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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.

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.

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.

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.

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.

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.

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.

Introduction: Antiproliferative effects of immunosuppressants used in human islet transplantation interfere with the capability of the beta cells to balance cell renewal and cell loss. Consequently, long-term use of these drugs might contribute to graft dysfunction in islet transplant recipients. New[for full text, please go to the a.m. URL], 127. Kongress der Deutschen Gesellschaft für Chirurgie

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.

High glucose concentrations trigger the translocation of polypyrimidine-tract-binding protein 1 (PTB1) from the nucleus to the cytosol, where it stabilizes the mRNA of insulin and secretory granule proteins, thereby increasing their translation. This mechanism is required for rapid restoration of the secretory granule pool upon glucose stimulation. Its functional importance suggests that PTB1 signal-ling might be a good candidate for islet quality assessment prior to transplantation. However, all data concerning the »PTB1 pathway« was obtained in insulinoma cell lines and isolated rodent islets. Therefore, we investigated the glucose stimulated nucleocytoplasmic PTB1 translocation in human islets

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.

Introduction: Since the recent introduction of the Edmonton experience by Shapiro et al. the trans-plantation of insulin producing tissue has become an attractive therapeutic approach for the treatment of type 1 diabetes. However, in order to transfer this therapy into clinical routine, the currently available pancreatic tissue has to be used more efficiently. An essential criterion for that is the assessment of islet quality which can vary dramatically depending on donor related factors and the isolation procedure. Still, up to now it is not possible surely to predict the metabolic performance of islets after transplantation. Material and methods: Pancreatic islets of male Lewis rats were cultured for 24 hours after isolation. Samples of these islets were exposed to 2 hours glucose stimulation (25 mM). After stimulation, the insulin secretion index was measured as well as the binding activity of polypyrimidine tract-binding protein (PTB) onto the m-RNA of selected markers of secretory granules (ICA-512, PC1). In a syngenic transplantation model the islets were transplanted under the kidney capsule of diabetic Lewis rats as 1500 islet equivalents (IEQ), 1800 IEQ, 2000 IEQ and 2500 IEQ. The metabolic performance of the transplanted islets was monitored by daily measurements of blood glucose values and body weight for 3 months. Results: We were able to show the stabilization of insu-lin-mRNA and further markers of secretory granules through a RNA binding protein (PTB). The stabilization effect occurs after the translocation of PTB from the nucleus into the cytoplasm through phosphorylation and binding on the specific mRNAs.

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.

Perfluorocarbons are known for their high oxygen-solubility coefficients and for their maintenance of high-oxygen partial pressures for extended time. They serve as oxygen „reservoirs“ for harvested organs in pancreas organ transplantation. The aim of our study was to assess the influence of oxygen saturated perfluorohexane on isolated pancreatic islets at different culture times. Methods: The islets were isolated by collagenase digestion with Liberase. After digestion islets were separated by density gradient centrifugation and hand-picked under a stereomicroscope. After pooling they were divided in different portions in Petri dishes and cultured in RPMI-1640 medium with or without oxygen saturated perfluorohexane at 37°C. During cultivation medium change and oxygen saturation was performed every second day. For assessment of islet cell viability we measured insulin secretion with RIA. Apoptosis was assessed by PARP-immunostaining and western blotting, necrosis with trypan blue-staining, gene expression of insulin and ICA 512 (marker of secretory granules) mRNAs by real time PCR. Results: After 1, 4, 7, 9 and 11 days of culture we found a significant decrease of insulin secretion in perfluorocarbone treated islets. We also detected a higher rate of necrosis. Measurements of insulin and ICA512 mRNAs expression and nucleocytoplasmic translocation of polypyrimidine tract binding protein upon glucose stimulation are on-going. Conclusions: Our preliminary studies suggest that perfluorohexane has a detrimental effect on viability of rat purified islets in culture. While advantageous for the transport of harvested organs, use of perfluorocarbone may not be recommended during the purification and culture of islets for transplantation.

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 β-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.

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 Electronic supplementary material The online version of this article (10.1007/s00125-020-05192-7) contains a slideset of the figures for download, which is available to authorised users.

Failure of pancreatic β-cells contributes to the development of type 2 diabetes. Besides evidence of reduced glucose-stimulated insulin secretion and β-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.