Fast Track Adaptation of Oncolytic Coxsackie B3 Virus to Resistant Colorectal Cancer Cells - a Method to Personalize Virotherapy.

BACKGROUND: The efficacy of oncolytic viruses (OV) in cancer treatment depends on their ability to successfully infect and destroy tumor cells. However, patients' tumors vary, and in the case of individual insensitivity to an OV, therapeutic efficacy is limited. Here, we present a protocol for rapid generation of tumor cell-specific adapted oncolytic coxsackievirus B3 (CVB3) with enhanced oncolytic potential and a satisfactory safety profile. This is achieved by combining directed viral evolution (DVE) with genetic modification of the viral genome and the use of a microRNA-dependent regulatory tool. METHODS: The oncolytic CVB3 variant PD-H was adapted to the refractory colorectal carcinoma cell line Colo320 through serial passaging. XTT assays and virus plaque assays were used to determine virus cytotoxicity and virus replication in vitro. Recombinant PD-H variants were generated through virus mutagenesis. Apoptosis was detected by Western blots, Caspase 3/7 assays, and DAPI staining. The therapeutic efficacy and safety of the adapted recombinant OV PD-SK-375TS were assessed in vivo using a subcutaneous Colo320 xenograft mouse model. RESULTS: PD-H was adapted to the colorectal cancer cell line Colo320 within 10 passages. Sequencing of passage 10 virus P-10 revealed a heterogenous virus population with five nucleotide mutations resulting in amino acid substitutions. The genotypically homogeneous OV PD-SK was generated by inserting the five detected mutations of P-10 into the genome of PD-H. PD-SK showed significantly stronger replication and cytotoxicity than PD-H in Colo320 cells, but not in other colorectal carcinoma cell lines. Increase of apoptosis induction was detected as key mechanisms of Colo320 cell-specific adaptation of PD-SK. For in vivo safety PD-SK was engineered with target sites of the miR-375 (miR-375TS) to exclude virus replication in normal tissues. PD-SK-375TS, unlike the PD-H-375TS not adapted homolog suppressed the growth of subcutaneous Colo320 tumors in nude mice without causing any side effects. CONCLUSION: Taken together, here we present an optimized protocol for the rapid generation of tumor cell-specific adapted oncolytic CVB3 based on the oncolytic CVB3 strain PD-H. The protocol is promising for the generation of personalized OV for tumor therapy and has the potential to be applied to other OV.

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs.

ß 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 ß 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.

miR-375- and miR-1-Regulated Coxsackievirus B3 Has No Pancreas and Heart Toxicity But Strong Antitumor Efficiency in Colorectal Carcinomas.

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.

The making of insulin in health and disease.

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes. Graphical abstract.

MiR-132 controls pancreatic beta cell proliferation and survival through Pten/Akt/Foxo3 signaling.

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-ßH1 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-ßH1 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.

Development of a new mouse model for coxsackievirus-induced myocarditis by attenuating coxsackievirus B3 virulence in the pancreas.

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 3'UTR 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-ßH1 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.

MiR-375-mediated suppression of engineered coxsackievirus B3 in pancreatic cells.

Coxsackievirus B3 (CVB3) has potential as a new oncolytic agent for the treatment of cancer but can induce severe pancreatitis. Here, we inserted target sequences of the microRNA miR-375 (miR-375TS) into the 5' terminus of the polyprotein encoding sequence or into the 3'UTR of the CVB3 strain rCVB3.1 to prevent viral replication in the pancreas. In pancreatic EndoC-ßH1 cells expressing miR-375 endogenously, replication of the 5'-miR-375TS virus and that of the 3'-miR-375TS virus was reduced by 4 × 103 -fold and 3.9 × 104 -fold, respectively, compared to the parental rCVB3.1. In colorectal carcinoma cells, replication and cytotoxicity of both viruses were slightly reduced compared to rCVB3.1, but less pronounced for the 3'-miR-375TS virus. Thus, CVB3 with miR-375TS in the 3'UTR of the viral genome may be suitable to avoid pancreatic toxicity.

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain.

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.

Virus-like infection induces human ß cell dedifferentiation.

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic ß cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than ß cell death, suggesting loss of ß cell identity. We undertook this study to examine whether viral infection could induce human ß cell dedifferentiation. Using the functional human ß cell line EndoC-ßH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in ß cell-specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-κB pathway and also in a paracrine non-cell-autonomous fashion through the secretion of IFN-α. Lastly, we identified SOX9 targets in human ß cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human ß cell dedifferentiation.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

A 4D view on insulin secretory granule turnover in the ß-cell.

Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper ß-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 ß-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.

Mechanisms of Beta Cell Dysfunction Associated With Viral Infection.

Type 1 diabetes (T1D) results from genetic predisposition and environmental factors leading to the autoimmune destruction of pancreatic beta cells. Recently, a rapid increase in the incidence of childhood T1D has been observed worldwide; this is too fast to be explained by genetic factors alone, pointing to the spreading of environmental factors linked to the disease. Enteroviruses (EVs) are perhaps the most investigated environmental agents in relationship to the pathogenesis of T1D. While several studies point to the likelihood of such correlation, epidemiological evidence in its support is inconclusive or in some instances even against it. Hence, it is still unknown if and how EVs are involved in the development of T1D. Here we review recent findings concerning the biology of EV in beta cells and the potential implications of this knowledge for the understanding of beta cell dysfunction and autoimmune destruction in T1D.

PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells.

Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3'-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5'-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure.

Polymorphism rs11085226 in the gene encoding polypyrimidine tract-binding protein 1 negatively affects glucose-stimulated insulin secretion.

OBJECTIVE: Polypyrimidine tract-binding protein 1 (PTBP1) promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in ß-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion. METHODS: We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs) within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698) covering 100% of genetic variation with an r(2)≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT. RESULTS: PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04). The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103). Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108). Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018), but the rs351974 was not. CONCLUSIONS: We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo.

Isolation of human islets from partially pancreatectomized patients.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction (1) have been hampered by the limited availability of type 2 diabetic islets from organ donors(2). Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area. To extend the applicability of our protocol to include the occasional case in which a large amount (>15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber(3) at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO(2;) at 37 °C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

Impaired insulin turnover in islets from type 2 diabetic patients.

Failure of pancreatic ß-cells contributes to the development of type 2 diabetes. Besides evidence of reduced glucose-stimulated insulin secretion and ß-cell mass, little information is available about the molecular deficits of human diabetic islets. Islets were isolated from macroscopically normal pancreatic tissue from 8 patients with type 2 diabetes and 17 matched non-diabetic patients who underwent pancreatic surgery. Insulin content and insulin secretion were measured before and after islet stimulation with 25 mM glucose for 2 hours. In parallel, we also investigated the subcellular localization of polypyrimidine tract-binding protein 1 (PTBP1), whose nucleocytoplasmic translocation is involved in the rapid posttranscriptional up-regulation of insulin biosynthesis following islet stimulation with glucose and GLP-1. Glucose stimulated insulin secretion was decreased, albeit not significantly, in type 2 diabetic islets compared to non-diabetic islets. Stimulation increased the total amount of insulin (islet insulin content + secreted insulin) in islet preparation from non-diabetic patients, but not from type 2 diabetic subjects. Furthermore, the nuclear levels of PTBP1 were decreased in stimulated non-diabetic islets, but not in type 2 diabetic islets. These results suggest that impairment of rapid insulin increase in response to glucose is a specific trait of type 2 diabetic islets. Nuclear retention of PTBP1 is likely to play a role in this deficit, which in turn can contribute to impaired insulin secretion in type 2 diabetes. Overall, these data highlight the importance of investigating mechanisms of insulin biosynthesis and degradation to gain insight into the pathogenesis of type 2 diabetes.

ß2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ß-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ß2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of ß2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-ß2-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 ß2-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 ß2-syntrophin phosphomutants we found that phosphorylation of ß2-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.

Rapid changes of mRNA-binding protein levels following glucose and 3-isobutyl-1-methylxanthine stimulation of insulinoma INS-1 cells.

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.

ICA512 signaling enhances pancreatic beta-cell proliferation by regulating cyclins D through STATs.

Changes in metabolic demands dynamically regulate the total mass of adult pancreatic beta-cells to adjust insulin secretion and preserve glucose homeostasis. Glucose itself is a major regulator of beta-cell proliferation by inducing insulin secretion and activating beta-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 beta-cell proliferation. On granule exocytosis, the ICA512 cytoplasmic domain is cleaved and the resulting cytosolic 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 beta-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 beta-cell proliferation through STATs and may have implication for diabetes therapy.

cAMP-dependent phosphorylation of PTB1 promotes the expression of insulin secretory granule proteins in beta cells.

Glucose stimulates the exocytosis of insulin secretory granules of pancreatic beta 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 beta 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.