cell lysosomes are hardly specific targets for alloxan.

Neogenesis is crucial for the maintenance of β-cell mass in the human pancreas and possibly for the outcome of clinical islet transplantation. To date, no studies have reported a stimulation of human β-cell neogenesis in vivo. Therefore, we investigated whether human α-, β-, and duct cell growth can be stimulated when human islets are xenotransplanted to obese hyperglycemic-hyperinsulinemic ob/ob mice immuno-suppressed with anti-lymphocyte serum. Moreover, we wanted to study whether β-cell growth and duct-to-β-cell differentiation were induced in the hepatocyte growth factor (HGF)-dependent compensatory kidney growth model. For that purpose, we evaluated human islets grafted to nude (nu/nu) mice before uninephrectomy of the contralateral kidney for DNA-synthesis and duct cell expression of the β-cell-specific transcription factor Nkx 6.1 as an estimate of differentiation. Human islet grafts were well preserved after 2 weeks when transplanted to ob/ob mice during anti-lymphocyte immunosuppression. Both human β-cells (P < 0.01) and duct cells (P < 0.001) were growth stimulated when islets were transplanted to ob/ob mice. We also observed a correlation between increased duct cell proliferation and increased organ donor age (P =0.02). Moreover, duct (P < 0.05) and β-cell (P

Nitric oxide (NO), synthesized by the inducible isoform of nitric oxide synthase (iNOS), has been proposed as a mediator of immune-induced beta-cell destruction in type 1 diabetes. To evaluate the role of iNOS for beta-cell dysfunction and death, we investigated the sensitivity of beta-cells from mice genetically deficient in this enzyme (iNOS-/-, background C57BL/6x129SvEv, H-2b) both to interleukin (IL)-1beta-induced beta-cell dysfunction in vitro and to multiple low-dose streptozotocin (MLDS)-induced diabetes in vivo. Exposure of islets isolated from C57BL/6 mice to IL-1beta for 24 h in vitro resulted in an induction of iNOS mRNA expression, an increase in nitrite formation, and a decrease in insulin release and proinsulin biosynthesis as compared with untreated C57BL/6 islets. IL-1beta failed to induce iNOS mRNA expression and increase nitrite formation by islets isolated from iNOS knockout mice (iNOS-/-), and no impairment in islet function was observed. The iNOS-/- mice showed a reduced incidence of hyperglycemia after treatment with MLDS as compared with wild-type C57BL/6 (H-2b) and 129 SvEv (H-2b) mice. On day 21 after the first streptozotocin (STZ) injection, 75% of the C57BL/6 mice and 100% of the 129SvEv mice had blood glucose levels >11 mmol/l, whereas the corresponding number for iNOS-/- mice was only 23%. This protection was not due to a delay in the onset of hyperglycemia, since no increase in number of hyperglycemic iNOS-/- mice was observed when the animals were followed up to 42 days. Moreover, islets isolated from iNOS-/- mice were susceptible to the in vitro deleterious effects of STZ. In conclusion, the present study provides evidence that iNOS may contribute to beta-cell damage after exposure to IL-1beta in vitro and treatment with MLDS in vivo.

Functional expression of sweet taste receptors (T1R2 and T1R3) has been reported in numerous metabolic tissues, including the gut, pancreas, and, more recently, in adipose tissue. It has been suggested that sweet taste receptors in these nongustatory tissues may play a role in systemic energy balance and metabolism. Smaller adipose depots have been reported in T1R3 knockout mice on a high carbohydrate diet, and sweet taste receptors have been reported to regulate adipogenesis in vitro. To assess the potential contribution of sweet taste receptors to adipose tissue biology, we investigated the adipose tissue phenotypes of T1R2 and T1R3 knockout mice. Here we provide data to demonstrate that when fed an obesogenic diet, both T1R2 and T1R3 knockout mice have reduced adiposity and smaller adipocytes. Although a mild glucose intolerance was observed with T1R3 deficiency, other metabolic variables analyzed were similar between genotypes. In addition, food intake, respiratory quotient, oxygen consumption, and physical activity were unchanged in T1R2 knockout mice. Although T1R2 deficiency did not affect adipocyte number in peripheral adipose depots, the number of bone marrow adipocytes is significantly reduced in these knockout animals. Finally, we present data demonstrating that T1R2 and T1R3 knockout mice have increased cortical bone mass and trabecular remodeling. This report identifies novel functions for sweet taste receptors in the regulation of adipose and bone biology, and suggests that in these contexts, T1R2 and T1R3 are either dependent on each other for activity or have common independent effects in vivo.

Human pancreatic beta-cell proliferation may be crucial for the success of islet transplantation. The aim of this study was to test the hypothesis that adult human beta-cells proliferate in vitro and in vivo and respond with increased rates of replication to factors known to promote rodent islet-cell proliferation, i.e. glucose, human recombinant GH, and FCS. For this purpose, human islets were prepared from a total of 19 adult heart-beating organ donors and cultured for 48 h with or without the additives described above. 3H-thymidine was added to the medium during the last 60 min of culture. After immunohistochemical staining for insulin and autoradiography, the labeling index (LI; i.e. % of labeled beta-cells over total number of beta-cells) was estimated by light microscopy. Islets also were transplanted under the kidney capsule of normal or alloxan-diabetic nude mice. After 2 weeks, 3H-thymidine was injected and the islet grafts prepared for determination of LI, as described above. Islets cultured at 5.6 mM glucose showed an increased beta-cell proliferation compared with islets cultured at 2.8 mM glucose (P0.05). However, culture at 11 mM glucose failed to further increase beta-cell proliferation. Addition of GH (1 microg/ml) to the medium, in the presence of 1% FCS and 5.6 mM glucose, did not influence the rate of beta-cell proliferation. In islets transplanted to hyperglycemic nude mice, beta-cell proliferation was similar to that observed in islets grafted into normoglycemic nude mice. Proliferation, however, decreased with increasing organ donor age. This study shows that pancreatic beta-cells from adult man are able to proliferate both in vitro and in vivo. Moreover, beta-cells from adult human donors respond with increased proliferation to glucose in vitro and show a decreased proliferation in vivo with increasing donor age.

Postprandial insulin release is regulated by glucose, but other circulating nutrients may target beta cells and potentiate glucose-stimulated insulin secretion via distinct signaling pathways. We demonstrate that fructose activates sweet taste receptors (TRs) on beta cells and synergizes with glucose to amplify insulin release in human and mouse islets. Genetic ablation of the sweet TR protein T1R2 obliterates fructose-induced insulin release and its potentiating effects on glucose-stimulated insulin secretion in vitro and in vivo. TR signaling in beta cells is triggered, at least in part, in parallel with the glucose metabolic pathway and leads to increases in intracellular calcium that are dependent on the activation of phospholipase C (PLC) and transient receptor potential cation channel, subfamily M, member 5 (TRPM5). Our results unveil a pathway for the regulation of insulin release by postprandial nutrients that involves beta cell sweet TR signaling.

Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human beta-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated beta-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on beta-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN.

To examine the mechanism by which growth-stimulated pancreatic beta-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and betalox5, a cell line derived from human beta-cells which progressively dedifferentiated in culture. MIN6/betalox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that betalox5 expresses a dominant transacting factor(s) that represses beta-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary beta-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in beta-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the beta-cell differentiated state.

The basic helix–loop–helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in β-cells and either absent or expressed below the limit of lacZ detection in mature α-, δ-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic δ-cell line, resulted in potent repression of somatostatin concomitant with induction of the β-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in β- but not δ-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously. Developmental Dynamics 233:946–953, 2005. © 2005 Wiley-Liss, Inc.

Reactive oxygen intermediates (ROI) may be involved in the destruction of pancreatic beta-cells during the development of insulin-dependent diabetes mellitus (IDDM). To investigate the possible role of lysosomes in this process, normal mouse beta-cells were cultured as monolayers at D-glucose concentrations of 1.6 (pronounced crinophagy), 11 or 28 mM (minimal crinophagy), subjected to a low level of oxidative stress and returned to standard culture conditions. Some cultures were exposed to desferrioxamine (Des) before the oxidative stress. As a result of such stress, many of the cells' lysosomes ruptured with consequent apoptosis or necrosis. Cells kept at 1.6 mM glucose were rich in secretory granules, showed crinophagy/autophagy, were very sensitive to oxidative stress, and had the least stable lysosomes. Cells kept at 28 mM glucose did not show crinophagy, contained fewer secretory granules, were less sensitive to oxidative stress, and had more stable lysosomes. Des-treated cells behaved almost as cells not exposed to oxidative stress at all. The findings suggest that iron may occur together with zinc within the secretory granules and that it sensitizes crinophagic lysosomes to oxidative stress. The stress that was applied in this study may be comparable to what occurs within the vicinity of activated macrophages during autoimmune insulitis.

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human β-cells, inducibly transformed pancreatic β-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of β-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional β-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human β-cells is a major step toward developing a cell transplantation therapy for diabetes.

We have previously shown that human beta-cells are resistant to the toxic effects of alloxan. In order to further clarify this characteristic of human islets, we investigated whether these cells might transfer their alloxan resistance to alloxan-sensitive rat or mouse islets. Islets from two species (human-mouse or rat-mouse) were mixed into one graft, which was implanted into the subcapsular kidney space of nude mice. Alloxan or saline was injected intravenously two weeks after implantation and one week thereafter the mice were killed. The number of grafted and endogenous beta-cells were evaluated by a semi-quantitative method after immunohistochemistry. Human islet production of the scavenging enzymes extracellular superoxide dismutase and plasma glutathione peroxidase were analyzed with ELISA-techniques, and mouse and human islet hydrogen peroxide breakdown activity were monitored with a horseradish peroxidase-dependent assay. Mouse beta-cells transplanted together with human islets were protected against alloxan cytotoxicity. Rat islets did not protect mouse beta-cells against alloxan, suggesting that the mixing procedure as such did not impose the protection. Production of extracellular superoxide dismutase and plasma glutathione peroxidase by human islets was very low. Moreover, H2O2 breakdown in vitro, did not differ between human and mouse islets. Alloxan-insensitive human islets protect mouse beta-cells against alloxan-induced lesions, suggesting that yet to be identified extracellular factors are involved in human islet resistance to alloxan toxicity.

Nitric oxide and peroxynitrite (generated by the reaction of nitric oxide with the superoxide anion) may both be mediators of β-cell damage in early insulin-dependent diabetes mellitus. We observed that acute exposure of primary cultured human pancreatic islets to peroxynitrite results in a significant decrease in glucose oxidation and islet retrieval. DNA strand breaks in single human and rat islet cells are detectable after acute peroxynitrite exposure, followed by a decrease in islet cell survival after 1 h and 24 h. Cell death appeared to occur via a toxic cell death mechanism (necrosis) rather than apoptosis, as suggested by vital staining and ultrastructural evidence of early membrane and organelle degradation, mitochondrial swelling and loss of matrix. This study demonstrates for the first time that cultured human pancreatic islets are susceptible to the noxious effects of peroxynitrite.