[Show abstract][Hide abstract] ABSTRACT: 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.
Current Diabetes Reports 10/2015; 15(10):654. DOI:10.1007/s11892-015-0654-x · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.
PLoS ONE 04/2015; 10(4):e0123930. DOI:10.1371/journal.pone.0123930 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin secretion from pancreatic β-cells in response to sudden glucose stimulation, is biphasic. Prolonged secretion in vivo requires synthesis, delivery to the plasma membrane (PM) and exocytosis of insulin secretory granules (SGs). Here, we provide the first agent-based space-resolved model for SG dynamics in pancreatic β-cells. Using recent experimental data, we consider a single β-cell with identical SGs moving on a phenomenologically represented cytoskeleton network. A single exocytotic machinery mediates SG exocytosis on the PM. This novel model reproduces the measured spatial organization of SGs and insulin secretion patterns under different stimulation protocols. It proposes that the insulin potentiation effect and the rising second-phase secretion are mainly due to the increasing number of docking sites on the PM. Furthermore, it shows that for 6 min after glucose stimulation, the "newcomer" SGs are recruited from a region within less than 600 nm from the PM.
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[Show abstract][Hide abstract] ABSTRACT: Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.
Proceedings of the National Academy of Sciences 02/2015; 112(7). DOI:10.1073/pnas.1409542112 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Partial pancreatic resection is accompanied not only by a reduction in the islet cell mass but also by a variety of other factors that are likely to interfere with glucose metabolism. The aim of this work was to characterize the patient dynamics of blood glucose homeostasis during the course of partial pancreatic resection and to specify the associated clinico-pathological variables.
In total, 84 individuals undergoing elective partial pancreatic resection were consecutively recruited into this observational trial. The individuals were assigned based on their fasting glucose or oral glucose tolerance testing results into one of the following groups: (I) deteriorated, (II) stable or (III) improved glucose homeostasis three months after surgery. Co-variables associated with blood glucose dynamics were identified.
Of the 84 participants, 25 (30%) displayed a normal oGTT, 17 (20%) showed impaired glucose tolerance, and 10 (12%) exhibited pathological glucose tolerance. Elevated fasting glucose was present in 32 (38%) individuals before partial pancreatic resection. Three months after partial pancreatic resection, 14 (17%) patients deteriorated, 16 (19%) improved, and 54 (64%) retained stable glucose homeostasis. Stability and improvement was associated with tumor resection and postoperative normalization of recently diagnosed glucose dysregulation, preoperatively elevated tumor markers and markers for common bile duct obstruction, acute pancreatitis and liver cell damage. Improvement was linked to preoperatively elevated insulin resistance, which normalized after resection and was accompanied by a decrease in fasting- and glucose-stimulated insulin secretion.
Surgically reversible blood glucose dysregulation diagnosed concomitantly with a (peri-) pancreatic tumor appears secondary to compromised liver function due to tumor compression of the common bile duct and the subsequent increase in insulin resistance. It can be categorized as "cholestasis-induced diabetes" and thereby distinguished from other forms of hyperglycemic disorders.
PLoS ONE 01/2015; 10(8):e0134140. DOI:10.1371/journal.pone.0134140 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.
[Show abstract][Hide abstract] ABSTRACT: Phogrin/IA-2β and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet β-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and β-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure-function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Similarly to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold: a sheet of four antiparallel β-strands packed against two α-helices. Sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.
Journal of Structural and Functional Genomics 11/2014; 16(1). DOI:10.1007/s10969-014-9191-0
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: IntroductionPancreatic islet transplantation is currently restricted to patients with critical metabolic lability due to long-term need for immunosuppression and a persistent shortage of donor organs [1–3]. To overcome these obstacles we have developed a strategy for islet macroencapsulation that provides sufficient immune-isolation whereas regulated islet graft function is maintained [4–8].Case Report and MethodsA 63 year old patient with type 1 diabetes and severe metabolic lability was transplanted with isolated islets (2,000 islets/kgBW) encapsulated in an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers. Via a small abdominal incision, a pre-peritoneal pocket for the chamber was dissected, connected oxygen ports were implanted subcutaneously. No immunosuppressive therapy was applied.ResultsThe procedure was surgically straightforward and without complications. We could demonstrate persistent graft function by detection of endogenous insulin and c-peptide secretion proving islet viability and function. This observation was accompanied by persistent lowering in HbA1c despite reduction in insulin requirement.For oxygenation of the non-vascularized and therefore immune-shielded islet graft, the chamber-integrated gas reservoir was replenished daily via the implanted ports without complications.Conclusion
This encapsulation strategy was for the first time applied to allogeneic human islet transplantation in man. We demonstrated a persistent graft function with regulated insulin secretion without any immunosuppressive therapy. This novel concept may allow for future widespread application for cell-based therapies.References 2007 update on allogeneic islet transplantation from the Collaborative Islet Transplant Registry (CITR). Cell Transplant 2009; 18: 753–767. Ludwig, B., Ludwig, S., Steffen, A., Saeger, H.D., Bornstein, S.R. Islet versus pancreas transplantation in type 1 diabetes: competitive or complementary? Curr Diab Rep 2010; 10: 506–511. Mccall, M., James Shapiro, A.M. Update on islet transplantation. Cold Spring Harb Perspect Med 2012; 2: a007823. Barkai, U., Weir G.C., Colton C.K. et al. Enhanced Oxygen Supply Improves Islet Viability in a New Bioartificial Pancreas. Cell Transplant 2013; 22(8): 1463–1476 Ludwig, B., Rotem A., Schmid J. et al. Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist. Proc Natl Acad Sci U S A 2012; 109: 5022–5027. Ludwig, B., Zimmermann B., Steffen A. et al. A novel device for islet transplantation providing immune protection and oxygen supply. Horm Metab Res 2010; 42: 918–922. Neufeld, T., Ludwig B., Barkai U. et al. The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs. PLoS One 2013; 8: e70150. Ludwig, B., Reichel A, Steffen A et al. Transplantation of human islets without immunosuppression. PNAS 2013; 110: 19054–19058.
[Show abstract][Hide abstract] ABSTRACT: β-cells of the pancreatic islets are highly specialized and high-throughput units for the production of insulin, the key hormone for maintenance of glucose homeostasis. Elevation of extracellular glucose and/or GLP-1 levels triggers a rapid upregulation of insulin biosynthesis through the activation of post-transcriptional mechanisms. RNA-binding proteins are emerging as key factors in the regulation of these mechanisms as well as in other aspects of β-cell function and glucose homeostasis at large, and thus may be implicated in the pathogenesis of diabetes. Here we review current research in the field, with a major emphasis on RNA-binding proteins that control biosynthesis of insulin and other components of the insulin secretory granules by modulating the stability and translation of their mRNAs.
[Show abstract][Hide abstract] ABSTRACT: Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.
Proceedings of the National Academy of Sciences 10/2013; 110(47). DOI:10.1073/pnas.1317561110 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR(-/-) mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR(-/-) mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in β cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.
The Journal of Immunology 09/2013; 191(8). DOI:10.4049/jimmunol.1300038 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin is stored within the secretory granules of pancreatic beta cells and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here we illustrate a technology that enables the study of granule aging in insulinoma cells and beta cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly-synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic beta cells. Furthermore, the method is of general interest for the study of membrane traffic and aging.