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Coxsackievirus B3 (CVB3) has strong oncolytic activity in colorectal carcinoma but it also infects the pancreas and the heart. To improve the safety of the virus, here we investigated whether pancreas and cardiac toxicity can be prevented by insertion of target sites (TS), which are complementary to miR-375 and miR-1 into the viral genome. Although miR-375 and miR-1 are abundantly expressed in the pancreas and in the heart, respectively, their expression levels are low in colorectal carcinomas, which allows the carcinomas to be selectively attacked. To investigate the importance of the microRNAs, two viruses were engineered, H3N-375TS containing only miR-375TS and H3N-375/1TS containing miR-375TS and miR-1TS. In vitro, both viruses replicated in and lysed colorectal carcinoma cells, similar to a nontargeted control virus H3N-39TS, whereas they were strongly attenuated in cell lines transiently or endogenously expressing the corresponding microRNAs. In vivo, the control virus H3N-39TS induced strong infection of the pancreas and the heart, which led to fatal disease within 4 days after a single intratumoral virus injection in mice xenografted with colorectal DLD-1 cell tumors. In contrast, three intratumoral injections of H3N-375TS or H3N-375/1TS failed to induce virus-induced sickness. In the animals, both viruses were completely ablated from the pancreas and H3N-375/1TS was also ablated from the heart, whereas the cardiac titers of H3N-375TS were strongly reduced. Long-term investigations of the DLD-1 tumor model confirmed lack of virus-induced adverse effects in H3N-375TS- and H3N-375/1TS-treated mice. There was no mortality, and the pancreas and the heart were free of pathological alterations. Regarding the therapeutic efficiency, the treated animals showed high and long-lasting H3N-375TS and H3N-375/1TS persistence in the tumor and significantly slower tumor growth. These data demonstrate that miR-375- and miR-1-mediated virus detargeting from the pancreas and heart is a highly effective strategy to prevent toxicity of oncolytic CVB3.

Objective MicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice. Methods RNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132−/− and control mice. Results Partial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-βH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212−/− mice than the control littermates. Conclusions This study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass., Highlights • miR-132 is induced in mouse islets upon partial pancreatectomy. • miR-132 promotes regeneration of β-cells in vivo following partial pancreatectomy. • miR-132 fosters in vitro proliferation/survival through Pten/Akt/Foxo3 signaling. • Downstream targets of miR-132 were identified in pancreatic β-cells. • miR-132−/− mice have impaired β-cell proliferation.

Significance The failure of β cells to secrete sufficient amounts of insulin is a key feature of diabetes mellitus. Each β cell secretes only a small amount of insulin upon stimulation in a highly regulated fashion: young insulin is preferentially released, whereas old insulin is mainly degraded within the β cell. How this process is regulated in vivo and likely altered in diabetes is currently unknown. We present here a transgenic pig model that allows the in vivo fluorescent labeling of age-distinct insulin secretory granule pools, hence providing a close-to-life readout of insulin turnover. This will enable the study of alterations in β cell function in an animal model close to humans., β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

Aims The coxsackievirus B3 (CVB3) mouse myocarditis model is the standard model for investigation of virus-induced myocarditis but the pancreas, rather than the heart, is the most susceptible organ in mouse. The aim of this study was to develop a CVB3 mouse myocarditis model in which animals develop myocarditis while attenuating viral infection of the pancreas and the development of severe pancreatitis. Methods and results We developed the recombinant CVB3 variant H3N-375TS by inserting target sites (TS) of miR-375, which is specifically expressed in the pancreas, into the 3ʹUTR of the genome of the pancreo- and cardiotropic CVB3 variant H3. In vitro evaluation showed that H3N-375TS was suppressed in pancreatic miR-375-expressing EndoC-βH1 cells >5 log10, whereas its replication was not suppressed in isolated primary embryonic mouse cardiomyocytes. In vivo, intraperitoneal (i.p.) administration of H3N-375TS to NMRI mice did not result in pancreatic or cardiac infection. In contrast, intravenous (i.v.) administration of H3N-375TS to NMRI and Balb/C mice resulted in myocardial infection and acute and chronic myocarditis, whereas the virus was not detected in the pancreas and the pancreatic tissue was not damaged. Acute myocarditis was characterized by myocardial injury, inflammation with mononuclear cells, induction of proinflammatory cytokines, and detection of replicating H3N-375TS in the heart. Mice with chronic myocarditis showed myocardial fibrosis and persistence of H3N-375TS genomic RNA but no replicating virus in the heart. Moreover, H3N-375TS infected mice showed distinctly less suffering compared with mice that developed pancreatitis and myocarditis after i.p. or i.v application of control virus. Conclusion In this study, we demonstrate that by use of the miR-375-sensitive CVB3 variant H3N-375TS, CVB3 myocarditis can be established without the animals developing severe systemic infection and pancreatitis. As the H3N-375TS myocarditis model depends on pancreas-attenuated H3N-375TS, it can easily be used in different mouse strains and for various applications.

Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper β-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the β-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24-hour sleep-wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR-KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.

Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic beta-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the beta-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in beta-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated beta-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.

Summary Glucose stimulates the exocytosis of insulin secretory granules of pancreatic β cells. Granule stores are quickly refilled by activation of posttranscriptional mechanisms that enhance the biosynthesis of granule components. Rapid replacement of granules is important to sustain insulin secretion, since new granules appear to be preferentially released. Posttranscriptional regulation of granule biogenesis includes the glucose-induced nucleocytoplasmic translocation of polypyrimidine tract binding protein 1 (PTB1), which binds mRNAs encoding granule proteins, and thus promotes their stabilization and translation. Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin gene expression and secretion by increasing cAMP levels in β cells. Here, we show that elevation of cAMP levels causes the protein kinase A-dependent phosphorylation and nucleocytoplasmic translocation of PTB1, thereby preventing the rapid degradation of insulin mRNA and enhancing the expression of various granule proteins. Taken together, these findings identify PTB1 as a common downstream target of glucose and GLP-1 for the posttranscriptional upregulation of granule biogenesis.

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Here we show that beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired.

Lung fibrosis is the end-point of numerous lung disorders induced by a pneumonia or by a variety of different noxes, one of which is the cytostatic drug bleomycin (BLM). Fibrosis is characterized by excessive extracellular matrix accumulation. Macrophage-fibroblast interactions are suggested to play an important role in the development of this disease. The present study was addressed to investigate one possible pathway of this interaction, the influence of soluble mediators produced by BLM-stimulated macrophages on lung fibroblast collagen synthesis and modification. Conditioned media (CM) of BLM-exposed macrophages of the cell line NR8383 submitted to rat lung fibroblast cultures increased the activity of prolyl 4-hydroxylase (P4H) in fibroblasts in a dose dependent manner. CM of stimulated macrophages increased the collagen concentration in fibroblast culture supernatant. The level of mRNAs specific for the alpha-subunit of P4H and that for alpha1(I) collagen were found to be increased by about two-fold, that for lysyloxidase (LO) by about 2.5-fold in fibroblasts cultured in CM of stimulated macrophages. Pre-incubation of CM of BLM-exposed macrophages with neutralizing antibodies against TGF-beta or against PDGF resulted in a partial reversal of the increasing effect of the CM on P4H- and LO-activities in fibroblasts. Both growth factors, TGF-beta and PDGF, added to fibroblast cultures led to significant increases of P4H activity in the treated cells. We conclude that TGF-beta and PDGF produced by stimulated macrophages are involved in the regulation of the expression of alpha1(I) collagen, of P4H-alpha-subunit and LO in lung fibroblasts. The results indicate that this is not a direct effect but involves the action of a so far unidentified mediator responsible for autocrine stimulation of collagen production.

Changes in the activities of several proteinases and their inhibitors were investigated during the development of bleomycin-induced pulmonary fibrosis in rat. Studies on the proteinase-antiproteinase-ratio may contribute to the understanding of the mechanism of the development of pulmonary fibrosis and may help to develop therapeutic strategies to prevent tissue damage by proteolytic attack. In the acute inflammatory period the activity of metalloelastase in lung tissue increased by about 10-fold. The time course of changes in the activity of 72 kD gelatinase indicates that this gelatinase accounts at least partially for the elastolytic activity. Elastase inhibitory activity in lung tissue showed maxima at days 1 and 5 and high levels in the fibrotic phase. The increase of the elastase inhibitory activity at the beginning of the fibrotic period corresponds with elevated activity of α2-macroglobulin. Alveolar fluid and alveolar macrophages did not contain elastase activity but contained high elastase inhibitory activity. During the period of chronic inflammation, the activities of the cathepsins L, B, H and S in lung tissue and in isolated alveolar macrophages were found to be strongly increased.

Immunosuppressive drugs used in human islet transplantation interfere with the balance between beta cell renewal and death, and thus may contribute to progressive graft dysfunction. We analysed the influence of immunosuppressants on the proliferation of transplanted alpha and beta cells after syngeneic islet transplantation in streptozotocin-induced diabetic mice.C57BL/6 diabetic mice were transplanted with syngeneic islets in the liver and simultaneously abdominally implanted with a mini-osmotic pump delivering BrdU alone or together with an immunosuppressant (tacrolimus, sirolimus, everolimus or mycophenolate mofetil [MMF]). Glycaemic control was assessed for 4 weeks. The area and proliferation of transplanted alpha and beta cells were subsequently quantified.After 4 weeks, glycaemia was significantly higher in treated mice than in controls. Insulinaemia was significantly lower in mice treated with everolimus, tacrolimus and sirolimus. MMF was the only immunosuppressant that did not significantly reduce beta cell area or proliferation, albeit its levels were in a lower range than those used in clinical settings.After transplantation in diabetic mice, syngeneic beta cells have a strong capacity for self-renewal. In contrast to other immunosuppressants, MMF neither impaired beta cell proliferation nor adversely affected the fractional beta cell area. Although human beta cells are less prone to proliferate compared with rodent beta cells, the use of MMF may improve the long-term outcome of islet transplantation.

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis. © 2010 Schubert et al.

Changes in metabolic demands dynamically regulate the total mass of adult pancreatic β-cells to adjust insulin secretion and preserve glucose homeostasis. Glucose itself is a major regulator of β-cell proliferation by inducing insulin secretion and activating β-cell insulin receptors. Here, we show that islet cell autoantigen 512 (ICA512)/IA-2, an intrinsic tyrosine phosphatase-like protein of the secretory granules, activates a complementary pathway for β-cell proliferation. On granule exocytosis, the ICA512 cytoplasmic domain is cleaved and the resulting c ytosolic fragment (ICA512-CCF) moves into the nucleus where it enhances the levels of phosphorylated STAT5 and STAT3, thereby inducing insulin gene transcription and granule biogenesis. We now show that knockdown of ICA512 decreases cyclin D1 levels and proliferation of insulinoma INS-1 cells, whereas β-cell regeneration is reduced in partially pancreatectomized ICA512 −/− mice. Conversely, overexpression of ICA512-CCF increases both cyclin D1 and D2 levels and INS-1 cell proliferation. Up-regulation of cyclin D1 and D2 by ICA512-CCF is affected by knockdown of STAT3 and STAT5, respectively, whereas it does not require insulin signaling. These results identify ICA512 as a regulator of cyclins D and β-cell proliferation through STATs and may have implication for diabetes therapy.

One impediment for a wider application of islet transplantation is the limited number of donor pancreata for islet isolation. A more efficient utilization of available organs could in part alleviate this problem. Perfluorocarbons (PFCs) have a high oxygen solubility coefficient and maintain high oxygen partial pressures for extended time. They serve also as oxygen “reservoirs” for harvested organs in pancreas organ transplantation. The aim of this study was to test whether the use of PFCs could also be beneficial for the secretory activity and overall viability of cultured purified islets before transplantation. Purified rat islets were cultured in static conditions with or without oxygen-saturated PFCs for 1 or 7 days. Cell death and apoptosis were assessed by trypan blue staining, DNA strand breaks, and caspase 3/7 activity. mRNA levels of insulin and ICA512/IA-2, a membrane marker of secretory granules (SGs), were quantitated by real-time PCR, whereas insulin content and secretion were measured by RIA. Polypyrimidine tract binding protein (PTB), which promotes SG biogenesis, was assessed by Western blotting. The number of SGs and the ultrastructural appearance of β-cells were analyzed by cryoimmunoelectronmicroscopy for insulin. Various parameters, including caspase activity, insulin and ICA512/IA-2 mRNA levels, PTB expression, number of secretory granules, and ultrastructural appearance did not significantly differ between control and PFC-cultured islets. On the other hand, PFC culture islets showed significantly increased DNA fragmentation and a reduced insulin stimulation index at both time points compared to control islets. While advantageous for the transport of human harvested organs, the use of PFH in the culture may be comparable to and/or not provide advantage over conventional protocols for culture of islets for transplantation.

Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic β-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca2+-dependent cleavage of ICA512 cytoplasmic domain by μ-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in β-cells, whose impaired insulin production and secretion causes diabetes.

Summary The cytostatic drug bleomycin (BLM) induces pulmonary fibrosis as its main side effect. Fibroblasts in fibrotic foci are the main cellular source for extracellular matrix accumulation that typifies fibrosis. In vitro studies demonstrated the ability of cytotoxic drugs to induce terminal differentiation of fibroblasts. These postmitotic cells are very active in regard to production of collagens. The present study was addressed to investigate the potential of BLM to induce terminal differentiation of rat lung fibroblasts in vitro and the consequences for collagen production and for the expression and activity of the collagen modifying enzyme prolyl 4-hydroxylase (P4H). The BLM effects were compared with those of mitomycin C (MMC), another cytotoxic agent with known potential for initiation of postmitotic differentiation of fibrobasts. BLM induced postmitotic differentiation of rat lung fibroblasts. The capacity of the cells to form clones was diminished by BLM or MMC in a concentration dependent manner. Both drugs initiated the formation of an increasing number of postmitotic cell clones. The postmitotic differentiation was accompanied by an increase in total collagen production by the cells. Administration of BLM to cultures of lung fibroblasts at concentrations of 1 or 10 mU/ml resulted in an increase of the collagen amount to about the 1.5-fold and 1.6-fold of controls, respectively. Treatment of fibroblasts with MMC elevated the collagen level to about the 2-fold. P4H activity and P4Hα mRNA levels in cells exposed to BLM or MMC were found to be increased. We conclude that terminally differentiated fibroblasts might be part of the heterogeneous population of fibroblast-like cells in fibrotic foci responsible for the increased production of collagen during the fibrotic phase of the development of pulmonary fibrosis.