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Sample records for allogeneic pancreatic islet

  1. Organ procurement organization compliance with 21 CFR 1271: a challenge for allogeneic pancreatic islet cell transplantation programs.

    PubMed

    Winters, J L; Tran, S A; Gastineau, D A; Padley, D J; Dean, P G; Kudva, Y C

    2009-06-01

    In order to protect tissue recipients, the Food and Drug Administration drafted Title 21, Section 1271 of the Code of Federal Regulations 1271 (21 CFR 1271) to address infectious disease risk. These regulations apply to tissues but not vascularized organs. Pancreatic islet cells are regulated under 21 CFR 1271. These regulations require qualification of suppliers of critical materials and services with regard to 21 CFR 1271 compliance. As part of supplier qualification, all organ procurement organizations (OPOs) in the United States were sent a questionnaire covering the key components of these regulations. Of the 57 OPOs, 29 (51%) were in compliance based upon survey results. Twelve (21%) were not compliant in one or more areas. All indicated plans to become compliant. The remaining 15 (27%) either failed or refused to complete the survey, some indicating 21 CFR 1271 did not apply to OPOs. Using 2006 data, OPOs compliant with 21 CFR 1271 recovered 50% of the organs procured in the United States. These findings represent a challenge for allogeneic islet cell transplant programs whose raw material must comply with 21 CFR 1271. OPOs should work toward understanding and complying with 21 CFR 1271. Regulatory agencies should work toward enhancing safety of the pancreas supply by facilitating compliance through harmonization of requirements.

  2. Pancreatic Islet Transplantation

    MedlinePlus

    ... allo-transplantation?" For each pancreatic islet allo-transplant infusion, researchers use specialized enzymes to remove islets from ... in a lab. Transplant patients typically receive two infusions with an average of 400,000 to 500, ...

  3. G-CSF and Exenatide Might Be Associated with Increased Long-Term Survival of Allogeneic Pancreatic Islet Grafts

    PubMed Central

    Peixoto, Eduardo; Messinger, Shari; Mantero, Alejandro; Padilla-Téllez, Nathalia D.; Baidal, David A.; Alejandro, Rodolfo; Ricordi, Camillo; Inverardi, Luca

    2016-01-01

    Background Allogeneic human islet transplantation is an effective therapy for the treatment of patients with Type 1 Diabetes (T1D). The low number of islet transplants performed worldwide and the different transplantation protocols used limit the identification of the most effective therapeutic options to improve the efficacy of this approach. Methods We present a retrospective analysis on the data collected from 44 patients with T1D who underwent islet transplantation at our institute between 2000 and 2007. Several variables were included: recipient demographics and immunological characteristics, donor and transplant characteristics, induction protocols, and additional medical treatment received. Immunosuppression was induced with anti-CD25 (Daclizumab), alone or in association with anti-tumor necrosis factor alpha (TNF-α) treatments (Etanercept or Infliximab), or with anti-CD52 (Alemtuzumab) in association with anti-TNF-α treatments (Etanercept or Infliximab). Subsets of patients were treated with Filgrastim for moderate/severe neutropenia and/or Exenatide for post prandial hyperglycemia. Results The analysis performed indicates a negative association between graft survival (c-peptide level ≥ 0.3 ng/ml) and islet infusion volume, with the caveat that, the progressive reduction of infusion volumes over the years has been paralleled by improved immunosuppressive protocols. A positive association is instead suggested between graft survival and administration of Exenatide and Filgrastim, alone or in combination. Conclusion This retrospective analysis may be of assistance to further improve long-term outcomes of protocols for transplant of islets and other organs. PMID:27285580

  4. Unraveling pancreatic islet biology by quantitative proteomics

    SciTech Connect

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  5. The Different Faces of the Pancreatic Islet.

    PubMed

    Abdulreda, Midhat H; Rodriguez-Diaz, Rayner; Cabrera, Over; Caicedo, Alejandro; Berggren, Per-Olof

    2016-01-01

    Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from

  6. Pancreatic islet blood flow and its measurement

    PubMed Central

    Jansson, Leif; Barbu, Andreea; Bodin, Birgitta; Drott, Carl Johan; Espes, Daniel; Gao, Xiang; Grapensparr, Liza; Källskog, Örjan; Lau, Joey; Liljebäck, Hanna; Palm, Fredrik; Quach, My; Sandberg, Monica; Strömberg, Victoria; Ullsten, Sara; Carlsson, Per-Ola

    2016-01-01

    Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future. PMID:27124642

  7. Isolation of Mouse Pancreatic Islets of Langerhans.

    PubMed

    Ramírez-Domínguez, Miriam

    2016-01-01

    The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol. PMID:27586420

  8. Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition

    PubMed Central

    Steiner, Donald J.; Kim, Abraham; Miller, Kevin; Hara, Manami

    2010-01-01

    The pancreatic islet displays diverse patterns of endocrine cell arrangement. The prototypic islet, with insulin-secreting β-cells forming the core surrounded by other endocrine cells in the periphery, is largely based on studies of normal rodent islets. Recent reports on large animals, including humans, show a difference in islet architecture, in which the endocrine cells are randomly distributed throughout the islet. This particular species difference has raised concerns regarding the interpretation of data based on rodent studies to humans. On the other hand, further variations have been reported in marsupials and some nonhuman primates, which possess an inverted ratio of β-cells to other endocrine cells. This review discusses the striking plasticity of islet architecture and cellular composition among various species including changes in response to metabolic states within a single species. We propose that this plasticity reflects evolutionary acquired adaptation induced by altered physiological conditions, rather than inherent disparities between species. PMID:20657742

  9. Mesenchymal Stem Cells as Feeder Cells for Pancreatic Islet Transplants

    PubMed Central

    Sordi, Valeria; Piemonti, Lorenzo

    2010-01-01

    Allogeneic islet transplantation serves as a source of insulin-secreting beta-cells for the maintenance of normal glucose levels and treatment of diabetes. However, limited availability of islets, high rates of islet graft failure, and the need for life-long non-specific immunosuppressive therapy are major obstacles to the widespread application of this therapeutic approach. To overcome these problems, pancreatic islet transplantation was recently suggested as a potential target of the "therapeutic plasticity" of adult stem cells. In fact, new results suggest that stem/precursor cells, and mesenchymal stem cells in particular, co-transplanted with islets can promote tissue engraftment and beta-cell survival via bystander mechanisms, mainly exerted by creating a milieu of cytoprotective and immunomodulatory molecules. This evidence consistently challenges the limited view that stem/precursor cells work exclusively through beta-cell replacement in diabetes therapy. It proposes that stem cells also act as "feeder" cells for islets, and supporter of graft protection, tissue revascularization, and immune acceptance. This article reviews the experience of using stem cell co-transplantation as strategy to improve islet transplantation. It highlights that comprehension of the mechanisms involved will help to identify new molecular targets and promote development of new pharmacological strategies to treat type 1 and type 2 diabetes patients. PMID:21060972

  10. Mesenchymal stem cells as feeder cells for pancreatic islet transplants.

    PubMed

    Sordi, Valeria; Piemonti, Lorenzo

    2010-01-01

    Allogeneic islet transplantation serves as a source of insulin-secreting beta-cells for the maintenance of normal glucose levels and treatment of diabetes. However, limited availability of islets, high rates of islet graft failure, and the need for life-long non-specific immunosuppressive therapy are major obstacles to the widespread application of this therapeutic approach. To overcome these problems, pancreatic islet transplantation was recently suggested as a potential target of the "therapeutic plasticity" of adult stem cells. In fact, new results suggest that stem/precursor cells, and mesenchymal stem cells in particular, co-transplanted with islets can promote tissue engraftment and beta-cell survival via bystander mechanisms, mainly exerted by creating a milieu of cytoprotective and immunomodulatory molecules. This evidence consistently challenges the limited view that stem/precursor cells work exclusively through beta-cell replacement in diabetes therapy. It proposes that stem cells also act as "feeder" cells for islets, and supporter of graft protection, tissue revascularization, and immune acceptance. This article reviews the experience of using stem cell co-transplantation as strategy to improve islet transplantation. It highlights that comprehension of the mechanisms involved will help to identify new molecular targets and promote development of new pharmacological strategies to treat type 1 and type 2 diabetes patients. PMID:21060972

  11. Pancreatic islet transplantation for treating diabetes.

    PubMed

    Matsumoto, Shinichi; Noguchi, Hirofumi; Yonekawa, Yukihide; Okitsu, Teru; Iwanaga, Yasuhiro; Liu, Xiaoling; Nagata, Hideo; Kobayashi, Naoya; Ricordi, Camillo

    2006-01-01

    Pancreatic islet transplantation is one of the options for treating diabetes and has been shown to improve the quality of life of severe diabetic patients. Since the Edmonton protocol was announced, islet transplantation have advanced considerably, including islet after kidney transplantation, utilisation of non-heart-beating donors, single-donor islet transplantation and living-donor islet transplantation. These advances were based on revised immunosuppression protocols, improved pancreas procurement and islet isolation methods, and enhanced islet engraftment. Further improvements are necessary to make islet transplantation a routine clinical treatment. To synergise efforts towards a cure for type 1 diabetes, a Diabetes Research Institute (DRI) Federation is currently being established to include leading diabetes research centres worldwide, including DRIs in Miami, Edmonton and Kyoto among others.

  12. Young capillary vessels rejuvenate aged pancreatic islets.

    PubMed

    Almaça, Joana; Molina, Judith; Arrojo E Drigo, Rafael; Abdulreda, Midhat H; Jeon, Won Bae; Berggren, Per-Olof; Caicedo, Alejandro; Nam, Hong Gil

    2014-12-01

    Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature. PMID:25404292

  13. Survival prolongation of microencapsulated allogeneic islet by nanosized nordihydroguaiaretic acid.

    PubMed

    Yang, T-Y; Chen, J-P; Ku, K-W; Fu, S-H; Hsu, B R-S

    2005-05-01

    Immunoisolation such as alginate-poly-L-lysine-alginate (APA) microencapsulation may protect entrapped islet graft cells from destruction by cellular and humoral immunities, but cannot avoid aggregation of macrophages and fibroblasts around microcapsules, which has been known to cause late dysfunction. Nordihydroguaiaretic acid (NDGA) is a lipoxygenase inhibitor that prevents the activation and chemotaxis of macrophages. In this study, we used the dialysis method without surfactant to prepare poly (DL-lactide-co-glycolide) (PLGA) nanoparticles to entrap NDGA. We determined the formulation conditions suitable for sustained release when coencapsulated with the islets. Nanoparticle sizes of 0.2-0.3 microm were suitable for sustained release in electromagnetic driven APA microcapsules. In the toxicity study, we coincubated islets with PLGA-NDGA nanoparticles in vitro for 2 and 4 weeks. The glucose stimulated insulin secretion and insulin contents of islets were not influenced significantly. To test whether nanosized NDGA provides extra protection for APA islets, about 160-200 allogeneic islets of C57BL/6 mice were either encapsulated alone using APA or coencapsulated with PLGA-NDGA. At 2 and 4 weeks after implantation into the peritoneal cavities of healthy BALB/c mice, the intraperitoneal islet grafts were recovered using lavage. Mice that received islets of APA-PLGA-NDGA preparations showed a higher recovery rate of functioning grafts than those that received islets prepared using APA alone (10.1%, n = 4 vs 5.2%, n = 3). In conclusion, nanosized NDGA prolonged the graft survival of APA microencapsulated allogeneic islets.

  14. The pancreatic islet as a signaling hub.

    PubMed

    Barker, Christopher J; Leibiger, Ingo B; Berggren, Per-Olof

    2013-01-01

    Over the last two decades we have focused on beta cell signal transduction, bringing many new insights, especially in the context of insulin signal transduction, the role of inositol polyphosphates and the regulation of cytoplasmic free Ca(2+) concentration. However, there has been a growing awareness that the beta cell, which is mandatory for insulin secretion, has a unique context within the micro-organ of the pancreatic Islet of Langerhans. In this environment the beta cell both mediates and receives paracrine regulation, critical for the control of blood glucose homeostasis. Failure of an appropriate beta cell function leads to the development of diabetes mellitus. In our quest to understand the molecular events maintaining beta cell function we have faced two key challenges. Firstly, whilst there are many similarities between signal transduction in pancreatic islets between the much used rodent models and humans there are some notable differences. Critical distinctions between rodent and primate can be made in the structure of the islet, including the arrangement of the islet cells, the innervation pattern and the microcirculation. This means that important signaling interactions between islets cells, mediated through for example insulin, glucagon, GABA, glutamate and ATP, will have a unique human framework. The second challenge was to be able to take the discoveries we have made using in vitro systems and examine them in an in vivo context. Advances in in vivo imaging achieved by utilizing the anterior chamber of the eye as a transplantation site for pancreatic islets make it possible for non-invasive, longitudinal studies at single cell resolution in real time of islet cell physiology and pathology. Thus it is becoming possible to study the insulin secreting pancreatic beta cell within the framework of the unique micro-organ, the Islet of Langerhans, for the first time in a physiological context, i.e. when being innervated and connected to the blood supply.

  15. Circadian variation of the pancreatic islet transcriptome.

    PubMed

    Rakshit, Kuntol; Qian, Jingyi; Ernst, Jason; Matveyenko, Aleksey V

    2016-09-01

    Pancreatic islet failure is a characteristic feature of impaired glucose control in diabetes mellitus. Circadian control of islet function is essential for maintaining proper glucose homeostasis. Circadian variations in transcriptional pathways have been described in diverse cell types and shown to be critical for optimization of cellular function in vivo. In the current study, we utilized Short Time Series Expression Miner (STEM) analysis to identify diurnally expressed transcripts and biological pathways from mouse islets isolated at 4 h intervals throughout the 24 h light-dark cycle. STEM analysis identified 19 distinct chronological model profiles, and genes belonging to each profile were subsequently annotated to significantly enriched Kyoto Encyclopedia of Genes and Genomes biological pathways. Several transcriptional pathways essential for proper islet function (e.g., insulin secretion, oxidative phosphorylation), cell survival (e.g., insulin signaling, apoptosis) and cell proliferation (DNA replication, homologous recombination) demonstrated significant time-dependent variations. Notably, KEGG pathway analysis revealed "protein processing in endoplasmic reticulum - mmu04141" as one of the most enriched time-dependent pathways in islets. This study provides unique data set on time-dependent diurnal profiles of islet gene expression and biological pathways, and suggests that diurnal variation of the islet transcriptome is an important feature of islet homeostasis, which should be taken into consideration for optimal experimental design and interpretation of future islet studies. PMID:27495157

  16. Human Pancreatic Islets and Diabetes Research

    PubMed Central

    Kaddis, John S.; Olack, Barbara J.; Sowinski, Janice; Cravens, James; Contreras, Juan L.; Niland, Joyce C.

    2013-01-01

    Human islet research is crucial to understanding the cellular biology of the pancreas in developing therapeutic options for diabetes patients and in attempting to prevent the development of this disease. The national Islet Cell Resource Center Consortium provides human pancreatic islets for diabetes research while simultaneously addressing the need to improve islet isolation and transplantation technologies. Since its inception in 2001, the consortium has supplied 297.6 million islet equivalents to 151 national and international scientists for use in clinical and laboratory projects. Data on the volume, quality, and frequency of shipments substantiate the importance of human islets for diabetes research, as do the number of funded grants for beta-cell projects and publications produced as a direct result of islets supplied by this resource. Limitations in using human islets are discussed, along with the future of islet distribution centers. The information presented here is instructive to clinicians, basic science investigators, and policy makers who determine the availability of funding for such work. Organ procurement coordinators also may find the information useful in explaining to donor families why research consent is so valuable. PMID:19366778

  17. Pancreatic Islet Cell Development and Regeneration

    PubMed Central

    Romer, Anthony I.; Sussel, Lori

    2015-01-01

    Purpose This review will discuss recent advances in understanding mouse and human pancreatic islet cell development, novel concepts related to β cell dysfunction and improved approaches for replenishing β cells to treat diabetes. Recent Findings Considerable knowledge about pancreatic islet development and function has been gained using model systems with subsequent validation in human tissues. Recently, several rodent studies have revealed that differentiated adult islet cells retain remarkable plasticity and can be converted to other islet cell types by perturbing their transcription factor profiles. Furthermore, significant advances have been made in the generation of β-like cells from stem cell populations. Therefore, the generation of functionally mature β cells by the in situ conversion of non-β cell populations or by the directed differentiation of human pluripotent stem cells could represent novel mechanisms for replenishing β cells in diabetic patients. Summary The overall conservation between mouse and human pancreatic development, islet physiology and etiology of diabetes encourages the translation of novel β cell replacement therapies to humans. Further deciphering the molecular mechanisms that direct islet cell regeneration, plasticity and function could improve and expand the β cell replacement strategies for treating diabetes. PMID:26087337

  18. In vivo imaging of pancreatic endocrine islets

    NASA Astrophysics Data System (ADS)

    Villiger, Martin; Goulley, Joan; Pache, Christophe; Friedrich, Michael; Grapin-Botton, Anne; Meda, Paolo; Leitgeb, Rainer; Lasser, Theo

    2009-07-01

    Extended focus optical coherence microscope (xfOCM) circumvents the compromise between lateral resolution and depth of field by us of a Bessel-like illumination beam. The high sensitivity and parallel depth profiling of Fourier domain optical coherence tomography are preserved, and combined with high isotropic resolution of 1.5 - 2 μm. To comply with the requirements for in vivo measurements, beam scanning had to be implemented. We then performed measurements, first of excised pancreas, validated by standard immunohistochemistry, to investigate the structures that can be observed. For a quantitative analysis, a semi-automatic islet detection algorithm evaluated the islet size, position, contrast and homogeneity. The influence of streptozotocin on the signature of the islets was investigated in a next step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo, visualizing pancreatic lobules, ducts, blood vessels and individual islets of Langerhans.

  19. General Information about Pancreatic Neuroendocrine Tumors (Islet Cell Tumors)

    MedlinePlus

    ... Islet Cell Tumors) Treatment (PDQ®)–Patient Version General Information About Pancreatic Neuroendocrine Tumors (Islet Cell Tumors) Go ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  20. Spontaneous pancreatic islet amyloidosis in 40 baboons.

    PubMed

    Hubbard, G B; Steele, K E; Davis, K J; Leland, M M

    2002-04-01

    Spontaneous amyloidosis occurs in many nonhuman primate species but remains difficult to diagnose and treat. Nonhuman primates continue to offer promise as animal models in which to study amyloidosis in humans. Amyloidosis was not diagnosed clinically but was found histologically in four male and 36 female baboons. The baboons averaged 18 years of age at death (range, 7-28 years). Clinical signs, if present, were hyperglycemia and cachexia. Blood glucose values were elevated in 12 of 30 baboons with available clinical pathology data. Four baboons had been clinically diagnosed as diabetic and three were treated with insulin. Amyloid was found in the islets of Langerhans of the pancreas in 40 baboons; 35 baboons had amyloid only in the islets of Langerhans. Amyloid was found in nonislet tissue of baboons as follows: five, nonislet pancreas; four, intestine and adrenal; three, kidney; two, prostate and spleen; and one each, lymph node, liver, gall bladder, stomach, tongue, urinary bladder, and salivary gland. Sections of paraffin-embedded tissues were evaluated for amyloid with hematoxylin and eosin (HE) and congo red (CR) staining, and using immunohistochemistry for human islet amyloid polypeptide (IAPP), calcitonin gene-related peptide (CGRP), glucagon, pancreatic polypeptide (PP), somatostatin (SS), and porcine insulin. Islet amyloid was positive with HE in 40 baboons, with CR in 39 baboons, and with IAPP and CGRP in 35 baboons. IAPP and CGRP only stained islet amyloid. PP, SS, glucagon, and porcine insulin did not stain amyloid. Islet amyloidosis in the baboon appears to be difficult to diagnose clinically, age-related, and similar to islet amyloidosis in other species. The baboon may be a good model for the study of islet amyloidosis in humans.

  1. [Xenogeneic cell therapeutics: Treatment of type 1 diabetes using porcine pancreatic islets and islet cells].

    PubMed

    Godehardt, Antonia W; Schilling-Leiß, Dagmar; Sanzenbacher, Ralf; Tönjes, Ralf R

    2015-11-01

    In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

  2. Staining Protocols for Human Pancreatic Islets

    PubMed Central

    Campbell-Thompson, Martha L.; Heiple, Tiffany; Montgomery, Emily; Zhang, Li; Schneider, Lynda

    2012-01-01

    Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database

  3. Historical Background of Pancreatic Islet Isolation.

    PubMed

    Ramírez-Domínguez, Miriam

    2016-01-01

    Until the discovery of insulin in the twentieth century, diabetes mellitus was a mortal disease with an unclear origin and physiology. Despite the appearance of the concept in an Egyptian papyrus dated c.1550 BC, and the documentation of its study by ancient Chinese, the term "diabetes" was only coined by the Greek Aretaeus in the second century AD. In Europe, the study of diabetes was largely ignored until the seventeenth century, when the characteristic sweet flavor of diabetic urine was first described. However, the link between diabetes and the pancreas was not discovered until 1889 by Minkowski and von Mering, long after the first description of the pancreatic islets by Paul Langerhans in 1869. One of the most significant milestones in the field was the discovery of insulin by Banting and collaborators in 1922, which led to the therapeutic development of insulin administration as a life-saving intervention for type 1 diabetic patients. On the other hand, the isolation of islets was first reported by Bensley in 1911, a critical technical achievement that paved the way for clinical islet transplantation. Here we discuss the history of islet isolation, since the firsts studies of diabetes by ancient civilizations to the birth and parallel evolution of islet isolation and transplantation. PMID:27586418

  4. Protein phosphatases in pancreatic islets

    PubMed Central

    Ortsäter, Henrik; Grankvist, Nina; Honkanen, Richard E.; Sjöholm1, Åke

    2014-01-01

    The prevalence of diabetes is increasing rapidly world-wide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis. PMID:24681827

  5. Transcriptional Regulation of the Pancreatic Islet: Implications for Islet Function

    PubMed Central

    Stitzel, Michael L.; Kycia, Ina; Kursawe, Romy; Ucar, Duygu

    2015-01-01

    Islets of Langerhans contain multiple hormone-producing endocrine cells controlling glucose homeostasis. Transcription establishes and maintains islet cellular fates and identities. Genetic and environmental disruption of islet transcription triggers cellular dysfunction and disease. Early transcriptional regulation studies of specific islet genes, including insulin (INS) and the transcription factor PDX1, identified the first cis-regulatory DNA sequences and trans-acting factors governing islet function. Here, we review how human islet “omics” studies are reshaping our understanding of transcriptional regulation in islet (dys)function and diabetes. First, we highlight the expansion of islet transcript number, form, and function and of DNA transcriptional regulatory elements controlling their production. Next, we cover islet transcriptional effects of genetic and environmental perturbation. Finally, we discuss how these studies’ emerging insights should empower our diabetes research community to build mechanistic understanding of diabetes pathophysiology and to equip clinicians with tailored, precision medicine options to prevent and treat islet dysfunction and diabetes. PMID:26272056

  6. Microfluidics-generated pancreatic islet microfibers for enhanced immunoprotection.

    PubMed

    Jun, Yesl; Kim, Min Jun; Hwang, Yong Hwa; Jeon, Eun Ae; Kang, Ah Ran; Lee, Sang-Hoon; Lee, Dong Yun

    2013-11-01

    Pancreatic islet transplantation is a promising method for treatment of type 1 diabetes mellitus. However, transplanted islets can be destroyed due to host immune reactions. To immunologically protect transplanted islets, here an immunoprotective microfiber including islets by using a polydimethylsiloxane (PDMS)-based microfluidic device is newly designed. A cylindrical-flow channel in the microfluidic platform is used for producing collagen-alginate composite (CAC) fibers. This enables mass production and uniform diameter distribution (<250 μm) without protruding islets. Collagen, which is the main extracellular matrix component, is added to alginate to mimic the native islet microenvironment. Compared to free islets (control) and alginate-fiber-encapsulated islets, CAC-fiber-encapsulated islets show higher viability and normal insulin secretion. When CAC-fiber-encapsulated islets (1200 islet equivalent) are implanted into the intraperitoneal cavity of streptozotocin-induced diabetic BALB/C mice, the blood glucose levels of all mice return to normoglycemia. Moreover, intraperitoneal glucose tolerance tests demonstrate that islets in the CAC-fiber have similar glucose responsiveness to those of non-diabetic normal mice. These results are attributed to the immunoprotection of the transplanted islets from host immune reactions. On the other hand, all free islets are completely rejected within a week due to severe immune responses. Collectively, fabrication of CAC fibers using microfluidic devices can be used for successful islet transplantation. PMID:23927952

  7. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  8. The microsomal glucose-6-phosphatase enzyme of pancreatic islets.

    PubMed Central

    Waddell, I D; Burchell, A

    1988-01-01

    Microsomal fractions isolated from pancreatic islet cells were shown to contain high specific glucose-6-phosphatase activity. The islet-cell glucose-6-phosphatase enzyme has the same Mr (36,500), similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme. Images Fig. 1. Fig. 2. PMID:2849415

  9. Imaging pancreatic islet cells by positron emission tomography

    PubMed Central

    Li, Junfeng; Karunananthan, Johann; Pelham, Bradley; Kandeel, Fouad

    2016-01-01

    It was estimated that every year more than 30000 persons in the United States - approximately 80 people per day - are diagnosed with type 1 diabetes (T1D). T1D is caused by autoimmune destruction of the pancreatic islet (β cells) cells. Islet transplantation has become a promising therapy option for T1D patients, while the lack of suitable tools is difficult to directly evaluate of the viability of the grafted islet over time. Positron emission tomography (PET) as an important non-invasive methodology providing high sensitivity and good resolution, is able to accurate detection of the disturbed biochemical processes and physiological abnormality in living organism. The successful PET imaging of islets would be able to localize the specific site where transplanted islets engraft in the liver, and to quantify the level of islets remain alive and functional over time. This information would be vital to establishing and evaluating the efficiency of pancreatic islet transplantation. Many novel imaging agents have been developed to improve the sensitivity and specificity of PET islet imaging. In this article, we summarize the latest developments in carbon-11, fluorine-18, copper-64, and gallium-68 labeled radioligands for the PET imaging of pancreatic islet cells. PMID:27721939

  10. Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

    PubMed Central

    Heileman, K.; Daoud, J.; Hasilo, C.; Gasparrini, M.; Paraskevas, S.; Tabrizian, M.

    2015-01-01

    Human pancreatic islets are seldom assessed for dynamic responses to external stimuli. Thus, the elucidation of human islet functionality would provide insights into the progression of diabetes mellitus, evaluation of preparations for clinical transplantation, as well as for the development of novel therapeutics. The objective of this study was to develop a microfluidic platform for in vitro islet culture, allowing the multi-parametric investigation of islet response to chemical and biochemical stimuli. This was accomplished through the fabrication and implementation of a microfluidic platform that allowed the perifusion of islet culture while integrating real-time monitoring using impedance spectroscopy, through microfabricated, interdigitated electrodes located along the microchamber arrays. Real-time impedance measurements provide important dielectric parameters, such as cell membrane capacitance and cytoplasmic conductivity, representing proliferation, differentiation, viability, and functionality. The perifusion of varying glucose concentrations and monitoring of the resulting impedance of pancreatic islets were performed as proof-of-concept validation of the lab-on-chip platform. This novel technique to elucidate the underlying mechanisms that dictate islet functionality is presented, providing new information regarding islet function that could improve the evaluation of islet preparations for transplantation. In addition, it will lead to a better understanding of fundamental diabetes-related islet dysfunction and the development of therapeutics through evaluation of potential drug effects. PMID:26339324

  11. Remodelling sympathetic innervation in rat pancreatic islets ontogeny

    PubMed Central

    Cabrera-Vásquez, Siraam; Navarro-Tableros, Víctor; Sánchez-Soto, Carmen; Gutiérrez-Ospina, Gabriel; Hiriart, Marcia

    2009-01-01

    Background Pancreatic islets are not fully developed at birth and it is not clear how they are vascularised and innervated. Nerve Growth Factor (NGF) is required to guide sympathetic neurons that innervate peripheral organs and also in cardiovascular system and ovary angiogenesis. Pancreatic beta cells of a transgenic mouse that over-expressed NGF in attracts sympathetic hyper-innervation towards them. Moreover, we have previously demonstrated that adult beta cells synthesize and secrete NGF; however, we do not know how is NGF secreted during development, nor if it might be trophic for sympathetic innervation and survival in the pancreas. We analyzed sympathetic innervation and vasculature development in rat pancreatic islets at different developmental stages; foetal (F19), early postnatal (P1), weaning period (P20) and adults. We temporarily correlated these events to NGF secretion by islet cells. Results Sympathetic fibres reached pancreatic islets in the early postnatal period, apparently following blood vessels. The maximal number of sympathetic fibres (TH immunopositive) in the periphery of the islets was observed at P20, and then fibres entered the islets and reached the core where beta cells are mainly located. The number of fibres decreased from that stage to adulthood. At all stages studied, islet cells secreted NGF and also expressed the high affinity receptor TrkA. Foetal and neonatal isolated islet cells secreted more NGF than adults. TrkA receptors were expressed at all stages in pancreatic sympathetic fibres and blood vessels. These last structures were NGF–immunoreactive only at early stages (foetal and P0). Conclusion The results suggest that NGF signalling play an important role in the guidance of blood vessels and sympathetic fibres toward the islets during foetal and neonatal stages and could also preserve innervation at later stages of life. PMID:19534767

  12. Autologous Pancreatic Islet Transplantation in Human Bone Marrow

    PubMed Central

    Maffi, Paola; Balzano, Gianpaolo; Ponzoni, Maurilio; Nano, Rita; Sordi, Valeria; Melzi, Raffaella; Mercalli, Alessia; Scavini, Marina; Esposito, Antonio; Peccatori, Jacopo; Cantarelli, Elisa; Messina, Carlo; Bernardi, Massimo; Del Maschio, Alessandro; Staudacher, Carlo; Doglioni, Claudio; Ciceri, Fabio; Secchi, Antonio; Piemonti, Lorenzo

    2013-01-01

    The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans. PMID:23733196

  13. Comparison of volume estimation methods for pancreatic islet cells

    NASA Astrophysics Data System (ADS)

    Dvořák, JiřÃ.­; Å vihlík, Jan; Habart, David; Kybic, Jan

    2016-03-01

    In this contribution we study different methods of automatic volume estimation for pancreatic islets which can be used in the quality control step prior to the islet transplantation. The total islet volume is an important criterion in the quality control. Also, the individual islet volume distribution is interesting -- it has been indicated that smaller islets can be more effective. A 2D image of a microscopy slice containing the islets is acquired. The input of the volume estimation methods are segmented images of individual islets. The segmentation step is not discussed here. We consider simple methods of volume estimation assuming that the islets have spherical or ellipsoidal shape. We also consider a local stereological method, namely the nucleator. The nucleator does not rely on any shape assumptions and provides unbiased estimates if isotropic sections through the islets are observed. We present a simulation study comparing the performance of the volume estimation methods in different scenarios and an experimental study comparing the methods on a real dataset.

  14. Immune responses to an encapsulated allogeneic islet {beta}-cell line in diabetic NOD mice

    SciTech Connect

    Black, Sasha P. . E-mail: Sasha.Black@ca.crl.com; Constantinidis, Ioannis; Cui, Hong; Tucker-Burden, Carol; Weber, Collin J.; Safley, Susan A.

    2006-02-03

    Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic {beta}-cell line ({beta}TC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of {beta}TC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic {beta}-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is First extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

  15. Mesobiliverdin IXα Enhances Rat Pancreatic Islet Yield and Function.

    PubMed

    Ito, Taihei; Chen, Dong; Chang, Cheng-Wei Tom; Kenmochi, Takashi; Saito, Tomonori; Suzuki, Satoshi; Takemoto, Jon Y

    2013-01-01

    The aims of this study were to produce mesobiliverdin IXα, an analog of anti-inflammatory biliverdin IXα, and to test its ability to enhance rat pancreatic islet yield for allograft transplantation into diabetic recipients. Mesobiliverdin IXα was synthesized from phycocyanobilin derived from cyanobacteria, and its identity and purity were analyzed by chromatographic and spectroscopic methods. Mesobiliverdin IXα was a substrate for human NADPH biliverdin reductase. Excised Lewis rat pancreata infused with mesobiliverdin IXα and biliverdin IXα-HCl (1-100 μM) yielded islet equivalents as high as 86.7 and 36.5%, respectively, above those from non-treated controls, and the islets showed a high degree of viability based on dithizone staining. When transplanted into livers of streptozotocin-induced diabetic rats, islets from pancreata infused with mesobiliverdin IXα lowered non-fasting blood glucose (BG) levels in 55.6% of the recipients and in 22.2% of control recipients. In intravenous glucose tolerance tests, fasting BG levels of 56 post-operative day recipients with islets from mesobiliverdin IXα infused pancreata were lower than those for controls and showed responses that indicate recovery of insulin-dependent function. In conclusion, mesobiliverdin IXα infusion of pancreata enhanced yields of functional islets capable of reversing insulin dysfunction in diabetic recipients. Since its production is scalable, mesobiliverdin IXα has clinical potential as a protectant of pancreatic islets for allograft transplantation. PMID:23630498

  16. Biomolecular Surface Engineering of Pancreatic Islets with Thrombomodulin

    PubMed Central

    Wilson, John T.; Haller, Carolyn A.; Qu, Zheng; Cui, Wanxing; Urlam, Murali K.; Chaikof, Elliot L.

    2010-01-01

    Islet transplantation has emerged as a promising treatment for Type 1 diabetes, but its clinical impact remains limited by early islet destruction mediated by prothrombotic and innate inflammatory responses elicited upon transplantation. Thrombomodulin (TM) acts as an important regulator of thrombosis and inflammation through its capacity to channel the catalytic activity of thrombin towards generation of activated protein C (APC), a potent anti-coagulant and anti-inflammatory agent. We describe herein a novel biomolecular strategy for re-engineering the surface of pancreatic islets with TM. A biosynthetic approach was employed to generate recombinant human TM (rTM) bearing a C-terminal azide group, which facilitated site-specific biotinylation of rTM through Staudinger ligation. Murine pancreatic islets were covalently biotinylated through targeting of cell surface amines and aldehydes, and both islet viability and the surface density of streptavidin were maximized through optimization of biotinylation conditions. rTM was immobilized on islet surfaces through streptavidin-biotin interactions, resulting in a nearly three-fold increase in the catalytic capacity of islets to generate APC. PMID:20102751

  17. Isolation of Pancreatic Islets from Nonhuman Primates.

    PubMed

    Berman, Dora M

    2016-01-01

    Nonhuman primates (NHP) constitute a highly relevant pre-clinical animal model to develop strategies for beta cell replacement. The close phylogenetic and immunologic relationship between NHP and humans results in cross-reactivity of various biological agents with NHP cells, as well as a very similar cytoarchitecture between islets from human and NHP that is strikingly different from that observed in rodent islets. The composition and location of endocrine cells in human or NHP islets, randomly distributed and associated with blood vessels, have functional consequences and a predisposition for paracrine interactions. Furthermore, translation of approaches that proved successful in rodent models to the clinic has been limited. Consequently, data collected from NHP studies can form the basis for an IND submission to the FDA. This chapter describes in detail the key aspects for isolation of islets from NHP, from organ procurement up to assessment of islet function, comparing and emphasizing the similarities between isolation procedures for human and NHP islets. PMID:27586422

  18. Demonstration of pepsinogen C in human pancreatic islets.

    PubMed Central

    Szecsi, P B; Halgreen, H; Poulsen, S S; Axelsson, C K; Damkjaer-Nielsen, M; Kjaer, T; Foltmann, B

    1987-01-01

    Pancreatic tissue from 16 post mortem kidney donors have been examined for the content of pepsinogens. A zymogen with electrophoretic mobility, isoelectric point and molecular weight equal to that of pepsinogen C of gastric origin was found in all specimens. A comparison between pepsinogen C extracted from pancreatic tissue and gastric mucosa demonstrated immunological identity. Quantitative measurements with a radioimmunoassay showed pepsinogen C concentrations in pancreatic tissue three to 80 times higher than those of blood serum. Immunohistochemical staining gave positive reaction for pepsinogen C only in the alpha cells of the pancreatic islets. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:3315877

  19. Metabolomics applied to the pancreatic islet

    PubMed Central

    Gooding, Jessica R.; Jensen, Mette V.; Newgard, Christopher B.

    2016-01-01

    Metabolomics, the characterization of the set of small molecules in a biological system, is advancing research in multiple areas of islet biology. Measuring a breadth of metabolites simultaneously provides a broad perspective on metabolic changes as the islets respond dynamically to metabolic fuels, hormones, or environmental stressors. As a result, metabolomics has the potential to provide new mechanistic insights into islet physiology and pathophysiology. Here we summarize advances in our understanding of islet physiology and the etiologies of type-1 and type-2 diabetes gained from metabolomics studies. PMID:26116790

  20. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture

    PubMed Central

    Malenczyk, Katarzyna; Keimpema, Erik; Piscitelli, Fabiana; Calvigioni, Daniela; Björklund, Peyman; Mackie, Kenneth; Di Marzo, Vincenzo; Hökfelt, Tomas G. M.; Dobrzyn, Agnieszka; Harkany, Tibor

    2015-01-01

    Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R−/− islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis. PMID:26494286

  1. Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

    PubMed Central

    Shimoda, Masayuki; Kanak, Mazhar A.; Shahbazov, Rauf; Kunnathodi, Faisal; Lawrence, Michael C.; Naziruddin, Bashoo; Levy, Marlon F.

    2015-01-01

    Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of two different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to three groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the three groups. All islet isolations in the PDP groups had >400,000 IEQ in total islet yield post-purification, a significant increase when compared with the control (P = 0.04 and <0.01). The islet quality assessments—including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation—also showed no significant differences. The proportion of TUNEL-positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusion Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions. PMID:25058879

  2. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    PubMed

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome.

  3. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    PubMed

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome. PMID:27586421

  4. Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets

    PubMed Central

    Mukhitov, Nikita; Roper, Michael G.; Bertram, Richard

    2016-01-01

    Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal’s ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet β-cells drives pulsatile insulin secretion. PMID:27788129

  5. Labeling and Tracking of Human Pancreatic Islets Using Carbon Nanotubes.

    PubMed

    Syed, Farooq; Riggio, Cristina; Masini, Matilde; Bugliani, Marco; Battaglia, Valentina; Novelli, Michela; Suleiman, Mara; Vittorio, Orazio; Boggi, Ugo; Filipponi, Franco; Marselli, Lorella; Bartolozzi, Carlo; Masiello, Pellegrino; Raffa, Vittoria; Marchetti, Piero

    2015-04-01

    Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies.

  6. Labeling and Tracking of Human Pancreatic Islets Using Carbon Nanotubes.

    PubMed

    Syed, Farooq; Riggio, Cristina; Masini, Matilde; Bugliani, Marco; Battaglia, Valentina; Novelli, Michela; Suleiman, Mara; Vittorio, Orazio; Boggi, Ugo; Filipponi, Franco; Marselli, Lorella; Bartolozzi, Carlo; Masiello, Pellegrino; Raffa, Vittoria; Marchetti, Piero

    2015-04-01

    Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies. PMID:26310079

  7. Hexose metabolism in pancreatic islets: the Pasteur effect.

    PubMed

    Malaisse, W J; Rasschaert, J; Zähner, D; Sener, A

    1988-02-01

    In rat pancreatic islets, hypoxia severely decreased both the oxidation of D-[U-14C]glucose and the release of insulin evoked by D-glucose. The production of [14C]lactate was increased in the hypoxic islets, the relative magnitude of such an increment being greater at low (2.8 mM) than high (8.3 and 16.7 mM) D-glucose concentrations. Hypoxia increased the detritiation of D-[5-3H]glucose at low glucose concentration (2.8 mM), failed to affect 3H2O production at an intermediate glucose level (8.3 mM), and inhibited the utilization of D-[5-3H]glucose at a higher hexose concentration (16.7 mM). In tumoral islet cells (RINm5F line) exposed to 16.7 mM D-glucose, hypoxia decreased D-[U-14C]glucose oxidation to the same extent as in normal islet cells, but increased the production of [14C]lactate and 3H2O to a greater extent than in normal islets. These findings indicate that the Pasteur effect is operative in islet cells. The experimental data also suggest that, under normal conditions of oxygenation, high concentrations of D-glucose lead to both activation of phosphofructokinase and stimulation of mitochondrial oxidative events in normal, but not tumoral, islet cells.

  8. RNA-sequencing of WFS1-deficient pancreatic islets.

    PubMed

    Ivask, Marilin; Hugill, Alison; Kõks, Sulev

    2016-04-01

    Wolfram syndrome, an autosomal recessive disorder characterized by juvenile-onset diabetes mellitus and optic atrophy, is caused by mutations in theWFS1gene.WFS1encodes an endoplasmic reticulum resident transmembrane protein. TheWfs1-null mice exhibit progressive insulin deficiency and diabetes. The aim of this study was to describe the insulin secretion and transcriptome of pancreatic islets inWFS1-deficient mice.WFS1-deficient (Wfs1KO) mice had considerably less pancreatic islets than heterozygous (Wfs1HZ) or wild-type (WT) mice. Wfs1KOpancreatic islets secreted less insulin after incubation in 2 and 10 mmol/L glucose and with tolbutamide solution compared toWTand Wfs1HZislets, but not after stimulation with 20 mmol/L glucose. Differences in proinsulin amount were not statistically significant although there was a trend that Wfs1KOhad an increased level of proinsulin. After incubation in 2 mmol/L glucose solution the proinsulin/insulin ratio in Wfs1KOwas significantly higher than that ofWTand Wfs1HZRNA-seq from pancreatic islets found melastatin-related transient receptor potential subfamily member 5 protein gene (Trpm5) to be downregulated inWFS1-deficient mice. Functional annotation ofRNAsequencing results showed thatWFS1 deficiency influenced significantly the pathways related to tissue morphology, endocrine system development and function, molecular transport network. PMID:27053292

  9. Autologous islet transplantation to prevent diabetes after pancreatic resection.

    PubMed Central

    Wahoff, D C; Papalois, B E; Najarian, J S; Kendall, D M; Farney, A C; Leone, J P; Jessurun, J; Dunn, D L; Robertson, R P; Sutherland, D E

    1995-01-01

    BACKGROUND: Extensive pancreatic resection for small-duct chronic pancreatitis is often required for pain relief, but the risk of diabetes is a major deterrent. OBJECTIVE: Incidence of pain relief, prevention of diabetes, and identification of factors predictive of success were the goals in this series of 48 patients who underwent pancreatectomy and islet autotransplantation for chronic pancreatitis. PATIENTS AND METHODS: Of the 48 patients, 43 underwent total or near-total (> 95%) pancreatectomy and 5 underwent partial pancreatectomy. The resected pancreas was dispersed by either old (n = 26) or new (n = 22) methods of collagenase digestion. Islets were injected into the portal vein of 46 of the 48 patients and under the kidney capsule in the remaining 2. Postoperative morbidity, mortality, pain relief, and need for exogenous insulin were determined, and actuarial probability of postoperative insulin independence was calculated based on several variables. RESULTS: One perioperative death occurred. Surgical complications occurred in 12 of the 48 patients (25%): of these, 3 had a total (n = 27); 8, a near-total (n = 16); and 1, a partial pancreatectomy (p = 0.02). Most of the 48 patients had a transient increase in portal venous pressure after islet infusion, but no serious sequelae developed. More than 80% of patients experienced significant pain relief after pancreatectomy. Of the 39 patients who underwent total or near-total pancreatectomy, 20 (51%) were initially insulin independent. Between 2 and 10 years after transplantation, 34% were insulin independent, with no grafts failing after 2 years. The main predictor of insulin independence was the number of islets transplanted (of 14 patients who received > 300,000 islets, 74% were insulin independent at > 2 years after transplantation). In turn, the number of islets recovered correlated with the degree of fibrosis (r = -0.52, p = 0.006) and the dispersion method (p = 0.005). CONCLUSION: Pancreatectomy can relieve

  10. Immune tolerance in pancreatic islet xenotransplantation

    PubMed Central

    Tang, Tian-Hua; Li, Chun-Lin; Li, Xin; Jiang, Feng-Qin; Zhang, Yu-Kun; Ren, Hai-Quan; Su, Shan-Shan; Jiang, Guo-Sheng

    2004-01-01

    AIM: To observe the effect of tail vein injection with donor hepatocytes and/or splenocytes on the islet xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islet xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK(natural killing cell) killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: The results showed that streptozotocin (STZ) could induce diabetes mellitus models of mice. The pre-injection of donor hepatocytes, splenocytes or their mixture by tail vein injection was effective in preventing donor islet transplantation from rejection, which was demonstrated by the above-mentioned immunological marks. Each group of transplantation could decrease blood glucose in recipients and increase survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection as compared with that of donor hepatocyte or splenocyte pre-injection respectively. CONCLUSION: Pre-injection of donor hepatocytes, splenocytes or their mixture before donor islet transplantation is a good way in preventing rejection. PMID:15133853

  11. Transplantation of islet cells across major histocompatibility barriers after total lymphoid irradiation and infusion of allogeneic bone marrow cells

    SciTech Connect

    Britt, L.D.; Scharp, D.W.; Lacy, P.E.; Slavin, S.

    1982-08-01

    Diabetic Lewis rats (AgB1/L) were evaluated as recipients of allogeneic Wistar-Furth (AgB2/2) isolated adult islets without the use of standard recipient immunosuppression. One group was treated with fractionated total lymphoid irradiation (TLI) and Wistar-Furth bone marrow cell reconstitution to proven chimerism prior to islet transplantation. This group returned to a prediabetic state following Wistar-Furth islet transplantation without any evidence of rejection for 100 days posttransplant. A second group of Lewis rats received only TLI without bone marrow treatment. They gave a varying result following islet transplantation with one recipient showing evidence of prolonged islet survival. A third chimeric control group did not receive isolated islets and did not alter their diabetic state. A fourth group was not given TLI nor donor bone marrow cells and uniformly rejected their allogeneic islets by 7 days. Thus, allogeneic adult islets will survive across major rat histocompatibility barriers using TLI and donor bone marrow chimerism as the only form of immunosuppression.

  12. Temporal decline in sirolimus elimination immediately after pancreatic islet transplantation.

    PubMed

    Sato, Eriko; Shimomura, Masahiro; Masuda, Satohiro; Yano, Ikuko; Katsura, Toshiya; Matsumoto, Shin-ichi; Okitsu, Teru; Iwanaga, Yasuhiro; Noguchi, Hirofumi; Nagata, Hideo; Yonekawa, Yukihide; Inui, Ken-ichi

    2006-12-01

    Pancreatic islet transplantation is a curable treatment for type 1 diabetes and has been put into practice in various countries. In this study, we analyzed the pharmacokinetic characteristics of sirolimus and tacrolimus in six Japanese patients with pancreatic islet transplants immediately after surgery, and monitored efficacy and toxicity. The patients were treated with immunosuppressive therapy based on the Edmonton protocol, that is, sirolimus and low-dose tacrolimus. Pharmacokinetic analyses were performed using the nonlinear mixed-effects modeling program NONMEM. Large inter- and intra-individual variability was observed in the pharmacokinetics of sirolimus and tacrolimus. A model with increased apparent clearance in the postoperative period explained well the intra-individual variability in the pharmacokinetics of both drugs. The most frequent drug-induced toxicity was a decrease in the white blood cell count, and two of six patients required the administration of granulocyte colony-stimulating factor. Clinical laboratory tests immediately before the transplantation and cytochrome P450 3A5 genotype were not related to the high blood concentrations of sirolimus after the loading dose. From these results, the apparent clearance of sirolimus and tacrolimus might temporally decline immediately after pancreatic islet transplantation. A high trough concentration of sirolimus might increase the risk of hematological toxicy, and adjustment of the dosage for immunosuppressive treatment will be necessary in Japanese patients.

  13. Protein-Mediated Interactions of Pancreatic Islet Cells

    PubMed Central

    Meda, Paolo

    2013-01-01

    The islets of Langerhans collectively form the endocrine pancreas, the organ that is soley responsible for insulin secretion in mammals, and which plays a prominent role in the control of circulating glucose and metabolism. Normal function of these islets implies the coordination of different types of endocrine cells, noticeably of the beta cells which produce insulin. Given that an appropriate secretion of this hormone is vital to the organism, a number of mechanisms have been selected during evolution, which now converge to coordinate beta cell functions. Among these, several mechanisms depend on different families of integral membrane proteins, which ensure direct (cadherins, N-CAM, occludin, and claudins) and paracrine communications (pannexins) between beta cells, and between these cells and the other islet cell types. Also, other proteins (integrins) provide communication of the different islet cell types with the materials that form the islet basal laminae and extracellular matrix. Here, we review what is known about these proteins and their signaling in pancreatic β-cells, with particular emphasis on the signaling provided by Cx36, given that this is the integral membrane protein involved in cell-to-cell communication, which has so far been mostly investigated for effects on beta cell functions. PMID:24278783

  14. Considerations for successful transplantation of encapsulated pancreatic islets.

    PubMed

    de Vos, P; Hamel, A F; Tatarkiewicz, K

    2002-02-01

    Encapsulation of pancreatic islets allows for transplantion in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. Encapsulation offers a solution to the shortage of donors in clinical islet transplantation because it allows animal islets or insulin-producing cells engineered from stem cells to be used. During the past two decades three major approaches to encapsulation have been studied. These include intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt; extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites; and extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of these three approaches are discussed and compared in the light of their applicability to clinical islet transplantation. All systems have been shown to be successful in preclinical studies but not all approaches meet the technical or physiological requirements for application in human beings. The extravascular approach has advantages over the intravascular because since it is associated with less complications such as thrombosis and infection. Microcapsules, due to their spatial characteristics, have a better diffusion capacity than macrocapsules. Recent progress in biocompatibility of microcapsules has brought this technology close to clinical application. Critical issues such as limitations in the functional performance and survival are being discussed. The latest results show that both issues can be solved by the transplantation of microencapsulated islets close to blood vessels in prevascularized solid supports. PMID:11935147

  15. Origin of induced pancreatic islet tumors: a radioautographic study

    SciTech Connect

    Michels, J.E.; Bauer, G.E.; Dixit, P.K.

    1987-02-01

    Endocrine tumors of the pancreas are induced in a high percentage of young rats by injections of streptozotocin and nicotinamide (SZ/NA). Benign tumors first appear 20 to 36 weeks after drug injections. To determine the possible site of their origin, the incorporation of (/sup 3/H)thymidine into islets, ducts, acini, microtumors, and gross tumors was examined by radioautography of histologic sections at 1 to 36 weeks after drug injection. Drug treatment led to early (1- to 6-week) increases in nuclear /sup 3/H labeling of exocrine pancreatic structures (ductal and acinar cells), which may involve DNA repair processes. A secondary increase in labeling of duct cells during the period of tumor emergence supports the assumption that SZ/NA-induced tumors are of ductal origin. Microtumors and gross tumors also exhibited markedly elevated rates of (/sup 3/H)thymidine incorporation compared to control islets. Nontumorous islet tissue, which exhibited a gradual decrease in volume due to B-cell destruction by the drug injection, showed about 10-fold higher /sup 3/H labeling than islets of controls at all time points. The results suggest that in addition to ductal precursors, islets that survive SZ/NA-induced injury may also provide sites of focal endocrine cell differentiation to tumor tissue. Once established, both microtumors and gross tumors continue to grow by accelerated cell division.

  16. Characterization of Pancreatic Ductal Cell in Human Islet Preparations

    PubMed Central

    Ichii, Hirohito; Miki, Atsushi; Yamamoto, Toshiyuki; Molano, R. Damaris; Barker, Scott; Mita, Atsuyoshi; Rodriguez-Diaz, Rayner; Klein, Dagmar; Pastori, Ricardo; Alejandro, Rodolfo; Inverardi, Luca; Pileggi, Antonello; Ricordi 6, Camillo

    2013-01-01

    Substantial amounts of non-endocrine cells are implanted as part of human islet grafts, and possible influence of non-endocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for non-endocrine cells due to lack of available methods. Aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDC) for clinical islet transplantation, and to characterize them regarding phenotype, viability and function. We assessed 161 human islet preparations using laser scanning cytometer (LSC/iCys) for phenotypic analysis of non-endocrine cells and flow cytometer (FACS) for PDC viability. PDC and β-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce pro-inflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF), relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDC, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than β-cells (PDC vs. β-cell: 75.5±13.9 and 62.7±18.7 %; p<0.0001). Although β-cells viability was independent from the density, that of PDC was higher as the density from which they were recovered increased. There was no correlation between PDC and β-cells viability (R2=0.0078). PDC sorted from high-density fractions produced significantly higher amount of pro-inflammatory mediators and VEGF, but not TF. PDC isolated from different fractions had different viability and function. The precise characterization and assessment of these cells in addition to β-cells in human islet cell products may be of assistance in understanding their contribution to islet

  17. Pancreatic mixed ductal-islet tumors. Is this an entity?

    PubMed

    Permert, J; Mogaki, M; Andrén-Sandberg, A; Kazakoff, K; Pour, P M

    1992-02-01

    Thirty-eight human pancreatic cancer specimens were studied for the reactivity of cancer cells with monoclonal antibodies against insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP), gastrin, calcitonin, and with argyrophilic reactivity. Immunoreactivity with one or several antibodies or argyrophilic reactivity were found in 30 (79%) cases. In 17 cases, the number of endocrine cells was excessive and morphologically consistent with the mixed ductal-islet tumor. Although most immunoreactive cells were located at the base of the malignant glands, some had intraepithelial location and were also present in the invasive portion of cancers, indicating their malignant nature. Endocrine cell proliferation were found in the pancreatic tissue adjacent to the carcinoma in 8 out of 12 specimens examined. In these cases, the immunoreactive cells were either distributed among the acinar cells or ductal cells. More endocrine cells were found in the hyperplastic ducts; however, no correlation was found between the degree of hyperplasia and the occurrence of any type of immunoreactive cells. Although several types of endocrine cells occurred in different pancreatic regions (head, body, and tail), PP cells were restricted to tissues taken from the head of the pancreas. Experimental data and similar observations by other investigators led us to conclude that participation of endocrine cells in ductal-type carcinomas is a general phenomenon and does not justify the classification of these lesions to mixed ductal-islet entity. However, because immunoreactive cells were more common and numerous in well-differentiated carcinomas, they may have some prognostic values. PMID:1316418

  18. 2007 update on allogeneic islet transplantation from the Collaborative Islet Transplant Registry (CITR).

    PubMed

    2009-01-01

    As of October 1, 2007, 25 North American medical institutions and one European islet transplant center reported detailed information to the Registry on 315 allograft recipients, of which 285 were islet alone (IA) and 30 were islet after kidney (IAK). Of the 114 IA recipients expected at 4 years after their last infusion, 12% were insulin independent, 16% were insulin dependent with detectable C-peptide, 40% had no detectable C-peptide, and 32% had missing C-peptide data or were lost to follow-up. Of the IA recipients, 72% achieved insulin independence at least once over 3 years and multiple infusions. Factors associated with achievement of insulin independence included islet size >1.0 expressed as IEQs per islet number [hazard ratio (HR) = 1.5, p = 0.06], additional infusions given (HR = 1.5, p = 0.01), lower pretransplant HbA(1c) (HR = 1.2 each %-age unit, p = 0.02), donor given insulin (HR = 2, p = 0.003), daclizumab given at any infusion (HR = 1.9, p = 0.06), and shorter cold storage time (HR = 1.04, p = 0.03), mutually adjusted in a multivariate model. Severe hypoglycemia prevalence was reduced from 78-83% preinfusion to less than 5% throughout the first year post-last infusion, and to 18% adjusted for missing data at 3 years post-last infusion. In Year 1 post-first infusion for IA recipients, 53% experienced a Grade 3-5 or serious adverse event (AE) and 35% experienced a severe AE related to either an infusion procedure or immunosuppression. In Year 1 post-first infusion, 33% of IA subjects and 35% of IAK subjects had an AE related to the infusion procedure, while 35% of IA subjects and only 27% of IAK subjects had an AE related to the immunosuppression therapy. Five deaths were reported, of which two were classified as probably related to the infusion procedure or immunosuppression, and 10 cases of neoplasm, of which two were classified as probably related to the procedure or immunosuppression. Islet transplantation continues to show short-term benefits of

  19. Transcriptional Regulation of Chemokine Genes: A Link to Pancreatic Islet Inflammation?

    PubMed Central

    Burke, Susan J.; Collier, J. Jason

    2015-01-01

    Enhanced expression of chemotactic cytokines (aka chemokines) within pancreatic islets likely contributes to islet inflammation by regulating the recruitment and activation of various leukocyte populations, including macrophages, neutrophils, and T-lymphocytes. Because of the powerful actions of these chemokines, precise transcriptional control is required. In this review, we highlight what is known about the signals and mechanisms that govern the transcription of genes encoding specific chemokine proteins in pancreatic islet β-cells, which include contributions from the NF-κB and STAT1 pathways. We further discuss increased chemokine expression in pancreatic islets during autoimmune-mediated and obesity-related development of diabetes. PMID:26018641

  20. Pancreatic islets and their roles in metabolic programming.

    PubMed

    Barella, Luiz Felipe; de Oliveira, Júlio Cezar; Mathias, Paulo Cezar de Freitas

    2014-04-01

    Experimental and epidemiologic data have confirmed that undernutrition or overnutrition during critical periods of life can result in metabolic dysfunction, leading to the development of obesity, hypertension, and type 2 diabetes, later in life. These studies have contributed to the concept of the developmental origins of health and disease (DOHaD), which involves metabolic programming patterns. Beyond the earlier phases of development, puberty can be an additional period of plasticity, during which any insult can lead to changes in metabolism. Impaired brain development, associated with imbalanced autonomous nervous system activity due to metabolic programming, is pivotal to the creation of pathophysiology. Excess glucocorticoid exposure, due to hypothalamic-pituitary-adrenal axis deregulation, is also involved in malprogramming in early life. Additionally, the pancreatic islets appear to play a decisive role in the setup and maintenance of these metabolic dysfunctions as key targets of metabolic programming, and epigenetic mechanisms may underlie these changes. Moreover, studies have indicated the possibility that deprogramming renders the islets able to recover their functioning after malprogramming. In this review, we discuss the key roles of the pancreatic islets as targets of malprogramming; however, we also discuss their roles as important targets for the treatment and prevention of metabolic diseases.

  1. Minireview: Dopaminergic Regulation of Insulin Secretion from the Pancreatic Islet

    PubMed Central

    Ustione, Alessandro

    2013-01-01

    Exogenous dopamine inhibits insulin secretion from pancreatic β-cells, but the lack of dopaminergic neurons in pancreatic islets has led to controversy regarding the importance of this effect. Recent data, however, suggest a plausible physiologic role for dopamine in the regulation of insulin secretion. We review the literature underlying our current understanding of dopaminergic signaling that can down-regulate glucose-stimulated insulin secretion from pancreatic islets. In this negative feedback loop, dopamine is synthesized in the β-cells from circulating l-dopa, serves as an autocrine signal that is cosecreted with insulin, and causes a tonic inhibition on glucose-stimulated insulin secretion. On the whole animal scale, l-dopa is produced by cells in the gastrointestinal tract, and its concentration in the blood plasma increases following a mixed meal. By reviewing the outcome of certain types of bariatric surgery that result in rapid amelioration of glucose tolerance, we hypothesize that dopamine serves as an “antiincretin” signal that counterbalances the stimulatory effect of glucagon-like peptide 1. PMID:23744894

  2. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants.

    PubMed

    Pasquali, Lorenzo; Gaulton, Kyle J; Rodríguez-Seguí, Santiago A; Mularoni, Loris; Miguel-Escalada, Irene; Akerman, Ildem; Tena, Juan J; Morán, Ignasi; Gómez-Marín, Carlos; van de Bunt, Martijn; Ponsa-Cobas, Joan; Castro, Natalia; Nammo, Takao; Cebola, Inês; García-Hurtado, Javier; Maestro, Miguel Angel; Pattou, François; Piemonti, Lorenzo; Berney, Thierry; Gloyn, Anna L; Ravassard, Philippe; Gómez-Skarmeta, José Luis; Müller, Ferenc; McCarthy, Mark I; Ferrer, Jorge

    2014-02-01

    Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes. PMID:24413736

  3. Pancreatic islet enhancer clusters enriched in type 2 diabetes risk–associated variants

    PubMed Central

    Mularoni, Loris; Miguel-Escalada, Irene; Akerman, İldem; Tena, Juan J.; Morán, Ignasi; Gómez-Marín, Carlos; van de Bunt, Martijn; Ponsa-Cobas, Joan; Castro, Natalia; Nammo, Takao; Cebola, Inês; García-Hurtado, Javier; Maestro, Miguel Angel; Pattou, François; Piemonti, Lorenzo; Berney, Thierry; Gloyn, Anna L.; Ravassard, Philippe; Skarmeta, José Luis Gómez; Müller, Ferenc; McCarthy, Mark I.; Ferrer, Jorge

    2013-01-01

    Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central for type 2 diabetes pathogenesis, and therefore understanding islet genome regulation could provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity, and show that most such sequences reside in clusters of enhancers that form physical 3D chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers, and identify trait-associated variants that disrupt DNA-binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome, and provide systematic evidence that dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes. PMID:24413736

  4. Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

    PubMed

    Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

    2007-01-01

    While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release. PMID:17201925

  5. Can pancreatic duct-derived progenitors be a source of islet regeneration?

    SciTech Connect

    Xia, Bing; Zhan, Xiao-Rong; Yi, Ran; Yang, Baofeng

    2009-06-12

    The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing {beta}-cells. The functional mass of {beta}-cells is decreased in type 1 diabetes, so replacing missing {beta}-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the {beta}-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding {beta}-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated. In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet {beta}-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the 'gold standard' of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal cells as

  6. Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane

    SciTech Connect

    Dunlop, M.E.; Malaisse, W.J.

    1986-02-01

    Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to (gamma-32P)ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with (U-14C)arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents.

  7. Weight loss in rats following intraventricular transplants of pancreatic islets.

    PubMed

    Richardson, R D; Ramsay, D S; Lernmark, A; Scheurink, A J; Baskin, D G; Woods, S C

    1994-01-01

    Because of the body's resistance to permanent weight change, obesity remains a major health problem in modern society. It is hypothesized that the regulatory system defending body weight utilizes pancreatic insulin as an indicator of adiposity to the brain. To take advantage of this negative feedback system, we transplanted neonatal (experiment 1) or adult (experiment 2) pancreatic islets containing insulin-secreting cells into the 3rd ventricle of syngeneic Lewis rats. This resulted in an elevation of the insulin signal within the brain and a significant long-term reduction of body weight. Changes in food intake were consistent with the changes of body weight. The implantation of more islets resulted in a greater reduction of body weight, and changes in weight were inversely correlated with the level of insulin achieved in the cerebrospinal fluid. After the two studies were completed, histological examination revealed the presence of insulin-containing cells within the 3rd ventricle and adjacent hypothalamus. These studies suggest that transplanted insulin-secreting cells may provide a potential therapeutic strategy for maintenance of weight loss.

  8. Modeling K,ATP-Dependent Excitability in Pancreatic Islets

    PubMed Central

    Silva, Jonathan R.; Cooper, Paige; Nichols, Colin G.

    2014-01-01

    In pancreatic β-cells, K,ATP channels respond to changes in glucose to regulate cell excitability and insulin release. Confirming a high sensitivity of electrical activity to K,ATP activity, mutations that cause gain of K,ATP function cause neonatal diabetes. Our aim was to quantitatively assess the contribution of K,ATP current to the regulation of glucose-dependent bursting by reproducing experimentally observed changes in excitability when K,ATP conductance is altered by genetic manipulation. A recent detailed computational model of single cell pancreatic β-cell excitability reproduces the β-cell response to varying glucose concentrations. However, initial simulations showed that the model underrepresents the significance of K,ATP activity and was unable to reproduce K,ATP conductance-dependent changes in excitability. By altering the ATP and glucose dependence of the L-type Ca2+ channel and the Na-K ATPase to better fit experiment, appropriate dependence of excitability on K,ATP conductance was reproduced. Because experiments were conducted in islets, which contain cell-to-cell variability, we extended the model from a single cell to a three-dimensional model (10×10×10 cell) islet with 1000 cells. For each cell, the conductance of the major currents was allowed to vary as was the gap junction conductance between cells. This showed that single cell glucose-dependent behavior was then highly variable, but was uniform in coupled islets. The study highlights the importance of parameterization of detailed models of β-cell excitability and suggests future experiments that will lead to improved characterization of β-cell excitability and the control of insulin secretion. PMID:25418087

  9. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices

    PubMed Central

    Johansson, Ulrika; Dekki Shalaly, Nancy; Zaitsev, Sergei V.; Berggren, Per-Olof; Hedhammar, My

    2015-01-01

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets

  10. Is Total Pancreatectomy with Islet Autotransplantation A Reasonable Choice for Pediatric Pancreatitis?

    PubMed

    Azhari, Hassan; Rahhal, Riad; Uc, Aliye

    2015-01-01

    Chronic pancreatitis is an emerging and poorly understood disease in childhood. Total pancreatectomy with islet cell autotransplantation is being proposed as a treatment for chronic pancreatitis and recent studies report a more favorable outcome in children compared to adults. Herein, we review the therapeutic alternatives for pediatric chronic pancreatitis, focusing primarily on TP/IAT. PMID:26523129

  11. The effectiveness of components of University of Wisconsin solution in improving human pancreatic islet purification.

    PubMed

    Robertson, G S; Chadwick, D R; Davies, J; Rose, S; Contractor, H; James, R F; Bell, P R; London, N J

    1994-02-01

    The purification of human pancreatic islets before transplantation relies on the density-dependent separation of islets from exocrine fragments after collagenase digestion of the donor pancreas. The results vary among pancreases despite increasing automation of the digestion and purification processes, reflecting variations in the overlapping densities of islets and contaminating exocrine tissue. Hypothermic storage of both the pancreas and the pancreatic digest alters cell volumes and tissue densities, thereby affecting islet purification. By biochemical analysis of the isopycnic distribution of islets and exocrine tissue fragments from 23 human pancreases on linear continuous density gradients, the effect of various solutions for cold storage of pancreatic digest was studied. The use of the University of Wisconsin cold storage solution, which resulted in a significant decrease in digest volume (P = 0.006) and increase in the densities of both exocrine tissue (P = 0.001) and islets (P = 0.005), produced a significant improvement in islet purity compared with tissue culture medium (P = 0.035), predominantly due to the inclusion of a colloid, which increased the difference in density between exocrine tissue and islets. The addition of large molecular weight cellular impermeants without alteration in the concentration of permeable anions produced no effect. The results of this study support the concept that the use of solutions that minimize cell swelling throughout the process of islet purification would result in significant improvements in density-dependent islet separation, and that such solutions should contain a colloid. PMID:8108869

  12. Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

    PubMed

    He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

    2015-04-01

    Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

  13. Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

    PubMed

    He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

    2015-04-01

    Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution. PMID:25682842

  14. Microfabricated biocapsules for the immunoisolation of pancreatic islets of Langerhans

    NASA Astrophysics Data System (ADS)

    Desai, Tejal Ashwin

    1998-08-01

    A silicon-based microfabricated biocapsule was developed and evaluated for use in the immunoisolation of transplanted cells, specifically pancreatic islets of Langerhans for the treatment of Type I diabetes. The transplantation of cells with specific functions is a promising therapy for a wide variety of pathologies including diabetes, Parkinson's, and hemophilia. Such transplanted cells, however, are sensitive to both cellular and humoral immune rejection as well as damage by autoimmune activity, without chronic immunosuppression. The research presented in this dissertation investigated whether microfabricated silicon-based biocapsules, with uniform membrane pore sizes in the tens of nanometer range, could provide an immunoprotective environment for pancreatic islets and other insulin-secreting cell lines, while maintaining cell viability and functionality. By utilizing fabrication techniques commonly employed in the microelectronics industry (MEMS), membranes were fabricated with precisely controlled and uniform pore sizes, allowing the optimization of biocapsule membrane parameters for the encapsulation of specific hormone-secreting cell types. The biocapsule-forming process employed bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes, which were surface micromachined to present a high density of uniform pores to allow sufficient permeability to oxygen, glucose, and insulin. Both in vitro and in vivo experiments established the biocompatibility of the microfabricated biocapsule, and demonstrated that encapsulated cells could live and function normally in terms of insulin-secretion within microfabricated environments for extended periods of time. This novel research shows the potential of using microfabricated biocapsules for the encapsulation of several different cell xenografts. The semipermeability

  15. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    PubMed Central

    Delalat, Bahman; Rojas-Canales, Darling M.; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J.; Penko, Daniella; Drogemuller, Christopher J.; Loudovaris, Thomas; Coates, Patrick T. H.; Voelcker, Nicolas H.

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  16. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

    PubMed

    Delalat, Bahman; Rojas-Canales, Darling M; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J; Penko, Daniella; Drogemuller, Christopher J; Loudovaris, Thomas; Coates, Patrick T H; Voelcker, Nicolas H

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  17. Effect of total lymphoid irradiation and pretransplant blood transfusion on pancreatic islet allograft survival

    SciTech Connect

    Mendez-Picon, G.; McGeorge, M.

    1983-05-01

    Total lymphoid irradiation (TLI) has been shown to have a strong immunosuppressive effect both experimentally and clinically. Pretransplant blood transfusions have also been shown to have a strong beneficial effect in the outcome of organ transplantation. A study was made of the effect of TLI and pretransplant blood transfusions, alone and in combination, as an immunosuppressive modality in the isolated pancreatic islet transplant in the rat model. Donor rats (Fischer RT1v1) were kept on a 50% DL-ethionine supplemented diet for 4-6 weeks prior to pancreas removal. Recipient rats (Lewis RT1) were made diabetics prior to transplantation by iv injection of streptozotocin (45 mg/kg). Transfusion protocol consisted of a biweekly transfusion of 2 ml of either donor specific or third party transfusions. Total lymphoid irradiation was carried out by daily administration of 200 rads during one week prior to transplantation. Transplantation of the isolated islets was performed by intraportal injection. Syngeneic transplant of one and a half donor pancreata in each recipient reverted the diabetic condition indefinitely (greater than 100 days). Untreated allogenic grafts had a mean survival time (MST) of 5.2 days. Total lymphoid irradiation in dosages of 800, 1000, and 1200 rads, as the only immunosuppressive regimen, prolonged the MST of allografts to 15.3, 16.5, and 21.8 days, respectively (P less than .05). Pretransplant third party blood transfusion had no effect on allograft survival (MST 6.0). When donor specific blood transfusions were given, the MST was prolonged to 25.3 days (P less than .05). When TLI was administered to recipients of donor specific transfusions, the MST of the allografts did not show any statistical significant difference when compared with untreated animals. This abrogation of the beneficial effect of specific blood transfusion was observed in all dosages of TLI employed: 800 rad (MST 3.0), 1000 rad (MST 8.0), 1200 rad (MST 5.18).

  18. Data on morphometric analysis of the pancreatic islets from C57BL/6 and BALB/c mice.

    PubMed

    da Silva, Thiago Aparecido; Lemes, Robertha Mariana; Oliveira, Carlo Jose Freire; Almeida, Aline da Silva; Chica, Javier Emílio Lazo

    2016-09-01

    The endocrine portion of the pancreas, which is characterized by pancreatic islets, has been widely investigated among different species. The BALB/c and C57BL/6 mice are extensively used in experimental research, and the morphometric differences in the pancreatic islets of these animals have not been evaluated so far. Thus, our data have a comparative perspective related to the morphometric analysis of area, diameters, circularity, and density of pancreatic islets from BALB/c and C57BL/6 mice. The data presented here are focused to evaluate the differences in morphology of pancreatic islets of two common laboratory mouse strains.

  19. Storage of human pancreatic digest in University of Wisconsin solution significantly improves subsequent islet purification.

    PubMed

    Robertson, G S; Chadwick, D; Contractor, H; Rose, S; Chamberlain, R; Clayton, H; Bell, P R; James, R F; London, N J

    1992-09-01

    Density-gradient purification of human pancreatic islets from the collagenase-digested pancreas relies on the exocrine tissue being denser than the islets. Cold storage of the pancreas before and after digestion causes cell swelling, which can decrease the density of pancreatic exocrine tissue and adversely affect subsequent purification. Using 14 human pancreata (seven perfused in situ with hyperosmolar citrate (HOC) and seven with University of Wisconsin solution (UW)), it is shown that storage of the pancreatic digest in UW significantly increases the density of pancreatic exocrine tissue compared with storage in minimal essential medium (MEM) (P = 0.009). This results in an improvement in islet purity (P = 0.036) for HOC- but not UW-perfused pancreata. Storage in UW for 1 h not only prevented the deterioration that occurred in MEM, but resulted in an improvement in islet purity for five of the seven HOC-perfused pancreata. Most pancreata in the UK are perfused with HOC, but storage of the digest in UW results in significantly better islet purity and, when islets cannot be purified immediately, a period of storage will often improve separation and allow islets to be purified. PMID:1422750

  20. Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion.

    PubMed

    Ilegems, E; van Krieken, P P; Edlund, P K; Dicker, A; Alanentalo, T; Eriksson, M; Mandic, S; Ahlgren, U; Berggren, P-O

    2015-01-01

    The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

  1. Selective expression of CYP2A13 in human pancreatic α-islet cells.

    PubMed

    Guo, Yu; Zhu, Liang-Ru; Lu, Gang; Wang, Hui; Hong, Jun-Yan

    2012-10-01

    Exposure to cigarette smoke is an etiological factor of human pancreatic cancer and has been associated with an increased risk of pancreatic diseases, including pancreatitis and diabetes. The toxicants in cigarette smoke can reach pancreatic tissue, and most of the toxicants require cytochrome P450 (P450)-mediated metabolic activation to exert their toxicity. Among all the human P450 enzymes, CYP2A13 is the most efficient enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific toxicant and a suspected human carcinogen. It also metabolically activates 4-aminobiphenyl, another toxicant in cigarette smoke. Immunohistochemical analysis in this study demonstrated that CYP2A13 was selectively expressed in the islets but not in the exocrine portion of adult human pancreas. Further study using dual immunofluorescence labeling technique showed that CYP2A13 protein was mainly expressed in the α-islet but not in β-islet cells. The selective expression of CYP2A13 in human pancreatic α-islet cells suggests that these islet cells could be damaged by the toxicants existing in cigarette smoke through CYP2A13-mediated in situ metabolic activation. Our result provides a mechanistic insight for human pancreatic diseases that have been associated with cigarette smoke exposure.

  2. Sympathetic innervation during development is necessary for pancreatic islet architecture and functional maturation

    PubMed Central

    Borden, Philip; Houtz, Jessica; Leach, Steven D.; Kuruvilla, Rejji

    2013-01-01

    Summary Sympathetic neurons depend on target-derived neurotrophic cues to control their survival and growth. However, whether sympathetic innervation contributes reciprocally to the development of target tissues is less clear. Here, we report that sympathetic innervation is necessary for the formation of the pancreatic islets of Langerhans and for their functional maturation. Genetic or pharmacological ablation of sympathetic innervation during development resulted in altered islet architecture, reduced insulin secretion and impaired glucose tolerance in mice. Similar defects were observed with pharmacological blockade of β-adrenergic signaling. Conversely, the administration of a β-adrenergic agonist restored islet morphology and glucose tolerance in de-innervated animals. Furthermore, in neuron-islet co-cultures, sympathetic neurons promoted islet cell migration in a β-adrenergic dependent manner. This study reveals that islet architecture requires extrinsic inductive cues from neighboring tissues such as sympathetic nerves, and suggests that early perturbations in sympathetic innervation might underlie metabolic disorders. PMID:23850289

  3. Seven consecutive successful clinical islet isolations with pancreatic ductal injection.

    PubMed

    Matsumoto, Shinichi; Noguichi, Hirofumi; Shimoda, Masayuki; Ikemoto, Tetsuya; Naziruddin, Bashoo; Jackson, Andrew; Tamura, Yoshiko; Olson, Greg; Fujita, Yasutaka; Chujo, Daisuke; Takita, Morihito; Kobayashi, Naoya; Onaca, Nicholas; Levy, Marlon

    2010-01-01

    Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 +/- 64,319 vs. 354,836 +/- 89,649 IE, p < 0.01) and viability (97.3 +/- 1.2% vs. 92.6 +/- 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

  4. Novel Stable Isotope Analyses Demonstrate Significant Rates of Glucose Cycling in Mouse Pancreatic Islets

    PubMed Central

    Pound, Lynley D.; Trenary, Irina; O’Brien, Richard M.

    2015-01-01

    A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat–fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans. PMID:25552595

  5. Effect of low temperature cultivation on insulin secretory of human pancreatic islets.

    PubMed

    Nikolic, D M; Djordjevic, P B; Lackovic, V B; Stojiljkovic, V; Stanojevic, B

    2013-01-01

    The experiment compared the physiological function (insulin secretory capacity) and membrane integrity of human adult pancreatic islets incubated in culture at 37°C and 24°C. Pancreatic tissue was digested with Collagenase XI, using a non-automated method. Cultures were incubated at 37°C and 24°C. Secretory capacity of the islets is determined by measuring of the stimulation index (SI) on the 1st, 3rd and 7th day of cultivation. Membrane integrity of the islets was determined by dithizone staining. Both groups of examined cultures show a slight increase in SI during the incubation. However islets incubated at 24°C show higher SI values than those incubated at 37°C on the 1st, 3rd and 7th day of incubation. And on the first day of incubation, this difference was statistically significant (p <0.05). Islets incubated at 37°C showed preservation of membrane integrity, the islets are regular spherical shape, while those incubated at 24°C lose such an organization. During the seven-day cultivation, islets incubated at a standard temperature of 37°C show less preserve physiological functions in relation to cultures incubated at 24°C, but islets incubated at 37°C show more regular morphological forms. PMID:23489685

  6. The detection of glycosaminoglycans in pancreatic islets and lymphoid tissues.

    PubMed

    Bogdani, Marika; Simeonovic, Charmaine; Nagy, Nadine; Johnson, Pamela Y; Chan, Christina K; Wight, Thomas N

    2015-01-01

    In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage, and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates. To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP. The hyaluronan-HABP complexes are extracted and the hyaluronan concentration in the tissue is determined using an ELISA-like assay. Heparan sulfate is identified in mouse tissues by Alcian blue histochemistry and indirect immunohistochemistry. In human tissues, heparan sulfate is best detected by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A biotinylated secondary antibody is then applied in conjunction with streptavidin-peroxidase and its binding to the anti-heparan sulfate antibody is visualized by enzymatic precipitation of chromogenic substrates.

  7. Diabetic Ketoacidosis with Concurrent Pancreatitis, Pancreatic β Islet Cell Tumor, and Adrenal Disease in an Obese Ferret (Mustela putorius furo)

    PubMed Central

    Phair, Kristen A; Carpenter, James W; Schermerhorn, Thomas; Ganta, Chanran K; DeBey, Brad M

    2011-01-01

    A 5.5-y-old spayed female ferret (Mustela putorius furo) with a history of adrenal disease, respiratory disease, and chronic obesity was evaluated for progressive lethargy and ataxia, diminished appetite, and possible polyuria and polydipsia. Physical examination revealed obesity, lethargy, tachypnea, dyspnea, a pendulous abdomen, significant weakness and ataxia of the hindlimbs, prolonged skin tenting, and mild tail-tip alopecia. Clinicopathologic analysis revealed severe hyperglycemia, azotemia, an increased anion gap, glucosuria, ketonuria, proteinuria, and hematuria. Abdominal ultrasonography showed hyperechoic hepatomegaly, bilateral adrenomegaly, splenic nodules, mild peritoneal effusion, and thickened and mildly hypoechoic limbs of the pancreas with surrounding hyperechoic mesentery. Fine-needle aspirates of the liver were highly suggestive of hepatic lipidosis. In light of a diagnosis of concurrent diabetic ketoacidosis and pancreatitis, the ferret was treated with fluid therapy, regular and long-acting insulin administration, and pain medication. However, electrolyte derangements, metabolic acidosis, dyspnea, and the clinical appearance of the ferret progressively worsened despite treatment, and euthanasia was elected. Necropsy revealed severe hepatic lipidosis, severe suppurative pancreatitis and vacuolar degeneration of pancreatic islet cells, a pancreatic β islet cell tumor, bilateral adrenal cortical adenomas, and myocardial fibrosis. To our knowledge, this case represents the first report of concurrent diabetes mellitus, pancreatitis, pancreatic β islet cell tumor (insulinoma), and adrenal disease in a domestic ferret. The simultaneous existence of 3 endocrine diseases, pancreatitis, and their associated complications is a unique and clinically challenging situation. PMID:21838985

  8. Dimethyl Fumarate Protects Pancreatic Islet Cells and Non-Endocrine Tissue in L-Arginine-Induced Chronic Pancreatitis

    PubMed Central

    Robles, Lourdes; Vaziri, Nosratola D.; Li, Shiri; Masuda, Yuichi; Takasu, Chie; Takasu, Mizuki; Vo, Kelly; Farzaneh, Seyed H.; Stamos, Michael J.; Ichii, Hirohito

    2014-01-01

    Background Chronic pancreatitis (CP) is a progressive disorder resulting in the destruction and fibrosis of the pancreatic parenchyma which ultimately leads to impairment of the endocrine and exocrine functions. Dimethyl Fumarate (DMF) was recently approved by FDA for treatment of patients with multiple sclerosis. DMF's unique anti-oxidant and anti-inflammatory properties make it an interesting drug to test on other inflammatory conditions. This study was undertaken to determine the effects of DMF on islet cells and non-endocrine tissue in a rodent model of L-Arginine-induced CP. Methods Male Wistar rats fed daily DMF (25 mg/kg) or vehicle by oral gavage were given 5 IP injections of L-Arginine (250 mg/100 g×2, 1 hr apart). Rats were assessed with weights and intra-peritoneal glucose tolerance tests (IPGTT, 2 g/kg). Islets were isolated and assessed for islet mass and viability with flow cytometry. Non-endocrine tissue was assessed for histology, myeloperoxidase (MPO), and lipid peroxidation level (MDA). In vitro assessments included determination of heme oxygenase (HO-1) protein expression by Western blot. Results Weight gain was significantly reduced in untreated CP group at 6 weeks. IPGTT revealed significant impairment in untreated CP group and its restoration with DMF therapy (P <0.05). Untreated CP rats had pancreatic atrophy, severe acinar architectural damage, edema, and fatty infiltration as well as elevated MDA and MPO levels, which were significantly improved by DMF treatment. After islet isolation, the volume of non-endocrine tissue was significantly smaller in untreated CP group. Although islet counts were similar in the two groups, islet viability was significantly reduced in untreated CP group and improved with DMF treatment. In vitro incubation of human pancreatic tissue with DMF significantly increased HO-1 expression. Conclusion Administration of DMF attenuated L-Arginine-induced CP and islet function in rats. DMF treatment could be a possible

  9. Anti-diabetic and neuroprotective effects of pancreatic islet transplantation into the central nervous system.

    PubMed

    Lazard, Daniel; Vardi, Pnina; Bloch, Konstantin

    2016-01-01

    During the last decades, the central nervous system (CNS) was intensively tested as a site for islet transplantation in different animal models of diabetes. Immunoprivilege properties of intracranial and intrathecal sites were found to delay and reduce rejection of transplanted allo-islets and xeno-islets, especially in the form of dispersed single cells. Insulin released from islets grafted in CNS was shown to cross the blood-brain barrier and to act as a regulator of peripheral glucose metabolism. In diabetic animals, sufficient nutrition and oxygen supply to islets grafted in the CNS provide adequate insulin response to increase glucose level resulting in rapid normoglycemia. In addition to insulin, pancreatic islets produce and secrete several other hormones, as well as neurotrophic and angiogenic factors with potential neuroprotective properties. Recent experimental studies and clinical trials provide a strong support for delivery of islet-derived macromolecules to CNS as a promising strategy to treat various brain disorders. This review article focuses mainly on analysis of current status of intracranial and intrathecal islet transplantations for treatment of experimental diabetes and discusses the possible neuroprotective properties of grafted islets into CNS as a novel therapeutic approach to brain disorders with cognitive dysfunctions characterized by impaired brain insulin signalling. Copyright © 2015 John Wiley & Sons, Ltd.

  10. Excitation wave propagation as a possible mechanism for signal transmission in pancreatic islets of Langerhans.

    PubMed Central

    Aslanidi, O V; Mornev, O A; Skyggebjerg, O; Arkhammar, P; Thastrup, O; Sørensen, M P; Christiansen, P L; Conradsen, K; Scott, A C

    2001-01-01

    In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets. PMID:11222284

  11. Outcomes of Pancreatic Islet Allotransplantation Using the Edmonton Protocol at the University of Chicago

    PubMed Central

    Tekin, Zehra; Garfinkel, Marc R.; Chon, W. James; Schenck, Lindsay; Golab, Karolina; Savari, Omid; Thistlethwaite, J. Richard; Philipson, Louis H.; Majewski, Colleen; Pannain, Silvana; Ramachandran, Sabarinathan; Rezania, Kourosh; Hariprasad, Seenu M.; Millis, J. Michael; Witkowski, Piotr

    2016-01-01

    Objective The aim of this study was to assess short-term and long-term results of the pancreatic islet transplantation using the Edmonton protocol at the University of Chicago. Materials and Methods Nine patients underwent pancreatic islet cell transplantation using the Edmonton Protocol; they were followed up for 10 years after initial islet transplant with up to 3 separate islet infusions. They were given induction treatment using an IL-2R antibody and their maintenance immunosuppression regimen consisted of sirolimus and tacrolimus. Results Nine patients received a total of 18 islet infusions. Five patients dropped out in the early phase of the study. Greater than 50% drop-out and noncompliance rate resulted from both poor islet function and recurrent side effects of immunosuppression. The remaining 4 (44%) patients stayed insulin free with intervals for at least over 5 years (cumulative time) after the first transplant. Each of them received 3 infusions, on average 445 000 islet equivalent per transplant. Immunosuppression regimen required multiple adjustments in all patients due to recurrent side effects. In the long-term follow up, kidney function remained stable, and diabetic retinopathy and polyneuropathy did not progress in any of the patients. Patients' panel reactive antibodies remained zero and anti-glutamic acid decarboxylase 65 antibody did not rise after the transplant. Results of metabolic tests including hemoglobin A1c, arginine stimulation, and mixed meal tolerance test were correlated with clinical islet function. Conclusions Pancreatic islet transplantation initiated according to Edmonton protocol offered durable long-term insulin-free glycemic control in only highly selected brittle diabetics providing stable control of diabetic neuropathy and retinopathy and without increased sensitization or impaired renal function. Immunosuppression adjustments and close follow-up were critical for patient retention and ultimate success.

  12. Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

    2008-08-01

    The pancreatic islets of Langerhans and insulin-producing beta cells in particular play a central role in the maintenance of glucose homeostasis and the islet dysfunction is associated with the pathogenesis of both type 1 and type 2 diabetes mellitus. To contribute to the understanding of the biology of the pancreatic islets we applied proteomic techniques based on liquid chromatography coupled with mass spectrometry. Here as an initial step we present the first comprehensive proteomic characterization of pancreas islets of the mouse, the commonly used animal model for diabetes research. Two-dimensional SCX LC/RP LC-MS/MS has been applied to characterize of the mouse islet proteome, resulting in the confident identification of 17,350 different tryptic peptides covering 2,612 proteins with at least two unique peptide identifications per protein. The dataset also allowed identification of a number of post-translational modifications including several modifications relevant to oxidative stress and phosphorylation. While many of the identified phosphorylation sites corroborates with previous known sites, the oxidative modifications observed on cysteinyl residues potentially reveal novel information related to the role of oxidation stress in islet functions. Comparative analysis of the islet proteome database with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 68 proteins uniquely detected only in the pancreatic islets. Besides proteins with known functions, like islet secreted peptide hormones, this unique set contains a number of proteins with yet unknown functions. The resulting peptide and protein database will be available at ncrr.pnl.gov web site of the NCRR proteomic center (ncrr.pnl.gov).

  13. A pumpless microfluidic device driven by surface tension for pancreatic islet analysis.

    PubMed

    Xing, Yuan; Nourmohammadzadeh, Mohammad; Elias, Joshua E Mendoza; Chan, Manwai; Chen, Zequn; McGarrigle, James J; Oberholzer, José; Wang, Yong

    2016-10-01

    We present a novel pumpless microfluidic array driven by surface tension for studying the physiology of pancreatic islets of Langerhans. Efficient fluid flow in the array is achieved by surface tension-generated pressure as a result of inlet and outlet size differences. Flow properties are characterized in numerical simulation and further confirmed by experimental measurements. Using this device, we perform a set of biological assays, which include real-time fluorescent imaging and insulin secretion kinetics for both mouse and human islets. Our results demonstrate that this system not only drastically simplifies previously published experimental protocols for islet study by eliminating the need for external pumps/tubing and reducing the volume of solution consumption, but it also achieves a higher analytical spatiotemporal resolution due to efficient flow exchanges and the extremely small volume of solutions required. Overall, the microfluidic platform presented can be used as a potential powerful tool for understanding islet physiology, antidiabetic drug development, and islet transplantation.

  14. Hypothyroidism Affects Vascularization and Promotes Immune Cells Infiltration into Pancreatic Islets of Female Rabbits

    PubMed Central

    Rodríguez-Castelán, Julia; Martínez-Gómez, Margarita; Castelán, Francisco; Cuevas, Estela

    2015-01-01

    Thyroidectomy induces pancreatic edema and immune cells infiltration similarly to that observed in pancreatitis. In spite of the controverted effects of hypothyroidism on serum glucose and insulin concentrations, the number and proliferation of Langerhans islet cells as well as the presence of extracellular matrix are affected depending on the islet size. In this study, we evaluated the effect of methimazole-induced hypothyroidism on the vascularization and immune cells infiltration into islets. A general observation of pancreas was also done. Twelve Chinchilla-breed female adult rabbits were divided into control (n = 6) and hypothyroid groups (n = 6, methimazole, 0.02% in drinking water for 30 days). After the treatment, rabbits were sacrificed and their pancreas was excised, histologically processed, and stained with Periodic Acid-Schiff (PAS) or Masson's Trichrome techniques. Islets were arbitrarily classified into large, medium, and small ones. The external and internal portions of each islet were also identified. Student-t-test and Mann-Whitney-U test or two-way ANOVAs were used to compare variables between groups. In comparison with control rabbits, hypothyroidism induced a strong infiltration of immune cells and a major presence of collagen and proteoglycans in the interlobular septa. Large islets showed a high vascularization and immune cells infiltration. The present results show that hypothyroidism induces pancreatitis and insulitis. PMID:26175757

  15. Hypothyroidism Affects Vascularization and Promotes Immune Cells Infiltration into Pancreatic Islets of Female Rabbits.

    PubMed

    Rodríguez-Castelán, Julia; Martínez-Gómez, Margarita; Castelán, Francisco; Cuevas, Estela

    2015-01-01

    Thyroidectomy induces pancreatic edema and immune cells infiltration similarly to that observed in pancreatitis. In spite of the controverted effects of hypothyroidism on serum glucose and insulin concentrations, the number and proliferation of Langerhans islet cells as well as the presence of extracellular matrix are affected depending on the islet size. In this study, we evaluated the effect of methimazole-induced hypothyroidism on the vascularization and immune cells infiltration into islets. A general observation of pancreas was also done. Twelve Chinchilla-breed female adult rabbits were divided into control (n = 6) and hypothyroid groups (n = 6, methimazole, 0.02% in drinking water for 30 days). After the treatment, rabbits were sacrificed and their pancreas was excised, histologically processed, and stained with Periodic Acid-Schiff (PAS) or Masson's Trichrome techniques. Islets were arbitrarily classified into large, medium, and small ones. The external and internal portions of each islet were also identified. Student-t-test and Mann-Whitney-U test or two-way ANOVAs were used to compare variables between groups. In comparison with control rabbits, hypothyroidism induced a strong infiltration of immune cells and a major presence of collagen and proteoglycans in the interlobular septa. Large islets showed a high vascularization and immune cells infiltration. The present results show that hypothyroidism induces pancreatitis and insulitis. PMID:26175757

  16. Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

    PubMed

    Yao, Virginia J; Ozawa, Michael G; Trepel, Martin; Arap, Wadih; McDonald, Donald M; Pasqualini, Renata

    2005-02-01

    Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

  17. Stem cells: a promising source of pancreatic islets for transplantation in type 1 diabetes.

    PubMed

    Street, Cale N; Rajotte, Ray V; Korbutt, Gregory S

    2003-01-01

    Diabetes is a disease that affects millions and causes a major burden on the health care system. Type 1 diabetes has traditionally been managed with exogenous insulin therapy, however factors such as cost, lifestyle restriction, and life threatening complications necessitate the development of a more efficient treatment alternative. Pancreas transplantation, and more recently transplant of purified pancreatic islets, has offered the potential for independence from insulin injections. Islet transplantation is gaining acceptance as it has been shown to be effective for certain patients with type 1 diabetes. One obstacle, however, is the fact that there is an inadequate supply of cadaveric human islets to implement this procedure on a widespread clinical basis. A promising source of transplantable islets in the future will come through the use of adult or embryonic stem cells. This chapter presents an overview of the advancements made in the development of a stem cell based application to islet transplantation. Advantages and limitations are discussed regarding the use of embryonic stem cells, adult pancreatic stem/progenitor cells, and the use of nonpancreatic tissues based on current experimental models in the literature. It is concluded that stem cells offer the greatest potential for the development of an abundant source of pancreatic islets, although specific obstacles must be overcome before this can become a reality. PMID:14711014

  18. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    SciTech Connect

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M. )

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub m}, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

  19. Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality.

    PubMed

    Chadwick, D R; Robertson, G S; Toomey, P; Contractor, H; Rose, S; James, R F; Bell, P R; London, N J

    1993-01-01

    Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7512874

  20. PDX-1 is a therapeutic target for pancreatic cancer, insulinoma and islet neoplasia using a novel RNA interference platform.

    PubMed

    Liu, Shi-He; Rao, Donald D; Nemunaitis, John; Senzer, Neil; Zhou, Guisheng; Dawson, David; Gingras, Marie-Claude; Wang, Zhaohui; Gibbs, Richard; Norman, Michael; Templeton, Nancy S; Demayo, Francesco J; O'Malley, Bert; Sanchez, Robbi; Fisher, William E; Brunicardi, F Charles

    2012-01-01

    Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a "drugable" target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNA(PDX-1), was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNA(humanPDX-1) lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNA(mousePDX-1) lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNA(mousePDX-1) lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.

  1. The matricellular protein CYR61 interferes with normal pancreatic islets architecture and promotes pancreatic neuroendocrine tumor progression.

    PubMed

    Huang, Yu-Ting; Lan, Qiang; Ponsonnet, Lionel; Blanquet, Marisa; Christofori, Gerhard; Zaric, Jelena; Rüegg, Curzio

    2016-01-12

    The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in β cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of α cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of β cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin α6β1-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic β cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors.

  2. B7-H4 as a protective shield for pancreatic islet beta cells

    PubMed Central

    Sun, Annika C; Ou, Dawei; Luciani, Dan S; Warnock, Garth L

    2014-01-01

    Auto- and alloreactive T cells are major culprits that damage β-cells in type 1 diabetes (T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell co-signaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin co-localization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1D. Future studies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1D. PMID:25512776

  3. Phase transitions in pancreatic islet cellular networks and implications for type-1 diabetes

    NASA Astrophysics Data System (ADS)

    Stamper, I. J.; Jackson, Elais; Wang, Xujing

    2014-01-01

    In many aspects the onset of a chronic disease resembles a phase transition in a complex dynamic system: Quantitative changes accumulate largely unnoticed until a critical threshold is reached, which causes abrupt qualitative changes of the system. In this study we examine a special case, the onset of type-1 diabetes (T1D), a disease that results from loss of the insulin-producing pancreatic islet β cells. Within each islet, the β cells are electrically coupled to each other via gap-junctional channels. This intercellular coupling enables the β cells to synchronize their insulin release, thereby generating the multiscale temporal rhythms in blood insulin that are critical to maintaining blood glucose homeostasis. Using percolation theory we show how normal islet function is intrinsically linked to network connectivity. In particular, the critical amount of β-cell death at which the islet cellular network loses site percolation is consistent with laboratory and clinical observations of the threshold loss of β cells that causes islet functional failure. In addition, numerical simulations confirm that the islet cellular network needs to be percolated for β cells to synchronize. Furthermore, the interplay between site percolation and bond strength predicts the existence of a transient phase of islet functional recovery after onset of T1D and introduction of treatment, potentially explaining the honeymoon phenomenon. Based on these results, we hypothesize that the onset of T1D may be the result of a phase transition of the islet β-cell network.

  4. Superficial necrolytic dermatitis in a dog with an insulin-producing pancreatic islet cell carcinoma.

    PubMed

    Isidoro-Ayza, M; Lloret, A; Bardagí, M; Ferrer, L; Martínez, J

    2014-07-01

    A 10-year-old dog presented with convulsive crisis and symmetrical hyperkeratotic cutaneous lesions affecting the abdomen, inguinal area, eyelids, muzzles, both pinnae, and all the paw pads. Hypoglycemia and hyperinsulinemia were the main biochemical findings. A mass 2 cm in diameter was detected within the left pancreatic lobe by ultrasonography. It was surgically removed and histologically and immunohistochemically diagnosed as an insulin-producing pancreatic islet cell carcinoma. The animal was eventually euthanized due to lack of clinical improvement. At necropsy, metastatic nodules were observed in the pancreatic lymph nodes and liver. Histopathological findings of cutaneous lesions were highly suggestive of superficial necrolytic dermatitis and were interpreted as a paraneoplastic syndrome derived from the islet cell carcinoma. To the authors' knowledge, this is the first report of superficial necrolytic dermatitis associated with an insulin-producing pancreatic neuroendocrine carcinoma in dogs.

  5. Isles within islets: The lattice origin of small-world networks in pancreatic tissues

    NASA Astrophysics Data System (ADS)

    Barua, Amlan K.; Goel, Pranay

    2016-02-01

    The traditional computational model of the pancreatic islets of Langerhans is a lattice of β-cells connected with gap junctions. Numerous studies have investigated the behavior of networks of coupled β-cells and have shown that gap junctions synchronize bursting strongly. This simplistic architecture of islets, however, seems increasingly untenable at the face of recent experimental advances. In a microfluidics experiment on isolated islets, Rocheleau et al. (2004) showed a failure of penetration of excitation when one end received high glucose and other end was not excited sufficiently; this suggested that gap junctions may not be efficient at inducing synchrony throughout the islet. Recently, Stozer et al. (2013) have argued that the functional networks of β-cells in an islet are small world. Their results implicate the existence of a few long-range connections among cells in the network. The physiological reason underlying this claim is not well understood. These studies cast doubt on the original lattice model that largely predict an all-or-none synchrony among the cells. Here we have attempted to reconcile these observations in a unified framework. We assume that cells in the islet are coupled randomly to their nearest neighbors with some probability, p. We simulated detailed β-cell bursting in such islets. By varying p systematically we were led to network parameters similar to those obtained by Stozer et al. (2013). We find that the networks within islets break up into components giving rise to smaller isles within the super structure-isles-within-islets, as it were. This structure can also account for the partial excitation seen by Rocheleau et al. (2004). Our updated view of islet architecture thus explains the paradox how islets can have strongly synchronizing gap junctions, and be weakly coordinated at the same time.

  6. Pancreatic islet and stem cell transplantation: new strategies in cell therapy of diabetes mellitus.

    PubMed

    Bretzel, R G; Eckhard, M; Brendel, M D

    2004-03-01

    Long-term studies strongly suggest that tight control of blood glucose can prevent the development and retard the progression of chronic complications of type 1 diabetes mellitus. In contrast to conventional insulin treatment, replacement of a patient's islets of Langerhans either by pancreas organ transplantation of by isolated islet transplantation is the only treatment to achieve a constant normoglycemic state and avoiding hypoglycemic episodes, a typical adverse event of multiple daily insulin injections. However, the expense of this benefit is still the need for immunosuppressive treatment of the recipient with all its potential risks. Islet cell transplantation offers the advantage of being performed as a minimally invasive procedure, in which islets can be perfused percutaneously into the liver via the portal vein. As of June 2003, 705 pancreatic islet transplants worldwide have been reported to the International Islet Transplant Registry (ITR) at our Third Medical Department, University of Giessen/Germany. Data analysis shows at 1 year after adult islet transplantation a patient survival rate of 97%, a functioning islet graft in 54% of the cases, whereas insulin independence was meanwhile achieved in 20% of the cases. However, using a novel protocol established by the Edmonton Center/Canada, the insulin independence rates have improved significantly reaching meanwhile a 50-80% level. Finally, the concept of islet cell or stem cell transplantation is most attractive since it offers many perspectives: islet cell availability could become unlimited and islet or stem cells my be transplanted without life-long immunosuppressive treatment of the recipient, just to mention 2 of them. PMID:15238879

  7. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

  8. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets. PMID:27539300

  9. The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets.

    PubMed

    Barbu, Andreea; Jansson, Leif; Sandberg, Monica; Quach, My; Palm, Fredrik

    2015-01-01

    The blood perfusion of pancreatic islets is regulated independently from that of the exocrine pancreas, and is of importance for multiple aspects of normal islet function, and probably also during impaired glucose tolerance. Single islet blood flow has been difficult to evaluate due to technical limitations. We therefore adapted a hydrogen gas washout technique using microelectrodes to allow such measurements. Platinum micro-electrodes monitored hydrogen gas clearance from individual endogenous and transplanted islets in the pancreas of male Lewis rats and in human and mouse islets implanted under the renal capsule of male athymic mice. Both in the rat endogenous pancreatic islets as well as in the intra-pancreatically transplanted islets, the vascular conductance and blood flow values displayed a highly heterogeneous distribution, varying by factors 6-10 within the same pancreas. The blood flow of human and mouse islet grafts transplanted in athymic mice was approximately 30% lower than that in the surrounding renal parenchyma. The present technique provides unique opportunities to study the islet vascular dysfunction seen after transplantation, but also allows for investigating the effects of genetic and environmental perturbations on islet blood flow at the single islet level in vivo.

  10. Antiapoptotic effects of cerium oxide and yttrium oxide nanoparticles in isolated rat pancreatic islets.

    PubMed

    Hosseini, A; Baeeri, M; Rahimifard, M; Navaei-Nigjeh, M; Mohammadirad, A; Pourkhalili, N; Hassani, S; Kamali, M; Abdollahi, M

    2013-05-01

    Type I diabetes mellitus is a metabolic disease caused by the impairment of pancreatic β-cells mainly mediated through oxidative stress and related apoptosis. Islets transplantation seems a promising treatment for these patients, but during islets transplant, various types of stresses related to the isolation and transplantation procedure compromise the function and viability of islets. We recently hypothesized that the combination of cerium oxide (CeO2) and yttrium oxide (Y2O3) nanoparticles with a potential free radical scavenger behavior should be useful to make isolated islets survive until transplanted. In the present study, oxidative stress-induced apoptosis in isolated rat pancreatic islets exposed to hydrogen peroxide (H2O2) and the protective effects of CeO2 and Y2O3 nanoparticles were investigated. Exposure of islets to H2O2 (50 µm, 2 h) increased intracellular oxidant formation such as reactive oxygen species and subsequently apoptosis and decreased viability, glucose-induced adenosine triphosphate (ATP) production and glucose-stimulated insulin secretion. Pretreatment with CeO2 and/or Y2O3 nanoparticles reduced the oxidant formation and apoptosis and increased viability, glucose-induced ATP production and glucose-stimulated insulin secretion. These results suggest that this combination may protect β-cell apoptosis by improving the oxidative stress-mediated apoptotic pathway.

  11. Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses.

    PubMed

    Hoang, Danh-Tai; Hara, Manami; Jo, Junghyo

    2016-01-01

    Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets. PMID:27035570

  12. Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses

    PubMed Central

    Hoang, Danh-Tai; Hara, Manami; Jo, Junghyo

    2016-01-01

    Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets. PMID:27035570

  13. Fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model

    PubMed Central

    Jo, Junghyo; Hörnblad, Andreas; Kilimnik, German; Hara, Manami; Ahlgren, Ulf; Periwal, Vipul

    2013-01-01

    The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, have not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension, 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with fractal dimension 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas. PMID:23629025

  14. The fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model

    NASA Astrophysics Data System (ADS)

    Jo, Junghyo; Hörnblad, Andreas; Kilimnik, German; Hara, Manami; Ahlgren, Ulf; Periwal, Vipul

    2013-06-01

    The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, has not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension of 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with a fractal dimension of 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas.

  15. Total Pancreatectomy with Islet Autologous Transplantation: The Cure for Chronic Pancreatitis?

    PubMed Central

    Kesseli, Samuel J; Smith, Kerrington A; Gardner, Timothy B

    2015-01-01

    Chronic pancreatitis (CP) is a debilitating disease that leads to varying degrees of pancreatic endocrine and exocrine dysfunction. One of the most difficult symptoms of CP is severe abdominal pain, which is often challenging to control with available analgesics and therapies. In the last decade, total pancreatectomy with autologous islet cell transplantation has emerged as a promising treatment for the refractory pain of CP and is currently performed at approximately a dozen centers in the United States. While total pancreatectomy is not a new procedure, the endocrine function-preserving autologous islet cell isolation and re-implantation have made the prospect of total pancreatectomy more acceptable to patients and clinicians. This review will focus on the current status of total pancreatectomy with autologous islet cell transplant including patient selection, technical considerations, and outcomes. As the procedure is performed at an increasing number of centers, this review will highlight opportunities for quality improvement and outcome optimization. PMID:25630865

  16. Assessment of benzene induced oxidative impairment in rat isolated pancreatic islets and effect on insulin secretion.

    PubMed

    Bahadar, Haji; Maqbool, Faheem; Mostafalou, Sara; Baeeri, Maryam; Rahimifard, Mahban; Navaei-Nigjeh, Mona; Abdollahi, Mohammad

    2015-05-01

    Benzene (C6H6) is an organic compound used in petrochemicals and numerous other industries. It is abundantly released to our environment as a chemical pollutant causing widespread human exposure. This study mainly focused on benzene induced toxicity on rat pancreatic islets with respect to oxidative damage, insulin secretion and glucokinase (GK) activity. Benzene was dissolved in corn oil and administered orally at doses 200, 400 and 800mg/kg/day, for 4 weeks. In rats, benzene significantly raised the concentration of plasma insulin. Also the effect of benzene on the release of glucose-induced insulin was pronounced in isolated islets. Benzene caused oxidative DNA damage and lipid peroxidation, and also reduced the cell viability and total thiols groups, in the islets of exposed rats. In conclusion, the current study revealed that pancreatic glucose metabolism is susceptible to benzene toxicity and the resultant oxidative stress could lead to functional abnormalities in the pancreas.

  17. Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice

    SciTech Connect

    Gao Xiaodong; Song Lujun; Shen Kuntang; Wang Hongshan; Niu Weixin Qin Xinyu

    2008-06-20

    The transplantation of bone marrow (BM) derived cells to initiate pancreatic regeneration is an attractive but as-yet unrealized strategy. Presently, BM derived cells from green fluorescent protein transgenic mice were transplanted into diabetic mice. Repair of diabetic islets was evidenced by reduction of hyperglycemia, increase in number of islets, and altered pancreatic histology. Cells in the pancreata of recipient mice co-expressed BrdU and insulin. Double staining revealed {beta} cells were in the process of proliferation. BrdU{sup +} insulin{sup -} PDX-1{sup +} cells, Ngn3{sup +} cells and insulin{sup +} glucagon{sup +} cells, which showed stem cells, were also found during {beta}-cell regeneration. The majority of transplanted cells were mobilized to the islet and ductal regions. In recipient pancreas, transplanted cells simultaneously expressed CD34 but did not express insulin, PDX-1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Pax6, and CD45. It is concluded that BM derived cells especially CD34{sup +} cells can promote repair of pancreatic islets. Moreover, both proliferation of {beta} cells and differentiation of pancreatic stem cells contribute to the regeneration of {beta} cells.

  18. Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation

    PubMed Central

    Shima, Hiroki; Inagaki, Akiko; Imura, Takehiro; Yamagata, Youhei; Watanabe, Kimiko; Igarashi, Kazuhiko; Goto, Masafumi; Murayama, Kazutaka

    2016-01-01

    The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans. PMID:27195301

  19. Kinetic analyses of peptidylglycine alpha-amidating monooxygenase from pancreatic islets.

    PubMed

    Noe, B D; Katopodis, A G; May, S W

    1991-08-01

    Peptidylglycine alpha-amidating monooxygenase (PAM) plays an important role in the post-translational processing of bioactive neuropeptides by participating in C-terminal amidation. We have examined PAM activity in the pancreatic islets of the anglerfish (AF), Lophius americanus. It was previously demonstrated that the cofactor requirements and pH optimum for the fish PAM are essentially identical to PAM obtained from other tissues and species. The present study was performed to examine the enzymatic characteristics of the fish islet PAM in more detail. One of the questions addressed was the suitability of the AF islet neuropeptide Y-like peptide, aPY-Gly, as a substrate for the islet PAM. Partially purified PAM from AF islet secretory granules was incubated with [125I] aPY-Gly and the resulting products were analyzed by HPLC. The islet PAM readily mediated the formation of aPY-amide from aPY-Gly. PAM purified from bovine adrenal chromaffin granules also catalyzed the amidation of [125I] aPY-Gly. The kinetic parameters of the islet PAM were examined using trinitrophenylated-D-Tyr-Val-Gly (TNP-D-YVG) and 4-nitrohippuric acid (4-NHA). The Km of the islet PAM was 25 +/- 5 microM for TNP-D-YVG and 3.4 +/- 1 mM for 4-NHA. The competitive inhibitor of mammalian PAM activity, 4-methoxybenzoxyacetic acid, proved to be a potent inhibitor of the islet PAM as well, with an apparent KI of 0.06 mM. These results demonstrate that the AF islet PAM exhibits substrate compatibility, kinetic parameters, and inhibitor susceptibility quite similar to the characteristics of PAM from other tissues and species. PMID:1916206

  20. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    SciTech Connect

    Dalgaard, Louise T.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  1. Prevention of murine autoimmune diabetes by CCL22-mediated Treg recruitment to the pancreatic islets.

    PubMed

    Montane, Joel; Bischoff, Loraine; Soukhatcheva, Galina; Dai, Derek L; Hardenberg, Gijs; Levings, Megan K; Orban, Paul C; Kieffer, Timothy J; Tan, Rusung; Verchere, C Bruce

    2011-08-01

    Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ–producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes. PMID:21737880

  2. FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.

    PubMed

    Kuznetsov, Andrey; Bindokas, Vytautas P; Marks, Jeremy D; Philipson, Louis H

    2005-07-01

    Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. PMID:15758044

  3. In vitro infection of mouse pancreatic islet cells with coxsackie viruses.

    PubMed

    Bopegamage, S A; Petrovicová, A

    1994-10-01

    We have demonstrated the ability of 4 standard coxsackie viruses (B4, B5, A7, and A9) and one fresh isolate (A7) from a newly diabetic child with homologous serological response, to infect in vitro grown mouse pancreatic islet cells. Up to the 9th day after infection the multiplication of viruses in the cells was proved using virus titration and immunofluorescence test. Isolated pancreatic cells proved to be a suitable model for detailed studies of experimental infection of pancreatic cells with coxsackie viruses.

  4. Adaptation of pancreatic islet cyto-architecture during development

    NASA Astrophysics Data System (ADS)

    Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

    2016-04-01

    Plasma glucose in mammals is regulated by hormones secreted by the islets of Langerhans embedded in the exocrine pancreas. Islets consist of endocrine cells, primarily α, β, and δ cells, which secrete glucagon, insulin, and somatostatin, respectively. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Varying demands and available nutrients during development produce changes in the local connectivity of β cells in an islet. We showed in earlier work that graph theory provides a framework for the quantification of the seemingly stochastic cyto-architecture of β cells in an islet. To quantify the dynamics of endocrine connectivity during development requires a framework for characterizing changes in the probability distribution on the space of possible graphs, essentially a Fokker-Planck formalism on graphs. With large-scale imaging data for hundreds of thousands of islets containing millions of cells from human specimens, we show that this dynamics can be determined quantitatively. Requiring that rearrangement and cell addition processes match the observed dynamic developmental changes in quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that there is a transient shift in preferred connectivity for β cells between 1-35 weeks and 12-24 months.

  5. Adaptation of pancreatic islet cyto-architecture during development

    NASA Astrophysics Data System (ADS)

    Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

    2016-04-01

    Plasma glucose in mammals is regulated by hormones secreted by the islets of Langerhans embedded in the exocrine pancreas. Islets consist of endocrine cells, primarily α, β, and δ cells, which secrete glucagon, insulin, and somatostatin, respectively. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Varying demands and available nutrients during development produce changes in the local connectivity of β cells in an islet. We showed in earlier work that graph theory provides a framework for the quantification of the seemingly stochastic cyto-architecture of β cells in an islet. To quantify the dynamics of endocrine connectivity during development requires a framework for characterizing changes in the probability distribution on the space of possible graphs, essentially a Fokker-Planck formalism on graphs. With large-scale imaging data for hundreds of thousands of islets containing millions of cells from human specimens, we show that this dynamics can be determined quantitatively. Requiring that rearrangement and cell addition processes match the observed dynamic developmental changes in quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that there is a transient shift in preferred connectivity for β cells between 1–35 weeks and 12–24 months.

  6. Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus.

    PubMed

    Xiong, Xiaoquan; Wang, Xiao; Li, Bing; Chowdhury, Subrata; Lu, Yarong; Srikant, Coimbatore B; Ning, Guang; Liu, Jun-Li

    2011-04-01

    Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. The expression of Reg3β (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed β-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3β was achieved in the pancreatic islets of RIP-I/Reg3β mice, which exhibited normal islet histology, β-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3β mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3β overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3β is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.

  7. Pancreatic islet hormone response to oral glucose in morbidly obese patients.

    PubMed Central

    Sirinek, K R; O'Dorisio, T M; Howe, B; McFee, A S

    1985-01-01

    Pancreatic islet peptides, as well as other gastrointestinal hormones, have been implicated in both the pathogenesis of obesity and the etiology of associated metabolic derangements. This study evaluated the pancreatic islet and gastrointestinal (GI) hormone response to oral glucose in 20 morbidly obese (151% above ideal body weight) patients. Glucose intolerance, hyperinsulinism, and exaggerated gastric inhibitory polypeptide (GIP) release occurred following glucose ingestion. Significant release of PP occurred in 14 patients, while only six patients had release of somatostatin. No significant changes in plasma concentrations of glucagon occurred. Since GIP is insulinotropic in the presence of hyperglycemia, the hyperinsulinism of morbid obesity may be secondary to the abnormally high glucose-stimulated GIP levels in these patients. Failure of glucagon suppression in response to oral glucose many contribute to the hyperglycemia noted. Somatostatin and pancreatic polypeptide may be responsible for some of the metabolic derangements of morbid obesity. PMID:2860876

  8. Copper addition prevents the inhibitory effects of interleukin 1-beta on rat pancreatic islets.

    PubMed

    Vinci, C; Caltabiano, V; Santoro, A M; Rabuazzo, A M; Buscema, M; Purrello, R; Rizzarelli, E; Vigneri, R; Purrello, F

    1995-01-01

    Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-I beta (IL-1 beta), we studied whether Cu(II) addition could prevent the inhibitory effect of IL-1 beta on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1 beta, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-L-histidyl-L-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO4 (1-1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0 +/- 11.4 pg.islet-1 h-1 (mean +/- SEM, n = 7) and glucose-induced release was 2824.0 +/- 249.0 pg.islet-1 h-1. In islets pre-exposed to 50 U/ml IL-1 beta, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2 +/- 76.9, n = 7, p < 0.005). In islets incubated with IL-1 beta and Cu-GHL (0.4 mumol/l, maximal effect) basal secretion was 119.0 +/- 13.1 pg.islet-1 h-1 and glucose-induced release was 2797.2 +/- 242.2, (n = 7, p < 0.01 in respect to islets exposed to IL-1 beta alone).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7744228

  9. [Treatment of type 1 diabetics by transplantation of isolated pancreatic islets].

    PubMed

    Ono, Junko

    2006-04-01

    Type 1 diabetes is an autoimmune disease with selective destruction of insulin-producing pancreatic beta cells. Since insulin plays pivotal roles in energy homeostasis by transferring glucose into cells, type 1 diabetic patients can not survive without insulin replacement. Insulin secretion is precisely controlled by ingested glucose as well as hormones and neural factors, therefore it is impossible to reproduce the physiological secretory pattern of insulin via exogenous insulin, even by multiple or continuous delivery by injection. Transplantation of beta cells has long been expected as the fundamental treatment to cure type 1 diabetics, and transplantation of the whole pancreas, both exocrine pancreas and islets, has been applied with success, resulting insulin independence. However, the exocrine pancreas, which releases amylase and trypsin to the digestive tract, is not indispensable for insulin replacement, so the interest in islet transplantation has increased enormously. In the past 20 years, the techniques for isolating large numbers of human islets have been advanced and more potent immunosuppressive agents have also been introduced, permitting newer attempts at islet transplantation. In 2000, insulin independence was first achieved in Canada using the Edmonton protocol. The success rates have increased gradually using this protocol, and 5 institutes in Japan have started to prepare human islet transplantation under the control of the Japan Pancreas and Islet Transplant Society. In 2004, insulin independence by islet transplantation was first achieved at Kyoto University Hospital and the number of islet transplantations has increased, though very slowly. By the end of 2005, approximately 100 patients were on the waiting list for islet transplantation in Japan. Many problems remain unsolved in islet transplantation to meet clinical practice: these are the shortage of insulin-producing cells, further progress in immunosuppressive agents that do not interfere

  10. Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

    SciTech Connect

    Antoine, M.H.; Hermann, M.; Herchuelz, A.; Lebrun, P. )

    1991-07-01

    Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the K+ channel openers. The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.

  11. Characterization of a pancreatic islet cell tumor in a polar bear (Ursus maritimus).

    PubMed

    Fortin, Jessica S; Benoit-Biancamano, Marie-Odile

    2014-01-01

    Herein, we report a 25-year-old male polar bear suffering from a pancreatic islet cell tumor. The aim of this report is to present a case of this rare tumor in a captive polar bear. The implication of potential risk factors such as high carbohydrate diet or the presence of amyloid fibril deposits was assessed. Necropsy examination revealed several other changes, including nodules observed in the liver, spleen, pancreas, intestine, and thyroid glands that were submitted for histopathologic analysis. Interestingly, the multiple neoplastic nodules were unrelated and included a pancreatic islet cell tumor. Immunohistochemistry of the pancreas confirmed the presence of insulin and islet amyloid polypeptide (IAPP) within the pancreatic islet cells. The IAPP gene was extracted from the paraffin-embedded liver tissue and sequenced. IAPP cDNA from the polar bear exhibits some differences as compared to the sequence published for several other species. Different factors responsible for neoplasms in bears such as diet, infectious agents, and industrial chemical exposure are reviewed. This case report raised several issues that further studies may address by evaluating the prevalence of cancers in captive or wild animals. PMID:25273481

  12. Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells

    PubMed Central

    Rekittke, Nadine E.; Ang, Meidjie; Rawat, Divya; Khatri, Rahul

    2016-01-01

    Type 1 diabetes is an autoimmune disease resulting in the permanent destruction of pancreatic islets. Islet transplantation to portal vein provides an approach to compensate for loss of insulin producing cells. Clinical trials demonstrated that even partial islet graft function reduces severe hypoglycemic events in patients. However, therapeutic impact is restrained due to shortage of pancreas organ donors and instant inflammation occurring in the hepatic environment of the graft. We summarize on what is known about regenerative therapy in type 1 diabetes focusing on pancreatic islet transplantation and new avenues of cell substitution. Metabolic pathways and energy production of transplanted cells are required to be balanced and protection from inflammation in their intravascular bed is desired. Mesenchymal stem cells (MSCs) have anti-inflammatory features, and so they are interesting as a therapy for type 1 diabetes. Recently, they were reported to reduce hyperglycemia in diabetic rodents, and they were even discussed as being turned into endodermal or pancreatic progenitor cells. MSCs are recognized to meet the demand of an individual therapy not raising the concerns of embryonic or induced pluripotent stem cells for therapy. PMID:27047547

  13. Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.

    PubMed

    Rekittke, Nadine E; Ang, Meidjie; Rawat, Divya; Khatri, Rahul; Linn, Thomas

    2016-01-01

    Type 1 diabetes is an autoimmune disease resulting in the permanent destruction of pancreatic islets. Islet transplantation to portal vein provides an approach to compensate for loss of insulin producing cells. Clinical trials demonstrated that even partial islet graft function reduces severe hypoglycemic events in patients. However, therapeutic impact is restrained due to shortage of pancreas organ donors and instant inflammation occurring in the hepatic environment of the graft. We summarize on what is known about regenerative therapy in type 1 diabetes focusing on pancreatic islet transplantation and new avenues of cell substitution. Metabolic pathways and energy production of transplanted cells are required to be balanced and protection from inflammation in their intravascular bed is desired. Mesenchymal stem cells (MSCs) have anti-inflammatory features, and so they are interesting as a therapy for type 1 diabetes. Recently, they were reported to reduce hyperglycemia in diabetic rodents, and they were even discussed as being turned into endodermal or pancreatic progenitor cells. MSCs are recognized to meet the demand of an individual therapy not raising the concerns of embryonic or induced pluripotent stem cells for therapy.

  14. Characterization of a pancreatic islet cell tumor in a polar bear (Ursus maritimus).

    PubMed

    Fortin, Jessica S; Benoit-Biancamano, Marie-Odile

    2014-01-01

    Herein, we report a 25-year-old male polar bear suffering from a pancreatic islet cell tumor. The aim of this report is to present a case of this rare tumor in a captive polar bear. The implication of potential risk factors such as high carbohydrate diet or the presence of amyloid fibril deposits was assessed. Necropsy examination revealed several other changes, including nodules observed in the liver, spleen, pancreas, intestine, and thyroid glands that were submitted for histopathologic analysis. Interestingly, the multiple neoplastic nodules were unrelated and included a pancreatic islet cell tumor. Immunohistochemistry of the pancreas confirmed the presence of insulin and islet amyloid polypeptide (IAPP) within the pancreatic islet cells. The IAPP gene was extracted from the paraffin-embedded liver tissue and sequenced. IAPP cDNA from the polar bear exhibits some differences as compared to the sequence published for several other species. Different factors responsible for neoplasms in bears such as diet, infectious agents, and industrial chemical exposure are reviewed. This case report raised several issues that further studies may address by evaluating the prevalence of cancers in captive or wild animals.

  15. Decreased basal insulin secretion from pancreatic islets of pups in a rat model of maternal obesity.

    PubMed

    Zambrano, Elena; Sosa-Larios, Tonantzin; Calzada, Lizbeth; Ibáñez, Carlos A; Mendoza-Rodríguez, Carmen A; Morales, Angélica; Morimoto, Sumiko

    2016-10-01

    Maternal obesity (MO) is a deleterious condition that enhances susceptibility of adult offspring to metabolic diseases such as type 2 diabetes. The objective is to study the effect of MO on in vitro insulin secretion and pancreatic cellular population in offspring. We hypothesize that a harmful antenatal metabolic environment due to MO diminishes the basal glucose-responsive secretory function of pancreatic beta cells in offspring. Mothers were fed a control (C) or high-fat diet from weaning through pregnancy (120 days) and lactation. At postnatal days (PNDs) 36 and 110, pups were killed, peripheral blood was collected and pancreatic islets were isolated. Basal insulin secretion was measured in vitro in islets for 60 min. It was found that blood insulin, glucose and homeostasis model assessment (HOMA) index were unaffected by maternal diet and age in females. However, male MO offspring at PND 110 showed hyperinsulinemia and insulin resistance compared with C. Body weight was not modified by MO, but fat content was higher in MO pups compared with C pups. Triglycerides and leptin concentrations were higher in MO than in C offspring in all groups except in females at PND 36. Pancreatic islet cytoarchitecture was unaffected by MO. At PND 36, islets of male and female C and MO offspring responded similarly to glucose, but at PND 110, male and female MO offspring islets showed a 50% decrease in insulin secretion. It was concluded that MO impairs basal insulin secretion of offspring with a greater impact on males than females, and this effect mainly manifests in adulthood. PMID:27496224

  16. Differentiation of chicken umbilical cord mesenchymal stem cells into beta-like pancreatic islet cells.

    PubMed

    Bai, Chunyu; Gao, Yuhua; Li, Qian; Feng, Yuan; Yu, Yanze; Meng, Gentong; Zhang, Minghai; Guan, Weijun

    2015-04-01

    In this study, we explored the possibility of using in vitro differentiation to create functional beta-like islet cells from chicken umbilical cord mesenchymal stem cells (UCMSCs). Passaged UCMSCs were induced to differentiate into pancreatic beta-like islet cells. Differentiated cells were observed through dithizone staining, and Pdx1 and insulin expressed in differentiated cells were detected with immunofluorescence. Insulin and C-peptide production from differentiated cells were analyzed using ELISA and western blotting. Differentiated cells were found to not only express Pdx1, insulin, and C-peptide, but also to display a glucose-responsive secretion of these hormones. PMID:24303870

  17. Evidence for the presence of glucose cycling in pancreatic islets of the ob/ob mouse

    SciTech Connect

    Khan, A.; Chandramouli, V.; Ostenson, C.G.; Ahren, B.; Schumann, W.C.; Loew, H.L.; Landau, B.R.; Efendic, S.

    1989-06-15

    Pancreatic islets from ob/ob mice incubated with /sub 3/H/sub 2/O and 5.5 mM glucose formed /sup 3/H-labeled glucose, 74 picoatoms incorporated/islet/h. Sixty-three percent of the 3H was bound to carbon 2 of the glucose. The amount of glucose-6-P dephosphorylated to glucose, determined from this incorporation, was 48 pmol/islet/h. Glucose utilization, measured by the formation of /sup 3/H/sub 2/O from (5-/sup 3/H)glucose, was 72 pmol/islet/h. The amount of glucose dephosphorylated was then about 40% of that phosphorylated. Thus, glucose-6-P is dephosphorylated to glucose to a significant extent by intact islets in vitro and presumably by the beta cells of the islets. The extent of this glucose cycling, i.e. glucose----glucose-6-P----glucose, may play a role in determining the extent of glucose-induced insulin secretion.

  18. PAX4 Defines an Expandable β-Cell Subpopulation in the Adult Pancreatic Islet

    PubMed Central

    Lorenzo, Petra I.; Fuente-Martín, Esther; Brun, Thierry; Cobo-Vuilleumier, Nadia; Jimenez-Moreno, Carmen María; G. Herrera Gomez, Irene; López Noriega, Livia; Mellado-Gil, José Manuel; Martin-Montalvo, Alejandro; Soria, Bernat; Gauthier, Benoit R.

    2015-01-01

    PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets. PMID:26503027

  19. Metabolism Regulates Exposure of Pancreatic Islets to Circulating Molecules In Vivo.

    PubMed

    Michau, Aurélien; Hodson, David J; Fontanaud, Pierre; Guillou, Anne; Espinosa-Carrasco, Gabriel; Molino, François; Peters, Catherine J; Robinson, Iain C; Le Tissier, Paul; Mollard, Patrice; Schaeffer, Marie

    2016-02-01

    Pancreatic β-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory β-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of β-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping β-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules. PMID:26581596

  20. Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets.

    PubMed

    Xu, Jianxiang; Han, Junying; Epstein, Paul N; Liu, Ye Q

    2006-06-01

    Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK. PMID:16631612

  1. The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes

    PubMed Central

    2010-01-01

    Over the past three decades the pancreatic islet of Langerhans has taken center stage as an endocrine microorgan whose glucoregulatory function is highly explicable on the basis of the increasingly well understood activities of three highly interactive secretory cells. Islet dysfunction underlies both type 1 and type 2 diabetes mellitus (DM); its protection from immune attack and gluco-and lipo-toxicity may prevent the development of DM; and its replacement by non-surgical transplantation may be curative of DM. During a career marked by vision, focus and tenacity, Paul Lacy contributed substantially to the development of each of these concepts. In this review we focus on Lacy's contribution to the development of the concept of the islet as a micro-organ, how this foreshadowed our current detailed understanding of single cell function and cell-cell interactions and how this led to a reduced model of islet function encouraging islet transplantation. Next, we examine how clinical allotransplantation, first undertaken by Lacy, has contributed to a more complex view of the interaction of islet endocrine cells with its circulation and neighboring tissues, both “in situ” and after transplantation. Lastly, we consider recent developments in some alternative approaches to treatment of DM that Lacy could glimpse on the horizon but did not have the chance to participate in. PMID:21099316

  2. Caprine pancreatic islet xenotransplantation into diabetic immunosuppressed BALB/c mice

    PubMed Central

    Hani, Homayoun; Allaudin, Zeenathul N; Mohd-Lila, Mohd-Azmi; Ibrahim, Tengku A Tengku; Othman, Abas M

    2014-01-01

    Background Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. Methods Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. Results The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (<11 mm glucose) for 30 days. In addition, weight gain negatively correlated with blood glucose level. The findings verified diabetes reversal in caprine islet recipient mice. A significant drop in non-fasting blood glucose level (from 23.3 ± 5.4 to 8.04 ± 0.44 mm) and simultaneous increase in serum insulin level (from 0.01 ± 0.001 to 0.56 ± 0.17 μg/l) and body weights (from 23.64 ± 0.31 to 25.85 ± 0.34 g) were observed (P < 0.05). Immunohistochemical analysis verified insulin production in the transplanted islets. Conclusions Purified caprine islets were demonstrated to successfully sustain viability and functionality for controlling blood glucose levels in an immunosuppressed mouse model of diabetes. These results suggest the use of caprine islets as an addition to the supply of xenogeneic islets for diabetes research. PMID:24645790

  3. FEM-based oxygen consumption and cell viability models for avascular pancreatic islets

    PubMed Central

    Buchwald, Peter

    2009-01-01

    Background The function and viability of cultured, transplanted, or encapsulated pancreatic islets is often limited by hypoxia because these islets have lost their vasculature during the isolation process and have to rely on gradient-driven passive diffusion, which cannot provide adequate oxygen transport. Pancreatic islets (islets of Langerhans) are particularly susceptible due to their relatively large size, large metabolic demand, and increased sensitivity to hypoxia. Here, finite element method (FEM) based multiphysics models are explored to describe oxygen transport and cell viability in avascular islets both in static and in moving culture media. Methods Two- and three-dimensional models were built in COMSOL Multiphysics using the convection and diffusion as well as the incompressible Navier-Stokes fluid dynamics application modes. Oxygen consumption was assumed to follow Michaelis-Menten-type kinetics and to cease when local concentrations fell below a critical threshold; in a dynamic model, it was also allowed to increase with increasing glucose concentration. Results Partial differential equation (PDE) based exploratory cellular-level oxygen consumption and cell viability models incorporating physiologically realistic assumptions have been implemented for fully scaled cell culture geometries with 100, 150, and 200 μm diameter islets as representative. Calculated oxygen concentrations and intra-islet regions likely to suffer from hypoxia-related necrosis obtained for traditional flask-type cultures, oxygen-permeable silicone-rubber membrane bottom cultures, and perifusion chambers with flowing media and varying incoming glucose levels are presented in detail illustrated with corresponding colour-coded figures and animations. Conclusion Results of the computational models are, as a first estimate, in good quantitative agreement with existing experimental evidence, and they confirm that during culture, hypoxia is often a problem for non-vascularised islet

  4. Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs

    PubMed Central

    Hellerström, Claes; Howell, Simon L.; Edwards, John C.; Andersson, Arne; Östenson, Claes-Göran

    1974-01-01

    1. The biosynthesis of glucagon in guinea-pig A2 cells was investigated by incubation of isolated islets of Langerhans in the presence of [3H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A2 cells in experiments using B-cell-depleted islets, and to A2-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis. PMID:4615708

  5. Total or near total pancreatectomy and islet autotransplantation for treatment of chronic pancreatitis.

    PubMed Central

    Najarian, J S; Sutherland, D E; Baumgartner, D; Burke, B; Rynasiewicz, J J; Matas, A J; Goetz, F C

    1980-01-01

    Total or near total pancreatectomy is the surest way to relieve the pain of chronic pancreatitis but is rarely applied because the metabolic consequences are so severe. For most patients drainage procedures are applicable, but pancreatectomy may be the only alternative for small duct disease or where procedures to improve duct drainage have failed. Preservation of endocrine function is a major problem in patients who require pancreatectomy. Experiments in pancreatectomized dogs have shown that intrasplenic or intraportal transplantation of unpurified pancreatic islet tissue dispersed by collagenase digestion can prevent diabetes. We have applied this technique to ten patients with chronic pancreatitis, small ducts, and intractable pain. The entire pancreas of > 95% of the pancrease was excised, minced, dispersed by collagenase digestion and infused into the portal vein < 2 1/2 hours after removal. Mean (+/- SD) rise in portal pressure was 17 +/- 8 cm of water. Liver function tests were altered minimally. All patients were relieved of pain. One patient died of a complication not related to the islet autotransplant; viable islets were identified in the liver at autopsy. Of the remaining nine patients, three have been insulin independent for 1, 9, and 38 months. One patient was insulin indpendent for 15 months and now takes 12 units of insulin daily. Three have nonketosis prone diabetes (tested by insulin withdrawal) and take 15--30 units of insulin per day. C-peptide studies in these patients show that functioning islets are present. Two patients are diabetic and require 35 and 60 units of insulin per day. In eight of nine patients tested serum insulin concentrations fell to undetectable levels during the interval between pancreatectomy and islet transplantation. Serum insulin levels during the first few hours after islet transplantation predicted success. In the insulin independent or in the patients with mild diabetes, insulin levels were persistently greater than

  6. Exercise at anaerobic threshold intensity and insulin secretion by isolated pancreatic islets of rats

    PubMed Central

    de Oliveira, Camila Aparecida Machado; Paiva, Mauricio Ferreira; Mota, Clécia Alencar Soares; Ribeiro, Carla; de Almeida Leme, José Alexandre Curiacos; Luciano, Eliete

    2010-01-01

    To evaluate the effect of acute exercise and exercise training at the anaerobic threshold (AT) intensity on aerobic conditioning and insulin secretion by pancreatic islets, adult male Wistar rats were submitted to the lactate minimum test (LMT) for AT determination. Half of the animals were submitted to swimming exercise training (trained), 1 h/day, 5 days/week during 8 weeks, with an overload equivalent to the AT. The other half was kept sedentary. At the end of the experimental period, the rats were submitted to an oral glucose tolerance test and to another LMT. Then, the animals were sacrificed at rest or immediately after 20 minutes of swimming exercise at the AT intensity for pancreatic islets isolation. At the end of the experiment mean workload (% bw) at AT was higher and blood lactate concentration (mmol/L) was lower in the trained than in the control group. Rats trained at the AT intensity showed no alteration in the areas under blood glucose and insulin during OGTT test. Islet insulin content of trained rats was higher than in the sedentary rats while islet glucose uptake did not differ among the groups. The static insulin secretion in response to the high glucose concentration (16.7 mM) of the sedentary group at rest was lower than the sedentary group submitted to the acute exercise and the inverse was observed in relation to the trained groups. Physical training at the AT intensity improved the aerobic condition and altered insulin secretory pattern by pancreatic islets. PMID:21099318

  7. Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets

    PubMed Central

    Luciani, Dan S; Misler, Stanley; Polonsky, Kenneth S

    2006-01-01

    Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca2+ concentration ([Ca2+]c). Evidence suggests that the oscillations in [Ca2+]c and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca2+ feedback. To address this question we explored the interaction of Ca2+ concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca2+]c, in parallel with measurements of mitochondrial membrane potential (ΔΨm). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of ∼5 min. This was observed in ∼90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca2+]c, but their upstroke was often accelerated during the increase in [Ca2+]c, and Ca2+ influx was a prerequisite for their generation. Prolonged elevations of [Ca2+]c augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca2+]c depolarized ΔΨm. The NAD(P)H lowering effect of an elevation of [Ca2+]c was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca2+ concentration in a dynamic balance between activation of NADH-generating mitochondrial dehydrogenases and a Ca2+-induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca2+, which reduces respiratory control and consequently accelerates oxidation of NADH. PMID:16455690

  8. Enzyme studies in the articular cartilage of diabetic rats and of rats bearing transplanted pancreatic islets.

    PubMed

    Silberberg, R; Hirshberg, G E; Lesker, P

    1977-08-01

    The articular cartilage of normal rats, of rats made diabetic with streptozotocin, and of rats made diabetic with streptozotocin and subsequently transplanted with isologous pancreatic islets was examined for the activities of enzymes engaged in the synthesis and degradation of glycosaminoglycans (mucopolysaccharides). The activities assayed were those of the degrading enzymes B-glucuronidase, B-acetyglucosaminidase, B-acetylgalactosaminidase, B-galactosidase, and those active in synthesis: uridine diphosphate dehydrogenase, glucose-6-phosphate dehyrogenase, and phosphofructokinase. In the diabetic animals all enzyme activities were increased, thos of the degrading enzymes more than those of the others. Implantation of pancreatic islets reversed the changes produced by diabetes, enzyme activities returning to near-normal levels. PMID:142034

  9. Mitochondrial priming modifies Ca2+ oscillations and insulin secretion in pancreatic islets.

    PubMed

    Ainscow, E K; Rutter, G A

    2001-01-15

    Increases in mitochondrial [Ca(2+)] ([Ca(2+)](m)) have recently been reported to cause long-term alterations in cellular ATP production [Jouaville, Bastianutto, Rutter and Rizzuto (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 13807-13812]. We have determined the importance of this phenomenon for nutrient sensing in pancreatic islets and beta-cells by imaging adenovirally expressed Ca(2+) and ATP sensors (aequorin and firefly luciferase). [Ca(2+)](m) increases provoked by KCl or tolbutamide evoked an immediate increase in cytosolic and mitochondrial free ATP concentration ([ATP](c) and [ATP](m) respectively) at 3 mM glucose. Subsequent increases in [glucose] (to 16 or 30 mM) then caused a substantially larger increase in [ATP](c) and [ATP](m) than in naïve cells, and pre-stimulation with tolbutamide led to a larger secretory response in response to glucose. Whereas pre-challenge of islets with KCl altered the response to high [glucose] of [Ca(2+)](m) from periodic oscillations to a sustained elevation, oscillations in [ATP](c) were observed neither in naïve nor in stimulated islets. Hence, long-term potentiation of mitochondrial ATP synthesis is a central element in nutrient recognition by pancreatic islets.

  10. Mitochondrial priming modifies Ca2+ oscillations and insulin secretion in pancreatic islets.

    PubMed Central

    Ainscow, E K; Rutter, G A

    2001-01-01

    Increases in mitochondrial [Ca(2+)] ([Ca(2+)](m)) have recently been reported to cause long-term alterations in cellular ATP production [Jouaville, Bastianutto, Rutter and Rizzuto (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 13807-13812]. We have determined the importance of this phenomenon for nutrient sensing in pancreatic islets and beta-cells by imaging adenovirally expressed Ca(2+) and ATP sensors (aequorin and firefly luciferase). [Ca(2+)](m) increases provoked by KCl or tolbutamide evoked an immediate increase in cytosolic and mitochondrial free ATP concentration ([ATP](c) and [ATP](m) respectively) at 3 mM glucose. Subsequent increases in [glucose] (to 16 or 30 mM) then caused a substantially larger increase in [ATP](c) and [ATP](m) than in naïve cells, and pre-stimulation with tolbutamide led to a larger secretory response in response to glucose. Whereas pre-challenge of islets with KCl altered the response to high [glucose] of [Ca(2+)](m) from periodic oscillations to a sustained elevation, oscillations in [ATP](c) were observed neither in naïve nor in stimulated islets. Hence, long-term potentiation of mitochondrial ATP synthesis is a central element in nutrient recognition by pancreatic islets. PMID:11139378

  11. Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

    PubMed Central

    Xin, Yurong; Kim, Jinrang; Ni, Min; Wei, Yi; Okamoto, Haruka; Lee, Joseph; Adler, Christina; Cavino, Katie; Murphy, Andrew J.; Yancopoulos, George D.; Lin, Hsin Chieh; Gromada, Jesper

    2016-01-01

    This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type–specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system. PMID:26951663

  12. The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.

    PubMed

    Krickhahn, Mareike; Bühler, Christoph; Meyer, Thomas; Thiede, Arnulf; Ulrichs, Karin

    2002-01-01

    Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the

  13. Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity

    PubMed Central

    Sarmiento, Luis; Frisk, Gun; Anagandula, Mahesh; Cabrera-Rode, Eduardo; Roivainen, Merja; Cilio, Corrado M.

    2013-01-01

    Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets. PMID:24223733

  14. Glucose activates prenyltransferases in pancreatic islet beta-cells.

    PubMed

    Goalstone, Marc; Kamath, Vasudeva; Kowluru, Anjaneyulu

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet beta-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 beta-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20mM] markedly stimulated the expression of the alpha-subunits of FTase/GGTase-1, but not the beta-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  15. Glucose activates prenyltransferases in pancreatic islet {beta}-cells

    SciTech Connect

    Goalstone, Marc; Kamath, Vasudeva; Kowluru, Anjaneyulu

    2010-01-01

    A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet {beta}-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 {beta}-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20 mM] markedly stimulated the expression of the {alpha}-subunits of FTase/GGTase-1, but not the {beta}-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

  16. Melatonin and Pancreatic Islets: Interrelationships between Melatonin, Insulin and Glucagon

    PubMed Central

    Peschke, Elmar; Bähr, Ina; Mühlbauer, Eckhard

    2013-01-01

    The pineal hormone melatonin exerts its influence in the periphery through activation of two specific trans-membrane receptors: MT1 and MT2. Both isoforms are expressed in the islet of Langerhans and are involved in the modulation of insulin secretion from β-cells and in glucagon secretion from α-cells. De-synchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genome-wide association studies identifying particularly the MT2 as a risk factor for this rapidly spreading metabolic disturbance. Since melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. This factor has hitherto been underestimated; the disruption of diurnal signaling within the islet may be one of the most important mechanisms leading to metabolic disturbances. The study of melatonin–insulin interactions in diabetic rat models has revealed an inverse relationship: an increase in melatonin levels leads to a down-regulation of insulin secretion and vice versa. Elucidation of the possible inverse interrelationship in man may open new avenues in the therapy of diabetes. PMID:23535335

  17. Galectin-3 is a regulator of metaflammation in adipose tissue and pancreatic islets

    PubMed Central

    Pejnovic, Nada N; Pantic, Jelena M; Jovanovic, Ivan P; Radosavljevic, Gordana D; Djukic, Aleksandar Lj; Arsenijevic, Nebojsa N; Lukic, Miodrag L

    2013-01-01

    The cells of the innate and adaptive immune systems have been implicated in the development of obesity-induced metaflammation and metabolic disorders including type 2 diabetes. Galectin-3, a β-galactoside-binding lectin, modulates immune/inflammatory responses and specifically binds to advanced glycation end products (AGE), modified lipoproteins, and endotoxin. In the recently published study we demonstrate proinflammatory changes in the visceral adipose tissue and pancreatic islets in galectin-3-deficient mice fed high-fat diet which also exhibited excess adiposity, hyperglycemia, insulin resistance and systemic inflammation compared with their diet matched wild-type controls. This was associated with the increased incidence of Type-1 T and NKT cells and pro-inflammatory CD11c+CD11b+ macrophages in the visceral adipose tissue. Severe insulitis, infiltration of macrophages expressing NLRP3 inflammasome and IL-1β, and enhanced accumulation of AGE were present within the pancreatic islets in obese LGALS3−/− mice. Moreover, increased caspase-1 dependent IL-1β secretion with increased expression of NLRP3 inflammasome and phospho-NFκBp65 were observed in LGALS3−/− peritoneal macrophages stimulated in vitro by lipopolysaccharide and/or saturated fatty acid palmitate. The amplified high-fat diet-induced obesity and hyperglycemia and exacerbated inflammation in adipose tissue and pancreatic islets in LGALS3−/− mice suggest an important role for galectin-3 in the regulation of adiposity, metaflammation and type 2 diabetes. PMID:24052904

  18. Galectin-3 is a regulator of metaflammation in adipose tissue and pancreatic islets.

    PubMed

    Pejnovic, Nada N; Pantic, Jelena M; Jovanovic, Ivan P; Radosavljevic, Gordana D; Djukic, Aleksandar Lj; Arsenijevic, Nebojsa N; Lukic, Miodrag L

    2013-10-01

    The cells of the innate and adaptive immune systems have been implicated in the development of obesity-induced metaflammation and metabolic disorders including type 2 diabetes. Galectin-3, a β-galactoside-binding lectin, modulates immune/inflammatory responses and specifically binds to advanced glycation end products (AGE), modified lipoproteins, and endotoxin. In the recently published study we demonstrate proinflammatory changes in the visceral adipose tissue and pancreatic islets in galectin-3-deficient mice fed high-fat diet which also exhibited excess adiposity, hyperglycemia, insulin resistance and systemic inflammation compared with their diet matched wild-type controls. This was associated with the increased incidence of Type-1 T and NKT cells and pro-inflammatory CD11c(+)CD11b(+) macrophages in the visceral adipose tissue. Severe insulitis, infiltration of macrophages expressing NLRP3 inflammasome and IL-1β, and enhanced accumulation of AGE were present within the pancreatic islets in obese LGALS3(-/-) mice. Moreover, increased caspase-1 dependent IL-1β secretion with increased expression of NLRP3 inflammasome and phospho-NFκBp65 were observed in LGALS3(-/-) peritoneal macrophages stimulated in vitro by lipopolysaccharide and/or saturated fatty acid palmitate. The amplified high-fat diet-induced obesity and hyperglycemia and exacerbated inflammation in adipose tissue and pancreatic islets in LGALS3(-/-) mice suggest an important role for galectin-3 in the regulation of adiposity, metaflammation and type 2 diabetes. PMID:24052904

  19. Production of islet-like structures from neonatal porcine pancreatic tissue in suspension bioreactors.

    PubMed

    Chawla, Meera; Bodnar, Cheryl A; Sen, Arindom; Kallos, Michael S; Behie, Leo A

    2006-01-01

    The aim of this study was to develop a scaleable process to expand pancreatic endocrine tissue (i.e., aggregates or islet-like structures) in suspension bioreactors. Key issues addressed included (i) serum-free media, (ii) cell inoculation density, (iii) medium pH, and (iv) aggregate dissociation. Suspension bioreactors were inoculated with pancreatic neonatal tissue and operated under controlled conditions for a 9-day period. Medium studies showed that a new serum-free medium developed in our laboratory was capable of supporting endocrine cell expansion. An inoculation density of 127,000 cells/mL resulted in more than a 7.5-fold increase in the number of insulin-positive cells after 9 days. The resulting population consisted of single cells and many islet-like aggregates that contained all of the endocrine cell types (including insulin-positive, glucagon-positive, somatostatin-positive, and pancreatic polypeptide-positive cells). Furthermore, the cell aggregates exhibited a glucose-responsive behavior. This study represents a significant milestone on the path to the effective expansion of human islet-like tissue in bioreactors that may be used for cell therapy to treat Type 1 diabetes.

  20. Diffusion of calcium and metabolites in pancreatic islets: killing oscillations with a pitchfork.

    PubMed

    Tsaneva-Atanasova, Krasimira; Zimliki, Charles L; Bertram, Richard; Sherman, Arthur

    2006-05-15

    Cell coupling is important for the normal function of the beta-cells of the pancreatic islet of Langerhans, which secrete insulin in response to elevated plasma glucose. In the islets, electrical and metabolic communications are mediated by gap junctions. Although electrical coupling is believed to account for synchronization of the islets, the role and significance of diffusion of calcium and metabolites are not clear. To address these questions we analyze two different mathematical models of islet calcium and electrical dynamics. To study diffusion of calcium, we use a modified Morris-Lecar model. Based on our analysis, we conclude that intercellular diffusion of calcium is not necessary for islet synchronization, at most supplementing electrical coupling. Metabolic coupling is investigated with a recent mathematical model incorporating glycolytic oscillations. Bifurcation analysis of the coupled system reveals several modes of behavior, depending on the relative strength of electrical and metabolic coupling. We find that whereas electrical coupling always produces synchrony, metabolic coupling can abolish both oscillations and synchrony, explaining some puzzling experimental observations. We suggest that these modes are generic features of square-wave bursters and relaxation oscillators coupled through either the activation or recovery variable.

  1. Diffusion of Calcium and Metabolites in Pancreatic Islets: Killing Oscillations with a Pitchfork

    PubMed Central

    Tsaneva-Atanasova, Krasimira; Zimliki, Charles L.; Bertram, Richard; Sherman, Arthur

    2006-01-01

    Cell coupling is important for the normal function of the β-cells of the pancreatic islet of Langerhans, which secrete insulin in response to elevated plasma glucose. In the islets, electrical and metabolic communications are mediated by gap junctions. Although electrical coupling is believed to account for synchronization of the islets, the role and significance of diffusion of calcium and metabolites are not clear. To address these questions we analyze two different mathematical models of islet calcium and electrical dynamics. To study diffusion of calcium, we use a modified Morris-Lecar model. Based on our analysis, we conclude that intercellular diffusion of calcium is not necessary for islet synchronization, at most supplementing electrical coupling. Metabolic coupling is investigated with a recent mathematical model incorporating glycolytic oscillations. Bifurcation analysis of the coupled system reveals several modes of behavior, depending on the relative strength of electrical and metabolic coupling. We find that whereas electrical coupling always produces synchrony, metabolic coupling can abolish both oscillations and synchrony, explaining some puzzling experimental observations. We suggest that these modes are generic features of square-wave bursters and relaxation oscillators coupled through either the activation or recovery variable. PMID:16500973

  2. Expression profiling of cell cycle genes in human pancreatic islets with and without type 2 diabetes.

    PubMed

    Taneera, Jalal; Fadista, Joao; Ahlqvist, Emma; Zhang, Mengze; Wierup, Nils; Renström, Erik; Groop, Leif

    2013-08-15

    Microarray gene expression data were used to analyze the expression pattern of cyclin, cyclin-dependent kinase (CDKs) and cyclin-dependent kinase inhibitor (CDKIs) genes from human pancreatic islets with and without type 2 diabetes (T2D). Of the cyclin genes, CCNI was the most expressed. Data obtained from microarray and qRT-PCR showed higher expression of CCND1 in diabetic islets. Among the CDKs, CDK4, CDK8 and CDK9 were highly expressed, while CDK1 was expressed at low level. High expression of CDK18 was observed in diabetic islets. Of the CDKIs, CDKN1A expression was higher in diabetic islets in both microarray and qRT-PCR. Expression of CDKN1A, CDKN2A, CCNI2, CDK3 and CDK16 was correlated with age. Finally, eight SNPs in these genes were associated with T2D in the DIAGRAM database. Our data provide a comprehensive expression pattern of cell cycle genes in human islets. More human studies are required to confirm and reproduce animal studies. PMID:23707792

  3. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion.

    PubMed

    Toomey, P; Chadwick, D R; Contractor, H; Bell, P R; James, R F; London, N J

    1993-02-01

    A prospective study was undertaken to compare an automated method of porcine pancreatic digestion with a simpler manual procedure. These techniques have not previously been compared directly. After intraductal distension with collagenase, seven porcine pancreata were divided longitudinally; half of each was digested by the automated method and half by the manual technique. Islet yield and purity were measured. Compared with the manual technique, the automated method isolated a significantly greater total volume of islet tissue (median (range) 3.56 (1.39-5.30) versus 1.07 (0.46-1.92) mm3/g, P = 0.022), increased the median (range) number of 105-microns islet equivalents isolated (5875 (2294-8746) versus 1766 (759-3168) per g, P = 0.022) and improved the islet cleavage index (median (range) 92 (89-99) versus 82 (78-92) per cent, P = 0.035). It is concluded that, although the automated method is more complicated to set up, it greatly improves the yield of intact islets from the porcine pancreas. PMID:8443669

  4. Mathematical model formulation and validation of water and solute transport in whole hamster pancreatic islets.

    PubMed

    Benson, James D; Benson, Charles T; Critser, John K

    2014-08-01

    Optimization of cryopreservation protocols for cells and tissues requires accurate models of heat and mass transport. Model selection often depends on the configuration of the tissue. Here, a mathematical and conceptual model of water and solute transport for whole hamster pancreatic islets has been developed and experimentally validated incorporating fundamental biophysical data from previous studies on individual hamster islet cells while retaining whole-islet structural information. It describes coupled transport of water and solutes through the islet by three methods: intracellularly, intercellularly, and in combination. In particular we use domain decomposition techniques to couple a transmembrane flux model with an interstitial mass transfer model. The only significant undetermined variable is the cellular surface area which is in contact with the intercellularly transported solutes, Ais. The model was validated and Ais determined using a 3×3 factorial experimental design blocked for experimental day. Whole islet physical experiments were compared with model predictions at three temperatures, three perfusing solutions, and three islet size groups. A mean of 4.4 islets were compared at each of the 27 experimental conditions and found to correlate with a coefficient of determination of 0.87±0.06 (mean ± SD). Only the treatment variable of perfusing solution was found to be significant (p<0.05). We have devised a model that retains much of the intrinsic geometric configuration of the system, and thus fewer laboratory experiments are needed to determine model parameters and thus to develop new optimized cryopreservation protocols. Additionally, extensions to ovarian follicles and other concentric tissue structures may be made. PMID:24950195

  5. Peptidergic hormones and neuropeptides, and aminergic neurotransmitters of the pancreatic islets of the Houbara bustard (Chlamydotis undulata).

    PubMed

    Mensah-Brown, E P; Bailey, T A; Pallot, D J; Garner, A

    2000-02-01

    Immunoreactivity to insulin (Ins), somatostatin (Som), glucagon (Glu) and pancreatic polypeptide (PP) was found in 70%, 22%, 15% and 11% respectively of Houbara pancreatic endocrine islet cells. Whilst Ins occurred centrally and SOM was observed both in peripherally and centrally located islets, the other hormones were localised in peripheral islet cells; Som was also observed in neuronal cell bodies and nerve fibres. In addition, the islet cells contained substance P (SP) (65%) in the centre and vasoactive intestinal polypeptide (VIP) (2%) at the periphery. Immunoreactivity to choline acetyltransferase (ChAT), VIP and galanin (Gal) occurred in the walls of blood vessels located mainly at the periphery of islets. Occasionally, VIP and Gal immunoreactive varicose nerve terminals and ChAT immunoreactive cell bodies were also observed in the centre of islets. SP neuronal cell bodies were not observed but prominent SP immunoreactive varicose terminals were discernible in capillary walls within the islets. Neuropeptide Y (NPY) immunoreactive neurons were detected in neuronal cell bodies located mainly peripherally. Neuronal nitric oxide synthase (nNOS) immunoreactivity occurred in neuronal cell bodies and nerve fibres mainly at the periphery and also in centrally located islet endocrine cells. Immunoreactivity to tyrosine hydroxylase (TH) was similar in distribution to that of ChAT. In comparison with other avian species, the islets of the dorsal pancreatic lobe of the bustard contain all the peptidergic hormones normally present in the islets of other avian species, but are not segregated into dark A and light B cells. Many of the insulin containing cells also contained SP. The islets also contained several neuropeptides which are probably involved in their regulation.

  6. Treated of type 1 diabetes mellitus in non-obese diabetic mice by transplantation of allogeneic bone marrow and pancreatic tissue

    SciTech Connect

    Yasumizu, R.; Sugiura, K.; Iwai, H.; Inaba, M.; Makino, S.; Ida, T.; Imura, H.; Hamashima, Y.; Good, R.A.; Ikehara, S.

    1987-09-01

    Non-obese diabetic (NOD) mice provide a model for type 1 diabetes mellitus. We previously showed that allogeneic bone marrow transplantation (ABMT) can prevent and treat insulitis and overt diabetes in NOD mice. However, ABMT alone could not be used to treat overt diabetes in NOD mice whose islets had been completely destroyed. To provide insulin-producing cells, pancreatic tissue from newborn mice was grafted under the renal capsules in combination with ABMT. The aims of concomitant ABMT are as follows. (i) It induces immunological tolerance to the donor-type major histocompatibility complex determinants and permits the host to accept subsequent pancreatic allografts from the bone marrow donor. (ii) ABMT replaces abnormal stem cells with normal stem cells. After transplantation of bone marrow plus newborn pancreas, NOD mice showed reduction of the glycosuria and a normal response in the glucose-tolerance test. Immunohistological study revealed the presence of clustered insulin-containing beta cells in the grafted pancreatic transplants. ABMT may become a viable treatment of established type 1 diabetes mellitus in humans.

  7. Methylated trivalent arsenicals are potent inhibitors of glucose stimulated insulin secretion by murine pancreatic islets

    SciTech Connect

    Douillet, Christelle; Currier, Jenna; Saunders, Jesse; Bodnar, Wanda M.; Matoušek, Tomáš; Stýblo, Miroslav

    2013-02-15

    Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAs{sup III}) or its methylated trivalent metabolites, methylarsonite (MAs{sup III}) and dimethylarsinite (DMAs{sup III}), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAs{sup III}, MAs{sup III} or DMAs{sup III} inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs{sup III} and DMAs{sup III} were more potent than iAs{sup III} as GSIS inhibitors with estimated IC{sub 50} ≤ 0.1 μM. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAs{sup III}, MAs{sup III} or DMAs{sup III} could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic β-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes. - Highlights: ► Trivalent arsenicals inhibit glucose stimulated insulin secretion by pancreatic islets. ► MAs{sup III} and DMAs{sup III} are more potent inhibitors than arsenite with IC{sub 50} ∼ 0.1 μM. ► The arsenicals have little or no effects on insulin expression in pancreatic islets. ► The inhibition of

  8. Efflux of 86Rb from rat and mouse pancreatic islets: the role of membrane depolarization.

    PubMed Central

    Matthews, E. K.; Shotton, P. A.

    1984-01-01

    The efflux of 86Rb from rat or mouse perifused islets preloaded with the isotope has been used as an index of the potassium permeability of the islet beta-cell membrane. Cellular transmembrane potentials were altered by changing [K]O or by direct electrical stimulation and the effects on potassium permeability examined. Omission of KCl from the medium perifusing rat islets induced a biphasic change in 86Rb efflux, a brief decline being superseded by a pronounced increase in efflux. Re-introduction of KCl, 4.7 mM, caused a further increase in 86Rb efflux preceding a return to control values. Increasing [K]O from 4.7 mM to 10 mM, 20 mM or 47 mM caused a phasic increase in 86Rb efflux with the magnitude of both the peak and average rate of efflux being dependent upon the extent of the change in [K]O. The increase in 86Rb efflux produced by [K]O, 47 mM, was attenuated by Co2+, 2.56 mM (51% inhibition) or quinine, 10 microM (47% inhibition), but efflux remained significantly (P less than 0.001) above control values. Electrical stimulation of single microdissected mouse pancreatic islets by currents of 0.1 to 0.5 mA evoked a rapid, phasic increase in 86Rb efflux. The magnitude of the response was unaffected by EGTA, 2 mM, or nupercaine, 100 microM. These observations are discussed in relation to the mechanisms controlling the potassium permeability, membrane potential and insulin secretion of the pancreatic islet beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6391599

  9. Niacin-induced hyperglycemia is partially mediated via niacin receptor GPR109a in pancreatic islets.

    PubMed

    Chen, Lihua; So, Wing Yan; Li, Stephen Y T; Cheng, Qianni; Boucher, Barbara J; Leung, Po Sing

    2015-03-15

    The widely used lipid-lowering drug niacin is reported to induce hyperglycemia during chronic and high-dose treatments, but the mechanism is poorly understood. Recently, the niacin receptor [G-protein-coupled receptor, (GPR) 109a], has been localized to islet cells while its potential role therein remains unclear. We, therefore, aimed at investigating how GPR109a regulates islet beta-cell function and its downstream signaling using high-fat diet-induced obese mice and INS-1E beta cells. Eight-week niacin treatment elevated blood glucose concentration in obese mice with increased areas under the curve at oral glucose and intraperitoneal insulin tolerance tests. Additionally, niacin treatment significantly decreased glucose-stimulated insulin secretion (GSIS) but induced peroxisome proliferator-activated receptor gamma (Pparg) and GPR109a expression in isolated pancreatic islets; concomitantly, reactive oxygen species (ROS) were transiently increased, with decreases in GSIS, intracellular cyclic adenosine monophosphate (cAMP) accumulation and mitochondrial membrane potential (ΔΨm), but with increased expression of uncoupling protein 2 (Ucp2), Pparg and Gpr109a in INS-1E cells. Corroborating these findings, the decreases in GSIS, ΔΨm and cAMP production and increases in ROS, Pparg and GPR109a expression were abolished in INS-1E cells by GPR109a knockdown. Our data indicate that niacin-induced pancreatic islet dysfunction is probably modulated through activation of the islet beta-cell GPR109a-induced ROS-PPARγ-UCP2 pathways.

  10. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  11. Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism.

    PubMed

    Shimodahira, Makiko; Fujimoto, Shimpei; Mukai, Eri; Nakamura, Yasuhiko; Nishi, Yuichi; Sasaki, Mayumi; Sato, Yuichi; Sato, Hiroki; Hosokawa, Masaya; Nagashima, Kazuaki; Seino, Yutaka; Inagaki, Nobuya

    2010-01-01

    Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity. PMID:19812126

  12. Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

    SciTech Connect

    Chow, S.A.; Falany, J.L.; Fischer, L.J. )

    1989-06-01

    The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic beta-cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of 3H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.

  13. Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism

    PubMed Central

    Fadista, João; Vikman, Petter; Laakso, Emilia Ottosson; Mollet, Inês Guerra; Esguerra, Jonathan Lou; Taneera, Jalal; Storm, Petter; Osmark, Peter; Ladenvall, Claes; Prasad, Rashmi B.; Hansson, Karin B.; Finotello, Francesca; Uvebrant, Kristina; Ofori, Jones K.; Di Camillo, Barbara; Krus, Ulrika; Cilio, Corrado M.; Hansson, Ola; Eliasson, Lena; Rosengren, Anders H.; Renström, Erik; Wollheim, Claes B.; Groop, Leif

    2014-01-01

    Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5′-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism. PMID:25201977

  14. Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism.

    PubMed

    Fadista, João; Vikman, Petter; Laakso, Emilia Ottosson; Mollet, Inês Guerra; Esguerra, Jonathan Lou; Taneera, Jalal; Storm, Petter; Osmark, Peter; Ladenvall, Claes; Prasad, Rashmi B; Hansson, Karin B; Finotello, Francesca; Uvebrant, Kristina; Ofori, Jones K; Di Camillo, Barbara; Krus, Ulrika; Cilio, Corrado M; Hansson, Ola; Eliasson, Lena; Rosengren, Anders H; Renström, Erik; Wollheim, Claes B; Groop, Leif

    2014-09-23

    Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

  15. Vitamin D receptor activation induces peptide YY transcription in pancreatic islets.

    PubMed

    Choi, Mihwa; Ozeki, Jun; Hashizume, Masami; Kato, Shigeaki; Ishihara, Hisamitsu; Makishima, Makoto

    2012-11-01

    Peptide YY (PYY) is a peptide hormone secreted from L cells in the intestine after food intake and regulates appetite and intestinal function. PYY is also expressed in the pancreas, but the mechanisms of regulation of pancreatic PYY expression have not been elucidated. The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D(3) and regulates numerous physiological processes. Because VDR is expressed in the pancreas, we investigated the role of pancreatic VDR activation and found that Pyy is a VDR target gene in the mouse pancreas. Treatment of mice with 1α-hydroxyvitamin D(3) increased plasma PYY levels. VDR activation increased mRNA and protein expression of PYY in the pancreatic islets of mice and pancreatic endocrine cell lines but did not change intestinal PYY expression. 1α-Hydroxyvitamin D(3)-dependent induction of pancreatic and plasma PYY was abolished in VDR-null mice. We identified a functional vitamin D-responsive element in the mouse Pyy promoter using chromatin immunoprecipitation assay, EMSA, and luciferase promoter assay. Thus, Pyy is a tissue-specific VDR target gene. The pancreatic VDR-PYY pathway may mediate a regulatory function of vitamin D in the neuroendocrine system.

  16. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    SciTech Connect

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O.

    1995-05-01

    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  17. Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro: implications for pancreatic islet transplantation.

    PubMed

    Qureshi, Khalid M; Oliver, Robert J; Paget, Michelle B; Murray, Hilary E; Bailey, Clifford J; Downing, Richard

    2011-01-01

    Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p < 0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 μg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of β-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close

  18. Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

    PubMed

    Jansson, L; Eizirik, D L; Pipeleers, D G; Borg, L A; Hellerström, C; Andersson, A

    1995-08-01

    Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores.

  19. Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

    PubMed

    Shin, Soojeong; Yoo, Young Je; Hong, Jong Wook

    2015-10-01

    Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules. PMID:26726432

  20. Paradoxical inhibitory effect of cromakalim on sup 86 Rb outflow from pancreatic islet cells

    SciTech Connect

    Lebrun, P.; Antoine, M.H.; Devreux, V.; Hermann, M.; Herchuelz, A. )

    1990-12-01

    Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the K+ channel openers. The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ channels.

  1. Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

    PubMed

    Shin, Soojeong; Yoo, Young Je; Hong, Jong Wook

    2015-10-01

    Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

  2. Protective efficacy of folic acid and vitamin B12 against nicotine-induced toxicity in pancreatic islets of the rat.

    PubMed

    Bhattacharjee, Ankita; Prasad, Shilpi Kumari; Pal, Swagata; Maji, Bithin; Syamal, Alak Kumar; Banerjee, Arnab; Mukherjee, Sandip

    2015-06-01

    Although cigarette smoking is associated with insulin resistance and an increased risk for type 2 diabetes, few studies have examined the effect of nicotine on the adult endocrine pancreas. In this study, male Wister rats were treated with nicotine (3 mg/kg body weight/ day) with or without supplementation of folic acid (36 μg/kg body weight/day) or vitamin B12 (0.63 μg/kg body weight/day) alone or in combination. Fasting blood glucose, insulin and HBA1C level and different oxidative and anti-oxidative stress parameters were measured and pancreatic tissue sections were stained with eosin-haematoxylene. Data were analysed by nonparametric statistics. The results revealed that nicotine induced prediabetes condition with subsequent damage to pancreatic islets in rats. Nicotine also caused oxidative stress in pancreatic tissue as evidenced by increased nitric oxide and malondialdehyde level and decreased superoxide dismutase, catalase and reduced glutathione level. Compared to vitamin B12 supplementation, folic acid blunted the nicotine-induced toxicity in pancreatic islets with higher efficacy. Further, folic acid and vitamin B12 in combination were able to confer significant protection on pancreatic islets against nicotine induced toxicity. These results suggest that supplementation of folic acid and vitamin B12 in combination may be a possible strategy of detoxification against nicotine-induced toxicity in pancreatic islets of the rat. PMID:27486368

  3. Protective efficacy of folic acid and vitamin B12 against nicotine-induced toxicity in pancreatic islets of the rat

    PubMed Central

    Bhattacharjee, Ankita; Prasad, Shilpi Kumari; Pal, Swagata; Maji, Bithin; Syamal, Alak Kumar; Banerjee, Arnab

    2015-01-01

    Although cigarette smoking is associated with insulin resistance and an increased risk for type 2 diabetes, few studies have examined the effect of nicotine on the adult endocrine pancreas. In this study, male Wister rats were treated with nicotine (3 mg/kg body weight/ day) with or without supplementation of folic acid (36 μg/kg body weight/day) or vitamin B12 (0.63 μg/kg body weight/day) alone or in combination. Fasting blood glucose, insulin and HBA1C level and different oxidative and anti-oxidative stress parameters were measured and pancreatic tissue sections were stained with eosin-haematoxylene. Data were analysed by nonparametric statistics. The results revealed that nicotine induced prediabetes condition with subsequent damage to pancreatic islets in rats. Nicotine also caused oxidative stress in pancreatic tissue as evidenced by increased nitric oxide and malondialdehyde level and decreased superoxide dismutase, catalase and reduced glutathione level. Compared to vitamin B12 supplementation, folic acid blunted the nicotine-induced toxicity in pancreatic islets with higher efficacy. Further, folic acid and vitamin B12 in combination were able to confer significant protection on pancreatic islets against nicotine induced toxicity. These results suggest that supplementation of folic acid and vitamin B12 in combination may be a possible strategy of detoxification against nicotine-induced toxicity in pancreatic islets of the rat. PMID:27486368

  4. Histogenesis and ultrastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial cells of host origin.

    PubMed Central

    Vajkoczy, P.; Olofsson, A. M.; Lehr, H. A.; Leiderer, R.; Hammersen, F.; Arfors, K. E.; Menger, M. D.

    1995-01-01

    In previous studies we have demonstrated that syngeneic and xenogeneic pancreatic islet grafts are revascularized within a 10 to 14-day period after transplantation. With the combined use of intravital and electron microscopy, as well as immunohistochemistry using a set of species-specific or -crossreacting antibodies to endothelial cell antigens, we investigated 1) the origin of the endothelium of the newly formed capillaries in free pancreatic islet isografts (hamster-->hamster) and xenografts (rat-->hamster), and 2) the ultrastructural characteristics of these microvessels. Intravital microscopy demonstrated that newly formed microvessels grow from the vascular bed of the host muscle tissue into the islet grafts. Immunohistochemical analysis of host tissue and transplanted islets with antibodies against factor VIII (recognizing both hamster and rat factor VIII), bovine PECAM-1 (CD31; endoCAM, crossreacting with hamster but not rat PECAM-1), and rat ICAM-1 (CD54, non-crossreacting with hamster ICAM-1) showed that the transplanted rat islets were revascularized by endothelium of hamster (host) origin. At an ultrastructural level, the endothelial lining of the newly formed microvessels showed diaphragmatic fenestration, a characteristic feature of endothelial cells of pancreatic islets in situ. On the basis of these findings we suggest that pancreatic islet transplantation may take a unique position in the field of organ transplantation, since the generally proposed mechanisms of endothelial cell-dependent antigen recognition as a trigger of graft rejection may not be transferred to islet grafts, containing microvessels lined by endothelial cells of host origin. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7539980

  5. Metabolic Oscillations in Pancreatic Islets Depend on the Intracellular Ca2+ Level but Not Ca2+ Oscillations

    PubMed Central

    Merrins, Matthew J.; Fendler, Bernard; Zhang, Min; Sherman, Arthur; Bertram, Richard; Satin, Leslie S.

    2010-01-01

    Abstract Plasma insulin is pulsatile and reflects oscillatory insulin secretion from pancreatic islets. Although both islet Ca2+ and metabolism oscillate, there is disagreement over their interrelationship, and whether they can be dissociated. In some models of islet oscillations, Ca2+ must oscillate for metabolic oscillations to occur, whereas in others metabolic oscillations can occur without Ca2+ oscillations. We used NAD(P)H fluorescence to assay oscillatory metabolism in mouse islets stimulated by 11.1 mM glucose. After abolishing Ca2+ oscillations with 200 μM diazoxide, we observed that oscillations in NAD(P)H persisted in 34% of islets (n = 101). In the remainder of the islets (66%) both Ca2+ and NAD(P)H oscillations were eliminated by diazoxide. However, in most of these islets NAD(P)H oscillations could be restored and amplified by raising extracellular KCl, which elevated the intracellular Ca2+ level but did not restore Ca2+ oscillations. Comparatively, we examined islets from ATP-sensitive K+ (KATP) channel-deficient SUR1−/− mice. Again NAD(P)H oscillations were evident even though Ca2+ and membrane potential oscillations were abolished. These observations are predicted by the dual oscillator model, in which intrinsic metabolic oscillations and Ca2+ feedback both contribute to the oscillatory islet behavior, but argue against other models that depend on Ca2+ oscillations for metabolic oscillations to occur. PMID:20655835

  6. /sup 3/H-cyclosporine internalization and secretion by human fetal pancreatic islets

    SciTech Connect

    Formby, B.; Walker, L.; Peterson, C.M.

    1988-10-01

    Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude.

  7. Chaotic electrical activity of living β-cells in the mouse pancreatic islet

    NASA Astrophysics Data System (ADS)

    Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

    2007-02-01

    To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

  8. A glucagon-secreting pancreatic alpha islet cell tumor presenting as spinal cord compression.

    PubMed

    Staren, E D; Steinecker, G A; Gould, V E

    1987-08-01

    We describe a patient with a pancreatic islet carcinoma presenting with spinal cord compression owing to vertebral metastases. Subsequent studies demonstrated a typical islet cell carcinoma by light microscopy. By electron microscopy, the neurosecretory granules were morphologically suggestive of glucagon production. Radioimmunoassay studies revealed markedly elevated levels of serum glucagon. Notably, the patient did not exhibit the characteristic glucagonoma syndrome. This case exemplifies clearly that elevated levels of immunoreactive neuropeptide hormones are not necessarily associated with overt hormonal syndromes. Possible mechanisms for explaining this apparent discrepancy include the production of immunoreactive molecules with weak or absent systemic biological activity. Nevertheless, the determination of immunoreactive hormone levels in neuroendocrine neoplasms is an extremely effective adjunct method for their diagnosis and monitoring.

  9. Reg3α Overexpression Protects Pancreatic β Cells from Cytokine-Induced Damage and Improves Islet Transplant Outcome

    PubMed Central

    Ding, Ying; Xu, Yuemei; Shuai, Xuanyu; Shi, Xuhui; Chen, Xiang; Huang, Wenbin; Liu, Yun; Liang, Xiubin; Zhang, Zhihong; Su, Dongming

    2014-01-01

    The process of islet transplantation for treating type 1 diabetes has been limited by the high level of graft failure. This may be overcome by locally delivering trophic factors to enhance engraftment. Regenerating islet-derived protein 3α (Reg3α) is a pancreatic secretory protein which functions as an antimicrobial peptide in control of inflammation and cell proliferation. In this study, to investigate whether Reg3α could improve islet engraftment, a marginal mass of syngeneic islets pretransduced with adenoviruses expressing Reg3α or control EGFP were transplanted under the renal capsule of streptozotocin-induced diabetic mice. Mice receiving islets with elevated Reg3α production exhibited significantly lower blood glucose levels (9.057 ± 0.59 mmol/L versus 13.48 ± 0.35 mmol/L, P < 0.05) and improved glucose-stimulated insulin secretion (1.80 ± 0.17 ng/mL versus 1.16 ± 0.16 ng/mL, P < 0.05) compared with the control group. The decline of apoptotic events (0.57% ± 0.15% versus 1.06% ± 0.07%, P < 0.05) and increased β-cell proliferation (0.70% ± 0.10% versus 0.36% ± 0.14%, P < 0.05) were confirmed in islet grafts overexpressing Reg3α by morphometric analysis. Further experiments showed that Reg3α production dramatically protected cultured islets and pancreatic β cells from cytokine-induced apoptosis and the impairment of glucose-stimulated insulin secretion. Moreover, exposure to cytokines led to the activation of MAPKs in pancreatic β cells, which was reversed by Reg3α overexpression in contrast to control group. These results strongly suggest that Reg3α could enhance islet engraftments through its cytoprotective effect and advance the therapeutic efficacy of islet transplantation. PMID:25826674

  10. Transport of ascorbic acid and dehydroascorbic acid by pancreatic islet cells from neonatal rats.

    PubMed Central

    Zhou, A; Nielsen, J H; Farver, O; Thorn, N A

    1991-01-01

    Several amidated biologically active peptides such as pancreastatin, thyrotropin-releasing hormone, pancreatic polypeptide and amylin are produced in endocrine pancreatic tissue which contains the enzyme necessary for their final processing, i.e. peptidylglycine alpha-amidating mono-oxygenase (EC 1.14.17.3). The enzyme needs ascorbic acid for activity as well as copper and molecular oxygen. The present work shows that pancreatic islet cells prepared from overnight cultures of isolated islets from 5-7-day-old rats accumulate 14C-labelled ascorbic acid by a Na(+)-dependent active transport mechanism which involves a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration. Dehydroascorbic acid was converted to ascorbic acid by an unknown mechanism after uptake. The uptake of both ascorbic acid and dehydroascorbic acid was inhibited by tri-iodothyronine, and uptake of ascorbic acid, but not of dehydroascorbic acid, was inhibited by glucocorticoids. Isolated secretory granules contained a fairly low concentration of iron but a high concentration of copper. Images Fig. 6. PMID:2012602

  11. Pancreatic-derived factor (FAM3B), a novel islet cytokine, induces apoptosis of insulin-secreting beta-cells.

    PubMed

    Cao, Xiaopei; Gao, Zhiyong; Robert, Claudia E; Greene, Scott; Xu, Gang; Xu, Weizhen; Bell, Ewan; Campbell, Don; Zhu, Yuan; Young, Robert; Trucco, Matteo; Markmann, James F; Naji, Ali; Wolf, Bryan A

    2003-09-01

    PANDER (PANcreatic DERived factor, FAM3B), a newly discovered secreted cytokine, is specifically expressed at high levels in the islets of Langerhans of the endocrine pancreas. To evaluate the role of PANDER in beta-cell function, we investigated the effects of PANDER on rat, mouse, and human pancreatic islets; the beta-TC3 cell line; and the alpha-TC cell line. PANDER protein was present in alpha- and beta-cells of pancreatic islets, insulin-secreting beta-TC3 cells, and glucagon-secreting alpha-TC cells. PANDER induced islet cell death in rat and human islets. Culture of beta-TC3 cells with recombinant PANDER had a dose-dependent inhibitory effect on cell viability. This effect was also time-dependent. PANDER caused apoptosis of beta-cells as assessed by electron microscopy, annexin V fluorescent staining, and flow-cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. PANDER did not affect cytosolic Ca(2+) levels or nitric oxide levels. However, PANDER activated caspase-3. Hence, PANDER may have a role in the process of pancreatic beta-cell apoptosis.

  12. Induction of beta-cell resistance to hypoxia and technologies for oxygen delivery to transplanted pancreatic islets.

    PubMed

    Lazard, Daniel; Vardi, Pnina; Bloch, Konstantin

    2012-09-01

    Hypoxia is believed to be a crucial factor involved in cell adaptation to environmental stress. Islet transplantation, especially with immunoisolated islets, interrupts vascular connections, resulting in the substantially decreased delivery of oxygen and nutrients to islet cells. Insulin-producing pancreatic beta cells are known to be highly susceptible to oxygen deficiency. Such susceptibility to hypoxia is believed to be one of the main causes of beta-cell death in the post-transplantation period. Different strategies have been developed for the protection of beta cells against hypoxic injury and for oxygen delivery to transplanted islets. The enhancement of beta-cell defense properties against hypoxia has been achieved using various techniques such as gene transfection, drug supplementation, co-culturing with stem cells and cell selection. Technologies for oxygen delivery to transplanted islets include local neovascularization of subcutaneous sites, electrochemical and photosynthetic oxygen generation, oxygen refuelling of bio-artificial pancreas and whole body oxygenation by using hyperbaric therapy. Progress in the field of oxygen technologies for islet transplantation requires a multidisciplinary approach to explore and optimize the interaction between components of the biological system and different technological processes. This review article focuses mainly on the recently developed strategies for oxygenation and protection from hypoxic injury - to achieve stable and long-term normoglycaemia in diabetic patients with transplanted pancreatic islets. PMID:22389124

  13. Excessive Food Intake, Obesity and Inflammation Process in Zucker fa/fa Rat Pancreatic Islets

    PubMed Central

    Chentouf, Myriam; Dubois, Gregor; Jahannaut, Céline; Castex, Françoise; Lajoix, Anne Dominique; Gross, René; Peraldi-Roux, Sylvie

    2011-01-01

    Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced β-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and β-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1β, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning β-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1β and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of β-cell function by IL-1β. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory

  14. Role of nucleolar protein NOM1 in pancreatic islet β cell apoptosis in diabetes

    PubMed Central

    Yu, Leilei; Wang, Huifeng; Guo, Zhongxiu; Li, Fenghua; Cui, Hong

    2016-01-01

    Diabetes is a metabolic disease that results from impairment in insulin secretion. The present study aimed to investigate the potential role of NOM1 in the function of pancreatic islet β cells and insulin secretion. MIN6 cells isolated from mice were transfected with siRNA-NOM1 to assess the influence of NOM1 on the expression of the cell apoptosis-associated proteins, such as caspase-3. In addition, MIN6 cells were cultured in medium containing different glucose concentrations in order to assess the sensitivity of MIN6 cells to glucose. The effect of NOM1 expression and glucose on MIN6 cell proliferation was also analyzed using an MTT assay. Furthermore, the mRNA expression levels of insulin 1 and 2 in MIN6 cells were detected using reverse transcription-quantitative polymerase chain reaction, while the expression levels of various cell apoptosis-associated proteins, Bcl-2 and Bax, were analyzed using western blot analysis. Compared with the control group, downregulation NOM1 and high glucose concentration of 25 mM significantly increased the cleaved caspase-3 level in MIN6 cells (P<0.05). In addition, downregulation of NOM1 significantly inhibited the MIN6 cell proliferation ability and reduced the insulin 2 mRNA expression (P<0.05). NOM1 knockdown also resulted in significantly increased Bax2 level and decreased Bcl-2 level in MIN6 cells (P<0.05). However no significant difference in insulin mRNA expression was observed between the control and siRNA-NOM1-transfected group (P>0.05). In conclusion, the present study suggested that NOM1 expression may be affected by blood glucose, and that NOM1 may be associated with pancreatic islet β cell apoptosis. In addition, NOM1 may serve a pivotal role in diabetes by affecting insulin synthesis and secretion in pancreatic islet β cells.

  15. One year of sitagliptin treatment protects against islet amyloid-associated β-cell loss and does not induce pancreatitis or pancreatic neoplasia in mice.

    PubMed

    Aston-Mourney, Kathryn; Subramanian, Shoba L; Zraika, Sakeneh; Samarasekera, Thanya; Meier, Daniel T; Goldstein, Lynn C; Hull, Rebecca L

    2013-08-15

    The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin is an attractive therapy for diabetes, as it increases insulin release and may preserve β-cell mass. However, sitagliptin also increases β-cell release of human islet amyloid polypeptide (hIAPP), the peptide component of islet amyloid, which is cosecreted with insulin. Thus, sitagliptin treatment may promote islet amyloid formation and its associated β-cell toxicity. Conversely, metformin treatment decreases islet amyloid formation by decreasing β-cell secretory demand and could therefore offset sitagliptin's potential proamyloidogenic effects. Sitagliptin treatment has also been reported to be detrimental to the exocrine pancreas. We investigated whether long-term sitagliptin treatment, alone or with metformin, increased islet amyloid deposition and β-cell toxicity and induced pancreatic ductal proliferation, pancreatitis, and/or pancreatic metaplasia/neoplasia. hIAPP transgenic and nontransgenic littermates were followed for 1 yr on no treatment, sitagliptin, metformin, or the combination. Islet amyloid deposition, β-cell mass, insulin release, and measures of exocrine pancreas pathology were determined. Relative to untreated mice, sitagliptin treatment did not increase amyloid deposition, despite increasing hIAPP release, and prevented amyloid-induced β-cell loss. Metformin treatment alone or with sitagliptin decreased islet amyloid deposition to a similar extent vs untreated mice. Ductal proliferation was not altered among treatment groups, and no evidence of pancreatitis, ductal metaplasia, or neoplasia were observed. Therefore, long-term sitagliptin treatment stimulates β-cell secretion without increasing amyloid formation and protects against amyloid-induced β-cell loss. This suggests a novel effect of sitagliptin to protect the β-cell in type 2 diabetes that appears to occur without adverse effects on the exocrine pancreas. PMID:23736544

  16. One year of sitagliptin treatment protects against islet amyloid-associated β-cell loss and does not induce pancreatitis or pancreatic neoplasia in mice

    PubMed Central

    Aston-Mourney, Kathryn; Subramanian, Shoba L.; Zraika, Sakeneh; Samarasekera, Thanya; Meier, Daniel T.; Goldstein, Lynn C.

    2013-01-01

    The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin is an attractive therapy for diabetes, as it increases insulin release and may preserve β-cell mass. However, sitagliptin also increases β-cell release of human islet amyloid polypeptide (hIAPP), the peptide component of islet amyloid, which is cosecreted with insulin. Thus, sitagliptin treatment may promote islet amyloid formation and its associated β-cell toxicity. Conversely, metformin treatment decreases islet amyloid formation by decreasing β-cell secretory demand and could therefore offset sitagliptin's potential proamyloidogenic effects. Sitagliptin treatment has also been reported to be detrimental to the exocrine pancreas. We investigated whether long-term sitagliptin treatment, alone or with metformin, increased islet amyloid deposition and β-cell toxicity and induced pancreatic ductal proliferation, pancreatitis, and/or pancreatic metaplasia/neoplasia. hIAPP transgenic and nontransgenic littermates were followed for 1 yr on no treatment, sitagliptin, metformin, or the combination. Islet amyloid deposition, β-cell mass, insulin release, and measures of exocrine pancreas pathology were determined. Relative to untreated mice, sitagliptin treatment did not increase amyloid deposition, despite increasing hIAPP release, and prevented amyloid-induced β-cell loss. Metformin treatment alone or with sitagliptin decreased islet amyloid deposition to a similar extent vs untreated mice. Ductal proliferation was not altered among treatment groups, and no evidence of pancreatitis, ductal metaplasia, or neoplasia were observed. Therefore, long-term sitagliptin treatment stimulates β-cell secretion without increasing amyloid formation and protects against amyloid-induced β-cell loss. This suggests a novel effect of sitagliptin to protect the β-cell in type 2 diabetes that appears to occur without adverse effects on the exocrine pancreas. PMID:23736544

  17. Geometric phase transition in the cellular network of the pancreatic islets may underlie the onset of type 1diabetes

    NASA Astrophysics Data System (ADS)

    Wang, Xujing

    Living systems are characterized by complexity in structure and emergent dynamic orders. In many aspects the onset of a chronic disease resembles phase transition in a dynamic system: quantitative changes accumulate largely unnoticed until a critical threshold is reached, which causes abrupt qualitative changes of the system. In this study we investigate this idea in a real example, the insulin-producing pancreatic islet β-cells and the onset of type 1 diabetes. Within each islet, the β-cells are electrically coupled to each other, and function as a network with synchronized actions. Using percolation theory we show how normal islet function is intrinsically linked to network connectivity, and the critical point where the islet cellular network loses site percolation, is consistent with laboratory and clinical observations of the threshold β-cell loss that causes islet functional failure. Numerical simulations confirm that the islet cellular network needs to be percolated for β-cells to synchronize. Furthermore, the interplay between site percolation and bond strength predicts the existence of a transient phase of islet functional recovery after disease onset and introduction of treatment, potentially explaining a long time mystery in the clinical study of type 1 diabetes: the honeymoon phenomenon. Based on these results, we hypothesized that the onset of T1D may be the result of a phase transition of the islet β-cell network. We further discuss the potential applications in identifying disease-driving factors, and the critical parameters that are predictive of disease onset.

  18. Histomorphological and morphometric studies of the pancreatic islet cells of diabetic rats treated with extracts of Annona muricata.

    PubMed

    Adeyemi, D O; Komolafe, O A; Adewole, O S; Obuotor, E M; Abiodun, A A; Adenowo, T K

    2010-05-01

    Microanatomical changes in the pancreatic islet cells of streptozotocin induced diabetic Wistar rats were studied after treatment with methanolic extracts of Annona muricata leaves. Thirty adult Wistar rats were randomly assigned into three groups (control, untreated diabetic group, and A. muricata-treated diabetic group) of ten rats each. Diabetes mellitus was experimentally induced in groups B and C by a single intra-peritoneal injection of 80 mg/kg streptozotocin dissolved in 0.1 M citrate buffer. The control rats were intraperitoneally injected with an equivalent volume of citrate buffer. Daily intra peritoneal injections of 100 mg/kg A. muricata were administered to group C rats for two weeks. Post sacrifice the pancreases of the rats were excised and fixed in Bouin's fluid. The tissues were processed for paraffin embedding and sections of 5 mum thickness were produced and stained with H & E, Gomori aldehyde fuchsin, and chrome alum haematoxylin-phloxine for demonstration of the beta-cells of islets of pancreatic islets. Histomorphological and morphometric examination of the stained pancreatic sections showed a significant increase in the number, diameter, and volume of the beta-cells of pancreatic islets of the A. muricata-treated group (5.67 +/- 0.184 N/1000 mum(2), 5.38 +/- 0.093 mum and 85.12 +/- 4.24 mum(3), respectively) when compared to that of the untreated diabetic group of rats (2.85 +/- 0.361 N/1000 mum(2), 2.85 +/- 0.362 mum and 69.56 +/- 5.216 mum(3), respectively). The results revealed regeneration of the beta-cells of islets of pancreatic islet of rats treated with extract of A. muricata.

  19. Analysis of morphological and functional maturation of neoislets generated in vitro from pancreatic ductal cells and their suitability for islet banking and transplantation.

    PubMed

    Katdare, M R; Bhonde, R R; Parab, P B

    2004-07-01

    The pancreatic ductal stem cells are known to differentiate into islets of Langerhans; however, their yield is limited and the islet population is not defined. Therefore, the aims of the present study were to improvise a methodology for obtaining large numbers of islets in vitro and to characterize their morphological and functional status for islet cell banking and transplantation. Pancreatic ductal epithelial cell cultures were set in serum-free medium. Monolayers of epithelial cells in culture gave rise to islet-like clusters within 3-4 weeks. The identity of neoislets was confirmed by dithizone staining and analysis of the gene expression for endocrine markers by reverse transcriptase-polymerase chain reaction (RT-PCR). The islet population obtained was analysed by image analysis and insulin secretion in response to secretagogues. The cellular extracts from neoislets were immunoreactive to anti-insulin antibody and expressed insulin, glucagon, GLUT-2, PDX-1 and Reg-1 genes. The islets generated within 3-4 weeks exhibited a mixed population of large- and small-sized islets with clear cut dichotomy in the pattern of their insulin secretion in response to L-arginine and glucose. These neoislets maintained their structural and functional integrity on cryopreservation and transplantation indicating their suitability for islet cell banking. Thus, the present study describes an improved method for obtaining a constant supply of large numbers of islets from pancreatic ductal stem cell cultures. The newly generated islets undergo functional maturation indicating their suitability for transplantation.

  20. Effect of copper deficiency on the content and secretion of pancreatic islet hormones

    SciTech Connect

    Bhathena, S.J.; Voyles, N.R.; Timmers, K.I.; Fields, M.; Kennedy, B.W.; Recant, L.

    1986-03-01

    Experimental copper (Cu) deficiency in rats is characterized by glucose intolerance and hyperlipemia. Its severity is increased by dietary fructose (F) as compared to starch (S). Since islet hormones are intimately involved in carbohydrate metabolism the authors studied the effects of Cu deficiency on their content and secretion. Rats were fed Cu deficient (CuD) (0.6 ..mu..g Cu/g) or Cu supplemented (6.0 ..mu..g Cu/g) diets with either 62% F or S for 7 weeks after weaning. Feeding CuD diets decreased plasma insulin (I) (P < 0.001) but not plasma glucagon (G). F feeding compared to S magnified the effects of Cu deficiency. Total pancreatic content of I in CuD rats was increased threefold (P < 0.001). Total somatostatin content increased significantly only in the pancreas of CuD rats fed F. Although total G content was not altered in CuD rats, when G was expressed per g protein or g wet weight, significant increases were found in CuD rats fed F. Thus, of the islet hormones, the major effect of Cu deficiency was on I. When pancreata were perfused in vitro with high glucose, pancreas from CuD rats had reduced insulin response. Thus, cellular functions dependent on Cu are involved in maintaining the ability of the islets of Langerhans to secrete I in a normal fashion.

  1. Effects of artificial sweeteners on insulin release and cationic fluxes in rat pancreatic islets.

    PubMed

    Malaisse, W J; Vanonderbergen, A; Louchami, K; Jijakli, H; Malaisse-Lagae, F

    1998-11-01

    Beta-L-glucose pentaacetate, but not alpha-D-galactose pentaacetate, was recently reported to taste bitter and to stimulate insulin release. This finding led, in the present study, to the investigation of the effects of both bitter and non-bitter artificial sweeteners on insulin release and cationic fluxes in isolated rat pancreatic islets. Sodium saccharin (1.0-10.0 mM), sodium cyclamate (5.0-10.0 mM), stevioside (1.0 mM) and acesulfame-K (1.0-15.0 mM), all of which display a bitter taste, augmented insulin release from islets incubated in the presence of 7.0 mM D-glucose. In contrast, aspartame (1.0-10.0 mM), which is devoid of bitter taste, failed to affect insulin secretion. A positive secretory response to acesulfame-K was still observed when the extracellular K+ concentration was adjusted to the same value as that in control media. No major changes in 86Rb and 45Ca outflow from pre-labelled perifused islets could be attributed to the saccharin, cyclamic or acesulfame anions. It is proposed that the insulinotropic action of some artificial sweeteners and, possibly, that of selected hexose pentaacetate esters may require G-protein-coupled receptors similar to those operative in the recognition of bitter compounds by taste buds.

  2. Restructuring of Pancreatic Islets and Insulin Secretion in a Postnatal Critical Window

    PubMed Central

    Aguayo-Mazzucato, Cristina; Sanchez-Soto, Carmen; Godinez-Puig, Victoria; Gutiérrez-Ospina, Gabriel; Hiriart, Marcia

    2006-01-01

    Function and structure of adult pancreatic islets are determined by early postnatal development, which in rats corresponds to the first month of life. We analyzed changes in blood glucose and hormones during this stage and their association with morphological and functional changes of alpha and beta cell populations during this period. At day 20 (d20), insulin and glucose plasma levels were two- and six-fold higher, respectively, as compared to d6. Interestingly, this period is characterized by physiological hyperglycemia and hyperinsulinemia, where peripheral insulin resistance and a high plasmatic concentration of glucagon are also observed. These functional changes were paralleled by reorganization of islet structure, cell mass and aggregate size of alpha and beta cells. Cultured beta cells from d20 secreted the same amount of insulin in 15.6 mM than in 5.6 mM glucose (basal conditions), and were characterized by a high basal insulin secretion. However, beta cells from d28 were already glucose sensitive. Understanding and establishing morphophysiological relationships in the developing endocrine pancreas may explain how events in early life are important in determining adult islet physiology and metabolism. PMID:17183663

  3. MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

    PubMed Central

    Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

    2013-01-01

    microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet

  4. Quantitative morphometric studies of pancreatic islets obtained from tolbutamide-treated rats.

    PubMed

    Yorde, D E; Kalkhoff, R K

    1986-09-01

    We have developed a computerized system for quantitative morphometric analysis of the number and position of secretory granules and organelles in pancreatic islet beta cells following tolbutamide treatment. Data from animals injected with tolbutamide for 1, 2, and 3 days were compared to tissues obtained from untreated control animals. Pancreatic islets removed by a collagenase technique were perfused with an appropriate medium to restore a basal state. After fixation and embedding, thick sections of beta cells were viewed by electron microscopy. Morphometric studies of randomly selected or serially cut cells were performed with computer programs for digitization, quantify, rotational, and perspective display. Tolbutamide treatment resulted in graded granule depletion which was maximal at 72 hr relative to control animals. Reduced granule density was associated with significant reduction in total cell area or cytoplasmic area, but was without effect on nuclear size. Since granule depletion improved visualization of subcellular structures, this will enable us to pursue studies of exocytosis under a variety of physiological conditions. PMID:3525667

  5. Bioorthogonal layer-by-layer encapsulation of pancreatic islets via hyperbranched polymers

    PubMed Central

    Gattás-Asfura, Kerim M.; Stabler, Cherie L.

    2013-01-01

    The encapsulation of viable tissues via layer-by-layer polymer assembly provides a versatile platform for cell surface engineering, with nanoscale control over capsule properties. Herein, we report the development of a hyperbranched polymer-based, ultrathin capsule architecture expressing bioorthogonal functionality and tailored physiochemical properties. Random carbodiimide-based condensation of 3,5-dicarboxyphenyl glycineamide on alginate yielded a highly branched polysaccharide with multiple, spatially restricted, and readily functionalizable terminal carboxylate moieties. Poly(ethylene glycol) (PEG) was utilized to link azido end groups to the structured alginate. Together with phosphine functionalized poly(amido amine) (PAMAM) dendrimer, nanoscale layer-by-layer coatings, covalently stabilized via Staudinger ligation, were assembled onto solid surfaces and pancreatic islets. The effects of electrostatic and/or bioorthogonal covalent interlayer interactions on the resulting coating efficiency and stability, as well as pancreatic islet viability and function, were studied. These hyperbranched polymers provide a flexible platform for the formation of covalently stabilized ultrathin coatings on viable cells and tissues. In addition, the hyperbranched nature of the polymers presents a highly functionalized surface capable of bioorthogonal conjugation of additional bioactive or labeling motifs. PMID:24063764

  6. Early pancreatic islet fate and maturation is controlled through RBP-Jκ

    PubMed Central

    Cras-Méneur, Corentin; Conlon, Megan; Zhang, Yaqing; Pasca Di Magliano, Marina; Bernal-Mizrachi, Ernesto

    2016-01-01

    Notch signaling is known to control early pancreatic differentiation through Ngn3 repression. In later stages, downstream of Notch, the Presenilins are still required to maintain the endocrine fate allocation. Amongst their multiple targets, it remains unclear which one actually controls the maintenance of the fate of the early islets. Conditional deletions of the Notch effector RBP-Jκ with lineage tracing in Presenilin-deficient endocrine progenitors, demonstrated that this factor is central to the control of the fate through a non-canonical Notch mechanism. RBP-Jκ mice exhibit normal islet morphogenesis and function, however, a fraction of the progenitors fails to differentiate and develop into disorganized masses resembling acinar to ductal metaplasia and chronic pancreatitis. A subsequent deletion of RBP-Jκ in forming β-cells led to the transdifferentiation into the other endocrine cells types, indicating that this factor still mediates the maintenance of the fate within the endocrine lineage itself. These results highlight the dual importance of Notch signaling for the endocrine lineage. Even after Ngn3 expression, Notch activity is required to maintain both fate and maturation of the Ngn3 progenitors. In a subset of the cells, these alterations of Notch signaling halt their differentiation and leads to acinar to ductal metaplasia. PMID:27240887

  7. The voltage-gated proton channel Hv1 is expressed in pancreatic islet β-cells and regulates insulin secretion.

    PubMed

    Zhao, Qing; Che, Yongzhe; Li, Qiang; Zhang, Shangrong; Gao, Ying-Tang; Wang, Yifan; Wang, Xudong; Xi, Wang; Zuo, Weiyan; Li, Shu Jie

    2015-12-25

    The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet β-cells, as well as β-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in β-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K(+)-induced insulin secretion in isolated islets and INS-1 (832/13) β-cells and has an impairment on glucose- and K(+)-induced intracellular Ca(2+) homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet β-cells regulates insulin secretion through regulating Ca(2+) homeostasis. PMID:26559003

  8. Different digestion enzymes used for human pancreatic islet isolation: a mixed treatment comparison (MTC) meta-analysis.

    PubMed

    Rheinheimer, Jakeline; Ziegelmann, Patrícia Klarmann; Carlessi, Rodrigo; Reck, Luciana Ross; Bauer, Andrea Carla; Leitão, Cristiane Bauermann; Crispim, Daisy

    2014-01-01

    Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval - CrI) = -2.19 (-4.25 to -0.21) and -2.28 (-4.49 to -0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = -1.69 (-2.87 to -0.51) and -1.07 (-1.79 to -0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF. PMID:25437379

  9. Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes.

    PubMed

    Yang, Beatrice T; Dayeh, Tasnim A; Volkov, Petr A; Kirkpatrick, Clare L; Malmgren, Siri; Jing, Xingjun; Renström, Erik; Wollheim, Claes B; Nitert, Marloes Dekker; Ling, Charlotte

    2012-07-01

    Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were

  10. Curcumin enhances recovery of pancreatic islets from cellular stress induced inflammation and apoptosis in diabetic rats

    SciTech Connect

    Rashid, Kahkashan; Sil, Parames C.

    2015-02-01

    The phytochemical, curcumin, has been reported to play many beneficial roles. However, under diabetic conditions, the detail mechanism of its beneficial action in the glucose homeostasis regulatory organ, pancreas, is poorly understood. The present study has been designed and carried out to explore the role of curcumin in the pancreatic tissue of STZ induced and cellular stress mediated diabetes in eight weeks old male Wistar rats. Diabetes was induced with a single intraperitoneal dose of STZ (65 mg/kg body weight). Post to diabetes induction, animals were treated with curcumin at a dose of 100 mg/kg body weight for eight weeks. Underlying molecular and cellular mechanism was determined using various biochemical assays, DNA fragmentation, FACS, histology, immunoblotting and ELISA. Treatment with curcumin reduced blood glucose level, increased plasma insulin and mitigated oxidative stress related markers. In vivo and in vitro experimental results revealed increased levels of proinflammatory cytokines (TNF-α, IL1-β and IFN-γ), reduced level of cellular defense proteins (Nrf-2 and HO-1) and glucose transporter (GLUT-2) along with enhanced levels of signaling molecules of ER stress dependent and independent apoptosis (cleaved Caspase-12/9/8/3) in STZ administered group. Treatment with curcumin ameliorated all the adverse changes and helps the organ back to its normal physiology. Results suggest that curcumin protects pancreatic beta-cells by attenuating inflammatory responses, and inhibiting ER/mitochondrial dependent and independent pathways of apoptosis and crosstalk between them. This uniqueness and absence of any detectable adverse effect proposes the possibility of using this molecule as an effective protector in the cellular stress mediated diabetes mellitus. - Highlights: • STZ induced cellular stress plays a vital role in pancreatic dysfunction. • Cellular stress causes inflammation, pancreatic islet cell death and diabetes. • Deregulation of Nrf-2

  11. EFFECTS OF GINSENG AND ITS FOUR PURIFED GINSENOSIDES (Rb2, Re, Rg1, Rd) ON HUMAN PANCREATIC ISLET β CELL IN VITRO

    PubMed Central

    Luo, John Z. Q.; Kim, Joseph W.; Luo, LuGuang

    2016-01-01

    Ginseng has attracted interest because of its potential therapeutic role in diabetes therapy. No direct evidence has shown the effects of ginseng and its components, ginsenosides, on human islet β cell. In this study, we evaluated ginseng extract and ginsenosides (Rb2, Re, Rg1, Rd) on human pancreatic β cell function. The results provide direct evidence that ginseng extract promotes human pancreatic β cell function. Ginsenoside Rb2 increased islet β cell insulin release and promoted β cell migration. Ginsenoside Re had some impact on cell migration, but had no effect on islet function by evaluating insulin release. The other ginsenosides had no effect on insulin release and islet migration. To date, this is the first study that examines the impact of ginsenosides on human pancreatic islets in vitro. PMID:27547829

  12. Pancreatic islet transplantation: utility of ductular obstruction and exocrine atrophy model?

    PubMed

    Verma, A; Dinda, A; Sarkar, C; Fotedar, R; Dhawan, I K; Sharma, L K; Khetarpal, K; Fotedar, A; Srikanta, S; Kochupillai, N

    1990-01-01

    Introduction of 'silent' exocrine atrophy (and endocrine 'enrichment') in pancreatic grafts following ductular blockade may have a role in human diabetes by circumventing currently elusive islet isolation/purification protocols. To explore this potential, pancreatic isografts were performed in 12 pairs of inbred Wistar NIN rats. Donor pancreatectomy was performed after distal clamping and canulation of common bile duct and injection of 0.5 ml. polyacrylamide gel (blocked n = 7) or normal saline (un-blocked n = 5) respectively. One to 2 m.m. fragments of the resulting mildly distended pancreases were transplanted in to 2 sites (renal capsule and iliac fossa subcutaneously) of cach recipient. Post-operative biopsies of the transplanted grafts (unilateral nephrectomy and iliac fossa biopsies) revealed macroscopic and microscopic evidence of necrotizing pancreatitis in both the groups at both the sites (histiocytic and giant cell infiltration, fat necrosis and focal calcification with destruction of exocrine and endocrine cells) as early as 1 and 3 weeks. Possible detrimental factors include: volume and pressure of ductal injection, graft sites (confined spaces), post-operative wound infection and bio-compatibility of the material used for ductular blockade.

  13. Impaired beta-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose.

    PubMed

    Marshak, S; Leibowitz, G; Bertuzzi, F; Socci, C; Kaiser, N; Gross, D J; Cerasi, E; Melloul, D

    1999-06-01

    In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients. PMID:10342809

  14. Simulated Microgravity Reduces TNF-Alpha Activity, Suppresses Glucose Uptake and Enhances Arginine Flux in Pancreatic Islets of Langerhans

    NASA Technical Reports Server (NTRS)

    Tobin, Brian W.; Leeper-Woodford, Sandra K.; Hashemi, Brian B.; Smith, Scott M.; Sams, Clarence F.; Paloski, W. H. (Technical Monitor)

    2000-01-01

    The present studies were designed to determine effects of microgravity upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF - alpha) activity and indices of insulin and fuel homeostasis of pancreatic islets of Langerhans. Islets (1726+/-117,150 u IEU) from Wistar Furth rats were treated as: 1) HARV (High Aspect Ratio Vessel cell culture) , 2) HARV plus LPS 3) static culture, 4) static culture plus LPS TNF-alpha (L929 cytotoxicity assay) was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (p<0.05). A decrease in insulin concentration was demonstrated in the LPS stimulated HARV culture (p<0.05). We observed a greater glucose concentration and increased disappearance of arginine in islets cultured in HARVs. While nitrogenous compound analysis indicated a ubiquitous reliance upon glutamine in all experimental groups, arginine was converted to ornithine at a two-fold greater rate in the islets cultured in the HARV microgravity paradigm (p<0.05). These studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF activity in the HARV paradigm. These alterations in fuel homeostasis may be promulgated by gravity averaged cell culture methods or by three dimensional cell assembly.

  15. Trimeprazine increases IRS2 in human islets and promotes pancreatic β cell growth and function in mice

    PubMed Central

    Kuznetsova, Alexandra; Yu, Yue; Hollister-Lock, Jennifer; Opare-Addo, Lynn; Rozzo, Aldo; Sadagurski, Marianna; Norquay, Lisa; Reed, Jessica E.; El Khattabi, Ilham; Bonner-Weir, Susan; Weir, Gordon C.; Sharma, Arun

    2016-01-01

    The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote β cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, β cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and β cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes. PMID:27152363

  16. Musa sapientum with exercises attenuates hyperglycemia and pancreatic islet cells degeneration in alloxan-diabetic rats

    PubMed Central

    Akinlolu, Adelaja Abdulazeez; Salau, Bamidele A.; Ekor, Martins; Otulana, Jubril

    2015-01-01

    Aim: We tested the hypothesis that administrations of methanolic extracts of Musa sapientum sucker (MEMS) with exercises attenuated hyperglycemia in alloxan-diabetic rats. Materials and Methods: A total of 40 adult male rats were divided into equal eight groups. Normoglycemic Group A was Control. Alloxan (180 mg/kg, i.p.) was administered to rats in Groups B - H to induce diabetes. Group B (diabetic control) received physiological saline. Groups C - H received MEMS (5 mg/kg), MEMS (10 mg/kg), Glibenclamide (5 mg/kg), MEMS (5 mg/kg) + exercises, MEMS (10 mg/kg) + exercises and Exercises only, respectively. Changes in body weight, blood glucose levels (BGL) and pancreatic histology were evaluated during or at the end of experiment. Body weights and BGL of rats were expressed as mean ± standard deviation and analyzed using the statistical software program SPSS 15. Statistical comparisons were done using the Student’s t-test for unpaired samples. Differences between groups were determined as significant at P ≤ 0.05. Results: Significantly (P < 0.05) decreased bodyweight was observed in B and H compared to A and C - G. Treatment with MEMS significantly (P < 0.05) decreased elevated BGL in C and D. Hypoglycemic effect of MEMS appeared enhanced with exercises in F and G. Exercises regimen alone (H) resulted in percentage reduction in BGL lower than those of C - G. Histopathological examinations revealed normal pancreas (A), atrophied islet cells (B), hyperplasia with adequate population of islet cells (C - G), and reduced hyperplasia of islet cells (H). Conclusion: MEMS with exercises attenuated hyperglycemia in alloxan-diabetic rats. PMID:26401408

  17. Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.

    PubMed

    Lavagnino, Zeno; Dwight, Jason; Ustione, Alessandro; Nguyen, Thuc-Uyen; Tkaczyk, Tomasz S; Piston, David W

    2016-07-26

    The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and β-cell function. Insulin secretion from β-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.

  18. Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.

    PubMed

    Lavagnino, Zeno; Dwight, Jason; Ustione, Alessandro; Nguyen, Thuc-Uyen; Tkaczyk, Tomasz S; Piston, David W

    2016-07-26

    The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and β-cell function. Insulin secretion from β-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and β-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and β-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels. PMID:27463142

  19. Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets.

    PubMed

    Nicolas, P; Delfour, A; Boussetta, H; Morel, A; Rholam, M; Cohen, P

    1986-10-30

    Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells. PMID:2877662

  20. Glucagon-Like Peptide 1 Analogs and their Effects on Pancreatic Islets.

    PubMed

    Tudurí, Eva; López, Miguel; Diéguez, Carlos; Nadal, Angel; Nogueiras, Rubén

    2016-05-01

    Glucagon-like peptide 1 (GLP-1) exerts many actions that improve glycemic control. GLP-1 stimulates glucose-stimulated insulin secretion and protects β cells, while its extrapancreatic effects include cardioprotection, reduction of hepatic glucose production, and regulation of satiety. Although an appealing antidiabetic drug candidate, the rapid degradation of GLP-1 by dipeptidyl peptidase 4 (DPP-4) means that its therapeutic use is unfeasible, and this prompted the development of two main GLP-1 therapies: long-acting GLP-1 analogs and DPP-4 inhibitors. In this review, we focus on the pancreatic effects exerted by current GLP-1 derivatives used to treat diabetes. Based on the results from in vitro and in vivo studies in humans and animal models, we describe the specific actions of GLP-1 analogs on the synthesis, processing, and secretion of insulin, islet morphology, and β cell proliferation and apoptosis. PMID:27062006

  1. Role of transcription factors in the transdifferentiation of pancreatic islet cells.

    PubMed

    van der Meulen, Talitha; Huising, Mark O

    2015-04-01

    The α and β cells act in concert to maintain blood glucose. The α cells release glucagon in response to low levels of glucose to stimulate glycogenolysis in the liver. In contrast, β cells release insulin in response to elevated levels of glucose to stimulate peripheral glucose disposal. Despite these opposing roles in glucose homeostasis, α and β cells are derived from a common progenitor and share many proteins important for glucose sensing and hormone secretion. Results from recent work have underlined these similarities between the two cell types by revealing that β-to-α as well as α-to-β transdifferentiation can take place under certain experimental circumstances. These exciting findings highlight unexpected plasticity of adult islets and offer hope of novel therapeutic paths to replenish β cells in diabetes. In this review, we focus on the transcription factor networks that establish and maintain pancreatic endocrine cell identity and how they may be perturbed to facilitate transdifferentiation. PMID:25791577

  2. Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

    PubMed

    Huang, Yao; Chang, Yongchang

    2014-01-01

    Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling.

  3. Modulation of the pancreatic islet-stress axis as a novel potential therapeutic target in diabetes mellitus.

    PubMed

    Ludwig, Barbara; Barthel, Andreas; Reichel, Andreas; Block, Norman L; Ludwig, Stefan; Schally, Andrew V; Bornstein, Stefan R

    2014-01-01

    Loss of pancreatic islet function and insulin-producing beta cell mass is a central hallmark in the pathogenesis of both type 1 and type 2 diabetes. While in type 1 diabetes this phenomenon is due to an extensive destruction of beta cells caused by an autoimmune process, the mechanisms resulting in beta cell failure in type 2 diabetes are different and less clear. Also, beta cell destruction in type 1 diabetes occurs early and is the initial step in the pathogenetic process, while beta cell loss in type 2 diabetes after an initial phase of hyperinsulinemia due to the underlying insulin resistance occurs relatively late and it is less pronounced. Since diabetes mellitus is the most frequent endocrine disease, with an increasing high prevalence worldwide, huge efforts have been made over the past many decades to identify predisposing genetic, environmental, and nutritional factors in order to develop effective strategies to prevent the disease. In parallel, extensive studies in different cell systems and animal models have helped to elucidate our understanding of the physiologic function of islets and to gain insight into the immunological and non-immunological mechanisms of beta cell destruction and failure. Furthermore, currently emerging concepts of beta cell regeneration (e.g., the restoration of the beta cell pool by regenerative, proliferative and antiapoptotic processes, and recovery of physiologic islet function) apparently is yielding the first promising results. Recent insights into the complex endocrine and paracrine mechanisms regulating the physiologic function of pancreatic islets, as well as beta cell life and death, constitute an essential part of this new and exciting area of diabetology. For example, understanding of the physiological role of glucagon-like peptide 1 has resulted in the successful clinical implementation of incretin-based therapies over the last years. Further, recent data suggesting paracrine effects of growth hormone

  4. mRNA analysis of intracytoplasmically-stained, FACS-purified pancreatic islet cells using the quantitative nuclease protection assay

    PubMed Central

    Pechhold, S; Stouffer, M; Walker, G; Martel, R; Seligmann, B; Hang, Y; Stein, R; Harlan, DM; Pechhold, K

    2009-01-01

    Exploring the pathophysiology underlying diabetes mellitus requires characterizing the cellular constituents of pancreatic islets, primarily insulin-producing β-cells. Such efforts have been limited by inadequate techniques for purifying islet cellular subsets for further biochemical and gene-expression studies. Using intracytoplasmic staining and fluorescence-activated cell-sorting (FACS) followed by quantitative nuclease protection assay (qNPA™) technology, we examined 30 relevant genes expressed by islet subpopulations. Purified islet cell subsets expressed all four tested “housekeeping” genes with a surprising variability, dependent on both cell lineage and developmental stage, suggesting caution when interpreting housekeeping gene-normalized mRNA quantifications. Our new approach confirmed expected islet cell lineage-specific gene expression patterns at the transcriptional level, but also detected new phenotypes, including mRNA-profiles (supported by immunohistology) demonstrating that during pregnancy, some β-cells express Mafb, previously found only in immature β-cells during embryonic development. Overall, qNPA™ gene expression analysis using intracellular-stained then FACS-sorted cells has broad applications beyond islet cell biology. PMID:19838197

  5. A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

    PubMed Central

    2015-01-01

    Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes. PMID:26216133

  6. A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques.

    PubMed

    Baskin, Denis G

    2015-08-01

    Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes.

  7. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

    PubMed Central

    Miller, C P; McGehee, R E; Habener, J F

    1994-01-01

    We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum. Images PMID:7907546

  8. Endothelial Cells Mediate Islet-Specific Maturation of Human Embryonic Stem Cell-Derived Pancreatic Progenitor Cells

    PubMed Central

    Jaramillo, Maria; Mathew, Shibin; Mamiya, Hikaru; Goh, Saik Kia

    2015-01-01

    It is well recognized that in vitro differentiation of embryonic stem cells (ESC) can be best achieved by closely recapitulating the in vivo developmental niche. Thus, implementation of directed differentiation strategies has yielded encouraging results in the area of pancreatic islet differentiation. These strategies have concentrated on direct addition of chemical signals, however, other aspect of the developmental niche are yet to be explored. During development, pancreatic progenitor (PP) cells grow as an epithelial sheet, which aggregates with endothelial cells (ECs) during the final stages of maturation. Several findings suggest that the interactions with EC play a role in pancreatic development. In this study, we recapitulated this phenomenon in an in vitro environment by maturing the human ESC (hESC)-derived PP cells in close contact with ECs. We find that co-culture with different ECs (but not fibroblast) alone results in pancreatic islet-specific differentiation of hESC-derived PP cells even in the absence of additional chemical induction. The differentiated cells responded to exogenous glucose levels by enhanced C-peptide synthesis. The co-culture system aligned well with endocrine development as determined by comprehensive analysis of involved signaling pathways. By recapitulating cell–cell interaction aspects of the developmental niche we achieved a differentiation model that aligns closely with islet organogenesis. PMID:24943736

  9. Pancreatic islet amyloidosis, β-cell apoptosis, and α-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons

    PubMed Central

    Guardado-Mendoza, Rodolfo; Davalli, Alberto M.; Chavez, Alberto O.; Hubbard, Gene B.; Dick, Edward J.; Majluf-Cruz, Abraham; Tene-Perez, Carlos E.; Goldschmidt, Lukasz; Hart, John; Perego, Carla; Comuzzie, Anthony G.; Tejero, Maria Elizabeth; Finzi, Giovanna; Placidi, Claudia; La Rosa, Stefano; Capella, Carlo; Halff, Glenn; Gastaldelli, Amalia; DeFronzo, Ralph A.; Folli, Franco

    2009-01-01

    β-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative β-cell volume, and increased relative α-cell volume and hyperglucagonemia. These results strongly support the concept that IA and β-cell apoptosis in concert with α-cell proliferation and hypertrophy are key determinants of islets of Langerhans “dysfunctional remodeling” and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R2 = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables. PMID:19666551

  10. Processing of immunoisolated pancreatic islets: implications for histological analyses of hydrated tissue.

    PubMed

    De Haan, Bart J; van Goor, Harry; De Vos, Paul

    2002-03-01

    Routine tissue processing is usually associated with histological artifacts as a consequence of shrinkage and distortion during dehydration required for embedding. With hydrated specimens such as lung, embryonic, and tissues in hydrophilic membranes, tissue processing can induce severe artifacts that interfere with adequate microscopic evaluation. Here we present a method for embedding hydrophilic alginate-polylysine microencapsulated pancreatic tissue that combines the absence of histological artifacts with a practical tissue processing method. We found that the glycol-methacrylate (GMA)-embedding method preserved the integrity of the encapsulated tissue better than snap-freezing or paraffin embedding, but the overall quality of the hydrophilic capsules remained poor Next, we modified the GMA method by introducing gradual dehydration to investigate whether the integrity of the sectioned capsules was better maintained by a more gradual pattern of water extraction. This modification resulted in well-preserved morphological details of the hydrophilic membranes, hydrogel-cell interface, and encapsulated pancreatic tissue. Subsequent routine staining gave excellent contrast between the islet tissue and hydrophilic components, which allowed adequate quantitative histological and pathological comparisons.

  11. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies

    PubMed Central

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0–120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48–120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs

  12. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

    PubMed

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

  13. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

    PubMed

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards

  14. Identification of microRNAs expressed highly in pancreatic islet-like cell clusters differentiated from human embryonic stem cells.

    PubMed

    Chen, Bo-Zhi; Yu, Sung-Liang; Singh, Sher; Kao, Li-Pin; Tsai, Zong-Yun; Yang, Pan-Chyr; Chen, Bai-Hsiun; Shoei-Lung Li, Steven

    2011-01-01

    Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self-renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES-T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet-like cell clusters derived from T3 cells), which expressed pancreatic islet cell-specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES-T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein-coding mRNAs. The T3pi showed very high expression of microRNAs, miR-186, miR-199a and miR-339, which down-regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics. PMID:20735361

  15. Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets.

    PubMed

    Zuellig, R A; Cavallari, G; Gerber, P; Tschopp, O; Spinas, G A; Moritz, W; Lehmann, R

    2014-04-16

    Previously we demonstrated the superiority of small islets vs large islets in terms of function and survival after transplantation, and we generated reaggregated rat islets (pseudo-islets) of standardized small dimensions by the hanging-drop culture method (HDCM). The aim of this study was to generate human pseudo-islets by HDCM and to evaluate and compare the physiological properties of rat and human pseudo-islets. Isolated rat and human islets were dissociated into single cells and incubated for 6-14 days by HDCM. Newly formed pseudo-islets were analysed for dimensions, morphology, glucose-stimulated insulin secretion (GSIS) and total insulin content. The morphology of reaggregated human islets was similar to that of native islets, while rat pseudo-islets had a reduced content of α and δ cells. GSIS of small rat and human pseudo-islets (250 cells) was increased up to 4.0-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, when compared to their native counterparts. Human pseudo-islets showed a more pronounced first-phase insulin secretion as compared to intact islets. GSIS was inversely correlated to islet size, and small islets (250 cells) contained up to six-fold more insulin/cell than large islets (1500 cells). Tissue loss with this new technology could be reduced to 49.2 ± 1.5% in rat islets, as compared to the starting amount. With HDCM, pseudo-islets of standardized size with similar cellular composition and improved biological function can be generated, which compensates for tissue loss during production. Transplantation of small pseudo-islets may represent an attractive strategy to improve graft survival and function, due to better oxygen and nutrient supply during the phase of revascularization. Copyright © 2014 John Wiley & Sons, Ltd.

  16. ISL-1 promotes pancreatic islet cell proliferation by forming an ISL-1/Set7/9/PDX-1 complex

    PubMed Central

    Yang, Zhe; Zhang, Qiao; Lu, Qin; Jia, Zhuqing; Chen, Ping; Ma, Kangtao; Wang, Weiping; Zhou, Chunyan

    2015-01-01

    Islet-1 (ISL-1), a LIM-homeodomain transcription factor, has been recently found to be essential for promoting postnatal pancreatic islet proliferation. However, the detailed mechanism has not yet been elucidated. In the present study, we investigated the mechanism by which ISL-1 promotes β-cell proliferation through regulation of CyclinD1 in HIT-T15 and NIT-1 cells, as well in rat islet mass. Our results provide the evidence that ISL-1 promotes adult pancreatic islet β-cell proliferation by activating CyclinD1 transcription through cooperation with Set7/9 and PDX-1 to form an ISL-1/Set7/9/PDX-1 complex. This complex functions in an ISL-1-dependent manner, with Set7/9 functioning not only as a histone methyltransferase, which increases the histone H3K4 tri-methylation of the CyclinD1 promoter region, but also an adaptor to bridge ISL-1 and PDX-1, while PDX-1 functions as a RNA pol II binding modulator. Furthermore, the formation of the ISL-1/Set7/9/PDX-1 complex is positively associated with insulin-like growth factor-1 treatment in NIT and HIT-T15 cells in vitro, while may be negatively correlated with age in vivo. PMID:26176407

  17. The Potential Protective Action of Vitamin D in Hepatic Insulin Resistance and Pancreatic Islet Dysfunction in Type 2 Diabetes Mellitus.

    PubMed

    Leung, Po Sing

    2016-03-01

    Vitamin D deficiency (i.e., hypovitaminosis D) is associated with increased insulin resistance, impaired insulin secretion, and poorly controlled glucose homeostasis, and thus is correlated with the risk of metabolic diseases, including type 2 diabetes mellitus (T2DM). The liver plays key roles in glucose and lipid metabolism, and its dysregulation leads to abnormalities in hepatic glucose output and triglyceride accumulation. Meanwhile, the pancreatic islets are constituted in large part by insulin-secreting β cells. Consequently, islet dysfunction, such as occurs in T2DM, produces hyperglycemia. In this review, we provide a critical appraisal of the modulatory actions of vitamin D in hepatic insulin sensitivity and islet insulin secretion, and we discuss the potential roles of a local vitamin D signaling in regulating hepatic and pancreatic islet functions. This information provides a scientific basis for establishing the benefits of the maintenance, or dietary manipulation, of adequate vitamin D status in the prevention and management of obesity-induced T2DM and non-alcoholic fatty liver disease. PMID:26959059

  18. ISL-1 promotes pancreatic islet cell proliferation by forming an ISL-1/Set7/9/PDX-1 complex.

    PubMed

    Yang, Zhe; Zhang, Qiao; Lu, Qin; Jia, Zhuqing; Chen, Ping; Ma, Kangtao; Wang, Weiping; Zhou, Chunyan

    2015-01-01

    Islet-1 (ISL-1), a LIM-homeodomain transcription factor, has been recently found to be essential for promoting postnatal pancreatic islet proliferation. However, the detailed mechanism has not yet been elucidated. In the present study, we investigated the mechanism by which ISL-1 promotes β-cell proliferation through regulation of CyclinD1 in HIT-T15 and NIT-1 cells, as well in rat islet mass. Our results provide the evidence that ISL-1 promotes adult pancreatic islet β-cell proliferation by activating CyclinD1 transcription through cooperation with Set7/9 and PDX-1 to form an ISL-1/Set7/9/PDX-1 complex. This complex functions in an ISL-1-dependent manner, with Set7/9 functioning not only as a histone methyltransferase, which increases the histone H3K4 tri-methylation of the CyclinD1 promoter region, but also an adaptor to bridge ISL-1 and PDX-1, while PDX-1 functions as a RNA pol II binding modulator. Furthermore, the formation of the ISL-1/Set7/9/PDX-1 complex is positively associated with insulin-like growth factor-1 treatment in NIT and HIT-T15 cells in vitro, while may be negatively correlated with age in vivo.

  19. The Potential Protective Action of Vitamin D in Hepatic Insulin Resistance and Pancreatic Islet Dysfunction in Type 2 Diabetes Mellitus.

    PubMed

    Leung, Po Sing

    2016-03-05

    Vitamin D deficiency (i.e., hypovitaminosis D) is associated with increased insulin resistance, impaired insulin secretion, and poorly controlled glucose homeostasis, and thus is correlated with the risk of metabolic diseases, including type 2 diabetes mellitus (T2DM). The liver plays key roles in glucose and lipid metabolism, and its dysregulation leads to abnormalities in hepatic glucose output and triglyceride accumulation. Meanwhile, the pancreatic islets are constituted in large part by insulin-secreting β cells. Consequently, islet dysfunction, such as occurs in T2DM, produces hyperglycemia. In this review, we provide a critical appraisal of the modulatory actions of vitamin D in hepatic insulin sensitivity and islet insulin secretion, and we discuss the potential roles of a local vitamin D signaling in regulating hepatic and pancreatic islet functions. This information provides a scientific basis for establishing the benefits of the maintenance, or dietary manipulation, of adequate vitamin D status in the prevention and management of obesity-induced T2DM and non-alcoholic fatty liver disease.

  20. The Potential Protective Action of Vitamin D in Hepatic Insulin Resistance and Pancreatic Islet Dysfunction in Type 2 Diabetes Mellitus

    PubMed Central

    Leung, Po Sing

    2016-01-01

    Vitamin D deficiency (i.e., hypovitaminosis D) is associated with increased insulin resistance, impaired insulin secretion, and poorly controlled glucose homeostasis, and thus is correlated with the risk of metabolic diseases, including type 2 diabetes mellitus (T2DM). The liver plays key roles in glucose and lipid metabolism, and its dysregulation leads to abnormalities in hepatic glucose output and triglyceride accumulation. Meanwhile, the pancreatic islets are constituted in large part by insulin-secreting β cells. Consequently, islet dysfunction, such as occurs in T2DM, produces hyperglycemia. In this review, we provide a critical appraisal of the modulatory actions of vitamin D in hepatic insulin sensitivity and islet insulin secretion, and we discuss the potential roles of a local vitamin D signaling in regulating hepatic and pancreatic islet functions. This information provides a scientific basis for establishing the benefits of the maintenance, or dietary manipulation, of adequate vitamin D status in the prevention and management of obesity-induced T2DM and non-alcoholic fatty liver disease. PMID:26959059

  1. Ventromedial hypothalamic lesions change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets

    PubMed Central

    Kiba, Takayoshi; Ishigaki, Yasuhito

    2014-01-01

    Studies in normal rats and ob/ob mice indicated that islet neogenesis does not occur in the intact rodent pancreas. We previously reported that ventromedial hypothalamic (VMH) lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism and examined how gene families involved in cell proliferation in total pancreatic tissue are regulated after VMH lesions formation. This study examined how gene families involved in cell proliferation in pancreatic islets alone are regulated after VMH lesions formation. Pancreatic islet RNA was extracted, and differences in gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time polymerase chain reaction. VMH lesions regulated genes that were involved in functions related to cell cycle and differentiation, growth, binding, apoptosis and morphology in pancreas islets. Real-time polymerase chain reaction also confirmed that gene expression of polo-like kinase 1 (Plk1) and topoisomerase (DNA) II α 170 kDa (Top2a), and stanniocalcin 1 (Stc1) were upregulated at day 3 after the VMH lesions. Ventromedial hypothalamic lesions may change the expression of cell proliferation-related genes and morphology-related genes in rat pancreatic islets. PMID:25658146

  2. Activation of the Wnt/β-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice.

    PubMed

    Maschio, D A; Oliveira, R B; Santos, M R; Carvalho, C P F; Barbosa-Sampaio, H C L; Collares-Buzato, C B

    2016-09-30

    The Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/β-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active β-catenin associated with significant increases in protein content and gene expression of β-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/β-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM. PMID:27576200

  3. Improvement in The Function of Isolated Rat Pancreatic Islets through Reduction of Oxidative Stress Using Traditional Iranian Medicine

    PubMed Central

    Mahroui, Neda; Mirzaei, Sanaz; Siahpoosh, Zahra; D.4, Pharm.; Nili-Ahmadabadi, Amir; Mohammadirad, Azadeh; Baeeri, Maryam; Hajiaghaie, Reza; Abdollahi, Mohammad

    2014-01-01

    Objective Pancreatic islets have fewer antioxidant enzymes than other tissues and thus are vulnerable to oxidative stress. In the present study, the effects of nine specifically selected Iranian medical plants on the mitochondria function and survival of isolated rat islets were examined. Materials and Methods In this experimental study, following laparotomy, pancreases of rats were removed and the islets isolated and incubated in vitro for 24 hours. Logarithmic doses of plant materials were added to the islets and incubated for an additional 24 hours after which the viability of the cells and production of reactive oxygen species (ROS) were measured. Levels of insulin production in relation to static and stimulated glucose concen- trations were also determined. Results The tested compounds markedly increased survival of the islet cells, their mi- tochondrial activity, and insulin levels at the same time as reducing production of ROS. Greatest effects were observed in the following order: Peganum harmala, Glycyrrhiza glabra, Satureja hortensis, Rosmarinus officinalis, Teucrium scordium, Aloe vera, Zingiber officinale, Silybum marianum, and Hypericum perforatum at doses of 10, 103, 104, 10, 102, 102, 10-1, 10 and 103μgmL-1, respectively. Conclusion Based on these results, we suggest that pretreatment with these select- ed Iranian medical plants can improve the outcomes of pancreas transplants and grafts through the control of oxidative stress damage. PMID:24567945

  4. Pancreatic tissue resident mesenchymal stromal cell (MSC)-like cells as a source of in vitro islet neogenesis.

    PubMed

    Gopurappilly, Renjitha; Bhat, Vijay; Bhonde, Ramesh

    2013-10-01

    Insufficient β-cell mass is a common denominator for both type 1 and type 2 diabetes. In vitro generation of β-cells from islet precursor cells, exocrine cells or ductal epithelia provide an alternative source of insulin-producing cells. However the presence of multipotent precursor cells within the pancreas is also deliberated. In this study we isolated mesenchymal stromal cell (MSC)-like cells from adult mouse pancreas by collagenase digestion. We used Knockout DMEM for our isolation procedure and the floating islets and acini were removed after 48 h. This strategy permitted the adhesion of stromal cells with typical mesenchymal morphology. These cells not only expressed MSC-specific markers like Sca-1, CD90.2, CD73, and CD44 but also generated osteocytes, adipocytes, and neurons when induced with specific growth media. Upon exposure to islet differentiation serum-free cocktail a significant upregulation of pancreatic markers like Nkx2.2, Nkx6.1, Pdx1, insulin, and somatostatin was seen. The differentiated islet-like cell aggregates (ICAs) secreted insulin which increased over the days in culture in presence of basal glucose levels. Taken together, our data strongly indicate that there is a tissue-resident precursor population within the pancreas that can be exploited for islet neogenesis in vitro. PMID:23606308

  5. Damage to pancreatic acinar cells and preservation of islets of Langerhans in a rat model of acute pancreatitis induced by Karwinskia humboldtiana (buckthorn).

    PubMed

    Carcano-Diaz, Katya; Garcia-Garcia, Aracely; Segoviano-Ramirez, Juan Carlos; Rodriguez-Rocha, Humberto; Loera-Arias, Maria de Jesus; Garcia-Juarez, Jaime

    2016-09-01

    Karwinskia humboldtiana (Kh) is a poisonous plant that grows in some regions of the American continent. Consuming large amounts of Kh fruit results in acute intoxication leading to respiratory failure, culminating in death within days. There is evidence of histological damage to the lungs, liver, and kidneys following accidental and experimental Kh intoxication. To date, the microscopic effect of Kh consumption on the pancreas has not been described. We examined the early effects of Kh fruit on pancreatic tissue at different stages of acute intoxication in the Wistar rat. We found progressive damage confined to the exocrine pancreas, starting with a reduction in the number of zymogen granules, loss of acinar architecture, the presence of autophagy-like vesicles, apoptosis and inflammatory infiltrate. The pancreatic pathology culminated in damaged acini characterized by necrosis and edema, with a complete loss of lobular architecture. Interestingly, the morphology of the islets of Langerhans was conserved throughout our evaluations. Taken together, our results indicate the damage induced by a high dose of Kh fruit in the Wistar rat is consistent with an early acute necrotizing pancreatitis that exclusively affects the exocrine pancreas. Therefore, this system might be useful as an animal model to study the treatment of pancreatic diseases. More importantly, as the islets of Langerhans were preserved, the active compounds of Kh fruit could be utilized for the treatment of acinar pancreatic cancer. Further studies might provide insight into the severity of acute Kh intoxication in humans and influence the design of treatments for pancreatic diseases and acinar pancreatic cancer. PMID:26877198

  6. Exocrine contamination impairs implantation of pancreatic islets transplanted beneath the kidney capsule.

    PubMed

    Gray, D W; Sutton, R; McShane, P; Peters, M; Morris, P J

    1988-11-01

    The effect of exocrine contamination on islets implanted under the kidney capsule has been studied by histological examination of pure or exocrine-contamination human, monkey, or rat islets transplanted to the kidney capsule of the nude rat, monkey, or rat, respectively. Exocrine contamination resulted in an appearance suggestive of impaired islet implantation, due to tissue necrosis and subsequent fibrosis. The effect of exocrine contamination was examined quantitatively in a rat islet isograft model in which handpicked DA rat islets were transplanted under the kidney capsule of normal DA rats. The islets were either pure or deliberately recontaminated with exocrine tissue (50 or 90% contamination). Four hundred pure islets were placed under one kidney capsule and 400 islets (of similar size and from the same islet preparation) were contaminated and then placed under the contralateral kidney capsule. After 2 weeks the kidneys were removed and extracted for insulin content. The insulin content of kidneys bearing islets contaminated by either 50 or 90% exocrine tissue was significantly reduced when compared to the contralateral kidney bearing pure islets. These findings support the view that exocrine contamination of islets resulted in impaired islet implantation when transplanted to a confined site such as the kidney subcapsule.

  7. Pancreatic islet cell transplantation using non-heart-beating donors (NHBDs).

    PubMed

    Matsumoto, Shinichi; Tanaka, Koichi

    2005-01-01

    Recent dramatic improvements in clinical islet cell transplantation demonstrated by the Edmonton group have increased the demand for this treatment, and donor shortage could become a major problem. Utilization of marginal donors could alleviate the donor shortage, and non-heart-beating donors (NHBDs) might be good resources. The University of Pennsylvania group demonstrated that it was possible to isolate islets from NHBDs, and the group actually transplanted islets from NHBDs, for the first time. The patient became insulin-independent; however, there had been no more cases using NHBDs until our group initiated islet transplantations from NHBDs in Japan. In order to utilize NHBDs effectively, we modified the standard islet isolation method. These modifications included minimizing the warm ischemic time, the use of trypsin inhibition during isolation, carrying out density measurement before purification and the use of a less toxic islet purification solution. With these modifications we were able to transplant nine of ten islet preparations from ten NHBDs (90%), into five type-1 diabetic patients. The first transplantation was performed on April 7, 2004 (the first time in Japan), and this patient became insulin-independent after the second islet transplantation (first time in Japan). All patients showed improved glycemic control and reduced insulin requirements, without hypoglycemic events. We also performed living-donor islet transplantation, with our modified islet isolation protocol, on January 19, 2005. The improved islet isolation protocol enabled us to perform effective islet transplantations from NHBDs, and it also enabled us to perform the living-donor islet transplantation.

  8. Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity.

    PubMed

    Carchia, E; Porreca, I; Almeida, P J; D'Angelo, F; Cuomo, D; Ceccarelli, M; De Felice, M; Mallardo, M; Ambrosino, C

    2015-01-01

    Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10(-9 )M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions. PMID:26512966

  9. Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity.

    PubMed

    Carchia, E; Porreca, I; Almeida, P J; D'Angelo, F; Cuomo, D; Ceccarelli, M; De Felice, M; Mallardo, M; Ambrosino, C

    2015-10-29

    Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10(-9 )M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

  10. Expression of PDX-1 is reduced in pancreatic islets from pups of rat dams fed a low protein diet during gestation and lactation.

    PubMed

    Arantes, Vanessa C; Teixeira, Vicente P A; Reis, Marise A B; Latorraca, Márcia Q; Leite, Adriana R; Carneiro, Everardo M; Yamada, Aureo T; Boschero, Antonio C

    2002-10-01

    Intrauterine and early postnatal malnutrition has profound consequences on fetal and postnatal development in both humans and animals. In addition, low birth weight has been reported to be associated with impaired insulin secretion, insulin resistance and diminished area of pancreatic islets. Because the transcription factor pancreatic and duodenal homeobox 1 (PDX-1) is important for the maintenance of B-cell physiology, PDX-1 expression and islet area were assessed in neonatal rats of dams fed low (6%) or normal (17%) protein diets during pregnancy. PDX-1 protein and mRNA levels, as well as insulin secretion and islet area, were measured after 28 d of life in normal, low protein and recovered rats whose dams consumed a normal protein diet after delivery. Insulin secretion by isolated islets in response to 2.8 and 16.7 mmol glucose/L was reduced in 28-d-old low protein rats compared with the control (P < 0.05). At birth and after 28 d of life, the islet area and PDX-1 protein expression were also reduced (P < 0.05). In contrast, PDX-1 mRNA levels in islets from 28-d-old low protein rats were not different from control rats. PDX-1 protein expression in pancreatic islets, the area of islets and insulin secretion were restored in recovered rats, whereas PDX-1 mRNA levels were higher than in normal rats (P < 0.05). These results suggest a link among diminished PDX-1 protein expression, a reduction in islet area and impaired insulin secretion in low protein rats. The reintroduction of a normal diet early in life restored islet area and cell physiology. PMID:12368391

  11. Synergistic Potentials of Coffee on Injured Pancreatic Islets and Insulin Action via KATP Channel Blocking in Zebrafish.

    PubMed

    Nam, Youn Hee; Hong, Bin Na; Rodriguez, Isabel; Ji, Min Gun; Kim, Keonwoo; Kim, Ung-Jin; Kang, Tong Ho

    2015-06-17

    Pancreatic islets (PIs) are damaged under diabetic conditions, resulting in decreased PI size. This study examined the regenerative effects of coffee and its components (caffeine, CFI; trigonelline, TRG; chlorogenic acid, CGA) on zebrafish larval PIs and β-cells damaged by administration of alloxan (AX). In addition, the influence of coffee and its active components on KATP channels was investigated using diazoxide (DZ) as a KATP channel activator. PI size and fluorescence intensity were significantly increased in the coffee-treated group relative to the no-treatment group (P < 0.0001). In addition, coffee exerted significant regenerative effects on pancreatic β-cells (p = 0.006). Treatment with TRG and CGA rescued PI damage, and the combination of TRG/CGA had a synergistic effect. In conclusion, the results indicate that coffee has beneficial effects on AX-damaged PIs and may also be useful as a blocker of pancreatic β-cell K(+) channels.

  12. Possible role for calmodulin in insulin release. Studies with trifluoperazine in rat pancreatic islets.

    PubMed

    Krausz, Y; Wollheim, C B; Siegel, E; Sharp, G W

    1980-09-01

    The role of calmodulin in insulin secretion from rat pancreatic islets has been examined by the use of trifluoperazine, an inhibitor of calmodulin-Ca++-directed functions. It was found that 30 microM trifluoperazine caused 50% inhibition, and 100 microM, up to 73% inhibition of 16.7 mM glucose-stimulated insulin release. 100 microM trifluoperazine caused a similar inhibition of 10 mM glyceraldehyde-stimulated release. Therefore, the site of action of trifluoperazine in glucose stimulus-secretion coupling appears to be after the trioses. As trifluoperazine had no effect upon insulin release stimulated by 1 mM 3-isobutyl-1-methylxanthine, the inhibitory effect of trifluoperazine appears to be rather specific. Further, the process of exocytosis per se is not affected. It was also found that although trifluoperazine inhibited the effect of glucose to stimulate insulin release, it did not affect the synergism between glucose and 3-isobutyl-1-methylxanthine to potentiate insulin release. It may be concluded that trifluoperazine selectively inhibits one part of the mechanism by which glucose stimulates insulin release. Calmodulin plays a role in the stimulation of insulin release by glucose at a site between metabolism of trioses and elevation of cytosol Ca++, but is not involved in the final process of exocytosis.

  13. Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species

    PubMed Central

    Pilkington, Emily H.; Gurzov, Esteban N.; Kakinen, Aleksandr; Litwak, Sara A.; Stanley, William J.; Davis, Thomas P.; Ke, Pu Chun

    2016-01-01

    Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders. PMID:26880502

  14. Vanadyl Sulfate Treatment Stimulates Proliferation and Regeneration of Beta Cells in Pancreatic Islets

    PubMed Central

    Missaoui, Samira; Ben Rhouma, Khémais; Yacoubi, Mohamed-Tahar; Sakly, Mohsen; Tebourbi, Olfa

    2014-01-01

    We examined the effects of vanadium sulfate (VOSO4) treatment at 5 and 10 mg/kg for 30 days on endocrine pancreas activity and histology in nondiabetic and STZ-induced diabetic rats. In diabetic group, blood glucose levels significantly increased while insulinemia level markedly decreased. At the end of treatment, VOSO4 at a dose of 10 mg/Kg normalized blood glucose level in diabetic group, restored insulinemia, and significantly improved insulin sensitivity. VOSO4 also increased in a dose-dependent manner the number of insulin immunopositive beta cells in pancreatic islets of nondiabetic rats. Furthermore, in the STZ-diabetic group, the decrease in the number of insulin immunopositive beta cells was corrected to reach the control level mainly with the higher dose of vanadium. Therefore, VOSO4 treatment normalized plasma glucose and insulin levels and improved insulin sensitivity in STZ-experimental diabetes and induced beta cells proliferation and/or regeneration in normal or diabetic rats. PMID:25215302

  15. The dynamic and geometric phase transition in the cellular network of pancreatic islet

    NASA Astrophysics Data System (ADS)

    Wang, Xujing

    2013-03-01

    The pancreatic islet is a micro-organ that contains several thousands of endocrine cells, majority of which being the insulin releasing β - cells . - cellsareexcitablecells , andarecoupledtoeachother through gap junctional channels. Here, using percolation theory, we investigate the role of network structure in determining the dynamics of the β-cell network. We show that the β-cell synchronization depends on network connectivity. More specifically, as the site occupancy is reducing, initially the β-cell synchronization is barely affected, until it reaches around a critical value, where the synchronization exhibit a sudden rapid decline, followed by an slow exponential tail. This critical value coincides with the critical site open probability for percolation transition. The dependence over bond strength is similar, exhibiting critical-behavior like dependence around a certain value of bond strength. These results suggest that the β-cell network undergoes a dynamic phase transition when the network is percolated. We further apply the findings to study diabetes. During the development of diabetes, the β - cellnetworkconnectivitydecreases . Siteoccupancyreducesfromthe reducing β-cell mass, and the bond strength is increasingly impaired from β-cell stress and chronic hyperglycemia. We demonstrate that the network dynamics around the percolation transition explain the disease dynamics around onset, including a long time mystery in diabetes, the honeymoon phenomenon.

  16. Vanadyl sulfate treatment stimulates proliferation and regeneration of beta cells in pancreatic islets.

    PubMed

    Missaoui, Samira; Ben Rhouma, Khémais; Yacoubi, Mohamed-Tahar; Sakly, Mohsen; Tebourbi, Olfa

    2014-01-01

    We examined the effects of vanadium sulfate (VOSO4) treatment at 5 and 10 mg/kg for 30 days on endocrine pancreas activity and histology in nondiabetic and STZ-induced diabetic rats. In diabetic group, blood glucose levels significantly increased while insulinemia level markedly decreased. At the end of treatment, VOSO4 at a dose of 10 mg/Kg normalized blood glucose level in diabetic group, restored insulinemia, and significantly improved insulin sensitivity. VOSO4 also increased in a dose-dependent manner the number of insulin immunopositive beta cells in pancreatic islets of nondiabetic rats. Furthermore, in the STZ-diabetic group, the decrease in the number of insulin immunopositive beta cells was corrected to reach the control level mainly with the higher dose of vanadium. Therefore, VOSO4 treatment normalized plasma glucose and insulin levels and improved insulin sensitivity in STZ-experimental diabetes and induced beta cells proliferation and/or regeneration in normal or diabetic rats.

  17. Insulinotropic action of Citrullus colocynthis seed extracts in rat pancreatic islets.

    PubMed

    Benariba, Nabila; Djaziri, Rabeh; Hupkens, Emeline; Louchami, Karim; Malaisse, Willy J; Sener, Abdullah

    2013-01-01

    The present study aimed to investigate the direct in vitro effects of several distinct Citrullus colocynthis seed extracts on glucose-stimulated insulin release from pancreatic islets isolated from rats. Six extracts were tested, a crude aqueous, defatted aqueous, ethyl acetate, H2O-methanol and n-butanol extract and an extract containing a major component (fraction A) identified by gel chromatography in the ethyl acetate, n-butanol and H2O-methanol extracts. Under selected experimental conditions, the majority of extracts exhibited a positive insulinotropic action, at least when tested in the presence of 8.3 mM D-glucose. The concentration-response correlation observed with distinct extracts revealed the participation of distinct chemical compounds, including compounds with an inhibitory insulinotropic potential, in the modulation of the insulin secretory response to D-glucose. The results of the present study are relevant for further investigations which aim to identify compounds exhibiting positive insulinotropic actions. These agents may be suitable for the treatment of human diabetic subjects.

  18. Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

    PubMed Central

    Sharivkin, Revital; Walker, Michael D.; Soen, Yoav

    2015-01-01

    The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+), glucagon-producing alpha cells (CD9- /CD56+) and trypsin-producing acinar cells (CD9- /CD56-). This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples. PMID:25706282

  19. Interleukin-21 receptor-mediated signals control autoreactive T cell infiltration in pancreatic islets.

    PubMed

    Van Belle, Tom L; Nierkens, Stefan; Arens, Ramon; von Herrath, Matthias G

    2012-06-29

    It remains unclear how interleukin-21 receptor (IL-21R) contributes to type 1 diabetes. Here we have shown that dendritic cells (DCs) in the pancreas required IL-21R not for antigen uptake, but to acquire the chemokine receptor CCR7 and migrate into the draining lymph node. Consequently, less antigen, major histocompatibility complex (MHC) class II, and CD86 was provided to autoreactive effector cells in Il21r(-/-) mice, impairing CD4(+) T cell activation, CD40:CD40L interactions, and pancreatic infiltration by autoreactive T cells. CD40 crosslinking restored defective CD4(+) cell expansion and CD4 independently expanded autoreactive CD8(+) cells, but CD8(+) cells still required CD4(+) cells to reach the pancreas and induce diabetes. Diabetes induction by transferred T cells required IL-21R-sufficient host antigen-presenting cells. Transferring IL-21R-sufficient DCs broke diabetes resistance in Il21r(-/-) mice. We conclude that IL-21R controls both antigen transport by DCs and the crucial beacon function of CD4(+) cells for autoreactive CD8(+) cells to reach the islets.

  20. Pancreatic β-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species.

    PubMed

    Pilkington, Emily H; Gurzov, Esteban N; Kakinen, Aleksandr; Litwak, Sara A; Stanley, William J; Davis, Thomas P; Ke, Pu Chun

    2016-02-16

    Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders.

  1. Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis.

    PubMed

    Kumar, Divya P; Asgharpour, Amon; Mirshahi, Faridoddin; Park, So Hyun; Liu, Sichen; Imai, Yumi; Nadler, Jerry L; Grider, John R; Murthy, Karnam S; Sanyal, Arun J

    2016-03-25

    The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic β cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases β cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic β cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and β cells by switching from glucagon to GLP-1 to restore β cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus. PMID:26757816

  2. Cooperation by Fibroblasts and Bone Marrow-Mesenchymal Stem Cells to Improve Pancreatic Rat-to-Mouse Islet Xenotransplantation

    PubMed Central

    Meana, Alvaro; Otero, Jesus; Esteban, Manuel M.

    2013-01-01

    Experimental and clinical experiences highlight the need to review some aspects of islet transplantation, especially with regard to site of grafting and control of the immune response. The subcutaneous space could be a good alternative to liver but its sparse vasculature is its main limitation. Induction of graft tolerance by using cells with immunoregulatory properties is a promising approach to avoid graft rejection. Both Fibroblasts and Mesenchymal Stem Cells (MSCs) have shown pro-angiogenic and immunomodulatory properties. Transplantation of islets into the subcutaneous space using plasma as scaffold and supplemented with fibroblasts and/or Bone Marrow-MSCs could be a promising strategy to achieve a functional extra-hepatic islet graft, without using immunosuppressive drugs. Xenogenic rat islets, autologous fibroblasts and/or allogenic BM-MSCs, were mixed with plasma, and coagulation was induced to constitute a Plasma-based Scaffold containing Islets (PSI), which was transplanted subcutaneously both in immunodeficient and immunocompetent diabetic mice. In immunodeficient diabetic mice, PSI itself allowed hyperglycemia reversion temporarily, but the presence of pro-angiogenic cells (fibroblasts or BM-MSCs) within PSI was necessary to improve graft re-vascularization and, thus, consistently maintain normoglycemia. In immunocompetent diabetic mice, only PSI containing BM-MSCs, but not those containing fibroblasts, normalized glycemia lasting up to one week after transplantation. Interestingly, when PSI contained both fibroblasts and BM-MSCs, the normoglycemia period showed an increase of 4-times with a physiological-like response in functional tests. Histology of immunocompetent mice showed an attenuation of the immune response in those grafts with BM-MSCs, which was improved by co-transplantation with fibroblasts, since they increased BM-MSC survival. In summary, fibroblasts and BM-MSCs showed similar pro-angiogenic properties in this model of islet

  3. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    SciTech Connect

    Cline, Gary W.; Zhao, Xiaojian; Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L.

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  4. Downregulation of Type II Diabetes Mellitus and Maturity Onset Diabetes of Young Pathways in Human Pancreatic Islets from Hyperglycemic Donors

    PubMed Central

    Groop, Leif

    2014-01-01

    Although several molecular pathways have been linked to type 2 diabetes (T2D) pathogenesis, it is uncertain which pathway has the most implication on the disease. Changes in the expression of an entire pathway might be more important for disease pathogenesis than changes in the expression of individual genes. To identify the molecular alterations in T2D, DNA microarrays of human pancreatic islets from donors with hyperglycemia (n = 20) and normoglycemia (n = 58) were subjected to Gene Set Enrichment Analysis (GSEA). About 178 KEGG pathways were investigated for gene expression changes between hyperglycemic donors compared to normoglycemic. Pathway enrichment analysis showed that type II diabetes mellitus (T2DM) and maturity onset diabetes of the young (MODY) pathways are downregulated in hyperglycemic donors, while proteasome and spliceosome pathways are upregulated. The mean centroid of gene expression of T2DM and MODY pathways was shown to be associated positively with insulin secretion and negatively with HbA1c level. To conclude, downregulation of T2DM and MODY pathways is involved in islet function and might be involved in T2D. Also, the study demonstrates that gene expression profiles from pancreatic islets can reveal some of the biological processes related to regulation of glucose hemostats and diabetes pathogenesis. PMID:25379510

  5. Heterogeneity and nearest-neighbor coupling can explain small-worldness and wave properties in pancreatic islets.

    PubMed

    Cappon, Giacomo; Pedersen, Morten Gram

    2016-05-01

    Many multicellular systems consist of coupled cells that work as a syncytium. The pancreatic islet of Langerhans is a well-studied example of such a microorgan. The islets are responsible for secretion of glucose-regulating hormones, mainly glucagon and insulin, which are released in distinct pulses. In order to observe pulsatile insulin secretion from the β-cells within the islets, the cellular responses must be synchronized. It is now well established that gap junctions provide the electrical nearest-neighbor coupling that allows excitation waves to spread across islets to synchronize the β-cell population. Surprisingly, functional coupling analysis of calcium responses in β-cells shows small-world properties, i.e., a high degree of local coupling with a few long-range "short-cut" connections that reduce the average path-length greatly. Here, we investigate how such long-range functional coupling can appear as a result of heterogeneity, nearest-neighbor coupling, and wave propagation. Heterogeneity is also able to explain a set of experimentally observed synchronization and wave properties without introducing all-or-none cell coupling and percolation theory. Our theoretical results highlight how local biological coupling can give rise to functional small-world properties via heterogeneity and wave propagation. PMID:27249943

  6. Oxytocin-like immunoreactive nerves are associated with insulin-containing cells in pancreatic islets of anglerfish (Lophius americanus).

    PubMed

    McDonald, J K; Greiner, F; Wood, J G; Noe, B D

    1987-07-01

    Recent reports indicate that oxytocin exerts direct effects on the release of insulin and glucagon from the endocrine pancreas of the rat. The purpose of this study was to determine whether oxytocin-like immunoreactivity is present in the anglerfish islet, and if it is associated with subsets of hormone-producing cells. Antisera against oxytocin, insulin, glucagon, somatostatin, neuropeptide Y, and the 200-kd neurofilament polypeptide were applied to serial 5 micrometers sections of pancreatic islets. The antiserum to the 200-kd neurofilament polypeptide labeled nerve bundles and axons, some of which were also stained with the oxytocin antiserum. Oxytocin immunoreactivity was observed in large nerves that branched into varicose fibers. These fibers were consistently associated only with clusters of insulin-producing cells. Successive application of oxytocin and insulin antisera to the same section provided additional verification of this relationship. Oxytocin-labeled nerves were not associated with cells immunoreactive to glucagon, somatostatin, or neuropeptide Y (anglerfish peptide Yg). The results demonstrate that oxytocin or an oxytocin-like peptide is located in fibers that surround only insulin-producing cells in the anglerfish islet. Although the functional significance of this observation remains to be determined, the results imply that oxytocin, or an oxytocin-like peptide, may affect the synthesis or release of insulin from anglerfish islets. PMID:3304646

  7. Heterogeneity and nearest-neighbor coupling can explain small-worldness and wave properties in pancreatic islets

    NASA Astrophysics Data System (ADS)

    Cappon, Giacomo; Pedersen, Morten Gram

    2016-05-01

    Many multicellular systems consist of coupled cells that work as a syncytium. The pancreatic islet of Langerhans is a well-studied example of such a microorgan. The islets are responsible for secretion of glucose-regulating hormones, mainly glucagon and insulin, which are released in distinct pulses. In order to observe pulsatile insulin secretion from the β-cells within the islets, the cellular responses must be synchronized. It is now well established that gap junctions provide the electrical nearest-neighbor coupling that allows excitation waves to spread across islets to synchronize the β-cell population. Surprisingly, functional coupling analysis of calcium responses in β-cells shows small-world properties, i.e., a high degree of local coupling with a few long-range "short-cut" connections that reduce the average path-length greatly. Here, we investigate how such long-range functional coupling can appear as a result of heterogeneity, nearest-neighbor coupling, and wave propagation. Heterogeneity is also able to explain a set of experimentally observed synchronization and wave properties without introducing all-or-none cell coupling and percolation theory. Our theoretical results highlight how local biological coupling can give rise to functional small-world properties via heterogeneity and wave propagation.

  8. Secretagogue-induced diacylglycerol accumulation in isolated pancreatic islets. Mass spectrometric characterization of the fatty acyl content indicates multiple mechanisms of generation

    SciTech Connect

    Wolf, B.A.; Easom, R.A.; Hughes, J.H.; McDaniel, M.L.; Turk, J. )

    1989-05-16

    Diacylglycerol accumulation has been examined in secretagogue-stimulated pancreatic islets with a newly developed negative ion chemical ionization mass spectrometric method. The muscarinic agonist carbachol induces islet accumulation of diacylglycerol rich in arachidonate and stearate, and a parallel accumulation of {sup 3}H-labeled diacylglycerol occurs in carbachol-stimulated islets that had been prelabeled with ({sup 3}H)glycerol. Islets so labeled do not accumulate {sup 3}H-labeled diacylglycerol in response to D-glucose, but D-glucose does induce islet accumulation of diacylglycerol by mass. This material is rich in palmitate and oleate and contains much smaller amounts of arachidonate. Neither secretagogue influences triacylglycerol labeling, and neither induces release of ({sup 3}H)choline or ({sup 3}H)phosphocholine from islets prelabeled with ({sup 3}H)choline. These observations indicate that the diacylglycerol that accumulates in islets in response to carbachol arises from hydrolysis of glycerolipids, probably including phosphoinositides. The bulk of the diacylglycerol which accumulates in response to glucose does not arise from glycerolipid hydrolysis and must therefore reflect de novo synthesis. The endogenous diacylglycerol which accumulates in secretagogue-stimulated islets may participate in insulin secretion because exogenous diacylglycerol induces insulin secretion from islets, and an inhibitor of diacylglycerol metabolism to phosphatidic acid augments glucose-induced insulin secretion.

  9. KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

    PubMed Central

    Deeney, Jude T.; Corkey, Barbara E.

    2015-01-01

    Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion. PMID:26444014

  10. Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression

    PubMed Central

    Chowdhury, Subrata; Grimm, Larson; Gong, Ying Jia Kate; Wang, Beixi; Li, Bing; Srikant, Coimbatore B.; Gao, Zu-hua; Liu, Jun-Li

    2015-01-01

    We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion. PMID:26305481

  11. Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression.

    PubMed

    Chowdhury, Subrata; Grimm, Larson; Gong, Ying Jia Kate; Wang, Beixi; Li, Bing; Srikant, Coimbatore B; Gao, Zu-hua; Liu, Jun-Li

    2015-01-01

    We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

  12. Quantitative analysis of cell composition and purity of human pancreatic islet preparations.

    PubMed

    Pisania, Anna; Weir, Gordon C; O'Neil, John J; Omer, Abdulkadir; Tchipashvili, Vaja; Lei, Ji; Colton, Clark K; Bonner-Weir, Susan

    2010-11-01

    Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light microscopy (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed an accurate identification and quantification of cell types with measured cell number fractions (mean±s.e.m.) of 35.6±2.1% β-cells, 12.6±1.0% non-β-islet cells (48.3±2.6% total islet cells), 22.7±1.5% duct cells, and 25.3±1.8% acinar cells. Of the islet cells, 73.6±1.7% were β-cells. For comparison with the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, β-cells were 86.5±1.1% of the total islet cell volume and 61.2±0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates produced 1560±20 cells in an islet equivalent (volume of 150-μm diameter sphere), of which 1140±15 were β-cells. To test whether LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction of the islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analyses correlated linearly with excellent agreement (R²=0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islet preparations and can help us understand many of the discrepancies of islet composition in the literature. PMID:20697378

  13. Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function.

    PubMed

    Lin, Huei-Min; Lee, Ji-Hyeon; Yadav, Hariom; Kamaraju, Anil K; Liu, Eric; Zhigang, Duan; Vieira, Anthony; Kim, Seong-Jin; Collins, Heather; Matschinsky, Franz; Harlan, David M; Roberts, Anita B; Rane, Sushil G

    2009-05-01

    Pancreatic islet beta-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate beta-cell function is of immense clinical relevance. Transforming growth factor (TGF)-beta signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-beta pathway in regulation of insulin gene transcription and beta-cell function. We identify insulin as a TGF-beta target gene and show that the TGF-beta signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-beta signaling regulates expression of genes involved in beta-cell function. Together, these studies emphasize TGF-beta/Smad3 signaling as an important regulator of insulin gene transcription and beta-cell function and suggest that components of the TGF-beta signaling pathway may be dysregulated in diabetes.

  14. What difference exists in the pancreas of mammals with sanguivorous diet? A morphological, stereological and immunohistochemical study of the pancreatic islets of the hematophagous bat Diphylla ecaudata.

    PubMed

    Machado-Santos, Clarice; Aquino, Júlio César Fraulob; Mikalauka, Jefferson Simanas; Abidu-Figueiredo, Marcelo; Mendes, Rosa Maria Marcos; Sales, Armando

    2013-05-10

    Diphylla ecaudata is a vampire bat that mainly feeds on the blood of birds. This highly specialized diet - hematophagy - is accompanied by a series of morphological changes in the gastro-entero-pancreatic system, since the distribution and relative proportions of different pancreatic endocrine cell types can vary between species due to different physiological conditions and eating habits. The aim of this study was to examine for the first time the pancreas of the vampire bat D. ecaudata using morphological, stereological and immunohistochemical techniques. The pancreas of the D. ecaudata has an exocrine acinar portion in which the highest concentration of pancreatic islets is scattered. These pancreatic islets have irregular size and a mean diameter of 56.94 μm. The total number of islets in the pancreas was 23,900, with a volumetric density of 4.1%. Insulin-immunoreactive (IR) cells were located in the central pancreatic islet region and had the largest density (54.8%). Glucagon-IR cells were located mainly in the peripheral mantle region (16.2%), along with somatostatin-IR (SS) cells (14.3%). Cells immunoreactive to insulin, glucagon and somatostatin were also observed to have spread in isolated places in the exocrine pancreas. In the connective tissue near the pancreatic ducts, a high concentration was identified of insulin-IR cells and a low concentration of glucagon-IR and somatostatin-IR cells. These results indicate that although the pancreas of D. ecaudata has morphological similarities with that of other mammals, it has a differentiated islet structure, because there were a large number of islets and different volumetric densities of α, β and δ cells. PMID:23500834

  15. Effect of C-peptide Alone or in Combination with Nicotinamide on Insulin Levels from Pancreatic Islets in Mouse

    PubMed Central

    Ahangarpour, Akram; Ali Akbari, Fatemeh Ramezani; Moghadam, Hadi Fathi

    2016-01-01

    Background Both c-peptide and nicotinamide are known to increase blood insulin in diabetes. In the present study, we examined the effect of c-peptide alone or in combination with nicotinamide on insulin levels in pancreatic islets in mice. Methods This study was conducted with 60 adult male Naval Medical Research Institute (NMARI) mice weighing 25 to 30 g. Pancreatic islets from normal mice were isolated by the collagenase digestion method. Mice were divided into ten groups of six (n = 6): control, glyburide (1 and 10 μM), C-peptide (50 and 100 nM), nicotinamide (10, 25, and 100 mM), nicotinamide + C-peptide (100 mM and 100 nM), and buffer in different glucose concentrations (2.8, 5.6, and 16.7 mM). Insulin secretion was measured using insulin radioimmunoassay method. Results Insulin secretion significantly increased at 16.7 mM glucose concentration compared with 2.8 and 5.6 mM glucose concentrations. Incubation of islets at 2.8 and 5.6 mM glucose concentrations and nicotinamide + C-peptide, nicotinamide 25 and 100 mM, and C-peptide 100 nM significantly increased insulin secretion compared with the control group. In addition, incubation of islets at 16.7 mM glucose with nicotinamide + C-peptide significantly increased insulin secretion. Glyburide at 10 μM concentration was more effective than nicotinamide at 10 and 100 mM, C-peptide 50 and 100 nM in the presence of 16.7 mM glucose concentration. However, the combination of nicotinamide + C-peptide was more effective than glyburide at a concentration of 10 μM in the presence of a 16.7 mM glucose concentration. Conclusions This paper suggests that c-peptide, nicotinamide, and the combination of c-peptide and nicotinamide in-creases insulin secretion from pancreatic islets. PMID:27540321

  16. A stereological study of effects of aqueous extract of Tamarindus indica seeds on pancreatic islets in streptozotocin-induced diabetic rats.

    PubMed

    Hamidreza, Hamidreza; Heidari, Zahra; Shahraki, Mohammadreza; Moudi, Bita

    2010-10-01

    Tamarindus indica Linn was used as a traditional medicine for the management of diabetes mellitus in human and experimental animals. This study investigated effects of aqueous extract of Tamarindus indica seeds (AETIS) against STZ-induced damages in pancreatic islands by means of stereological methods. sixty matured normoglycemic male Wistar rats, weighing 200-250 gr, were selected and randomly divided into 6 groups (n=10). Control, STZ-induced diabetic; by intraperitoneal injection of 55 mg/Kg streptozotocin, Treated control group (TC); received AETIS at a dose of 200mg/kg/day, and AETIS treated diabetic groups (TD1-3); received respectively AETIS at the dose of 50, 100,and 200 mg/kg/day by gavage from one week after induction of diabetes by STZ. After 8 weeks of experiment, stereological estimation of volume density and total volume of islets and beta cells, volume weighted mean islets volume, mass of beta cells, islets, and pancreas and total number of islets were done. Volume density and total volume of islets, volume weighted mean islets volume, volume density islets/pancreas, volume density beta cells/islet, mass of islets and pancreas of treated diabetic groups (TD1-3) were significantly higher than untreated diabetic group (P<0.001), and in TD3 group these values were comparable to controls. Although total volume and mass of beta cells in TD1-3 were significantly higher than D group but they were significantly lower than control group (P>0.05). Total number of islets, pancreas wet weight and volume did not show any significant changes between control and experimental groups (P>0.05). Results suggested that AETIS partially restores pancreatic beta cells and repairs STZ-induced damages in rats.

  17. Layer-by-Layer Assembly of a Conformal Nanothin PEG Coating for Intraportal Islet Transplantation

    PubMed Central

    Wilson, John T.; Cui, Wanxing; Chaikof, Elliot L.

    2009-01-01

    Encapsulation of cells and tissue offers a rational approach for attenuating deleterious host responses toward transplanted cells, but a need exists to develop cell encapsulation strategies that minimize transplant volume. In this report, we describe the formation of nanothin, PEG-rich conformal coatings on individual pancreatic islets via layer-by-layer self-assembly of poly(l-lysine)-g-poly(ethylene glycol)(biotin) (PPB) and streptavidin (SA). Through control of grafting ratio, PPB could be rendered nontoxic and facilitated growth of PPB/SA multilayer thin films that conformed to the heterogeneous islet surface. (PPB/SA)8 multilayer films could be assembled without loss of islet viability or function, and coated islets performed comparably to untreated controls in vivo in a murine model of allogenic intraportal islet transplantation. PMID:18547122

  18. Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.

    PubMed

    Fueger, Patrick T; Schisler, Jonathan C; Lu, Danhong; Babu, Daniella A; Mirmira, Raghavendra G; Newgard, Christopher B; Hohmeier, Hans E

    2008-05-01

    Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.

  19. Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media.

    PubMed

    Holmes, M A; Clayton, H A; Chadwick, D R; Bell, P R; London, N J; James, R F

    1995-10-27

    There have been no extensive studies investigating the effect of tissue culture media on the in vitro functional characteristics of rat, porcine and human Islets of Langerhans. We therefore aimed to compare ten commercially available tissue culture media on the basis of their ability to maintain islet viability. Following isolation, islets were cultured free-floating in the ten media (RPMI 1640-11mM glucose (control), RPMI 1640-2.2mM glucose, Dulbecco's MEM, TCM 199, CMRL 1066, Iscove's MEM, Waymouth's MEM, Serum-Free medium, Ex-cell 300, Ham's F-12) and viability was assessed after 24 hr, 3 days, and 7 days on the basis of macroscopic appearance, cell membrane integrity, and insulin secretion in response to glucose stimulation both by dynamic incubation and by perifusion. Each islet species demonstrated physiological insulin release characteristics in all media--however, it was possible to distinguish between the media by comparing the stimulation indices calculated from the insulin release studies. Significantly higher stimulation indices were produced in Iscove's MEM for rat islets, in Ham's F-12 for porcine islets and in CMRL 1066 for human islets. Over the entire culture period a significant deterioration in function was observed in all species cultured in the control media, although this was reversed when islets were cultured in the optimal media. Furthermore, in the case of porcine and human islets a significant improvement in function over the seven-day period was noted in the optimal media. In conclusion, of the commercially available media, the optimal tissue culture medium for rat islets is Iscove's MEM, for porcine islets is Ham's F-12, and for human islets is CMRL 1066. PMID:7482747

  20. Islet Autoimmunity Identifies a Unique Pattern of Impaired Pancreatic Beta-Cell Function, Markedly Reduced Pancreatic Beta Cell Mass and Insulin Resistance in Clinically Diagnosed Type 2 Diabetes

    PubMed Central

    Subauste, Angela; Gianani, Roberto; Chang, Annette M.; Plunkett, Cynthia; Pietropaolo, Susan L.; Zhang, Ying-Jian; Barinas-Mitchell, Emma; Kuller, Lewis H.; Galecki, Andrzej; Halter, Jeffrey B.; Pietropaolo, Massimo

    2014-01-01

    There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired β-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic β-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the

  1. Impact of adverse pancreatic injury at surgical procurement upon islet isolation outcome.

    PubMed

    Andres, Axel; Kin, Tatsuya; O'Gorman, Doug; Bigam, David; Kneteman, Norman; Senior, Peter; Shapiro, Am James

    2014-11-01

    The consequence of a pancreas injury during the procurement for islet isolation purpose is unknown. The goal of this work was to assess the injuries of the pancreata procured for islet isolation, and to determine their effect on the islet yield. Between January 2007 and October 2013, we prospectively documented every injury of the pancreata processed in our centre for islet isolation. Injuries involving the main duct were classified as major, the others as minor. Donors' characteristics and islet yields were compared between the groups of injuries. A pancreas injury was identified in 42 of 452 pancreata received for islet isolation (9.3%). In 15 cases, the injury was major (3.3% of all pancreata). Although a minor injury did not affect the islet yield, a major injury was significantly associated with unfavourable outcomes (postpurification mean islet equivalent of 364 ± 181, 405 ± 190 and 230 ± 115 × 10(3) for absence of injury, minor injury and major injury, respectively). A major injury was significantly more prevalent in lean and short donors. We recommend assessing the quality of the pancreas in the islet isolation centre before starting the isolation procedure. Each centre should determine its own policy based on its financial resources and on the wait list.

  2. Magnetic resonance imaging and biological properties of pancreatic islets labeled with iron oxide nanoparticles.

    PubMed

    Kim, Hoe Suk; Choi, YoonSeok; Song, In Chan; Moon, Woo Kyung

    2009-10-01

    This study was undertaken to investigate the in vitro effect of islet labeling with iron oxide nanoparticles for MRI on islet viability, insulin secretion, and gene expression. Isolated rat islets were labeled with Resovist (25-200 microg Fe/mL, a clinically approved MRI contrast agent) in the presence or absence of poly-l-Lysine (PLL, 1.5 microg/mL) for 48 h. The iron content of labeled islets was found to increase in a dose-dependent manner. More than 90% of the islets were labeled with 100 microg Fe/mL. We confirmed the localizations of iron oxide nanoparticles within islet beta-cells by insulin immunostaining. As the concentration of Resovist increased, T(2) values as determined by T(2)-weighted MRI on a 1.5 Tesla MR scanner decreased. Labeling of 100 islets in a medium containing 100 microg Fe/mL of Resovist in the absence of PLL provided sufficient contrast for islet visualization on T(2)-weighted MRI. MTT assays showed that the viability of labeled islets was not different from that of unlabeled islets. No statistical difference was observed between labeled (2.91 +/- 0.36) and unlabeled islets (2.83 +/- 0.61) in terms of the ability to secrete insulin, as determined by the glucose stimulation index. We also evaluated the effect of iron oxide incorporation on the gene expressions in islet cells using RT-PCR (reverse transcriptase PCR). Insulin expression in labeled islets was significantly elevated (1.83 +/- 0.25 fold vs. unlabeled; p = 0.005), but not the expression of somatostatin (1.39 +/- 0.18 fold vs. unlabeled; p = 0.085) or glucagons (1.28 +/- 0.13 fold vs. unlabeled; p = 0.09). Expression of an important transcription factor for insulin gene transcription, BETA2 (beta-cell E-box trans-activator), was increased in labeled islets (1.67 +/- 0.15 fold vs. unlabeled; p = 0.029). The findings of this study indicate that Resovist provides a satisfactory means to image islets and has no deleterious effect on islet function or gene expression.

  3. Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia

    PubMed Central

    Rodriguez-Brotons, A.; Bietiger, W.; Peronet, C.; Magisson, J.; Sookhareea, C.; Langlois, A.; Mura, C.; Jeandidier, N.; Pinget, M.; Sigrist, S.; Maillard, E.

    2016-01-01

    In bioartificial pancreases (BP), the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2) in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ)/cm2) and cultured in normal atmospheric pressure (160 mmHg) as well as hypoxic conditions (15 mmHg) for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance. PMID:26824040

  4. Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia.

    PubMed

    Rodriguez-Brotons, A; Bietiger, W; Peronet, C; Magisson, J; Sookhareea, C; Langlois, A; Mura, C; Jeandidier, N; Pinget, M; Sigrist, S; Maillard, E

    2016-01-01

    In bioartificial pancreases (BP), the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2) in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ)/cm(2)) and cultured in normal atmospheric pressure (160 mmHg) as well as hypoxic conditions (15 mmHg) for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance. PMID:26824040

  5. Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

    PubMed

    Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

    2016-08-01

    Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

  6. Role of glucose in IRS signaling in rat pancreatic islets: specific effects and interplay with insulin.

    PubMed

    Paris, Maryline; Bernard-Kargar, Catherine; Vilar, José; Kassis, Nadim; Ktorza, Alain

    2004-01-01

    We investigated the possible interplay between insulin and glucose signaling pathways in rat pancreatic beta-cell with a special focus on the role of glucose in IRS signaling in vivo. Three groups of rats were constituted by combining simultaneous infusion during 48 h either of glucose and/or insulin, or glucose+diazoxide: Hyperglycemic-Hyperinsulinemic (HGHI), euglycemic-Hyperinsulinemic (eGHI), Hyperglycemic-euinsulinemic (HGeI). Control rats were infused with 0,9%NaCl. In HGHI and HGeI rats plasma glucose levels were maintained at 20-22 mmol/l. In eGHI rats, plasma glucose was not different from that of controls, whereas plasma insulin was much higher than in controls. In HGHI rats, IRS-2 mRNA expression, total protein and phosphorylated protein amounts were increased compared to controls. In HGeI rats, only IRS-2 mRNA expression was increased. No change was observed in eGHI rats whatever the parameter considered. In all groups, mRNA concentration of IRS-1 was similar to that of controls. The quantity of total and phosphorylated IRS-1 protein was dramatically increased in HGHI rats and to a lesser extent in eGHI rats. Neither mRNA nor IRS-1 protein expression were modified in HGeI rats. The data suggest that glucose and insulin play at once a specific and a complementary role in islet IRSs signaling. Especially, glucose stimulates IRS-2 mRNA expression whatever the insulin status and independently of the secretory process. The differential regulation of IRS-1 and IRS-2 expressions is in agreement with their supposed different involvement in the control of beta-cell growth and function.

  7. Pro-inflammatory and pro-oxidant status of pancreatic islet in vitro is controlled by TLR-4 and HO-1 pathways.

    PubMed

    Vivot, Kevin; Langlois, Allan; Bietiger, William; Dal, Stéphanie; Seyfritz, Elodie; Pinget, Michel; Jeandidier, Nathalie; Maillard, Elisa; Gies, Jean-Pierre; Sigrist, Séverine

    2014-01-01

    Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation. PMID:25343247

  8. Co-Transplantation of Endothelial Progenitor Cells and Pancreatic Islets to Induce Long-Lasting Normoglycemia in Streptozotocin-Treated Diabetic Rats

    PubMed Central

    Spiga, Saturnino; Mazzanti, Benedetta; Curcio, Michele; Mulas, Giovanna; Diana, Marco; Marzola, Pasquina; Mosca, Franco; Longoni, Biancamaria

    2014-01-01

    Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3–5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards. PMID:24733186

  9. The CB1 antagonist rimonabant decreases insulin hypersecretion in rat pancreatic islets.

    PubMed

    Getty-Kaushik, Lisa; Richard, Ann-Marie T; Deeney, Jude T; Krawczyk, Sarah; Shirihai, Orian; Corkey, Barbara E

    2009-10-01

    Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose-stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL-treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL-treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL-exposed islets. PMID:19644453

  10. Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS

    SciTech Connect

    Metz, Thomas O.; Jacobs, Jon M.; Gritsenko, Marina A.; Fontes, Ghislaine; Qian, Weijun; Camp, David G.; Poitout, Vincent J.; Smith, Richard D.

    2006-12-01

    Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.

  11. Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

    PubMed

    Taylor, Michael J; Baicu, Simona C

    2014-01-01

    Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze

  12. Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

    PubMed

    Taylor, Michael J; Baicu, Simona C

    2014-01-01

    Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze

  13. Hormone-sensitive lipase deficiency suppresses insulin secretion from pancreatic islets of Lep{sup ob/ob} mice

    SciTech Connect

    Sekiya, Motohiro; Yahagi, Naoya; Tamura, Yoshiaki; Okazaki, Hiroaki; Igarashi, Masaki; Ohta, Keisuke; Takanashi, Mikio; Kumagai, Masayoshi; Takase, Satoru; Nishi, Makiko; Takeuchi, Yoshinori; Izumida, Yoshihiko; Kubota, Midori; Ohashi, Ken; Iizuka, Yoko; Yagyu, Hiroaki; Gotoda, Takanari; Nagai, Ryozo; Shimano, Hitoshi; Yamada, Nobuhiro; and others

    2009-09-25

    It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic {beta}-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL (Lep{sup ob/ob}/HSL{sup -/-}) and explored the role of HSL in pancreatic {beta}-cells in the setting of obesity. Lep{sup ob/ob}/HSL{sup -/-} developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep{sup ob/ob}/HSL{sup +/+} in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep{sup +/+} background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep{sup ob/ob} islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep{sup ob/ob} mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.

  14. Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.

    PubMed

    Buckingham, R E; Al-Barazanji, K A; Toseland, C D; Slaughter, M; Connor, S C; West, A; Bond, B; Turner, N C; Clapham, J C

    1998-08-01

    Rosiglitazone (BRL 49653), a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist and potent insulin action-enhancing agent, was given in the diet (50 micromol/kg of diet) to male Zucker rats ages 6-7 weeks for 9 months (prevention group). In this treatment mode, rosiglitazone prolonged the time to onset of proteinuria from 3 to 6 months and markedly reduced the rate of its subsequent progression. Progression was also retarded when treatment was commenced (intervention group) after proteinuria had become established (4 months; ages 24-25 weeks). In either treatment mode, rosiglitazone normalized urinary N-acetyl-beta-D-glucosaminidase activity, a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure that occurred coincidentally with the development of proteinuria in Zucker fatty control rats. The renal protective action of rosiglitazone was verified morphologically. Thus in the prevention group there was an absence of the various indexes of chronic nephropathy that were prominent in the Zucker fatty control group, namely, glomerulosclerosis, dilated tubules containing proteinaceous casts, a loss of functional microvilli on the tubular epithelium, and varying degrees of chronic interstitial nephritis. An intermediate pathology was observed in the intervention group. Also, pancreatic islet hyperplasia, ultrastructural evidence of beta-cell work hypertrophy, and derangement of alpha-cell distribution within the islet were prominent features of Zucker fatty control rats, but these adaptive changes were ameliorated (intervention group) or prevented (prevention group) by rosiglitazone treatment. These data demonstrate that treatment of Zucker fatty rats with rosiglitazone produced substantial protection over a prolonged period against the development and progression of renal injury and the adaptive changes to pancreatic islet morphology caused by sustained hyperinsulinemia. PMID:9703335

  15. Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans.

    PubMed Central

    Srivastava, Meera; Eidelman, Ofer; Leighton, Ximena; Glasman, Mirta; Goping, Gertrude; Pollard, Harvey B.

    2002-01-01

    BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control

  16. New insights into the role of connexins in pancreatic islet function and diabetes

    PubMed Central

    Farnsworth, Nikki L.; Benninger, Richard K.P.

    2014-01-01

    Multi-cellular systems require complex signaling mechanisms for proper tissue function, to mediate signaling between cells in close proximity and at distances. This holds true for the islets of Langerhans, which are multicellular micro-organs located in the pancreas responsible for glycemic control, through secretion of insulin and other hormones. Coupling of electrical and metabolic signaling between islet β-cells is required for proper insulin secretion and effective glycemic control. β-cell specific coupling is established through gap junctions composed of connexin36, which results in coordinated insulin release across the islet. Islet connexins have been implicated in both Type-1 and Type-2 diabetes; however a clear link remains to be determined. The goal of this review is to discuss recent discoveries regarding the role of connexins in regulating insulin secretion, the regulation of connexins within the islet, and recent studies which support a role for connexins in diabetes. Further studies which investigate the regulation of connexins in the islet and their role in diabetes may lead to novel diabetes therapies which regulate islet function and β-cell survival through modulation of gap junction coupling. PMID:24583073

  17. Pancreatic islet transplantation in cynomolgus monkeys. Initial studies and evidence that cyclosporine impairs glucose tolerance in normal monkeys.

    PubMed

    Stegall, M D; Chabot, J; Weber, C; Reemtsma, K; Hardy, M A

    1989-12-01

    Using a model of streptozotocin-induced, ketosis-prone, insulin-dependent diabetes mellitus (IDDM) in the cynomolgus monkey, we performed 11 intraportal transplants of collagenase-digested, Ficoll-purified pancreatic islets (9 ABO-compatible allografts and 2 concordant baboon xenografts). Islets were pretreated with ultraviolet-B irradiation and recipients received cyclosporine A immunosuppression. Two grafts never functioned, five grafts showed evidence of partial function, and four grafts (three allografts and one xenograft) showed evidence of good function, with the animals independent of exogenous insulin with morning fasting blood glucose levels less than 200 mg/dl. Because two grafts functioned only after CsA was either tapered or discontinued, we performed a related study that showed that therapeutic doses of CsA (morning trough serum level 150-250 ng/ml) impaired intravenous glucose tolerance tests (IVGTT) of normal monkeys and may contributed to graft dysfunction in our islet transplantation model. The results show that there is a decrease in release of serum insulin during an IVGTT leading to impairment of glucose utilization, while serum glucagon remains unaffected. After cessation of CsA, the IVGTT did not return to normal for 28 days. Oral glucose tolerance tests were unaffected in CsA-treated monkeys. These initial studies show that the streptozotocin-diabetic monkey is a valuable model to study IDDM and islet transplantation in nonhuman primates. We also confirm studies in rodents, dogs, and sheep by showing that CsA partially inhibits beta cell function in normal monkeys.

  18. Human fetal pancreatic islet-like structures as source material to treat type 1 diabetes.

    PubMed

    Ikeda, Yasuhiro; Kudva, Yogish C

    2013-01-01

    The incidence of type 1 diabetes is increasing worldwide. Current therapy continues to be suboptimal. An exciting therapeutic advance in the short term is closed loop technology development and application. However, cell and tissue therapy continues to be an unmet need for the disorder. Human islets isolated from deceased donors will be clinically available to treat type 1 diabetes within the next 1 to 2 years. Other approaches such as xenotransplantation and islet products derived from human embryonic stem cells and induced pluripotent stem cells are currently being pursued. The current commentary provides context and discusses future endeavors for transplantation of islet-like structures derived from fetal pancreas. PMID:24377429

  19. Comparison of therapeutic characteristics of islet cell transplantation simultaneous with pancreatic mesenchymal stem cell transplantation in rats with Type 1 diabetes mellitus.

    PubMed

    Unsal, Ilknur Ozturk; Ginis, Zeynep; Pinarli, Ferda Alparslan; Albayrak, Aynur; Cakal, Erman; Sahin, Mustafa; Delibasi, Tuncay

    2015-06-01

    Although, pancreas islet call transplantation is a new, promising method for type 1 diabetic patients, it remains as an experimental procedure applied in selected patients. The present study aimed to investigate effect of pancreatic mesenchymal stem cell transplantation simultaneous with islet cell transplantation on islet liveliness and thus on the treatment of diabetes in type 1 diabetic rats. The study used Wistar Albino Rats and was performed in a total of four groups [control (G1), mesenchymal stem cell (G2), islet (G3) and islet + mesencymal stem cell (G4)] each including 8 rats. Blood glucose level of the rats, in which diabetes model has been created using streptozotocin, was measured after 72 h. Blood samples were obtained from the rats 30 days after transplantation and then, their livers and pancreases were kept in 10% formaldehyde and the experiment was ended. Following staining with H&E, they were morphologically evaluated under a light microscope. Change in mean blood glucose level was statistically significant in G3 and G4 versus G1 and G2 (p = 0.001, p < 0.001, p < 0.001, and p < 0.001 respectively). Histological examination revealed that mean number of islet cells in the pancreases of the rats was higher in G4; difference between the groups was statistically significant (p < 0.001). Transplantation of islet cells together with mesenchymal stem cells showed beneficial effects in terms of prolonging survival of islet grafts suggesting that transplantation of mesenchymal stem cells together with islet cells during clinical islet transplantation may be beneficial in increasing the number of noninsulin-dependent patients in Type 1 diabetes.

  20. Characterization of pancreatic islets in two selectively bred mouse lines with different susceptibilities to high-fat diet-induced glucose intolerance.

    PubMed

    Nagao, Mototsugu; Asai, Akira; Inaba, Wataru; Kawahara, Momoyo; Shuto, Yuki; Kobayashi, Shunsuke; Sanoyama, Daisuke; Sugihara, Hitoshi; Yagihashi, Soroku; Oikawa, Shinichi

    2014-01-01

    Hereditary predisposition to diet-induced type 2 diabetes has not yet been fully elucidated. We recently established 2 mouse lines with different susceptibilities (resistant and prone) to high-fat diet (HFD)-induced glucose intolerance by selective breeding (designated selectively bred diet-induced glucose intolerance-resistant [SDG-R] and -prone [SDG-P], respectively). To investigate the predisposition to HFD-induced glucose intolerance in pancreatic islets, we examined the islet morphological features and functions in these novel mouse lines. Male SDG-P and SDG-R mice were fed a HFD for 5 weeks. Before and after HFD feeding, glucose tolerance was evaluated by oral glucose tolerance test (OGTT). Morphometry and functional analyses of the pancreatic islets were also performed before and after the feeding period. Before HFD feeding, SDG-P mice showed modestly higher postchallenge blood glucose levels and lower insulin increments in OGTT than SDG-R mice. Although SDG-P mice showed greater β cell proliferation than SDG-R mice under HFD feeding, SDG-P mice developed overt glucose intolerance, whereas SDG-R mice maintained normal glucose tolerance. Regardless of whether it was before or after HFD feeding, the isolated islets from SDG-P mice showed impaired glucose- and KCl-stimulated insulin secretion relative to those from SDG-R mice; accordingly, the expression levels of the insulin secretion-related genes in SDG-P islets were significantly lower than those in SDG-R islets. These findings suggest that the innate predispositions in pancreatic islets may determine the susceptibility to diet-induced diabetes. SDG-R and SDG-P mice may therefore be useful polygenic animal models to study the gene-environment interactions in the development of type 2 diabetes.

  1. Effects of Tocotrienols on Insulin Secretion-Associated Genes Expression of Rat Pancreatic Islets in a Dynamic Culture.

    PubMed

    Chia, Ling L; Jantan, Ibrahim; Chua, Kien H; Lam, Kok W; Rullah, Kamal; Aluwi, Mohd F M

    2016-01-01

    Tocotrienols (T3) are well-known for their antioxidant properties besides showing therapeutic potential in clinical complications such as hyperlipidemia induced by diabetes. The aim of this study was to determine the effects of δ-T3, γ-T3, and α-T3 on insulin secretion-associated genes expression of rat pancreatic islets in a dynamic culture. Pancreatic islets freshly isolated from male Wistar rats were treated with T3 for 1 h at 37°C in a microfluidic system with continuous operation. The cells were collected for total RNA extraction and reverse-transcribed, followed by measurement of insulin secretion-associated genes expression using quantitative real-time polymerase chain reaction. Molecular docking experiments were performed to gain insights on how the T3 bind to the receptors. Short-term exposure of δ- and γ-T3 to pancreatic β cells in a stimulant glucose condition (16.7 mM) significantly regulated preproinsulin mRNA levels and insulin gene transcription. In contrast, α-T3 possessed less ability in the activation of insulin synthesis level. Essentially, potassium chloride (KCl), a β cell membrane depolarising agent added into the treatment further enhanced the insulin production. δ- and γ-T3 revealed significantly higher quantitative expression in most of the insulin secretion-associated genes groups containing 16.7 mM glucose alone and 16.7 mM glucose with 30 mM KCl ranging from 600 to 1200 μM (p < 0.05). The findings suggest the potential of δ-T3 in regulating insulin synthesis and glucose-stimulated insulin secretion through triggering pathway especially in the presence of KCl. PMID:27625609

  2. Effects of Tocotrienols on Insulin Secretion-Associated Genes Expression of Rat Pancreatic Islets in a Dynamic Culture

    PubMed Central

    Chia, Ling L.; Jantan, Ibrahim; Chua, Kien H.; Lam, Kok W.; Rullah, Kamal; Aluwi, Mohd F. M.

    2016-01-01

    Tocotrienols (T3) are well-known for their antioxidant properties besides showing therapeutic potential in clinical complications such as hyperlipidemia induced by diabetes. The aim of this study was to determine the effects of δ-T3, γ-T3, and α-T3 on insulin secretion-associated genes expression of rat pancreatic islets in a dynamic culture. Pancreatic islets freshly isolated from male Wistar rats were treated with T3 for 1 h at 37°C in a microfluidic system with continuous operation. The cells were collected for total RNA extraction and reverse-transcribed, followed by measurement of insulin secretion-associated genes expression using quantitative real-time polymerase chain reaction. Molecular docking experiments were performed to gain insights on how the T3 bind to the receptors. Short-term exposure of δ- and γ-T3 to pancreatic β cells in a stimulant glucose condition (16.7 mM) significantly regulated preproinsulin mRNA levels and insulin gene transcription. In contrast, α-T3 possessed less ability in the activation of insulin synthesis level. Essentially, potassium chloride (KCl), a β cell membrane depolarising agent added into the treatment further enhanced the insulin production. δ- and γ-T3 revealed significantly higher quantitative expression in most of the insulin secretion-associated genes groups containing 16.7 mM glucose alone and 16.7 mM glucose with 30 mM KCl ranging from 600 to 1200 μM (p < 0.05). The findings suggest the potential of δ-T3 in regulating insulin synthesis and glucose-stimulated insulin secretion through triggering pathway especially in the presence of KCl. PMID:27625609

  3. Effects of Tocotrienols on Insulin Secretion-Associated Genes Expression of Rat Pancreatic Islets in a Dynamic Culture

    PubMed Central

    Chia, Ling L.; Jantan, Ibrahim; Chua, Kien H.; Lam, Kok W.; Rullah, Kamal; Aluwi, Mohd F. M.

    2016-01-01

    Tocotrienols (T3) are well-known for their antioxidant properties besides showing therapeutic potential in clinical complications such as hyperlipidemia induced by diabetes. The aim of this study was to determine the effects of δ-T3, γ-T3, and α-T3 on insulin secretion-associated genes expression of rat pancreatic islets in a dynamic culture. Pancreatic islets freshly isolated from male Wistar rats were treated with T3 for 1 h at 37°C in a microfluidic system with continuous operation. The cells were collected for total RNA extraction and reverse-transcribed, followed by measurement of insulin secretion-associated genes expression using quantitative real-time polymerase chain reaction. Molecular docking experiments were performed to gain insights on how the T3 bind to the receptors. Short-term exposure of δ- and γ-T3 to pancreatic β cells in a stimulant glucose condition (16.7 mM) significantly regulated preproinsulin mRNA levels and insulin gene transcription. In contrast, α-T3 possessed less ability in the activation of insulin synthesis level. Essentially, potassium chloride (KCl), a β cell membrane depolarising agent added into the treatment further enhanced the insulin production. δ- and γ-T3 revealed significantly higher quantitative expression in most of the insulin secretion-associated genes groups containing 16.7 mM glucose alone and 16.7 mM glucose with 30 mM KCl ranging from 600 to 1200 μM (p < 0.05). The findings suggest the potential of δ-T3 in regulating insulin synthesis and glucose-stimulated insulin secretion through triggering pathway especially in the presence of KCl.

  4. Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells

    PubMed Central

    Tapia-Limonchi, Rafael; Díaz, Irene; Cahuana, Gladys M; Bautista, Mario; Martín, Franz; Soria, Bernat; Tejedo, Juan R; Bedoya, Francisco J

    2014-01-01

    Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic β cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic β cell proteome. Species differences in the proteins involved are apparent. PMID:25658244

  5. Storage of porcine pancreatic digest prior to islet purification. The benefits of UW solution and the roles of its individual components.

    PubMed

    Chadwick, D R; Robertson, G S; Rose, S; Contractor, H; James, R F; Bell, P R; London, N J

    1993-08-01

    The aim of this study was to establish the beneficial effect of storage of pancreatic digest in University of Wisconsin solution on porcine islet purification, the mechanism of this effect, and the components of UW responsible. Ten porcine pancreata were collagenase-digested, and samples of digest were washed and stored for 1 hr in either UW or minimum essential medium at 4 degrees C, prior to separation on continuous linear density gradients of bovine serum albumin. Samples of digest from a further ten pancreata were similarly treated, comparing storage in MEM, UW, and five solutions varying in lactobionate:chloride ratio and raffinose content. The purity of the islet preparations and the densities of islets and exocrine tissue were determined from insulin and amylase assay of aliquots aspirated from these gradients. Washing and storage of digest in UW markedly improved islet purity, compared with MEM, due to an increase in the density of exocrine tissue. Exocrine tissue density following storage was dependent upon the control of acinar cell volume, rather than exocrine enzyme discharge, and was determined primarily by the chloride:lactobionate ratio of the storage solution. Raffinose was of little additional benefit, while the beneficial effect of UW was greater than that due to its lactobionate and raffinose content alone. In conclusion, inadequate purification of islets results from exocrine tissue swelling. This swelling is reduced by storage of the pancreatic digest in UW solution, due primarily to the replacement of chloride by lactobionate in UW. PMID:8356582

  6. Islet Culture/Preservation Before Islet Transplantation.

    PubMed

    Noguchi, Hirofumi; Miyagi-Shiohira, Chika; Kurima, Kiyoto; Kobayashi, Naoya; Saitoh, Issei; Watanabe, Masami; Noguchi, Yasufumi; Matsushita, Masayuki

    2015-12-17

    Although islet culture prior to transplantation provides flexibility for the evaluation of isolated islets and the pretreatment of patients, it is well known that isolated islets deteriorate rapidly in culture. Human serum albumin (HSA) is used for medium supplementation instead of fetal bovine serum (FBS), which is typically used for islet culture research, to avoid the introduction of xenogeneic materials. However, FBS contains several factors that are beneficial to islet viability and which also neutralize the endogenous pancreatic enzymes or exogenous enzymes left over from the isolation process. Several groups have reported the comparison of cultures at 22°C and 37°C. Recent studies have demonstrated the superiority of 4°C preservation to 22°C and 37°C cultures. We herein review the current research on islet culture/preservation for clinical islet transplantation. PMID:26858905

  7. St. John's wort extract and hyperforin protect rat and human pancreatic islets against cytokine toxicity.

    PubMed

    Novelli, Michela; Beffy, Pascale; Menegazzi, Marta; De Tata, Vincenzo; Martino, Luisa; Sgarbossa, Anna; Porozov, Svetlana; Pippa, Anna; Masini, Matilde; Marchetti, Piero; Masiello, Pellegrino

    2014-02-01

    The extract of Hypericum perforatum (St. John's wort, SJW) and its component hyperforin (HPF) were previously shown to inhibit cytokine-induced activation of signal transducer and activator of transcription-1 and nuclear factor κB and prevent apoptosis in a cultured β-cell line. Objective of this study was to assess the protection exerted by SJW and HPF on isolated rat and human islets exposed to cytokines in vitro. Functional, ultrastructural, biomolecular and cell death evaluation studies were performed. In both rat and human islets, SJW and HPF counteracted cytokine-induced functional impairment and down-regulated mRNA expression of pro-inflammatory target genes, such as iNOS, CXCL9, CXCL10, COX2. Cytokine-induced NO production from cultured islets, evaluated by nitrites measurement in the medium, was significantly reduced in the presence of the vegetal compounds. Noteworthy, the increase in apoptosis and necrosis following 48-h exposure to cytokines was fully prevented by SJW and partially by HPF. Ultrastructural morphometric analysis in human islets exposed to cytokines for 20 h showed that SJW or HPF avoided early β-cell damage (e.g., mitochondrial alterations and loss of insulin granules). In conclusion, SJW compounds protect rat and human islets against cytokine effects by counteracting key mechanisms of cytokine-mediated β-cell injury and represent promising pharmacological tools for prevention or limitation of β-cell dysfunction and loss in type 1 diabetes.

  8. Pancreatic stem cells: building blocks for a better surrogate islet to treat type 1 diabetes.

    PubMed

    Peck, A B; Chaudhari, M; Cornelius, J G; Ramiya, V K

    2001-04-01

    Type 1, insulin-dependent, diabetes is one of the more costly chronic diseases of children, adolescents and adults in Europe and North America. While routine insulin injections currently provide diabetic patients with their daily insulin requirements, blood glucose excursions are common, leading eventually to microvascular and macrovascular complications and early death. A 'cure' for Type 1 diabetes relies on replacement of the beta-cell mass which, today, is accomplished by pancreas transplants or islets of Langerhans implants. Recent advances in the isolation of stem cells that possess the capacity to differentiate to functional endocrine pancreas provide new opportunities to produce large numbers of islets, even autologous islets, that can be used as implants. We discuss briefly this new technology and its meaning for diabetes. PMID:11370772

  9. Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes.

    PubMed

    Stiles, Bangyan L; Kuralwalla-Martinez, Christine; Guo, Wei; Gregorian, Caroline; Wang, Ying; Tian, Jide; Magnuson, Mark A; Wu, Hong

    2006-04-01

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies. PMID:16537919

  10. Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes.

    PubMed

    Stiles, Bangyan L; Kuralwalla-Martinez, Christine; Guo, Wei; Gregorian, Caroline; Wang, Ying; Tian, Jide; Magnuson, Mark A; Wu, Hong

    2006-04-01

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies.

  11. Results of open and robot-assisted pancreatectomies with autologous islet transplantations: treating chronic pancreatitis and preventing surgically induced diabetes.

    PubMed

    Gruessner, R W G; Cercone, R; Galvani, C; Rana, A; Porubsky, M; Gruessner, A C; Rilo, H

    2014-01-01

    For patients with chronic pancreatitis (CP), standard surgical procedures (eg, partial or total resections, drainage procedures) are inadequate treatment options, because they do not confer pain relief and they leave patients prone to brittle diabetes and hypoglycemia. The combination of total pancreatectomy and islet autotransplantation (TP-IAT), however, can create insulin-independent and pain-free states. At our center, from August 2009 through August 2013, 61 patients with CP underwent either open or robot-assisted TP-IAT. The 30-day mortality rate was 0%. The transplanted islet equivalents per body weight ranged from 10,000 to 17,770. In all, 19% of the patients became insulin independent (after a range of 1-24 months); 27% of patients required <10 units of insulin. Moreover, at 12 months after surgery, 71% of the patients were pain free and no longer required analgesics. Our metabolic outcomes could have been even better if most patients had been referred at an earlier disease stage; instead, ∼80% had already undergone surgical procedures, and 91% had abnormal results on preoperative continuous glucose monitoring tests. Only if patients with CP are referred early for a TP-IAT-rather than being subjected to additional inadequate endoscopic and surgical procedures-can insulin-independent and pain-free states be accomplished in most. PMID:25131087

  12. Specific localization of membrane dipeptidase and dipeptidyl peptidase IV in secretion granules of two different pancreatic islet cells.

    PubMed

    Grondin, G; Hooper, N M; LeBel, D

    1999-04-01

    Endocrine cells require several protein convertases to process the precursors of hormonal peptides that they secrete. In addition to the convertases, which have a crucial role in the maturation of prohormones, many other proteases are present in endocrine cells, the roles of which are less well established. Two of these proteases, dipeptidyl peptidase IV (EC 3.4.14.5) and membrane dipeptidase (EC 3.4.13.19), have been immunocytochemically localized in the endocrine pancreas of the pig. Membrane dipeptidase was present exclusively in cells of the islet of Langerhans that were positive for the pancreatic polypeptide, whereas dipeptidyl peptidase IV was restricted to cells positive for glucagon. Both enzymes were observed in the content of secretory granules and therefore would be released into the interstitial space as the granules undergo exocytosis. At this location they could act on secretions of other islet cells. The relative concentration of dipeptidyl peptidase IV was lower in dense glucagon granules, where the immunoreactivity to glucagon was higher, and vice versa for light granules. This suggests that, in A-cells, dipeptidyl peptidase IV could be sent for degradation in the endosomal/lysosomal compartment during the process of granule maturation or could be removed from granules for continuous release into the interstitial space. The intense proteolytic activity that takes place in the endocrine pancreas could produce many potential dipeptide substrates for membrane dipeptidase. (J Histochem Cytochem 47:489-497, 1999)

  13. Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

    PubMed Central

    Sturm, Dorothée; Marselli, Lorella; Ehehalt, Florian; Richter, Daniela; Distler, Marius; Kersting, Stephan; Grützmann, Robert; Bokvist, Krister; Froguel, Philippe; Liechti, Robin; Jörns, Anne; Meda, Paolo; Baretton, Gustavo Bruno; Saeger, Hans-Detlev; Schulte, Anke M.; Marchetti, Piero; Solimena, Michele

    2013-01-01

    Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 °C until sectioning. Forty serial sections of 10 μm thickness were cut on a cryostat under a -20 °C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 °C. Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For

  14. A new approach for pancreatic tissue engineering: human endometrial stem cells encapsulated in fibrin gel can differentiate to pancreatic islet beta-cell.

    PubMed

    Niknamasl, Azadeh; Ostad, Seyed Nasser; Soleimani, Mansoureh; Azami, Mahmoud; Salmani, Maryam Kabir; Lotfibakhshaiesh, Nasrin; Ebrahimi-Barough, Somayeh; Karimi, Roya; Roozafzoon, Reza; Ai, Jafar

    2014-10-01

    Metabolic diabetes mellitus as the most serious and prevalent metabolic disease in the world has various complications. The most effective treatment of type I diabetes seems to be islet cell transplantation. Shortage of donors and difficult procedures and high rate of rejection have always restricted this approach. Tissue engineering is a novel effective solution to many medical problems such as diabetes. Endometrial mesenchymal stem cells as a lineage which have the potential to differentiate to mesodermal and endodermal tissues seem to be suitable for this purpose. Fibrin hydrogel with a high degree of biocompatibility and specific properties making it similar to normal pancreas seems to be an ideal scaffold. After successfully isolating stem cells (hEnSCs) from human endometrium, a three-step protocol was used to differentiate them into pancreatic beta cells. Fibrin was used as 3D scaffold. After 2 weeks, cells formed clusters like islets cells, and secretion of insulin was measured by chemiluminescence. PDX1, proinsulin, and c-peptide as special markers of β cells were detected by immunofluorescence. Expression of glucagon, PDX1, and insulin genes in mRNA level was detected by Real time PCR and gel electrophoresis. The former showed higher levels of gene expression in 3D cultures. SEM analysis showed good integrity between cells and scaffold. No toxicity was detected with fibrin scaffold by MTT assay.

  15. Calcium-signaling components in rat insulinoma β-cells (INS-1) and pancreatic islets are differentially influenced by melatonin.

    PubMed

    Bazwinsky-Wutschke, Ivonne; Mühlbauer, Eckhard; Albrecht, Elke; Peschke, Elmar

    2014-05-01

    The pineal secretory product melatonin exerts its influence on the insulin secretion of pancreatic islets by different signaling pathways. The purpose of this study was to analyze the impact of melatonin on calcium-signaling components under different conditions. In a transfected INS-1 cell line overexpressing the human MT2 receptor (hMT2-INS-1), melatonin treatment induced even stronger depressive effects on calcium/calmodulin-dependent kinase 2d and IV (Camk2d, CamkIV) transcripts during 3-isobutyl-1-methylxanthine (IBMX) treatment than in normal INS-1 cells, indicating a crucial influence of melatonin receptor density on transcript-level regulation. In addition, melatonin induced a significant downregulation of calmodulin (Calm1) in IBMX-treated hMT2-INS-1 cells. Long-term administration of melatonin alone reduced CamkIV transcript levels in INS-1 cells; however, transcript levels of Camk2d remained unchanged. The release of insulin was diminished under long-term melatonin treatment. The impact of melatonin also involved reductions in CAMK2D protein during IBMX or forskolin treatments in INS-1 cells, as measured by an enzyme-linked immunosorbent assay, indicating a functional significance of transcriptional changes in pancreatic islets. Furthermore, analysis of melatonin receptor knockout mice showed that the transcript levels of Camk2d, CamkIV, and Calm1 were differentially influenced according to the melatonin receptor subtype deleted. In conclusion, this study provides evidence that melatonin has different impacts on the regulation of Calm1 and Camk. These calcium-signaling components are known as participants in the calcium/calmodulin pathway, which plays an important functional role in the modulation of the β-cell signaling pathways leading to insulin secretion.

  16. HMGB1 binds to the rs7903146 locus in TCF7L2 in human pancreatic islets.

    PubMed

    Zhou, Yuedan; Oskolkov, Nikolay; Shcherbina, Liliya; Ratti, Joyce; Kock, Kian-Hong; Su, Jing; Martin, Brian; Oskolkova, Malin Zackrisson; Göransson, Olga; Bacon, Julie; Li, Weimin; Bucciarelli, Saskia; Cilio, Corrado; Brazma, Alvis; Thatcher, Bradley; Rung, Johan; Wierup, Nils; Renström, Erik; Groop, Leif; Hansson, Ola

    2016-07-15

    The intronic SNP rs7903146 in the T-cell factor 7-like 2 gene (TCF7L2) is the common genetic variant most highly associated with Type 2 diabetes known to date. The risk T-allele is located in an open chromatin region specific to human pancreatic islets of Langerhans, thereby accessible for binding of regulatory proteins. The risk T-allele locus exhibits stronger enhancer activity compared to the non-risk C-allele. The aim of this study was to identify transcriptional regulators that bind the open chromatin region in the rs7903146 locus and thereby potentially regulate TCF7L2 expression and activity. Using affinity chromatography followed by Edman sequencing, we identified one candidate regulatory protein, i.e. high-mobility group protein B1 (HMGB1). The binding of HMGB1 to the rs7903146 locus was confirmed in pancreatic islets from human deceased donors, in HCT116 and in HEK293 cell lines using: (i) protein purification on affinity columns followed by Western blot, (ii) chromatin immunoprecipitation followed by qPCR and (iii) electrophoretic mobility shift assay. The results also suggested that HMGB1 might have higher binding affinity to the C-allele of rs7903146 compared to the T-allele, which was supported in vitro using Dynamic Light Scattering, possibly in a tissue-specific manner. The functional consequence of HMGB1 depletion in HCT116 and INS1 cells was reduced insulin and TCF7L2 mRNA expression, TCF7L2 transcriptional activity and glucose stimulated insulin secretion. These findings suggest that the rs7903146 locus might exert its enhancer function by interacting with HMGB1 in an allele dependent manner. PMID:26845344

  17. Total pancreatectomy with islet cell transplantation vs intrathecal narcotic pump infusion for pain control in chronic pancreatitis

    PubMed Central

    Mokadem, Mohamad; Noureddine, Lama; Howard, Thomas; McHenry, Lee; Sherman, Stuart; Fogel, Evan L; Watkins, James L; Lehman, Glen A

    2016-01-01

    AIM: To evaluate pain control in chronic pancreatitis patients who underwent total pancreatectomy with islet cell transplantation or intrathecal narcotic pump infusion. METHODS: We recognized 13 patients who underwent intrathecal narcotic pump (ITNP) infusion and 57 patients who underwent total pancreatectomy with autologous islet cell transplantation (TP + ICT) for chronic pancreatitis (CP) pain control between 1998 and 2008 at Indiana University Hospital. All patients had already failed multiple other modalities for pain control and the decision to proceed with either intervention was made at the discretion of the patients and their treating physicians. All patients were evaluated retrospectively using a questionnaire inquiring about their pain control (using a 0-10 pain scale), daily narcotic dose usage, and hospital admission days for pain control before each intervention and during their last follow-up. RESULTS: All 13 ITNP patients and 30 available TP + ICT patients were evaluated. The mean age was approximately 40 years in both groups. The median duration of pain before intervention was 6 years and 7 years in the ITNP and TP + ICT groups, respectively. The median pain score dropped from 8 to 2.5 (on a scale of 0-10) in both groups on their last follow up. The median daily dose of narcotics also decreased from 393 mg equivalent of morphine sulfate to 8 mg in the ITNP group and from 300 mg to 40 mg in the TP + ICT group. No patient had diabetes mellitus (DM) before either procedure whereas 85% of those who underwent pancreatectomy were insulin dependent on their last evaluation despite ICT. CONCLUSION: ITNP and TP + ICT are comparable for pain control in patients with CP however with high incidence of DM among those who underwent TP + ICT. Prospective comparative studies and longer follow up are needed to better define treatment outcomes. PMID:27122666

  18. Galectin-3 Deficiency Accelerates High-Fat Diet–Induced Obesity and Amplifies Inflammation in Adipose Tissue and Pancreatic Islets

    PubMed Central

    Pejnovic, Nada N.; Pantic, Jelena M.; Jovanovic, Ivan P.; Radosavljevic, Gordana D.; Milovanovic, Marija Z.; Nikolic, Ivana G.; Zdravkovic, Nemanja S.; Djukic, Aleksandar L.; Arsenijevic, Nebojsa N.; Lukic, Miodrag L.

    2013-01-01

    Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet–induced obesity and diabetes. Obese LGALS3−/− mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3−/− mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c+CD11b+ macrophages and decreased CD4+CD25+FoxP3+ regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3−/− animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3−/− peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1–dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3−/− macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes. PMID:23349493

  19. Galectin-3 deficiency accelerates high-fat diet-induced obesity and amplifies inflammation in adipose tissue and pancreatic islets.

    PubMed

    Pejnovic, Nada N; Pantic, Jelena M; Jovanovic, Ivan P; Radosavljevic, Gordana D; Milovanovic, Marija Z; Nikolic, Ivana G; Zdravkovic, Nemanja S; Djukic, Aleksandar L; Arsenijevic, Nebojsa N; Lukic, Miodrag L

    2013-06-01

    Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet-induced obesity and diabetes. Obese LGALS3(-/-) mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3(-/-) mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c(+)CD11b(+) macrophages and decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3(-/-) animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3(-/-) peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1-dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3(-/-) macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes. PMID:23349493

  20. Abnormal anxiety- and depression-like behaviors in mice lacking both central serotonergic neurons and pancreatic islet cells.

    PubMed

    Jia, Yun-Fang; Song, Ning-Ning; Mao, Rong-Rong; Li, Jin-Nan; Zhang, Qiong; Huang, Ying; Zhang, Lei; Han, Hui-Li; Ding, Yu-Qiang; Xu, Lin

    2014-01-01

    Dysfunction of central serotonin (5-HT) system has been proposed to be one of the underlying mechanisms for anxiety and depression, and the association of diabetes mellitus and psychiatric disorders has been noticed by the high prevalence of anxiety/depression in patients with diabetes mellitus. This promoted us to examine these behaviors in central 5-HT-deficient mice and those also suffering with diabetes mellitus. Mice lacking either 5-HT or central serotonergic neurons were generated by conditional deletion of Tph2 or Lmx1b respectively. Simultaneous depletion of both central serotonergic neurons and pancreatic islet cells was achieved by administration of diphtheria toxin (DT) in Pet1-Cre;Rosa26-DT receptor (DTR) mice. The central 5-HT-deficient mice showed reduced anxiety-like behaviors as they spent more time in and entered more often into the light box in the light/dark box test compared with controls; similar results were observed in the elevated plus maze test. However, they displayed no differences in the immobility time of the forced swimming and tail suspension tests suggesting normal depression-like behaviors in central 5-HT-deficient mice. As expected, DT-treated Pet1-Cre;Rosa26-DTR mice lacking both central serotonergic neurons and pancreatic islet endocrine cells exhibited several classic diabetic symptoms. Interestingly, they displayed increased anxiety-like behaviors but reduced immobility time in the forced swimming and tail suspension tests. Furthermore, the hippocampal neurogenesis was dramatically enhanced in these mice. These results suggest that the deficiency of central 5-HT may not be sufficient to induce anxiety/depression-like behaviors in mice, and the enhanced hippocampal neurogenesis may contribute to the altered depression-like behaviors in the 5-HT-deficient mice with diabetes. Our current investigation provides understanding the relationship between diabetes mellitus and psychiatric disorders.

  1. Gadolinium- and manganite-based contrast agents with fluorescent probes for both magnetic resonance and fluorescence imaging of pancreatic islets: a comparative study.

    PubMed

    Berkova, Zuzana; Jirak, Daniel; Zacharovova, Klara; Lukes, Ivan; Kotkova, Zuzana; Kotek, Jan; Kacenka, Michal; Kaman, Ondrej; Rehor, Ivan; Hajek, Milan; Saudek, Frantisek

    2013-04-01

    Three magnetic resonance (MR)/fluorescence imaging probes were tested for visualization, cellular distribution, and survival of labeled pancreatic islets in vitro and following transplantation. As T(1) contrast agents (CAs), gadolinium(III) complexes linked to β-cyclodextrin (Gd-F-βCD) or bound to titanium dioxide (TiO2 @RhdGd) were tested. As a T(2) CA, perovskite manganite nanoparticles (LSMO@siF@si) were examined. Fluorescein or rhodamine was incorporated as a fluorescent marker in all probes. Islets labeled with gadolinium(III) CAs were visible as hyperintense spots on MR in vitro, but detection in vivo was inconclusive. Islets labeled with LSMO@siF@si CA were clearly visible as hypointense spots or areas on MR scans in vitro as well as in vivo. All CAs were detected inside the islet cells by fluorescence. Although the vitality and function of the labeled islets was not impaired by any of the tested CAs, results indicate that LSMO@siF@si CA is a superior marker for islet labeling, as it provides better contrast enhancement within a shorter scan time. PMID:23316021

  2. The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity.

    PubMed

    Wali, J A; Rondas, D; McKenzie, M D; Zhao, Y; Elkerbout, L; Fynch, S; Gurzov, E N; Akira, S; Mathieu, C; Kay, T W H; Overbergh, L; Strasser, A; Thomas, H E

    2014-03-13

    Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

  3. JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-{kappa}B activation and the JAK/STAT pathway

    SciTech Connect

    Lv, Na; Kim, Eun-Kyung; Song, Mi-Young; Choi, Ha-Na; Moon, Woo Sung; Park, Sung-Joo; Park, Jin-Woo; Kwon, Kang-Beom; Park, Byung-Hyun

    2009-07-15

    JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic {beta}-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1{beta} and interferon (IFN)-{gamma}-induced {beta}-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor {kappa}B (NF-{kappa}B) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3{sup -/-} mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects {beta}-cells from cytokine toxicity through suppression of the NF-{kappa}B and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional {beta}-cell mass.

  4. Influence of High Aspect Ratio Vessel Cell Culture on TNF-Alpha, Insulin Secretion and Glucose Homeostasis in Pancreatic Islets of Langerhans from Wistar Furth Rats

    NASA Technical Reports Server (NTRS)

    Tobin, Brian W.a; Leeper-Woodford, Sandra K.

    1999-01-01

    The present studies were carried out to determine the influence of a ground based microgravity paradigm, utilizing the High Aspect Ratio Vessel (HARV) cell culture upon lipopolysaccharide (LPS) stimulated tumor necrosis factor alpha (TNF-alpha) production of pancreatic islets of Langerhans. An additional aim was to elucidate alterations in insulin secretion and glucose utilization using the HARV low shear, gravity averaged vector, cell culture technique. Islets were isolated (1726 +/- 117, 150 micron islet equivalent units) from Wistar Furth rats and assigned to four treatment groups: 1) HARV, 2) HARV plus LPS, 3) static culture, 4) static culture plus LPS. Following 48 hours of culture, insulin concentration was increased in both HARV and static cultures (p<0.05). Islet medium from HARV and static cultures were assayed for TNF-alpha (L929 cytotoxicity assay) and was measured at selected time points for 48 hours. TNF-alpha was significantly increased in LPS-induced HARV and static cultures, yet the increase was more pronounced in the static culture group (p<0.05). This is a novel observation and indicates that TNF producing cells are present in islets and that LPS stimulates TNF secretion in isolated islets. A decrease in insulin concentration was demonstrated in the islet medium of the LPS stimulated HARV culture (p<0.05). That TNF-alpha is associated with a decreased insulin secretion is intriguing, both as it relates to in-flight investigations, and as it may provide insight into the pathophysiology of Type I and Type 11 diabetes. Glucose concentration in islet medium was lesser throughout the experiment in static cultures, suggesting a decreased reliance upon glucose as a metabolic substrate in the islets cultured in HARVS. In conclusion, the present studies demonstrate alterations in LPS induced TNF-alpha production of pancreatic islets of Langerhans, favoring a lesser TNF production in the microgravity HARV paradigm. Additionally, alterations in fuel

  5. Cross talk between the extracellular matrix and the immune system in the context of endocrine pancreatic islet transplantation. A review article.

    PubMed

    Kuehn, C; Vermette, P; Fülöp, T

    2014-04-01

    This review aims to highlight the importance of the bidirectional influence of the extracellular matrix (ECM) and immune cells in the context of type 1 diabetes mellitus (T1DM) and endocrine pancreatic islet transplantation. We introduced the main classes of molecules and proteins constituting the ECM as well as cells and cytokines of the immune system with the aim to further examine their roles in T1DM and islet transplantation. Integrins expressed by immune cells and their functions are detailed. Finally, this article reviews the roles of the ECM and the immune system in islet transplantation as well as ECM-related cytokines and their influence on the ECM and immune cells. PMID:24679589

  6. Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult pancreatic progenitors and mediates islet regeneration

    PubMed Central

    Chen, Shuyuan; Shimoda, Masyuki; Chen, Jiaxi; Matsumodo, Shinichi

    2012-01-01

    The molecular mechanism of β-cell regeneration remains poorly understood. Cyclin D2/CDK4 expresses in normal β cells and maintains adult β-cell growth. We hypothesized that gene therapy with cyclin D2/CDK4/GLP-1 plasmids targeted to the pancreas of STZ-treated rats by ultrasound-targeted microbubble destruction (UTMD) would force cell cycle re-entry of residual G0-phase islet cells into G1/S phase to regenerate β cells. A single UTMD treatment induced β-cell regeneration with reversal of diabetes for 6 mo without evidence of toxicity. We observed that this β-cell regeneration was not mediated by self-replication of pre-existing β cells. Instead, cyclin D2/CDK4/GLP-1 initiated robust proliferation of adult pancreatic progenitor cells that exist within islets and terminally differentiate to mature islets with β cells and α cells. PMID:22373529

  7. Genome-Wide DNA Methylation Analysis of Human Pancreatic Islets from Type 2 Diabetic and Non-Diabetic Donors Identifies Candidate Genes That Influence Insulin Secretion

    PubMed Central

    Dayeh, Tasnim; Volkov, Petr; Salö, Sofia; Hall, Elin; Nilsson, Emma; Olsson, Anders H.; Kirkpatrick, Clare L.; Wollheim, Claes B.; Eliasson, Lena; Rönn, Tina; Bacos, Karl; Ling, Charlotte

    2014-01-01

    Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, β- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3′UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, β- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼7%) and overrepresented in the open sea (∼60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic β- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal β- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight

  8. Adult Human Biliary Tree Stem Cells Differentiate to β-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide

    PubMed Central

    Scafetta, Gaia; Renzi, Anastasia; De Canio, Michele; Sicilia, Francesca; Nevi, Lorenzo; Casa, Domenico; Panetta, Rocco; Berloco, Pasquale Bartolomeo; Reid, Lola M.; Federici, Giorgio; Gaudio, Eugenio; Maroder, Marella; Alvaro, Domenico

    2015-01-01

    Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells. PMID:26252949

  9. Adult Human Biliary Tree Stem Cells Differentiate to β-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide.

    PubMed

    Cardinale, Vincenzo; Puca, Rosa; Carpino, Guido; Scafetta, Gaia; Renzi, Anastasia; De Canio, Michele; Sicilia, Francesca; Nevi, Lorenzo; Casa, Domenico; Panetta, Rocco; Berloco, Pasquale Bartolomeo; Reid, Lola M; Federici, Giorgio; Gaudio, Eugenio; Maroder, Marella; Alvaro, Domenico

    2015-01-01

    Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells. PMID:26252949

  10. Gene expression changes in human islets exposed to type 1 diabetic serum

    PubMed Central

    Jackson, Andrew M.; Kanak, Mazhar A.; Grishman, Ellen K.; Chaussabel, Damien; Levy, Marlon F.; Naziruddin, Bashoo

    2012-01-01

    A major obstacle to the success of islet cell transplantation as a standard treatment for labile type 1 diabetes mellitus is the immediate loss of up to 70% of the transplanted islet mass. Activation of the complement cascade and coagulation factors has been implicated in initiating the destruction of the islet graft. In this study, we analyzed the gene expression changes in islet cells following exposure to type 1 diabetes mellitus serum (T1DM). Isolated human pancreatic islet cells were cultured for 2 d to stabilize islet cell gene expression. Cultured islets were divided into three groups for treatment as follows: group 1 was treated with autologous donor serum, while groups two and three were treated with sera from ABO-matched allogeneic donors or autoantibody positive type 1 diabetic patient, respectively. Complement was detected using anti-C3 FITC and CH50 assay. Islet gene expression was analyzed using Illumina micro-array technology. Results were confirmed using real-time PCR. Immunofluorescent imaging demonstrated complement deposition only in the T1DM condition. Gene array and class prediction analysis generated a list of 50 genes that were able to predict the effect of T1DM serum on islets. Quantitative PCR corroborated microarray results. Both techniques demonstrated upregulation of MMP9 (243%), IL-1β (255%), IL-11 (220%), IL-12A (132%), RAD (343%) and a concomitant downregulation of IL-1RN (64%) in islets treated with T1DM serum. Islets treated with T1DM serum overexpressed genes associated with angiogenesis while decreasing transcription of genes that protect islets from inflammatory cytokines and reactive oxygen species. PMID:22885994

  11. Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

    PubMed Central

    Rezania, Alireza; Bruin, Jennifer E.; Riedel, Michael J.; Mojibian, Majid; Asadi, Ali; Xu, Jean; Gauvin, Rebecca; Narayan, Kavitha; Karanu, Francis; O’Neil, John J.; Ao, Ziliang; Warnock, Garth L.

    2012-01-01

    Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes. PMID:22740171

  12. A second glucagon in the pancreatic islets of the daddy sculpin Cottus scorpius.

    PubMed

    Cutfield, S M; Cutfield, J F

    1993-09-01

    The peptide hormone glucagon has been isolated from the islet tissue (Brockmann bodies) of the teleost Cottus scorpius (daddy sculpin) and sequenced. The sequence is HSEGTSNDYSKYLEDRKAQDFVQWLMNN differing at four positions from the glucagon found earlier in the same species by Conlon and coworkers (1987b, Eur. J. Biochem, 164, 117-122). Thus sculpin, in common with anglerfish, possesses two distinct glucagons. Comparative sequence data are presented as a phylogenetic tree. PMID:8224771

  13. Feasibility of Amylin Imaging in Pancreatic Islets with β-Amyloid Imaging Probes

    PubMed Central

    Yoshimura, Masashi; Ono, Masahiro; Watanabe, Hiroyuki; Kimura, Hiroyuki; Saji, Hideo

    2014-01-01

    Islet amyloid deposition composed of amylin aggregates is regarded as one of the hallmarks of type 2 diabetes mellitus (T2DM). For the diagnosis of T2DM, several nuclear medical imaging probes have been developed. However, there have been no reports regarding the development of imaging probes targeting amylin. In this report, we investigated the feasibility of amylin imaging using [125I]IPBF as one of the model compounds of β-amyloid (Aβ) imaging probes. In in vitro experiments, [125I]IPBF exhibited high binding affinity for amylin aggregates (Kd = 8.31 nM). Moreover, autoradiographic images showed that [125I]IPBF specifically bound to islet amyloid composed of amylin. These results suggest the potential application of Aβ imaging probes to amylin imaging. In addition, [125I]IPBF is one of the promising lead compounds for amylin imaging, and further structural optimization based on [125I]IPBF may lead to useful tracers for the in vivo imaging of islet amyloids in the pancreas. PMID:25142178

  14. Fibroblast growth factor receptor-1 signaling in pancreatic islet beta-cells is modulated by the extracellular matrix.

    PubMed

    Kilkenny, Dawn M; Rocheleau, Jonathan V

    2008-01-01

    Maintenance of pancreatic beta-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the beta-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal beta-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote beta-cell survival and proliferation. To examine beta-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1betav) and kinase-deficient (KDR1betav) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in betaTC3 cells, dispersed beta-cells, and beta-cells in intact islets. This response was enhanced by R1betav expression and reduced by KDR1betav expression. Plating-dispersed beta-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit beta-cell attachment to laminin via alpha(6)-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated beta-cell response is negatively affected by alpha(6)-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling. PMID:17916654

  15. Accurate measurement of pancreatic islet beta-cell mass using a second-generation fluorescent exendin-4 analog.

    PubMed

    Reiner, Thomas; Thurber, Greg; Gaglia, Jason; Vinegoni, Claudio; Liew, Chong Wee; Upadhyay, Rabi; Kohler, Rainer H; Li, Li; Kulkarni, Rohit N; Benoist, Christophe; Mathis, Diane; Weissleder, Ralph

    2011-08-01

    The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts. PMID:21768367

  16. Accurate measurement of pancreatic islet β-cell mass using a second-generation fluorescent exendin-4 analog

    PubMed Central

    Reiner, Thomas; Thurber, Greg; Gaglia, Jason; Vinegoni, Claudio; Liew, Chong Wee; Upadhyay, Rabi; Kohler, Rainer H.; Kulkarni, Rohit N.; Benoist, Christophe; Mathis, Diane; Weissleder, Ralph

    2011-01-01

    The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K12 position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4×12-VT750) had a high binding affinity (∼3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4×12-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts. PMID:21768367

  17. Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

    PubMed

    Pinton, Paolo; Tsuboi, Takashi; Ainscow, Edward K; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A

    2002-10-01

    The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.

  18. DISCOVERY OF NOVEL GLUCOSE-REGULATED PROTEINS IN ISOLATED HUMAN PANCREATIC ISLETS USING LC-MS/MS-BASED PROTEOMICS

    PubMed Central

    Schrimpe-Rutledge, Alexandra C.; Fontès, Ghislaine; Gritsenko, Marina A.; Norbeck, Angela D.; Anderson, David J.; Waters, Katrina M.; Adkins, Joshua N.; Smith, Richard D.; Poitout, Vincent; Metz, Thomas O.

    2012-01-01

    The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (~p<0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Up-regulation of Dicer 1 and SLC27A2 and down-regulation of Phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles. PMID:22578083

  19. An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet β-cells

    PubMed Central

    Suckow, Arthur T.; Craige, Branch; Faundez, Victor; Cain, William J.

    2010-01-01

    Pancreatic islet β-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since β-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in β-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates β-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 β-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits β3B and μ3B are expressed in β-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that β-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to β-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in β-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 δ-subunit expression. Our findings suggest that β-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation. PMID:20442321

  20. Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics

    SciTech Connect

    Rutledge, Alexandra C.; Fontes, Ghislaine; Gritsenko, Marina A.; Norbeck, Angela D.; Anderson, David J.; Waters, Katrina M.; Adkins, Joshua N.; Smith, Richard D.; Poitout, Vincent; Metz, Thomas O.

    2012-07-06

    The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is due in part to insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent and physiologically important regulators of beta-cell function under physiological conditions, understanding the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins ({approx}p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Many proteins found to be differentially abundant after high glucose stimulation were uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.

  1. Control of Insulin Secretion by Production of Reactive Oxygen Species: Study Performed in Pancreatic Islets from Fed and 48-Hour Fasted Wistar Rats

    PubMed Central

    Riva, Patrícia; Simões, Daniel; Curi, Rui; Carpinelli, Angelo Rafael

    2016-01-01

    Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic β-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870–20 μM or diphenylene iodonium—DPI—5 μM). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 μM) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses

  2. Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells

    SciTech Connect

    Nakabayashi, Hiroko; Ohta, Yasuharu Yamamoto, Masayoshi; Susuki, Yosuke; Taguchi, Akihiko; Tanabe, Katsuya; Kondo, Manabu; Hatanaka, Masayuki; Nagao, Yuko; Tanizawa, Yukio

    2013-05-03

    Highlights: •Arnt mRNA expressed in a circadian manner in mouse pancreatic islets. •Expressions of Dbp and Arnt damped in the islets of a diabetic model mouse. •DBP and E4BP4 regulate Arnt promoter activity by direct binding. •Arnt may have a role in connecting circadian rhythm and metabolism. -- Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1{sup −/−} A{sup y}/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the

  3. Expression and function of Set7/9 in pancreatic islets.

    PubMed

    Ogihara, Takeshi; Vanderford, Nathan L; Maier, Bernhard; Stein, Roland W; Mirmira, Raghavendra G

    2009-01-01

    Histone tail acetylation and methylation are known to enhance accessibility of islet genes to transcription factors and the basal transcriptional machinery.  In this brief report, we follow up on a recent study in which we identified the islet enriched factor Set7/9 as a potentially important histone methyltransferase in β-cells (Deering, et al. Diabetes 2009; 58:185-93).  We had suggested that the methylation of H3-Lys4 by Set7/9 enhances accessibility of the insulin gene to the basal transcriptional machinery.  Consistent with this hypothesis, we show here that RNA polymerase II occupancy at the insulin and IAPP genes is considerably enhanced in β-cells compared to α cells (or NIH3T3 cells), and that the converse is true for RNA polymerase II occupancy at the glucagon gene. The enrichment of Set7/9 in β-cells appears to be dependent upon Pdx1, as knockdown of Pdx1 in INS-1 β-cells using small hairpin RNAs almost completely abolishes Set7/9 expression.  A LacZ expression vector driven by the -6.5 kilobase pair Set7/9 promoter that contains putative Pdx1 binding sites shows β-cell-line-specific expression.  Taken together, our data support further the hypothesis that Pdx1-dependent Set7/9 expression may be crucial to enhancing chromatin accessibility and transcription of β-cell genes. PMID:21099283

  4. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

    PubMed Central

    Llanos, Paola; Contreras-Ferrat, Ariel; Barrientos, Genaro; Valencia, Marco; Mears, David; Hidalgo, Cecilia

    2015-01-01

    Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS. PMID:26046640

  5. Storage of pancreatic digest before islet purification. The influence of colloids and the sodium to potassium ratio in University of Wisconsin-based preservation solutions.

    PubMed

    Chadwick, D R; Robertson, G S; Contractor, H H; Rose, S; Johnson, P R; James, R F; Bell, P R; London, N J

    1994-07-15

    The density-dependent purification of islets from several species of mammalian pancreata is improved by prior storage of the dispersed, collagenase-digested pancreas in suitable storage solutions, such as University of Wisconsin (UW) solution. The optimal composition of such solutions, however, is not fully established, although previous investigations have suggested separately that cellular impermeants and colloids are important components. To investigate this issue further, dispersed tissues from 7 porcine and 7 human pancreata were stored in UW or in solutions containing the impermeants lactobionate and raffinose, with either no added colloid or in the presence of the colloids hydroxyethyl starch, dextran 40, dextran 250, or Ficoll 400; hydroxyethyl starch-containing solutions in which the principal cation was sodium, rather than potassium, were also studied. Subsequent purification of islets on continuous linear density gradients of BSA was then assessed by insulin/amylase assay of gradient fractions. Islet purity was slightly reduced using solutions containing impermeants but lacking a colloid, compared with using UW. In the combined presence of impermeants and a colloid, however, islet purity was similar to that obtained with UW, and for porcine pancreata, solutions containing Ficoll 400 or dextran 40 were slightly superior to UW. Purity was not, however, influenced by the sodium to potassium ratio of storage media. In conclusion, impermeants and colloids are both essential components of solutions used to preserve pancreatic tissue before islet purification, findings which may be relevant when designing media for use during other phases of islet isolation, e.g., during collagenase digestion/density gradient purification. PMID:8036714

  6. Gastrointestinal Symptoms Before and After Total Pancreatectomy With Islet Autotransplantation: The Role of Pancreatic Enzyme Dosing and Adherence

    PubMed Central

    Crosby, Jill; Bellin, Melena D.; Radosevich, David M.; Chinnakotla, Srinath; Dunn, Ty B.; Pruett, Timothy L.; Freeman, Martin L.; Beilman, Greg J.; Schwarzenberg, Sarah J.

    2014-01-01

    Objectives In a large cohort of subjects undergoing total pancreatectomy with islet autotransplantation (TPIAT), we assessed the prevalence and duration of gastrointestinal symptoms before and after the procedure and to determine the impact of enzyme adherence on gastrointestinal symptoms. Methods 356 pre- and post-operative questionnaires were collected from 184 subjects between ages of 5 and 66 years who underwent TPIAT between 2008 and 2011 at University of Minnesota. Questionnaires were analyzed for self-reported frequency and severity of gastrointestinal symptoms, pancreatic enzyme usage, and glycemic variability index (GVI). Results After surgery, patient-reported steatorrhea increased; constipation decreased. Gastrointestinal symptoms interfered with daily activity in 44–69% of subjects, before and after surgery, despite high reported enzyme adherence. Post-operatively, ≥79% of subjects reported consistent use of enzymes at all meals. Presence of gastrointestinal symptoms did not vary with adherence. GVI of 2 had a 2.8 fold increased odds of steatorrhea (95% confidence interval 1.1– 7.0) compared to GVI of 0. Conclusions Gastrointestinal symptoms were common after TPIAT; ongoing management is needed. Enzyme non-adherence was not a major contributor to diarrhea/steatorrhea in this cohort. Glycemic variability was closely associated with steatorrhea; poor response to enzyme replacement may complicate diabetes management. PMID:25486528

  7. Carbohydrate-Responsive Element-Binding Protein (ChREBP) Is a Negative Regulator of ARNT/HIF-1β Gene Expression in Pancreatic Islet β-Cells

    PubMed Central

    Noordeen, Nafeesa A.; Khera, Tarnjit K.; Sun, Gao; Longbottom, E. Rebecca; Pullen, Timothy J.; da Silva Xavier, Gabriela; Rutter, Guy A.; Leclerc, Isabelle

    2010-01-01

    OBJECTIVE Carbohydrate-responsive element-binding protein (ChREBP) is a transcription factor that has been shown to regulate carbohydrate metabolism in the liver and pancreatic β-cells in response to elevated glucose concentrations. Because few genes have been identified so far as bona fide ChREBP-target genes, we have performed a genome-wide analysis of the ChREBP transcriptome in pancreatic β-cells. RESEARCH DESIGN AND METHODS Chromatin immunoprecipitation and high-density oligonucleotide tiling arrays (ChIP-chip; Agilent Technologies) using MIN6 pancreatic β-cell extracts were performed together with transcriptional and other analysis using standard techniques. RESULTS One of the genes identified by ChIP-chip and linked to glucose sensing and insulin secretion was aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor-1β (HIF-1β), a transcription factor implicated in altered gene expression and pancreatic-islet dysfunction in type 2 diabetes. We first confirmed that elevated glucose concentrations decreased ARNT/HIF-1β levels in INS-1 (832/13) cells and primary mouse islets. Demonstrating a role for ChREBP in ARNT gene regulation, ChREBP silencing increased ARNT mRNA levels in INS-1 (832/13) cells, and ChREBP overexpression decreased ARNT mRNA in INS-1 (832/13) cells and primary mouse islets. We demonstrated that ChREBP and Max-like protein X (MLX) bind on the ARNT/HIF-1β promoter on the proximal region that also confers the negative glucose responsiveness. CONCLUSIONS These results demonstrate that ChREBP acts as a novel repressor of the ARNT/HIF-1β gene and might contribute to β-cell dysfunction induced by glucotoxicity. PMID:19833882

  8. Total Pancreatectomy and Islet Auto-Transplantation in Children for Chronic Pancreatitis. Indication, Surgical Techniques, Post Operative Management and Long-Term Outcomes

    PubMed Central

    Chinnakotla, Srinath; Bellin, Melena D.; Schwarzenberg, Sarah J.; Radosevich, David M.; Cook, Marie; Dunn, Ty B.; Beilman, Gregory J.; Freeman, Martin L.; Balamurugan, A.N.; Wilhelm, Josh; Bland, Barbara; Jimenez-Vega, Jose M; Hering, Bernhard J.; Vickers, Selwyn M.; Pruett, Timothy L.; Sutherland, David E.R.

    2014-01-01

    Objective Describe the surgical technique, complications and long term outcomes of total pancreatectomy and islet auto transplantation (TP-IAT) in a large series of pediatric patients. Summary Background Data Surgical management of childhood pancreatitis is not clear; partial resection or drainage procedures often provide transient pain relief, but long term recurrence is common due to the diffuse involvement of the pancreas. Total pancreatectomy (TP) removes the source of the pain, while islet auto transplantation (IAT) potentially can prevent or minimize TP-related diabetes. Methods Retrospective review of 75 children undergoing TP-IAT for chronic pancreatitis who had failed medical, endoscopic or surgical treatment between 1989–2012. Results Pancreatitis pain and the severity of pain statistically improved in 90% of patients after TP-IAT (p =<0.001). The relief from narcotics was sustained. Of the 75 patients undergoing TP-IAT, 31 (41.3%) achieved insulin independence. Younger age (p=0.032), lack of prior Puestow (p=0.018), lower body surface area (p=0.048), IEQ per Kg Body Weight (p=0.001) and total IEQ (100,000) (0.004) were associated with insulin independence. By multivariate analysis, 3 factors were associated with insulin independence after TP-IAT:(1) male gender, (2) lower body surface area and the (3) higher total IEQ per kilogram body weight. Total IEQ (100,000) was the single factor most strongly associated with insulin independence (OR = 2.62; p value < 0.001). Conclusions TP-IAT provides sustained pain relief and improved quality of life. The β cell function is dependent on islet yield. TP-IAT is an effective therapy for children with painful pancreatitis that fail medical and or endoscopic management PMID:24509206

  9. In vivo synchronous membrane potential oscillations in mouse pancreatic beta-cells: lack of co-ordination between islets.

    PubMed Central

    Valdeolmillos, M; Gomis, A; Sánchez-Andrés, J V

    1996-01-01

    1. The properties of the oscillations in electrical activity of different beta-cells within the same islet of Langerhans, and of different islets within the same pancreas, recorded in vivo, are described. 2. Simultaneous recordings of two cells within the same islet showed that the oscillations were synchronous. A rapid increase in blood glucose led to the simultaneous appearance of a transitory phase of continuous electrical activity in both cells. These results indicate that under physiological conditions, the islets operate as a functional syncytium. 3. Simultaneous recordings of cells from two different islets within the same pancreas showed that the oscillations in the electrical activity were not synchronous, which suggests that each islet is a functionally independent unit. Rapid changes in blood glucose led to the appearance of a transitory phase of increased electrical activity in both islets, although of different duration. These results suggest that the endocrine pancreas lacks a pacemaker driving the electrical activity of all the islets. 4. The comparison of the degree of activation of different islets, simultaneously recorded at different glucose concentrations, indicated that all the islets had a similar sensitivity to glucose. Furthermore, when the glucose concentration was increased, the electrical activity in both islets increased in parallel, suggesting that the amount of insulin released due to the increase in glycaemia was produced by the simultaneous response of all the islets and not by the recruitment of islets with different sensitivities to glucose. 5. Our results predict that the synchronous electrical activity of all the cells within an islet will result in widespread intracellular calcium oscillations and pulsatile insulin secretion. The periodicity of the pulses of insulin secretion in different islets is suggested to be of slightly different length and asynchronous. PMID:8735691

  10. Palmitic acid increase levels of pancreatic duodenal homeobox-1 and p38/stress-activated protein kinase in islets from rats maintained on a low protein diet.

    PubMed

    Arantes, Vanessa C; Reis, Marise A B; Latorraca, Márcia Q; Ferreira, Fabiano; Stoppiglia, Luiz Fabrízio; Carneiro, Everardo M; Boschero, Antonio C

    2006-12-01

    A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of beta-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6%) or normal (17%; control) protein diets and maintained for 48 h in culture medium containing 5.6 mmol/l glucose, with or without 0.6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2.8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways. PMID:17181874

  11. The use of biomaterials in islet transplantation.

    PubMed

    Borg, Danielle J; Bonifacio, Ezio

    2011-10-01

    Pancreatic islet transplantation is a therapeutic option to replace destroyed β cells in autoimmune diabetes. Islets are transplanted into the liver via the portal vein; however, inflammation, the required immunosuppression, and lack of vasculature decrease early islet viability and function. Therefore, the use of accessory therapy and biomaterials to protect islets and improve islet function has definite therapeutic potential. Here we review the application of niche accessory cells and factors, as well as the use of biomaterials as carriers or capsules, for pancreatic islet transplantation. PMID:21748257

  12. Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets.

    PubMed

    Kashio, Makiko; Tominaga, Makoto

    2015-05-01

    Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca(2+)-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H2O2, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca(2+)]i increases were likely caused by Ca(2+) influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca(2+). In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H2O2 application at 37 °C induced [Ca(2+)]i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H2O2 on KATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals.

  13. Three-dimensional culture of mouse pancreatic islet on a liver-derived perfusion-decellularized bioscaffold for potential clinical application.

    PubMed

    Xu, Tianxin; Zhu, Mingyan; Guo, Yibing; Wu, Di; Huang, Yan; Fan, Xiangjun; Zhu, Shajun; Lin, Changchun; Li, Xiaohong; Lu, Jingjing; Zhu, Hui; Zhou, Pengcheng; Lu, Yuhua; Wang, Zhiwei

    2015-10-01

    The cutting-edge technology of three-dimensional liver decellularized bioscaffold has a potential to provide a microenvironment that is suitable for the resident cells and even develop a new functional organ. Liver decellularized bioscaffold preserved the native extracellular matrix and three-dimensional architecture in support of the cell culture. The goal of this study was to discover if three-dimensional extracellular matrix derived from mouse liver could facilitate the growth and maintenance of physiological functions of mouse isolated islets. We generated a whole organ liver decellularized bioscaffold which could successfully preserve extracellular matrix proteins and the native vascular channels using 1% Triton X-100/0.1% ammonium protocol. To evaluate the potential of decellularized liver as a scaffold for islets transplantation, the liver decellularized bioscaffold was infused with mouse primary pancreatic islets which were obtained through Collagenase P digestion protocol. Its yield, morphology, and quality were estimated by microscopic analysis, dithizone staining, insulin immunofluorescence and glucose stimulation experiments. Comparing the three-dimensional culture in liver decellularized bioscaffold with the orthodoxy two-dimensional plate culture, hematoxylin-eosin staining, immunohistochemistry, and insulin gene expression were tested. Our results demonstrated that the liver decellularized bioscaffold could support cellular culture and maintenance of cell functions. In contrast with the conventional two-dimensional culture, three-dimensional culture system could give rise to an up-regulated insulin gene expression. These findings demonstrated that the liver bioscaffold by a perfusion-decellularized technique could serve as a platform to support the survival and function of the pancreatic islets in vitro. Meanwhile three-dimensional culture system had a superior role in contrast with the two-dimensional culture. This study advanced the field of

  14. Capecitabine, Temozolomide and Bevacizumab for Metastatic or Unresectable Pancreatic Neuroendocrine Tumors

    ClinicalTrials.gov

    2016-09-21

    Gastrinoma; Glucagonoma; Insulinoma; Pancreatic Polypeptide Tumor; Recurrent Islet Cell Carcinoma; Recurrent Pancreatic Cancer; Somatostatinoma; Stage III Pancreatic Cancer; Stage IV Pancreatic Cancer

  15. GLP-1R–Targeting Magnetic Nanoparticles for Pancreatic Islet Imaging

    PubMed Central

    Wang, Ping; Yoo, Byunghee; Yang, Jingsheng; Zhang, Xueli; Ross, Alana; Pantazopoulos, Pamela; Dai, Guangping; Moore, Anna

    2014-01-01

    Noninvasive assessment of pancreatic β-cell mass would tremendously aid in managing type 1 diabetes (T1D). Toward this goal, we synthesized an exendin-4 conjugated magnetic iron oxide–based nanoparticle probe targeting glucagon-like peptide 1 receptor (GLP-1R), which is highly expressed on the surface of pancreatic β-cells. In vitro studies in βTC-6, the β-cell line, showed specific accumulation of the targeted probe (termed MN-Ex10-Cy5.5) compared with nontargeted (termed MN-Cy5.5). In vivo magnetic resonance imaging showed a significant transverse relaxation time (T2) shortening in the pancreata of mice injected with the MN-Ex10-Cy5.5 probe compared with control animals injected with the nontargeted probe at 7.5 and 24 h after injection. Furthermore, ΔT2 of the pancreata of prediabetic NOD mice was significantly higher than that of diabetic NOD mice after the injection of MN-Ex10-Cy5.5, indicating the decrease of probe accumulation in these animals due to β-cell loss. Of note, ΔT2 of prediabetic and diabetic NOD mice injected with MN-Cy5.5 was not significantly changed, reflecting the nonspecific mode of accumulation of nontargeted probe. We believe our results point to the potential for using this agent for monitoring the disease development and response of T1D to therapy. PMID:24458362

  16. Expression of calbindin-D(28k) in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis.

    PubMed

    Rabinovitch, A; Suarez-Pinzon, W L; Sooy, K; Strynadka, K; Christakos, S

    2001-08-01

    Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.

  17. Detection of autoantibodies to the pancreatic islet heat shock protein 60 in insulin-dependent diabetes mellitus.

    PubMed

    Ozawa, Y; Kasuga, A; Nomaguchi, H; Maruyama, T; Kasatani, T; Shimada, A; Takei, I; Miyazaki, J; Saruta, T

    1996-08-01

    Autoantibodies against heat shock protein (hsp) 60 have been reported to be detected in sera of non-obese diabetic mice, in an experimental model of IDDM. However, there are only a few studies which have examined IDDM patients for antibodies against mammalian hsp60. We produced murine hsp60 derived from pancreatic beta cells which has high homology to human hsp60 and examined antibodies against the hsp60 in IDDM patients using an enzyme-linked immunosorbent assay. We extended the analysis to patients with other immune-mediated diseases and non-insulin-dependent diabetes mellitus (NIDDM). Positive sera for hsp60 antibody were more frequently detected in 13 out of 84 IDDM (15.5%) and 5 out of 25 rheumatoid arthritis patients (20%), when compared to healthy subjects (1/85; 1.2%, P < 0.001 and P < 0.01, respectively). The levels of hsp60 antibodies of IDDM (0.218 +/- 0.227) and rheumatoid arthritis patients (0.259 +/- 0.191) were significantly higher than those of healthy subjects (0.076 +/- 0.131, P < 0.001, P < 0.01, respectively). Patients with slowly progressive IDDM (n = 26), autoimmune thyroid disease (n = 42), or NIDDM (n = 40) had levels of hsp60 antibodies similar to those in healthy subjects. We found no relationship between the levels of hsp60 antibodies and islet cell antibodies (ICA) or antibodies to glutamic acid decarboxylase (GAD65) in IDDM patients. In conclusion, hsp60 antibodies were detected in Japanese IDDM as well as in rheumatoid arthritis patients. Although the positivity was low, the detection of hsp60 antibodies may be helpful for diagnosis of IDDM especially in GAD65 Ab- or JCA-negative Japanese patients. PMID:8864827

  18. Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment

    PubMed Central

    1992-01-01

    We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C- peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway. PMID:1639842

  19. Apoptosis in pancreatic β-islet cells in Type 2 diabetes

    PubMed Central

    Tomita, Tatsuo

    2016-01-01

    Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications.

  20. Apoptosis in pancreatic β-islet cells in Type 2 diabetes.

    PubMed

    Tomita, Tatsuo

    2016-08-01

    Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications. PMID:27209071

  1. Apoptosis in pancreatic β-islet cells in Type 2 diabetes.

    PubMed

    Tomita, Tatsuo

    2016-08-01

    Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications. PMID:27483174

  2. Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets.

    PubMed

    Alenkvist, Ida; Dyachok, Oleg; Tian, Geng; Li, Jia; Mehrabanfar, Saba; Jin, Yang; Birnir, Bryndis; Tengholm, Anders; Welsh, Michael

    2014-12-01

    The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression of Shb in β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereas Shb deficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics in Shb-knockout mice. Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor was Shb deficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca(2+) handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in the Shb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion, Shb deficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

  3. Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells via p-38 MAP Kinase-SMAD Pathway: An In-Vitro and In-Vivo Study

    PubMed Central

    Dadheech, Nidheesh; Srivastava, Abhay; Paranjape, Neha; Gupta, Shivika; Dave, Arpita; Shah, Girish M.; Bhonde, Ramesh R.; Gupta, Sarita

    2015-01-01

    Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes

  4. Acetylcholine and muscarinic receptor function in cerebral cortex of diabetic young and old male Wistar rats and the role of muscarinic receptors in calcium release from pancreatic islets.

    PubMed

    Savitha, Balakrishnan; Joseph, Binoy; Peeyush Kumar, T; Paulose, C S

    2010-04-01

    We investigated acetylcholine esterase (AChE) activity, acetylcholine and muscarinic M1, M3 receptors kinetics in the cerebral cortex of young and old streptozotocin induced and insulin treated diabetic rats. The role of muscarinic receptors in intracellular calcium release from pancreatic islets was studied in vitro. Wistar rats of 7 and 90-weeks old were used. All studies were done in cerebral cortex. AChE assay was done by spectrophotometric method. Radioreceptor binding assays were done for Acetylcholine, Muscarinic M1 and M3 receptors using specific ligands. Calcium imaging was done using fluo4-AM in pancreatic cells. Ninety-weeks old control rats showed significantly decreased Vmax and increased Km for AChE compared to 7-weeks old control rats. An increased Vmax observed in both 7 and 90-weeks old diabetic groups with significant decrease in Km. Scatchard analysis using specific agonists showed significant decrease in the B (max) and K (d) of acetylcholine and muscarinic M1 receptors in 90-weeks old control rats compared to 7-weeks old control. Binding studies for M3 receptors showed no significant change compared to 7-weeks old control. Acetylcholine, muscarinic M1 and M3 receptor number significantly increased in 90-weeks old diabetic rat groups compared to their respective controls. Insulin treatment significantly reversed the binding parameters to near control compared to diabetic group. In vitro studies showed that acetylcholine through muscarinic M1 and M3 receptors' stimulated calcium release from the pancreatic islets. Thus our studies suggest that Insulin signaling play an important part in differentially regulating pancreatic cholinergic activity, and the diabetes mediated cortical dysfunctions with age.

  5. β-cell-targeted blockage of PD1 and CTLA4 pathways prevents development of autoimmune diabetes and acute allogeneic islets rejection

    PubMed Central

    El Khatib, Moustafa; Sakuma, Toshie; Tonne, Jason M.; Mohamed, Magid S.; Holditch, Sara J.; Lu, Brian; Kudva, Yogish C.; Ikeda, Yasuhiro

    2015-01-01

    Protection of beta cells from autoimmune destruction potentially cures type 1 diabetes mellitus (T1D). During antigen presentation, interactions between cytotoxic T-lymphocyte antigen-4 (CTLA4) and B7 molecules, or programmed death 1 (PD1) and its ligand PDL1, negatively regulate immune responses in a non-redundant manner. Here, we employed beta cell-targeted adeno-associated virus serotype 8 (AAV8)-based vectors to over-express an artificial PDL1-CTLA4Ig polyprotein or IL10. Beta cell-targeted expression of PDL1-CTLA4Ig or IL10 preserved beta cell mass and protected NOD mice from T1D development. When NOD mice were treated with vectors at early onset of hyperglycemia, PDL1-CTLA4Ig or IL10 alone failed to normalize the early onset of hyperglycemia. When drug-induced diabetic mice received MHC-matched allo-islets, with or without pretreatment of the PDL1-CTLA4Ig-expressing vector, PDL1-CTLA4Ig-expressing islets were protected from rejection for at least 120 days. Similarly, transplantation of PDL1-CTLA4Ig-expressing MHC-matched islets into mice with established T1D resulted in protection of allo-islets from acute rejection, although islet grafts were eventually rejected. Thus, the present study demonstrates the potent immuno-suppressive effects of beta cell-targeted PDL1-CTLA4Ig overexpression against T1D development and allo-islet rejection. The gene-based simultaneous inhibition of PD1 and CTLA4 pathways provides a unique strategy for immunosuppression-free tissue/organ transplantation, especially in the setting of no established autoimmunity. PMID:25786871

  6. Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

    PubMed

    Conlon, J M; Davis, M S; Falkmer, S; Thim, L

    1987-11-01

    The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I. PMID:2889597

  7. Paternal high-fat diet consumption induces common changes in the transcriptomes of retroperitoneal adipose and pancreatic islet tissues in female rat offspring.

    PubMed

    Ng, Sheau-Fang; Lin, Ruby C Y; Maloney, Christopher A; Youngson, Neil A; Owens, Julie A; Morris, Margaret J

    2014-04-01

    We previously showed that paternal high-fat diet (HFD) consumption programs β-cell dysfunction in female rat offspring, together with transcriptome alterations in islets. Here we investigated the retroperitoneal white adipose tissue (RpWAT) transcriptome using gene and pathway enrichment and pathway analysis to determine whether commonly affected network topologies exist between these two metabolically related tissues. In RpWAT, 5108 genes were differentially expressed due to a paternal HFD; the top 5 significantly enriched networks identified by pathway analysis in offspring of HFD fathers compared with those of fathers fed control diet were: mitochondrial and cellular response to stress, telomerase signaling, cell death and survival, cell cycle, cellular growth and proliferation, and cancer. A total of 187 adipose olfactory receptor genes were down-regulated. Interrogation against the islet transcriptome identified specific gene networks and pathways, including olfactory receptor genes that were similarly affected in both tissues (411 common genes, P<0.05). In particular, we highlight a common molecular network, cell cycle and cancer, with the same hub gene, Myc, suggesting early onset developmental changes that persist, shared responses to programmed systemic factors, or crosstalk between tissues. Thus, paternal HFD consumption triggers unique gene signatures, consistent with premature aging and chronic degenerative disorders, in both RpWAT and pancreatic islets of daughters. PMID:24421403

  8. Alterations of pancreatic islet structure, metabolism and gene expression in diet-induced obese C57BL/6J mice.

    PubMed

    Roat, Regan; Rao, Vandana; Doliba, Nicolai M; Matschinsky, Franz M; Tobias, John W; Garcia, Eden; Ahima, Rexford S; Imai, Yumi

    2014-01-01

    The reduction of functional β cell mass is a key feature of type 2 diabetes. Here, we studied metabolic functions and islet gene expression profiles of C57BL/6J mice with naturally occurring nicotinamide nucleotide transhydrogenase (NNT) deletion mutation, a widely used model of diet-induced obesity and diabetes. On high fat diet (HF), the mice developed obesity and hyperinsulinemia, while blood glucose levels were only mildly elevated indicating a substantial capacity to compensate for insulin resistance. The basal serum insulin levels were elevated in HF mice, but insulin secretion in response to glucose load was significantly blunted. Hyperinsulinemia in HF fed mice was associated with an increase in islet mass and size along with higher BrdU incorporation to β cells. The temporal profiles of glucose-stimulated insulin secretion (GSIS) of isolated islets were comparable in HF and normal chow fed mice. Islets isolated from HF fed mice had elevated basal oxygen consumption per islet but failed to increase oxygen consumption further in response to glucose or carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP). To obtain an unbiased assessment of metabolic pathways in islets, we performed microarray analysis comparing gene expression in islets from HF to normal chow-fed mice. A few genes, for example, those genes involved in the protection against oxidative stress (hypoxia upregulated protein 1) and Pgc1α were up-regulated in HF islets. In contrast, several genes in extracellular matrix and other pathways were suppressed in HF islets. These results indicate that islets from C57BL/6J mice with NNT deletion mutation develop structural, metabolic and gene expression features consistent with compensation and decompensation in response to HF diet. PMID:24505268

  9. Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.

    PubMed

    Benninger, R K P; Head, W Steven; Zhang, Min; Satin, Leslie S; Piston, David W

    2011-11-15

    Cell-cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca(2+)](i)) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca(2+)](i) ceases when glucose is at a basal levels. We determine the roles of gap-junctions and other cell-cell communication pathways in the suppression of insulin secretion under basal conditions. Metabolic, electrical and insulin secretion levels were measured from islets lacking gap-junction coupling following deletion of connexion36 (Cx36(-/-)), and these results were compared to those obtained using fully isolated β-cells. K(ATP) loss-of-function islets provide a further experimental model to specifically study gap-junction mediated suppression of electrical activity. In isolated β-cells or Cx36(-/-) islets, elevations in [Ca(2+)](i) persisted in a subset of cells even at basal glucose. Isolated β-cells showed elevated insulin secretion at basal glucose; however, insulin secretion from Cx36(-/-) islets was minimally altered. [Ca(2+)](i) was further elevated under basal conditions, but insulin release still suppressed in K(ATP) loss-of-function islets. Forced elevation of cAMP led to PKA-mediated increases in insulin secretion from islets lacking gap-junctions, but not from islets expressing Cx36 gap junctions. We conclude there is a redundancy in how cell-cell communication in the islet suppresses insulin release. Gap junctions suppress cellular heterogeneity and spontaneous [Ca(2+)](i) signals, while other juxtacrine mechanisms, regulated by PKA and glucose, suppress more distal steps in exocytosis. Each mechanism is sufficiently robust to compensate for a loss of the other and still suppress basal insulin secretion.

  10. Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets.

    PubMed

    Olsson, Anders H; Volkov, Petr; Bacos, Karl; Dayeh, Tasnim; Hall, Elin; Nilsson, Emma A; Ladenvall, Claes; Rönn, Tina; Ling, Charlotte

    2014-11-01

    Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic

  11. Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets.

    PubMed

    Olsson, Anders H; Volkov, Petr; Bacos, Karl; Dayeh, Tasnim; Hall, Elin; Nilsson, Emma A; Ladenvall, Claes; Rönn, Tina; Ling, Charlotte

    2014-11-01

    Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic

  12. Genome-Wide Associations between Genetic and Epigenetic Variation Influence mRNA Expression and Insulin Secretion in Human Pancreatic Islets

    PubMed Central

    Olsson, Anders H.; Volkov, Petr; Bacos, Karl; Dayeh, Tasnim; Hall, Elin; Nilsson, Emma A.; Ladenvall, Claes; Rönn, Tina; Ling, Charlotte

    2014-01-01

    Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic

  13. The angiotensin-converting enzyme 2/angiotensin (1-7)/Mas axis protects the function of pancreatic β cells by improving the function of islet microvascular endothelial cells.

    PubMed

    Lu, Chun-Li; Wang, Ying; Yuan, Li; Li, Yang; Li, Xiao-Ya

    2014-11-01

    In the diabetic state, the local rennin-angiotensin system (RAS) is activated in the pancreas, and is strongly associated with islet dysfunction. The angiotensin-converting enzyme 2 (ACE2)/angiotensin (1-7) [Ang(1-7)]/Mas axis is a protective, negative regulator of the classical renin-angiotensin system. In this study, we assessed the role of the ACE2/Ang(1‑7)/Mas axis in pancreatic β cell survival and function. ACE2 knockout and wild-type mice were fed a high-fat diet for 16 weeks. We then performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, and determined the expression levels of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) in the pancreatic islets. The effects of Ang(1-7) or Mas receptor silencing on endothelial function were assessed in MS-1 cells. MIN6 cells were then co-cultured with the MS-1 cells to evaluate the effects of ACE2 on insulin secretion. The ACE2 knockout mice were more susceptible than the wild-type mice to high-fat diet-induced β cell dysfunction. The TUNEL-positive area of the pancreatic islets and the expression levels of IL-1β and iNOS were markedly increased in the ACE2 knockout mice compared with their wild-type littermates. The Mas-silenced MS-1 cells were more sensitive to palmitate-induced dysfunction and apoptosis in vitro. Ang(1-7) increased the activity of the Akt/endothelial NOS/nitric oxide (NO) pathway in the MS-1 cells, protected MIN6 cells against palmitate-induced apoptosis, and improved MIN6 insulin secretory function in the co-culture system. In conclusion, this study demonstrates that the ACE2/Ang(1-7)/Mas axis is a potential target for protecting the funcion of β cells by improving the function of islet microvascular endothelial cells. PMID:25175177

  14. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    SciTech Connect

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  15. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets.

    PubMed

    Sordi, Valeria; Malosio, Maria Luisa; Marchesi, Federica; Mercalli, Alessia; Melzi, Raffaella; Giordano, Tiziana; Belmonte, Nathalie; Ferrari, Giuliana; Leone, Biagio Eugenio; Bertuzzi, Federico; Zerbini, Gianpaolo; Allavena, Paola; Bonifacio, Ezio; Piemonti, Lorenzo

    2005-07-15

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)-positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs. PMID:15784733

  16. Small human islets comprised of more β-cells with higher insulin content than large islets

    PubMed Central

    Farhat, Bilal; Almelkar, Akshay; Ramachandran, Karthik; Williams, S. Janette; Huang, Han-Hung; Zamierowksi, David; Novikova, Lesya; Stehno-Bittel, Lisa

    2013-01-01

    For the past 30 y, data have suggested that unique islet populations exist, based on morphology and glucose sensitivity. Yet little has been done to determine the mechanism of these functional differences. The purpose of this study was to determine whether human islets were comprised functionally unique populations, and to elucidate a possible mechanism. Islets or pancreatic sections from 29 human donors were analyzed. Islets were isolated and measured for insulin secretion, cell composition and organization, insulin and glucagon granule density and insulin content. Insulin secretion was significantly greater in small compared with large islets. In sectioned human pancreata, β-cells comprised a higher proportion of the total endocrine cells in small islets (63%) than large islets (39%). A higher percentage of β-cells in small islets contacted blood vessels (44%) compared with large islets (31%). Total insulin content of isolated human islets was significantly greater in the small (1323 ± 512 μIU/IE) compared with large islets (126 ± 48 μIU/IE). There was less immunostaining for insulin in the large islets from human pancreatic sections, especially in the core of the islet, compared with small islets. The results suggest that differences in insulin secretion between large and small islets may be due to a higher percentage of β-cells in small islets with more β-cells in contact with blood vessels and a higher concentration of insulin/β-cell in small islets. PMID:23648896

  17. Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes.

    PubMed

    Radichev, Ilian A; Maneva-Radicheva, Lilia V; Amatya, Christina; Salehi, Maryam; Parker, Camille; Ellefson, Jacob; Burn, Paul; Savinov, Alexei Y

    2016-02-15

    Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and β cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D. PMID:26773144

  18. Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets.

    PubMed

    Takahashi, Rui; Ishihara, Hisamitsu; Tamura, Akira; Yamaguchi, Suguru; Yamada, Takahiro; Takei, Daisuke; Katagiri, Hideki; Endou, Hitoshi; Oka, Yoshitomo

    2006-02-01

    Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.

  19. Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

    PubMed

    Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

    2016-01-01

    Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.

  20. Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

    PubMed

    Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

    2016-01-01

    Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. PMID:26578518

  1. COMBINED PANCREATIC ISLET AND KIDNEY TRANSPLANTATION IN A CHILD WITH UNSTABLE TYPE 1 DIABETES AND END-STAGE RENAL DISEASE

    PubMed Central

    Benedict, K.A.; Moassesfar, S.; Adi, S.; Gitelman, S.E.; Brennan, J.L.; McEnhill, M.; Stock, P.G.; Portale, A.A.; Posselt, A.M.

    2014-01-01

    Islet transplantation after successful kidney transplantation is a recognized treatment for adults with diabetes and end-stage renal disease (ESRD), but has not been considered an option in the pediatric population. To our knowledge, we report the first combined islet and kidney transplant in a child. The patient was born with bilateral renal hypoplasia and was diagnosed with type 1 diabetes mellitus at age 13 months. He had erratic glycemic control and hypoglycemia unawareness. At 6 years of age, the child safely underwent simultaneous islet and live donor kidney transplantation. Although function of the islet graft was transient, the combined transplant provided significant benefits in terms of glucose control and overall growth and development. Such an approach represents a viable treatment option for pediatric patients with ESRD and unstable diabetes. PMID:23763601

  2. Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).

    PubMed Central

    Diraison, Frédérique; Parton, Laura; Ferré, Pascal; Foufelle, Fabienne; Briscoe, Celia P; Leclerc, Isabelle; Rutter, Guy A

    2004-01-01

    Accumulation of intracellular lipid by pancreatic islet beta-cells has been proposed to inhibit normal glucose-regulated insulin secretion ('glucolipotoxicity'). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for beta-cells at the islet periphery. Real-time PCR (TaqMan) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-gamma (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of beta-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes. PMID:14690455

  3. Is Dynamic Autocrine Insulin Signaling Possible? A Mathematical Model Predicts Picomolar Concentrations of Extracellular Monomeric Insulin within Human Pancreatic Islets

    PubMed Central

    Wang, Minghu; Li, Jiaxu; Lim, Gareth E.; Johnson, James D.

    2013-01-01

    Insulin signaling is essential for -cell survival and proliferation in vivo. Insulin also has potent mitogenic and anti-apoptotic actions on cultured -cells, with maximum effect in the high picomolar range and diminishing effect at high nanomolar doses. In order to understand whether these effects of insulin are constitutive or can be subjected to physiological modulation, it is essential to estimate the extracellular concentration of monomeric insulin within an intact islet. Unfortunately, the in vivo concentration of insulin monomers within the islet cannot be measured directly with current technology. Here, we present the first mathematical model designed to estimate the levels of monomeric insulin within the islet extracellular space. Insulin is released as insoluble crystals that exhibit a delayed dissociation into hexamers, dimers, and eventually monomers, which only then can act as signaling ligands. The rates at which different forms of insulin dissolve in vivo have been estimated from studies of peripheral insulin injection sites. We used this and other information to formulate a mathematical model to estimate the local insulin concentration within a single islet as a function of glucose. Model parameters were estimated from existing literature. Components of the model were validated using experimental data, if available. Model analysis predicted that the majority of monomeric insulin in the islet is that which has been returned from the periphery, and the concentration of intra-islet monomeric insulin varies from 50–300 pM when glucose is in the physiological range. Thus, our results suggest that the local concentration of monomeric insulin within the islet is in the picomolar ‘sweet spot’ range of insulin doses that activate the insulin receptor and have the most potent effects on -cells in vitro. Together with experimental data, these estimations support the concept that autocrine/paracrine insulin signalling within the islet is dynamic, rather

  4. Low protein diet confers resistance to the inhibitory effects of interleukin 1beta on insulin secretion in pancreatic islets*

    PubMed

    Vieira, E C.; Carneiro, E M.; Latorraca, M Q.; Delguingaro-Augusto, V; Amaral, M E.C.; Bosqueiro, J R.; Boschero, A C.

    2001-05-01

    High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1beta (IL-1beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1beta for 1 h or 24 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1beta-induced nitric oxide (NO) production, lower inhibition of D-[U(14)C]-glucose oxidation to (14)CO(2) and less pronounced effect of IL-1beta on alpha-ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K(+) the inhibitory effect of IL-1beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta-cells of the deleterious effect of IL-1beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca(2+) homeostasis. PMID:11382546

  5. VEGF-conjugated alginate hydrogel prompt angiogenesis and improve pancreatic islet engraftment and function in type 1 diabetes.

    PubMed

    Yin, Nina; Han, Yongming; Xu, Hanlin; Gao, Yisen; Yi, Tao; Yao, Jiale; Dong, Li; Cheng, Dejun; Chen, Zebin

    2016-02-01

    Type 1 diabetes was a life-long disease that affected numerous people around the world. Insulin therapy has its limitations that may involve hyperglycemia and heavy burden of patient by repeated dose. Islet transplantation emerged as a promising approach to reach periodical reverse of diabetes, however, transplanted islets suffer from foreign body reaction and lack of nutrition and oxygen supply, especially in the blood-vessel-shortage subcutaneous site which was preferred by patient and surgeon. In this study, we designed and synthesized a vascular endothelial growth factor (VEGF) conjugated alginate material to encapsulate the transplanted islets via 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) reaction, and successful conjugation was confirmed by Nuclear Magnetic Resonance H1 spectrum. The best VEGF concentration (100ng/ml) was determined by the combined studies of the mechanical property and endothelial cell growth assay. In vivo study, conjugated VEGF on alginate exhibited sustained promoting angiogenesis property after subcutaneous transplantation by histology study and islets encapsulated in this material achieved long term therapeutic effect (up to 50days) in the diabetic mice model. In conclusion, this study establishes a simple biomaterial strategy for islet transplantation to enhance islet survival and function, which could be a feasible therapeutic alternative for type 1 diabetes. PMID:26652453

  6. Pancreatic islet regeneration and some liver biochemical parameters of leaf extracts of Vitex doniana in normal and streptozotocin-induced diabetic albino rats

    PubMed Central

    Oche, Okpe; Sani, Ibrahim; Chilaka, Njoku Godwin; Samuel, Ndidi Uche; Samuel, Atabo

    2014-01-01

    Objective To test two water soluble extracts (aqueous and ethanolic) obtained from the leaves of Vitex doniana in normal and streptozotocin-induced diabetic rats for their effects on pancreatic endocrine tissues and serum marker enzymes for a period of 21 d. Methods A total of 55 rats divided into 11 groups of 5 rats each were assigned into diabetic and non-diabetic groups and followed by a daily administration of ethanolic and aqueous extracts for 21 d. Group 1 was the normal control while group 7 was treated with standard drug. Results The histopathological studies of the diabetic rats indicated increase in the volume density of islets, percent of β-cells and size of islet in the groups that received the plant extracts, which suggested regeneration of β-cells along with β-cells repairs, as compared with the non-treated diabetic control which showed complete degeneration of the islet cells. There was significant reduction (P<0.05) in the serum activities of marker enzymes, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in diabetes treated rats, whereas an insignificant increase (P>0.01) in the serum activities of marker enzymes was observed for non-diabetic treated rats. Results of total bilirubin, direct bilirubin and unconjugated bilirubin showed that diabetic control group was significantly higher (P<0.05) in total bilirubin and unconjugated bilirubin compared with treated groups while non-diabetic treated groups showed no significant increase (P>0.01) in total bilirubin and direct bilirubin compared with the normal control. Conclusion This herbal therapy appears to bring about repair/regeneration of the endocrine pancreas and hepatic cells protection in the diabetic rat. PMID:25182283

  7. Yapsin 1 immunoreactivity in alpha cells of human pancreatic Islets: Implications for the processing of human proglucagon by mammalian aspartic proteases

    PubMed Central

    Cawley, Niamh X.; Portela-Gomes, Guida; Lou, Hong; Loh, Y. Peng

    2013-01-01

    Yapsin 1 is an aspartic protease from Saccharomyces cerevisiae and belongs to a class of aspartic proteases that demonstrate specificity for basic amino acids. It is capable of processing prohormone substrates at specific basic residue cleavage sites, similar to that of the prohormone convertases, to generate bioactive peptide hormones. An antibody raised against yapsin 1 was previously shown to immuno-stain endocrine cells of rat pituitary and brain as well as lysates from bovine pituitary secretory granules demonstrating the existence of yapsin 1-like aspartic proteases in mammalian endocrine tissues, potentially involved in peptide hormone production. Here we show the specific staining of yapsin 1 immunoreactivity in the alpha cells of human pancreatic islets. No staining was observed in the beta or delta cells indicating a specificity of the staining for glucagon producing and not insulin or somatostatin producing cells. Purified yapsin 1 was also shown to process proglucagon into authentic glucagon in vitro demonstrating that the prototypical enzyme of this sub-class of enzymes can correctly process proglucagon to glucagon. These findings suggest the existence of a yapsin 1-like enzyme exclusively in the alpha cells of the islets of Langerhans in humans and may play a role in the production of glucagon in that tissue. PMID:21632904

  8. Preptin derived from proinsulin-like growth factor II (proIGF-II) is secreted from pancreatic islet beta-cells and enhances insulin secretion.

    PubMed Central

    Buchanan, C M; Phillips, A R; Cooper, G J

    2001-01-01

    Pancreatic islet beta-cells secrete the hormones insulin, amylin and pancreastatin. To search for further beta-cell hormones, we purified peptides from secretory granules isolated from cultured murine beta TC6-F7 beta-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp(69)-Leu(102) of the proinsulin-like growth factor II E-peptide, which we have termed 'preptin'. Preptin, is present in islet beta-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion. PMID:11716772

  9. Pancreatic islet cell tumor

    MedlinePlus

    Complications of these tumors include: Diabetes Hormone crises (if the tumor releases certain types of hormones) Severe low blood sugar (from insulinomas) Severe ulcers in the stomach and small intestine (from gastrinomas) Spread of the tumor to the liver

  10. Increased L-CPT-1 activity and altered gene expression in pancreatic islets of malnourished adult rats: a possible relationship between elevated free fatty acid levels and impaired insulin secretion.

    PubMed

    de Barros Reis, Marise Auxiliadora; Arantes, Vanessa Cristina; Cunha, Daniel Andrade; Latorraca, Márcia Queiroz; Toyama, Marcos Hikari; Carneiro, Everardo Magalhães; Boschero, Antonio Carlos

    2008-02-01

    Intrauterine growth restriction is associated with chronically elevated levels of serum fatty acids and reduced glucose-stimulated insulin secretion. Lipid metabolism in pancreatic beta cells is critical for the regulation of insulin secretion, and the chronic exposure to fatty acids results in higher palmitate oxidation rates and an altered insulin response to glucose. Using a rat model of isocaloric protein restriction, we examined whether pre- and postnatal protein malnutrition influences the properties of pancreatic islet carnitine palmitoyltransferase-1 (liver isoform, L-CPT-1), a rate-limiting enzyme that regulates fatty acid oxidation in mitochondria. The activity of L-CPT-1 in pancreatic islets increased in the low protein (LP), although the L-CPT-1 mRNA levels were unaffected by malnutrition. The susceptibility of enzyme to inhibition by malonyl-CoA was unaltered and the content of malonyl-CoA was reduced in LP cells. Because the mitochondrial oxidation of fatty acids is related to the altered expression of a number of genes encoding proteins involved in insulin secretion, the levels of expression of insulin and GLUT-2 mRNA were assessed. A reduced expression of both genes was observed in malnourished rats. These results provide further evidence that increased L-CPT-1 activity and changes in gene expression in pancreatic islets may be involved in the reduced insulin secretion seen in malnourished rats. PMID:17531461

  11. Islet cell transplantation.

    PubMed

    Srinivasan, P; Huang, G C; Amiel, S A; Heaton, N D

    2007-04-01

    People with type 1 diabetes have normal exocrine pancreatic function, making islet cell rather than whole organ transplantation an attractive option. Achieving insulin independence in type 1 diabetes was the perceived goal of islet cell transplantation. The success of the Edmonton group in achieving this in a selected group of type 1 patients has led to renewed optimism that this treatment could eventually replace whole organ pancreas transplantation. However the long-term results of this treatment indicate that insulin independence is lost with time in a significant proportion of patients, although they may retain glycaemic stability. In this context, the indications for islet cell transplantation, which have evolved over the last 5 years, indicate that the patients who benefit most are those who experience severe hypoglycaemic reactions despite optimal insulin therapy. This review will summarise the history of islet cell transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, indications for islet cell transplantation, current clinical trials, the early UK islet cell transplant experience using the Edmonton protocol, and some of the challenges that lie ahead. PMID:17403947

  12. Human islet isolation--a prospective randomized comparison of pancreatic vascular perfusion with hyperosmolar citrate or University of Wisconsin solution.

    PubMed

    Robertson, G S; Chadwick, D; Thirdborough, S; Swift, S; Davies, J; James, R; Bell, P R; London, N J

    1993-09-01

    University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC.

  13. Human islet isolation--a prospective randomized comparison of pancreatic vascular perfusion with hyperosmolar citrate or University of Wisconsin solution.

    PubMed

    Robertson, G S; Chadwick, D; Thirdborough, S; Swift, S; Davies, J; James, R; Bell, P R; London, N J

    1993-09-01

    University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC. PMID:8212148

  14. Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

    PubMed

    Hernández-Fisac, Inés; Fernández-Pascual, Sergio; Ortsäter, Henrik; Pizarro-Delgado, Javier; Martín del Río, Rafael; Bergsten, Peter; Tamarit-Rodriguez, Jorge

    2006-11-15

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

  15. Quantitative Assessment of Proliferative Effects of Oral Vanadium on Pancreatic Islet Volumes and Beta Cell Numbers of Diabetic Rats

    PubMed Central

    Pirmoradi, Leila; Noorafshan, Ali; Safaee, Akbar; Dehghani, Gholam Abbas

    2016-01-01

    Background: Oral vanadyl sulfate (vanadium) induces normoglycemia, proliferates beta cells and prevents pancreatic islet atrophy in streptozotocin-induced diabetic rats. Soteriological method is used to quantitate the proliferative effects of vanadium on beta-cell numbers and islet volumes of normal and diabetic rats. Methods: Adult male Sprague-Dawley rats were made diabetic with intravenous streptozotocin injection (40 mg/kg). Normal and diabetic rats were divided into four groups. While control normal and diabetic (CD) groups used water, vanadium-treated normal (VTN) and diabetic (VTD) groups used solutions containing vanadyl sulfate (0.5-1 mg/mL, VOSO4+5H2O). Tail blood samples were used to measure blood glucose (BG) and plasma insulin. Two months after treatment, rats were sacrificed, pancreata prepared, and stereology method was used to quantitatively evaluate total beta cell numbers (TBCN) and total islet volumes (TISVOL). Results: Normoglycemia persisted in VTN with significantly decreased plasma insulin (0.190.08 vs. 0.970.27 ng/dL, P<0.002). The respective high BG (53249 vs. 14446 mg/dL, P<0.0001) and reduced plasma insulin (0.260.15 vs. 0.540.19 ng/dL, P<0.002) seen in CD were reversed in VTD during vanadium treatment or withdrawal. While the induction of diabetes, compared to their control, significantly decreased TISVOL (1.90.2 vs. 3.030.6 mm3, P<0.003) and TBCN (0.990.1 vs. 3.20.2 x 106, P<0.003), vanadium treatment significantly increased TISVOL (2.90.8 and 4.071.0 mm3, P<0.003) and TBCN (1.50.3 and 3.80.6 x 106, P<0.03). Conclusion: Two-month oral vanadium therapy in STZ-diabetic rats ameliorated hyperglycemia by partially restoring plasma insulin. This action was through proliferative actions of vanadium in preventing islet atrophy by increasing beta-cell numbers. PMID:26459400

  16. Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence.

    PubMed

    de Vos, Paul; Smink, Alexandra M; Paredes, Genaro; Lakey, Jonathan R T; Kuipers, Jeroen; Giepmans, Ben N G; de Haan, Bart J; Faas, Marijke M

    2016-01-01

    The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet

  17. Polyphenol-Rich Extract of Syzygium cumini Leaf Dually Improves Peripheral Insulin Sensitivity and Pancreatic Islet Function in Monosodium L-Glutamate-Induced Obese Rats.

    PubMed

    Sanches, Jonas R; França, Lucas M; Chagas, Vinicyus T; Gaspar, Renato S; Dos Santos, Kayque A; Gonçalves, Luciana M; Sloboda, Deborah M; Holloway, Alison C; Dutra, Richard P; Carneiro, Everardo M; Cappelli, Ana Paula G; Paes, Antonio Marcus de A

    2016-01-01

    Syzygium cumini (L.) Skeels (Myrtaceae) has been traditionally used to treat a number of illnesses. Ethnopharmacological studies have particularly addressed antidiabetic and metabolic-related effects of extracts prepared from its different parts, especially seed, and pulp-fruit, however. there is a lack of studies on phytochemical profile and biological properties of its leaf. As there is considerable interest in bioactive compounds to treat metabolic syndrome and its clustered risk factors, we sought to characterize the metabolic effects of hydroethanolic extract of S. cumini leaf (HESc) on lean and monosodium L-glutamate (MSG)-induced obese rats. HPLC-MS/MS characterization of the HESc polyphenolic profile, at 254 nm, identified 15 compounds pertaining to hydrolysable tannin and flavanol subclasses. At 60 days of age, both groups were randomly assigned to receive HESc (500 mg/kg) or vehicle for 30 days. At the end of treatment, obese+HESc exhibited significantly lower body weight gain, body mass index, and white adipose tissue mass, compared to obese rats receiving vehicle. Obese rats treated with HESc showed a twofold increase in lipolytic activity in the periepididymal fat pad, as well as, brought triglyceride levels in serum, liver and skeletal muscle back to levels close those found in lean animals. Furthermore, HESc also improved hyperinsulinemia and insulin resistance in obese+HESc rats, which resulted in partial reversal of glucose intolerance, as compared to obese rats. HESc had no effect in lean rats. Assessment of ex vivo glucose-stimulated insulin secretion showed HESc potentiated pancreatic function in islets isolated from both lean and obese rats treated with HESc. In addition, HESc (10-1000 μg/mL) increased glucose stimulated insulin secretion from both isolated rat islets and INS-1E β-cells. These data demonstrate that S. cumini leaf improved peripheral insulin sensitivity via stimulating/modulating β-cell insulin release, which was associated

  18. Polyphenol-Rich Extract of Syzygium cumini Leaf Dually Improves Peripheral Insulin Sensitivity and Pancreatic Islet Function in Monosodium L-Glutamate-Induced Obese Rats

    PubMed Central

    Sanches, Jonas R.; França, Lucas M.; Chagas, Vinicyus T.; Gaspar, Renato S.; dos Santos, Kayque A.; Gonçalves, Luciana M.; Sloboda, Deborah M.; Holloway, Alison C.; Dutra, Richard P.; Carneiro, Everardo M.; Cappelli, Ana Paula G.; Paes, Antonio Marcus de A.

    2016-01-01

    Syzygium cumini (L.) Skeels (Myrtaceae) has been traditionally used to treat a number of illnesses. Ethnopharmacological studies have particularly addressed antidiabetic and metabolic-related effects of extracts prepared from its different parts, especially seed, and pulp-fruit, however. there is a lack of studies on phytochemical profile and biological properties of its leaf. As there is considerable interest in bioactive compounds to treat metabolic syndrome and its clustered risk factors, we sought to characterize the metabolic effects of hydroethanolic extract of S. cumini leaf (HESc) on lean and monosodium L-glutamate (MSG)-induced obese rats. HPLC-MS/MS characterization of the HESc polyphenolic profile, at 254 nm, identified 15 compounds pertaining to hydrolysable tannin and flavanol subclasses. At 60 days of age, both groups were randomly assigned to receive HESc (500 mg/kg) or vehicle for 30 days. At the end of treatment, obese+HESc exhibited significantly lower body weight gain, body mass index, and white adipose tissue mass, compared to obese rats receiving vehicle. Obese rats treated with HESc showed a twofold increase in lipolytic activity in the periepididymal fat pad, as well as, brought triglyceride levels in serum, liver and skeletal muscle back to levels close those found in lean animals. Furthermore, HESc also improved hyperinsulinemia and insulin resistance in obese+HESc rats, which resulted in partial reversal of glucose intolerance, as compared to obese rats. HESc had no effect in lean rats. Assessment of ex vivo glucose-stimulated insulin secretion showed HESc potentiated pancreatic function in islets isolated from both lean and obese rats treated with HESc. In addition, HESc (10–1000 μg/mL) increased glucose stimulated insulin secretion from both isolated rat islets and INS-1E β-cells. These data demonstrate that S. cumini leaf improved peripheral insulin sensitivity via stimulating/modulating β-cell insulin release, which was associated

  19. Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

    PubMed Central

    Reinert, Rachel B.; Cai, Qing; Hong, Ji-Young; Plank, Jennifer L.; Aamodt, Kristie; Prasad, Nripesh; Aramandla, Radhika; Dai, Chunhua; Levy, Shawn E.; Pozzi, Ambra; Labosky, Patricia A.; Wright, Christopher V. E.; Brissova, Marcela; Powers, Alvin C.

    2014-01-01

    Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers. PMID:24574008

  20. Effects of ethanolic extract of Syzygium cumini (Linn) seed powder on pancreatic islets of alloxan diabetic rats.

    PubMed

    Singh, N; Gupta, M

    2007-10-01

    The ethanolic extract of seeds of S. cumini increased body weight and decreased blood sugar level in alloxan diabetic albino rats. Level of significance for decrease in blood sugar after feeding alcoholic extract of S. cumini seeds in various doses was highly significant. The extract feeding showed definite improvement in the histopathology of islets. The most important finding is that the blood sugar level, which once dropped to normal levels after extract feeding was not elevated when extract feeding was discontinued for 15 days. PMID:17948734

  1. Imaging of the islet neural network.

    PubMed

    Tang, S-C; Peng, S-J; Chien, H-J

    2014-09-01

    The islets of Langerhans receive signals from the circulation and nerves to modulate hormone secretion in response to physiological cues. Although the rich islet innervation has been documented in the literature dating as far back as Paul Langerhans' discovery of islets in the pancreas, it remains a challenging task for researchers to acquire detailed islet innervation patterns in health and disease due to the dispersed nature of the islet neurovascular network. In this article, we discuss the recent development of 3-dimensional (3D) islet neurohistology, in which transparent pancreatic specimens were prepared by optical clearing to visualize the islet microstructure, vasculature and innervation with deep-tissue microscopy. Mouse islets were used as an example to illustrate how to apply this 3D imaging approach to characterize (i) the islet parasympathetic innervation, (ii) the islet sympathetic innervation and its reinnervation after transplantation under the kidney capsule and (iii) the reactive cellular response of the Schwann cell network in islet injury. While presenting and characterizing the innervation patterns, we also discuss how to apply the signals derived from transmitted light microscopy, vessel painting and immunostaining of neural markers to verify the location and source of tissue information. In summary, the systematic development of tissue labelling, clearing and imaging methods to reveal the islet neuroanatomy offers insights to help study the neural-islet regulatory mechanisms and the role of neural tissue remodelling in the development of diabetes.

  2. Cocaine- and amphetamine-regulated transcript: a novel regulator of energy homeostasis expressed in a subpopulation of pancreatic islet cells.

    PubMed

    Gilon, Patrick

    2016-09-01

    Type 2 diabetes is characterised by chronic hyperglycaemia and its incidence is highly increased by exaggerated food consumption. It results from a lack of insulin action/production, but growing evidence suggests that it might also involve hyperglucagonaemia and impaired control of glucose homeostasis by the brain. In recent years, the cocaine and amphetamine-regulated transcript (CART) peptides have generated a lot of interest in the battle against obesity because, via the brain, they exert anorexic effects and they increase energy expenditure. They are also localised, outside the brain, in discrete regions of the body and play a hormonal role in controlling various functions. In this issue of Diabetologia, the Wierup group (doi: 10.1007/s00125-016-4020-6 ) shows that CART peptides are expressed heterogeneously in islet cells of various species, including humans, and that their expression is upregulated in diabetes. The authors also shine a spotlight on some interesting effects of CART peptides on islet function, including stimulation of insulin secretion and inhibition of glucagon release. CART peptides would thus be at the centre of a cooperation between the brain and the endocrine pancreas to control glucose homeostasis. Although the mechanisms of action of CART peptides remain enigmatic because no specific receptor for these peptides has so far been discovered, their potential therapeutic use is evident and represents a new challenge for future research. PMID:27421727

  3. Alginate Encapsulation of Human Embryonic Stem Cells to Enhance Directed Differentiation to Pancreatic Islet-Like Cells

    PubMed Central

    Richardson, Thomas; Kumta, Prashant N.

    2014-01-01

    The pluripotent property of human embryonic stem cells (hESCs) makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs, which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate, along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand, encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel two-dimensional cultures, resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation, investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation, the ratio of pSMAD/pAKT was significantly higher, indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cell types provides a potentially translatable treatment option for type 1 diabetes. PMID:24881778

  4. Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence

    PubMed Central

    de Vos, Paul; Smink, Alexandra M.; Paredes, Genaro; Lakey, Jonathan R. T.; Kuipers, Jeroen; Giepmans, Ben N. G.; de Haan, Bart J.; Faas, Marijke M.

    2016-01-01

    The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet

  5. Use of the BacT/alert system for rapid detection of microbial contamination in a pilot study using pancreatic islet cell products.

    PubMed

    Murray, Laura; McGowan, Neil; Fleming, John; Bailey, Laura

    2014-10-01

    At the Islet Isolation Laboratory of the Scottish National Blood Transfusion Service, manual sterility testing data show that contamination rates are 57.7% for pancreas transport fluid, 4.3% for postpurification islet samples, and 0% for pretransplant islet samples. This pilot study presents the BacT/Alert System as an alternative to manual testing to provide more rapid and sensitive sterility results for islet cell products.

  6. Protective Effects of the Mushroom Lactarius deterrimus Extract on Systemic Oxidative Stress and Pancreatic Islets in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Mihailović, Mirjana; Arambašić Јovanović, Jelena; Uskoković, Aleksandra; Grdović, Nevena; Dinić, Svetlana; Vidović, Senka; Poznanović, Goran; Mujić, Ibrahim; Vidaković, Melita

    2015-01-01

    The aim of this study was to assess the in vivo effects of the extract of the medicinal mushroom, Lactarius deterrimus, when administered (60 mg/kg, i.p.) daily for four weeks to streptozotocin- (STZ-) induced diabetic rats. Diabetic rats treated with the L. deterrimus extract displayed several improved biochemical parameters in the circulation: reduced hyperglycemia, lower triglyceride concentration and reduced glycated hemoglobin, glycated serum protein, and advanced glycation end product (AGE) levels. This treatment also adjusted the diabetes-induced redox imbalance. Thus, higher activities of the antioxidative enzymes, superoxide dismutase, and catalase in the circulation were accompanied by increased levels of free intracellular thiols and glutathionylated proteins after treatment with the L. deterrimus extract. In addition to a systemic antioxidant effect, the administration of the extract to diabetic rats also had a positive localized effect on pancreatic islets where it decreased AGE formation, and increased the expression of chemokine CXCL12 protein that mediates the restoration of β-cell population through the activation of the serine/threonine-specific Akt protein kinase prosurvival pathway. As a result, the numbers of proliferating cell nuclear antigen- (PCNA-) and insulin-positive β-cells were increased. These results show that the ability of the L. deterrimus extract to alleviate oxidative stress and increase β-cell mass represents a therapeutic potential for diabetes management. PMID:26221612

  7. Protective Effects of the Mushroom Lactarius deterrimus Extract on Systemic Oxidative Stress and Pancreatic Islets in Streptozotocin-Induced Diabetic Rats.

    PubMed

    Mihailović, Mirjana; Arambašić Јovanović, Jelena; Uskoković, Aleksandra; Grdović, Nevena; Dinić, Svetlana; Vidović, Senka; Poznanović, Goran; Mujić, Ibrahim; Vidaković, Melita

    2015-01-01

    The aim of this study was to assess the in vivo effects of the extract of the medicinal mushroom, Lactarius deterrimus, when administered (60 mg/kg, i.p.) daily for four weeks to streptozotocin- (STZ-) induced diabetic rats. Diabetic rats treated with the L. deterrimus extract displayed several improved biochemical parameters in the circulation: reduced hyperglycemia, lower triglyceride concentration and reduced glycated hemoglobin, glycated serum protein, and advanced glycation end product (AGE) levels. This treatment also adjusted the diabetes-induced redox imbalance. Thus, higher activities of the antioxidative enzymes, superoxide dismutase, and catalase in the circulation were accompanied by increased levels of free intracellular thiols and glutathionylated proteins after treatment with the L. deterrimus extract. In addition to a systemic antioxidant effect, the administration of the extract to diabetic rats also had a positive localized effect on pancreatic islets where it decreased AGE formation, and increased the expression of chemokine CXCL12 protein that mediates the restoration of β-cell population through the activation of the serine/threonine-specific Akt protein kinase prosurvival pathway. As a result, the numbers of proliferating cell nuclear antigen- (PCNA-) and insulin-positive β-cells were increased. These results show that the ability of the L. deterrimus extract to alleviate oxidative stress and increase β-cell mass represents a therapeutic potential for diabetes management.

  8. Carbonyl stress-induced 5-hydroxytriptamine secretion from RIN-14B, rat pancreatic islet tumor cells, via the activation of transient receptor potential ankyrin 1.

    PubMed

    Suzawa, Sayaka; Takahashi, Kenji; Shimada, Takahisa; Ohta, Toshio

    2016-07-01

    Methylglyoxal (MG), a highly reactive dicarbonyl substance, is known as an endogenous carbonyl stress-inducing substance related to various disease states. Irritable bowel syndrome (IBS) is one of the most frequently encountered gastrointestinal disorders and MG is considered to be its causal substance. An increased serum 5-hydroxytryptamine (5-HT) level is related to IBS symptoms and the majority of 5-HT originates from enterochromaffin (EC) cells in the intestine. Here we examine the mechanisms of MG-induced 5-HT secretion using RIN-14B cells derived from a rat pancreatic islet tumor since these cells are used as a model for EC cells. MG increased the intracellular Ca(2+) concentration ([Ca(2+)]i) and 5-HT secretion, both of which were inhibited by the removal of extracellular Ca(2+) and specific transient receptor potential ankyrin 1 (TRPA1) antagonists. MG elicited an inward current under voltage-clamped conditions. Prior application of MG evoked reciprocal suppression of subsequent [Ca(2+)]i responses to allylisothiocyanate, a TRPA1 agonist, and vice versa. Glyoxal, an analog of MG, also evoked [Ca(2+)]i and secretory responses but its potency was much lower than that of MG. The present results suggest that MG promotes 5-HT secretion through the activation of TRPA1 in RIN-14B cells. These results may indicate that TRPA1 is a promising target for the treatment of IBS and that the RIN-14B cell line is a useful model for investigation of IBS. PMID:27423812

  9. Pancreatitis.

    PubMed

    Mitchell, R M S; Byrne, M F; Baillie, J

    2003-04-26

    In the past decade, our understanding of the genetic basis, pathogenesis, and natural history of pancreatitis has grown strikingly. In severe acute pancreatitis, intensive medical support and non-surgical intervention for complications keeps patients alive; surgical drainage (necrosectomy) is reserved for patients with infected necrosis for whom supportive measures have failed. Enteral feeding has largely replaced the parenteral route; controversy remains with respect to use of prophylactic antibiotics. Although gene therapy for chronic pancreatitis is years away, our understanding of the roles of gene mutations in hereditary and sporadic pancreatitis offers tantalising clues about the disorder's pathogenesis. The division between acute and chronic pancreatitis has always been blurred: now, genetics of the disorder suggest a continuous range of disease rather than two separate entities. With recognition of pancreatic intraepithelial neoplasia, we see that chronic pancreatitis is a premalignant disorder in some patients. Magnetic resonance cholangiopancreatography and endoscopic ultrasound are destined to replace endoscopic retrograde cholangiopancreatography for many diagnostic indications in pancreatic disease.

  10. Pancreatitis

    MedlinePlus

    ... the hormones insulin and glucagon into the bloodstream. Pancreatitis is inflammation of the pancreas. It happens when digestive enzymes start digesting the pancreas itself. Pancreatitis can be acute or chronic. Either form is ...

  11. The role of endothelial cells on islet function and revascularization after islet transplantation

    PubMed Central

    Del Toro-Arreola, Alicia; Robles-Murillo, Ana Karina; Daneri-Navarro, Adrian; Rivas-Carrillo, Jorge David

    2016-01-01

    ABSTRACT Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation. PMID:27002241

  12. The use of continuous density gradients for the assessment of islet and exocrine tissue densities and islet purification.

    PubMed

    Robertson, G S; Chadwick, D R; Contractor, H; James, R F; Bell, P R; London, N J

    1993-01-01

    The purification of large numbers of human pancreatic islets remains one of the limiting factors in islet transplantation. This paper describes and validates a method for accurately and reproducibly determining the density of islets and exocrine tissue in pancreatic digest on the basis of their isopycnic distribution on linear continuous density gradients. The use of this data to analyse and compare the purity of a standard 60% islet yield is described. The results obtained using such gradients will enable factors responsible for the variation in yield between pancreases to be determined and optimized, improving the results and reliability of islet purification. PMID:8111080

  13. Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

    PubMed

    Assouline-Thomas, Béatrice; Ellis, Daniel; Petropavlovskaia, Maria; Makhlin, Julia; Ding, Jieping; Rosenberg, Lawrence

    2015-01-01

    Regeneration of β-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

  14. Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

    PubMed

    Assouline-Thomas, Béatrice; Ellis, Daniel; Petropavlovskaia, Maria; Makhlin, Julia; Ding, Jieping; Rosenberg, Lawrence

    2015-01-01

    Regeneration of β-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate. PMID:26558987

  15. What is the origin of pancreatic adenocarcinoma?

    PubMed Central

    Pour, Parviz M; Pandey, Krishan K; Batra, Surinder K

    2003-01-01

    The concept of pancreatic cancer origin is controversial. Acinar, ductal or islet cells have been hypothesized as the cell of origin. The pros and cons of each of these hypotheses are discussed. Based on the world literature and recent observations, pancreatic cells seem to have potential for phenotypical transdifferentiation, i.e ductal-islet, ductal-acinar, acinar-ductal, acinar-islet, islet-acinar and islet-ductal cells. Although the possibility is discussed that cancer may arise from either islet, ductal or acinar cells, the circumstances favoring the islet cells as the tumor cell origin include their greater transdifferentiation potency into both pancreatic and extrapancreatic cells, the presence of a variety of carcinogen-metabolizing enzymes, some of which are present exclusively in islet cells and the growth factor-rich environment of islets. PMID:12636873

  16. Portal Vein Embolization with Radiolabeled Polyvinyl Alcohol Particles in a Swine Model: Hepatic Distribution and Implications for Pancreatic Islet Cell Transplantation

    SciTech Connect

    Owen, Richard J.; Mercer, John R.; Al-Saif, Faisal; Molinari, Michele; Ashforth, Robert A.; Rajotte, Ray V.; Conner-Spady, Barbara; Shapiro, A. M. James

    2009-05-15

    The distribution of radiolabeled polyvinyl alcohol microspheres (PVAMs) when infused into the portal vein of domestic swine was investigated, with the purpose of assessing implications for pancreatic islet cell transplantation. PVAMs measuring 100-300 {mu}m (Contour SE) and labeled with {sup 99m}Tc were infused into the main portal vein of 12 swine, with intermittent portal venous pressure measurements. The infusion catheter was introduced antegradely via direct or indirect cannulation of the portal vein. The liver was subsequently divided into anatomical segments. Radioactivity (decay corrected) was measured for {sup 99m}Tc microsphere synthesis, dose preparation, gross organ activities, tissue samples, and blood. Particulate labeling, catheter positioning, and infusion were successful in all cases. The number of particles used was (185,000 {+-} 24,000) with a volume of 1 ml. Mean portal pressure at 5 min was significantly higher than baseline, but without a significant difference at 15 min. Extrahepatic tissue and serum radioactivity was negligible. A significant difference in number of radioactive particles per gram was detected between segments 6/7 and segments 5/8. Intrasegmental activity was analyzed, and for segments 2/3 a significant difference in the percentage dose per gram across samples was demonstrated (P = 0.001). Effective and stable radiolabeling of PVAMs with {sup 99m}Tc-sulfur colloid was demonstrated. Portal venous infusion of 100- to 300-{mu}m particles showed entrapment in the sinusoidal hepatic system with transient portal pressure elevation. Preferential embolization into the right lateral and posterior segments occurs, suggesting that flow dynamics/catheter tip position plays a role in particle distribution.

  17. Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies.

    PubMed

    Nguyen, T M; Wright, J R; Nielsen, P F; Conlon, J M

    1995-05-01

    The Brockmann body of the teleost fish, the tilapia (Oreochromis nilotica) has been considered as a potential source of islet xenograft tissue for patients with insulin-dependent diabetes. This study describes the purification from an extract of tilapia Brockmann bodies of insulin and several peptides arising from different pathways of post-translational processing of two proglucagons, two prosomatostatins and proPYY. The primary structure of tilapia insulin is similar to insulins from other teleosts (particularly the anglerfish, Lophius americanus) except that the strongly conserved glutamine residue at position 5 in the A-chain, a residue that is important in the binding of insulin to its receptor, is replaced by glutamic acid. In common with other teleosts, the tilapia Brockmann body expresses two non-allelic glucagon genes. Alternative pathways of post-translational processing lead to glucagons with 29 and 36 amino acid residues derived from proglucagon I and glucagons with 29 and 32 residues derived from proglucagon II. Glucagon-like peptides with 30 and 34 residues derived from proglucagon II were also isolated. In each case, the longer peptide is a C-terminally extended form of the shorter. Tilapia peptide tyrosine-tyrosine (PYY) was isolated in a C-terminally alpha-amidated from with 36 amino acid residues that is structurally similar (89% sequence identity) to anglerfish PYY. A 30-amino acid peptide, representing the C-terminal flanking peptide of PYY, was also isolated that shows only 53% sequence identity with the corresponding anglerfish peptide. Tilapia somatostatin-14 is identical to mammalian somatostatin but the [Tyr7, Gly10] somatostatin-containing peptide derived from prosomatostatin II contains the additional substitution (Phe11-->Leu) compared with the corresponding peptide from other teleosts. PMID:7656183

  18. Effects of calcium buffering on glucose-induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor

    PubMed Central

    Pertusa, José A G; Sanchez-Andrés, Juan V; Martín, Franz; Soria, Bernat

    1999-01-01

    The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. Slow Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 μm), their effects being observed in the innermost shells dependent on Ca2+ affinity. In contrast, fast Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50% inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. These data are consistent with the existence in pancreatic β-cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) ‘primed’ vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) ‘reserved pool’ vesicles, not colocalized and responsible for the second phase. PMID:10523416

  19. Though Active on RINm5F Insulinoma Cells and Cultured Pancreatic Islets, Recombinant IL-22 Fails to Modulate Cytotoxicity and Disease in a Protocol of Streptozotocin-Induced Experimental Diabetes

    PubMed Central

    Berner, Anika; Bachmann, Malte; Bender, Christine; Pfeilschifter, Josef; Christen, Urs; Mühl, Heiko

    2016-01-01

    Interleukin (IL)-22 is a cytokine displaying tissue protective and pro-regenerative functions in various preclinical disease models. Anti-bacterial, pro-proliferative, and anti-apoptotic properties mediated by activation of the transcription factor signal transducer and activator of transcription (STAT)-3 are key to biological functions of this IL-10 family member. Herein, we introduce RINm5F insulinoma cells as rat β-cell line that, under the influence of IL-22, displays activation of STAT3 with induction of its downstream gene targets Socs3, Bcl3, and Reg3b. In addition, IL-22 also activates STAT1 in this cell type. To refine those observations, IL-22 biological activity was evaluated using ex vivo cultivated murine pancreatic islets. In accord with data on RINm5F cells, islet exposure to IL-22 activated STAT3 and upregulation of STAT3-inducible Socs3, Bcl3, and Steap4 was evident under those conditions. As these observations supported the hypothesis that IL-22 may exert protective functions in toxic β-cell injury, application of IL-22 was investigated in murine multiple-low-dose streptozotocin (STZ)-induced diabetes. For that purpose, recombinant IL-22 was administered thrice either immediately before and at disease onset (at d4, d6, d8) or closely thereafter (at d8, d10, d12). These two IL-22-treatment periods coincide with two early peaks of β-cell injury detectable in this model. Notably, none of the two IL-22-treatment strategies affected diabetes incidence or blood glucose levels in STZ-treated mice. Moreover, pathological changes in islet morphology analyzed 28 days after disease induction were not ameliorated by IL-22 administration. Taken together, despite being active on rat RINm5F insulinoma cells and murine pancreatic islets, recombinant IL-22 fails to protect pancreatic β-cells in the tested protocols from toxic effects of STZ and thus is unable to ameliorate disease in the widely used model of STZ-induced diabetes. PMID:26793108

  20. Deficiency in type I interferon signaling prevents the early interferon-induced gene signature in pancreatic islets but not type 1 diabetes in NOD mice.

    PubMed

    Quah, Hong Sheng; Miranda-Hernandez, Socorro; Khoo, Aimee; Harding, Ashley; Fynch, Stacey; Elkerbout, Lorraine; Brodnicki, Thomas C; Baxter, Alan G; Kay, Thomas W H; Thomas, Helen E; Graham, Kate L

    2014-03-01

    Type I interferons (IFNs) have been implicated in the initiation of islet autoimmunity and development of type 1 diabetes. To directly test their involvement, we generated NOD mice deficient in type I IFN receptors (NOD.IFNAR1(-/-)). Expression of the type I IFN-induced genes Mx1, Isg15, Ifit1, Oas1a, and Cxcr4 was detectable in NOD islets as early as 1 week of age. Of these five genes, expression of Isg15, Ifit1, Oas1a, and Mx1 peaked at 3-4 weeks of age, corresponding with an increase in Ifnα mRNA, declined at 5-6 weeks of age, and increased again at 10-14 weeks of age. Increased IFN-induced gene expression was ablated in NOD.IFNAR1(-/-) islets. Loss of Toll-like receptor 2 (TLR2) resulted in reduced islet expression of Mx1 at 2 weeks of age, but TLR2 or TLR9 deficiency did not change the expression of other IFN-induced genes in islets compared with wild-type NOD islets. We observed increased β-cell major histocompatibility complex class I expression with age in NOD and NOD.IFNAR1(-/-) mice. NOD.IFNAR1(-/-) mice developed insulitis and diabetes at a similar rate to NOD controls. These results indicate type I IFN is produced within islets in young mice but is not essential for the initiation and progression of diabetes in NOD mice.

  1. The role of islet neogenesis-associated protein (INGAP) in islet neogenesis.

    PubMed

    Lipsett, Mark; Hanley, Stephen; Castellarin, Mauro; Austin, Emily; Suarez-Pinzon, Wilma L; Rabinovitch, Alex; Rosenberg, Lawrence

    2007-01-01

    Islet Neogenesis-Associated Protein (INGAP) is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked temporally and spatially with the induction of islet neogenesis in animal models of disease and regeneration. Furthermore, administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. Cultured human pancreatic tissue has also been shown to be responsive to INGAP peptide, producing islet-like structures with function, architecture and gene expression matching that of freshly isolated islets. Likewise, studies in normoglycemic animals show evidence of islet neogenesis. Finally, recent clinical studies suggest an effect of INGAP peptide to improve insulin production in type 1 diabetes and glycemic control in type 2 diabetes.

  2. Is there a role for locally produced interleukin-1 in the deleterious effects of high glucose or the type 2 diabetes milieu to human pancreatic islets?

    PubMed

    Welsh, Nils; Cnop, Miriam; Kharroubi, Ilham; Bugliani, Marco; Lupi, Roberto; Marchetti, Piero; Eizirik, Décio L

    2005-11-01

    Different degrees of beta-cell failure and apoptosis are present in type 1 and type 2 diabetes. It has been recently suggested that high glucose-induced beta-cell apoptosis in type 2 diabetes shares a final common pathway with type 1 diabetes, involving interleukin-1beta (IL-1beta) production by beta-cells, nuclear factor-kappaB (NF-kappaB) activation, and death via Fas-FasL. The aim of this study was to test whether human islet exposure to high glucose in vitro, or to the type 2 diabetes environment in vivo, induces IL-1beta expression and consequent activation of NF-kappaB-dependent genes. Human islets were isolated from five normoglycemic organ donors. The islets were cultured for 48 h to 7 days at 5.6, 11, or 28 mmol/l glucose. For comparative purposes, islets were also exposed to IL-1beta. Gene mRNA expression levels were assessed by real-time RT-PCR in a blinded fashion. Culture of the human islets at 11 and 28 mmol/l glucose induced a four- to fivefold increase in medium insulin as compared with 5.6 mmol/l glucose, but neither IL-1beta nor IL-1 receptor antagonist (IL-1ra) expression changed. IL-1beta and IL-1ra protein release to the medium was also unchanged. Stimulated human monocytes, studied in parallel, released >50-fold more IL-1beta than the islets. There was also no glucose-induced islet Fas expression. Expression of the NF-kappaB-dependent genes IkappaB-alpha and monocyte chemoattractant protein (MCP)-1 was induced in human islets by IL-1beta but not by high glucose. In a second set of experiments, human islets were isolated from seven type 2 diabetic patients and eight control subjects. The findings on mRNA levels were essentially the same as in the in vitro experiments, namely the in vivo diabetic state did not induce IL-1beta, Fas, or MCP-1 expression in human islets, and also did not modify IL-1ra expression. The present findings suggest that high glucose in vitro, or the diabetic milieu in vivo, does not induce IL-1beta production or NF

  3. Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function.

    PubMed

    Noguchi, Hirofumi; Nakai, Yusuke; Matsumoto, Shinichi; Kawaguchi, Miho; Ueda, Michiko; Okitsu, Teru; Iwanaga, Yasuhiro; Yonekawa, Yukihide; Nagata, Hideo; Minami, Kohtaro; Masui, Yumi; Futaki, Shiroh; Tanaka, Koichi

    2005-08-01

    Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.

  4. Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human {beta}-cells from hyperglycemia-induced apoptosis

    SciTech Connect

    Mohanty, S.; Spinas, G.A.; Maedler, K.; Zuellig, R.A.; Lehmann, R.; Donath, M.Y.; Trueb, T.; Niessen, M. . E-mail: markus.niessen@usz.ch

    2005-02-01

    Studies in vivo indicate that IRS2 plays an important role in maintaining functional {beta}-cell mass. To investigate if IRS2 autonomously affects {beta}-cells, we have studied proliferation, apoptosis, and {beta}-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that {beta}-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a {beta}-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of {beta}-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human {beta}-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve {beta}-cell function. Our results indicate that IRS2 acts autonomously in {beta}-cells in maintenance and expansion of functional {beta}-cell mass in vivo.

  5. Microwell Scaffolds for the Extrahepatic Transplantation of Islets of Langerhans

    PubMed Central

    Buitinga, Mijke; Truckenmüller, Roman; Engelse, Marten A.; Moroni, Lorenzo; Ten Hoopen, Hetty W. M.; van Blitterswijk, Clemens A.; de Koning, Eelco JP.; van Apeldoorn, Aart A.; Karperien, Marcel

    2013-01-01

    Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet’s native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation. PMID:23737999

  6. [Prolonged acute pancreatitis after bone marrow transplantation].

    PubMed

    De Singly, B; Simon, M; Bennani, J; Wittnebel, S; Zagadanski, A-M; Pacault, V; Gornet, J-M; Allez, M; Lémann, M

    2008-04-01

    Acute pancreatitis is not infrequent after allogenic marrow transplantation. Several causes can predispose to pancreatitis, including Graft-Versus-Host Disease (GVHD), a condition which is probably underestimated. In the literature, few description of pancreatic GVHD can be found. Pancreatic GVHD diagnosis can be difficult if pancreatic involvement occurs without other typical manifestations of GVHD. We report the case of a woman, 54 years old, suffering from prolonged, painful pancreatitis two months after allogenic bone marrow transplantation for acute myeloid leucemia. Pancreatic GVHD diagnosis was performed after five weeks on duodenal biopsies despite the absence of diarrheoa. The patient dramatically improved within few days on corticosteroids.

  7. Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

    PubMed Central

    Kowluru, A; Seavey, S E; Li, G; Sorenson, R L; Weinhaus, A J; Nesher, R; Rabaglia, M E; Vadakekalam, J; Metz, S A

    1996-01-01

    Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in

  8. Pancreatitis

    MedlinePlus

    ... to the abdomen. In 1 out of 4 childhood cases, a cause is never found. What are the symptoms of pancreatitis? Inflammation of the pancreas is often associated with pain in the upper abdomen and/or the back which may develop slowly, ...

  9. In vitro effects of bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxidovanadium(IV), or VO(dmpp)2, on insulin secretion in pancreatic islets of type 2 diabetic Goto-Kakizaki rats.

    PubMed

    Pelletier, Julien; Domingues, Neuza; Castro, M Margarida C A; Östenson, Claes-Göran

    2016-01-01

    Vanadium compounds have been explored as therapy of diabetes, and most studies have focussed on insulin mimetic effects, i.e. reducing hyperglycemia by improving glucose sensitivity and thus glucose uptake in sensitive tissues. We have recently shown that bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxidovanadium(IV), VO(dmpp)2, has promising effects when compared to another vanadium compound, bis(maltolato)oxidovanadium(IV), BMOV, and insulin itself, in isolated adipocytes and in vivo in Goto-Kakizaki (GK) rats, an animal model of hereditary type 2 diabetes (T2D).We now have investigated in GK rats whether VO(dmpp)2 also modulates another important defect in T2D, impaired insulin secretion. VO(dmpp)2, but not BMOV, stimulated insulin secretion from isolated GK rat pancreatic islets at high, 16.7mM, but not at low–normal, 3.3 mM, glucose concentration. Mechanistic studies demonstrate that the insulin releasing effect of VO(dmpp)2 is due to its interaction with several steps in the stimulus-secretion coupling for glucose, including islet glucose metabolism and K-ATP channels, L-type Ca2+ channels, modulation by protein kinases A and C, as well as the exocytotic machinery. In conclusion, VO(dmpp)2 exhibits properties of interest for treatment of the insulin secretory defect in T2D, in addition to its well-described insulin mimetic activity.

  10. Chronic pancreatitis.

    PubMed

    Chari, S T; DiMagno, E P

    2001-09-01

    An increasing number of novel mutations are associated with chronic pancreatitis. Some cause a high-penetrance, autosomal dominant type of clinical picture (eg, mutations at codons 29 and 122 of the cationic trypsinogen gene), whereas others have a low penetrance or are frequent in the general population (eg, mutations in Kazal type 1 [SPINK1] and in codons 16, 22, and 23 of the cationic trypsinogen gene) and act as disease modifiers. The results of recent studies indicate that smoking adversely affects the course and complications of chronic pancreatitis (more frequent and faster rate of calcification and higher risk of development of pancreatic cancer). Thus, regardless of the cause of chronic pancreatis, patients with this condition should not smoke. Using current diagnostic criteria, the accuracy of endoscopic ultrasound for the diagnosis of chronic pancreatitis is not good. For example, 39% of dyspeptic persons without any other evidence of chronic pancreatitis fulfilled the endoscopic ultrasound criteria for chronic pancreatitis. Diabetes frequently occurs in chronic pancreatitis, but it is not prevented or increased by pancreatic surgery. Islet cell autotransplantation holds promise for the prevention of diabetes in patients requiring total pancreatectomy if the pancreas is not extensively fibrotic. Splenic vein occlusion is present in 7% of patients undergoing surgery for chronic pancreatitis, but fewer than one fifth of these patients have variceal bleeding before or after surgery.

  11. Macroporous Three Dimensional PDMS Scaffolds for Extrahepatic Islet Transplantation

    PubMed Central

    Pedraza, Eileen; Brady, Ann-Christina; Fraker, Christopher A.; Molano, R. Damaris; Sukert, Steven; Berman, Dora M.; Kenyon, Norma S.; Pileggi, Antonello; Ricordi, Camillo; Stabler, Cherie L.

    2015-01-01

    Clinical islet transplantation has demonstrated success in treating type 1 diabetes. A current limitation is the intrahepatic portal vein transplant site, which is prone to mechanical stress and inflammation. Transplantation of pancreatic islets into alternative sites is preferable, but challenging, as it may require a three-dimensional vehicle to confer mechanical protection and to confine islets to a well-defined, retrievable space where islet neovascularization can occur. We have fabricated biostable, macroporous scaffolds from poly(dimethylsiloxane) (PDMS) and investigated islet retention and distribution, metabolic function, and glucose-dependent insulin secretion within these materials. Islets from multiple sources, including rodents, non-human primates, and humans, were tested in vitro. We observed high islet retention and distribution within PDMS scaffolds, with retention of small islets (< 100 µm) improved through the post-loading addition of fibrin gel. Islets loaded within PDMS scaffolds exhibited viability and function comparable to standard culture conditions when incubated under normal oxygen tensions, but displayed improved viability compared to standard two-dimensional culture controls under low oxygen tensions. In vivo efficacy of scaffolds to support islet grafts was evaluated after transplantation in the omental pouch of chemically-induced diabetic syngeneic rats, which promptly achieved normoglycemia. Collectively, these results are promising in that they indicate the potential for transplanting islets into a clinically relevant, extrahepatic site that provides spatial distribution of islets, as well as intra-device vascularization. PMID:23031502

  12. Glucose cycling in islets from healthy and diabetic rats

    SciTech Connect

    Khan, A.; Chandramouli, V.; Ostenson, C.G.; Loew, H.L.; Landau, B.R.; Efendic, S. )

    1990-04-01

    Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with {sup 3}H{sub 2}O and 5.5 or 16.7 mM glucose. At 5.5 mM glucose, no detectable ({sup 3}H)glucose was formed. At 16.7 mM, 2.2 patom.islet-1.h-1 of {sup 3}H was incorporated into glucose by the control islets and 5.4 patom.islet-1.h-1 by STZ-D islets. About 75% of the {sup 3}H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol.islet-1.h-1 by