Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic β 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 β cell death, suggesting loss of β cell identity. We undertook this study to examine whether viral infection could induce human β cell dedifferentiation. Using the functional human β cell line EndoC-βH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in β 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 β cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human β cell dedifferentiation.

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.

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.

No abstract

No abstract

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‐βH1 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.

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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2962/ Protocol 1. Pancreatic tissue collection in the operating room 1. The surgeon performs a partial resection of the pancreas. 2. After placing the pancreatic specimen in a box on ice, deliver it immediately to the pathologist. Tissue selection for islet isolation1. The pathologist selects tissue that appears soft and healthy, retaining the damaged tissue for diagnostic purposes. Fibrotic tissue and specimens offering less than 2 g of usable tissue are excluded from islet isolation. 2. Immerse the pancreatic tissue in Euro Collins Solution and deliver on ice to the laboratory. Isolation of human islets1. Weigh the pancreatic tissue, then place it into a 10 cm dish. 2. Put 150 ml RPMI media in a 500 ml flask. 3. In a 250 ml flask, prepare the digestive enzyme solution by combining 130 ml RPMI media with 100 mg/ml DNase and 20 ml of 5mg/ml Liberase RI. Dr...

Pi3k -Phosphatidylinositol-4,5-bisphosphate 3-kinase; Ras -Rat sarcoma; Raf -rapidly accelerated fibrosarcoma; Mapk -mitogen-activated protein kinase; Sos1 -Son of sevenless homolog 1; Gnb1 -G Protein Subunit Beta 1; Nras -Neuroblastoma RAS viral oncogene homolog; Pik3r1 -Phosphoinositide-3-Kinase Regulatory Subunit 1; Gnb -G Protein Subunit Beta; Fgfr3 -fibroblast growth factor receptor 3; Creb -cAMP response element-binding protein peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/233098 doi: bioRxiv preprint first posted online Dec. 12, 2017; Mziaut et al, 2018 3 Abstract Aim and hypothesis: microRNAs (miRNAs) play an integral role in maintaining beta cell function and identity.Deciphering their targets and precise role, however, remains a challenge. In this study we aimed to identify miRNAs and their downstream targets involved in 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 control of beta cell regeneration. Altered expression of selected miRNAs, including miR- peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/233098 doi: bioRxiv preprint first posted online Dec. 12, 2017; Mziaut et al, 2018 4 Based on previous knowledge of its function, we chose to focus our attention on miR-132. Downregulation of miR-132 Conclusions/Interpretations:Our study provides compelling evidence for upregulation of miR-132 being critical for regeneration of mouse islet beta cells in vivo through downregulation of its target Pten.Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.Keywords miR-132; beta cell function; beta cell regeneration; apoptosis; Pten; Akt; Foxo3; pancreatectomy, diabetes.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/233098 doi: bioRxiv preprint first posted online Dec. 12, 2017; Mziaut et al, 2018 5 Research in ContextWhat is already known?• Several miRNAs, including miR-132, are known to regulate beta cell function and mass in several mouse models of diabetes db/db, ob/ob and high fat-diet. What is the key question?• Which are the miRNAs implicated in control of beta cell regeneration upon partial pancreatectomy and how?What are the new findings?• miR-132 is critical to promote regeneration of mouse beta cells in vivo following partial pancreatectomy• In vitro studies in mouse MIN6 cells indicate that miR-132 fosters beta cell proliferation by down-regulating the expression of phosphatase Pten, thereby tilting the balance between anti-apoptotic ...