Loss Of Islet Cells Research Articles

Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans.

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.

The development of diabetes in E2f1/E2f2 mutant mice reveals important roles for bone marrow-derived cells in preventing islet cell loss.

Our studies of mice deficient for the E2F1 and E2F2 transcription factors have revealed essential roles for these proteins in the cell cycle control of pancreatic exocrine cells and the regulation of pancreatic beta cell maintenance. Pancreatic exocrine cells in E2f1-/-E2f2 mutant mice become increasingly polyploid with age, coinciding with severe exocrine atrophy. Furthermore, mice deficient for both E2F1 and E2F2 develop nonautoimmune, insulin-dependent diabetes with high penetrance. Surprisingly, transplantation of wild-type bone marrow can prevent or rescue diabetes in E2f1-/-E2f2-/-mice. We hypothesize that exocrine degeneration results in a destructive environment for beta cells, which can be alleviated by restoration of the hematopoietic system that is also defective in E2f1-/-E2f2-/-mice The demonstration that beta cell maintenance under conditions of stress is influenced by bone marrow-derived cells may provide important insight into the design of therapies to boost islet mass and function in diabetic patients.

Molecular detection of circulating beta-cells after islet transplantation.

Islet transplantation is a promising treatment for type 1 diabetes. However, islet grafts are submitted to multiple injuries, including immunosuppressive drug toxicity, hyperglycemia, hypoxia, unspecific inflammatory reactions, as well as allo- and autoimmune destruction. Therapeutic approaches to these damage mechanisms require early detection of islet injury, which is currently not feasible because of the lack of efficient markers. Based on the hypothesis of islet dissociation and release of islet cells into the circulation during islet injury, we designed a highly sensitive and specific molecular assay, able to detect two beta-cells per milliliter of venous blood by RT-PCR of insulin mRNA. We report that circulating beta-cells can be demonstrated up to 10 weeks after intraportal islet transplantation, as assessed after six islet grafts in four type 1 diabetic patients. Furthermore, our results suggest that the time during which circulating islet cells can be detected may depend on the graft environment and the immunosuppressive regimen. This test may allow better estimation of islet cell loss and identification of factors involved in islet graft injury.

Cell cycle regulation: Mechanisms and therapeutics

Cyclin-dependent kinase 4 (Cdk4) is an important regulator of G1/S cell cycle progression of mammalian cells. To understand the role of Cdk4in vivo we generated strains of mice that either lack Cdk4 or express an activated form of this enzyme (Cdk4R24C), which cannot interact with the cycli kinase inhibitor, p16INK4A. Cdk4R24C mutations that abolish the interaction of pl6INK4A and CDK predispose humans to familial melanoma. Cdk4-/- mutant mice are viable but express defects in growth, fertility and are diabetic due to loss of insulin-producing islet cells. In contrast, CDK4 (R24C/R24C) mice exhibit growth advantage and their fibroblasts have a shorter cycle with high proliferative rates. The Cdk4 (R24C/R24C) fibroblasts fail to undergo senescence and are capable long-term growth in culture. Moreover, approximate 80% of Cdk4 (R24C/R24C) mice developed spontaneous tumors of varying cellular origin such as breast, pituitary, brain, skin, pancreas, liver, lung, testes, ovarian and hematopoietic tumors by 12 month. In addition, cdk4(R24C/R24C) mice are highly susceptible to TPA or DMBA carcinogen-induced tumors and Cdk4(R24C/R24C) mice developed skin tumors with average latency of 10–12 weeks. In contrast, wild type mice had a reduced frequency and latency of tumor formation. These studies indicated that the cdk4(R24C) mutation could exacerbate de-regulated Ras-pathways and accelerate tumorigenesisIn addition, Cdk4 (R24C/R24C) mice exhibit severe hyperplasia of the immune system and are highly susceptible to the development of hematopoietic malignancies. These results suggest that Cdk4 is an important modulator of cell cycle progression and abnormalities associated with this gene results in the development of abnormalities in multiple organs including the hematopoietic compartment.

Islet Cryopreservation: Improved Recovery following Taurine Pretreatment

Simple and efficient freezing methods with maximal postthawing recovery form the basis of ideal cryopreservation. Taurine (2-amino ethanesulfonic acid), an end-product of sulphur amino acid metabolism, is one of the most abundant free amino acids in the body. The membrane stabilizing, free radical scavenging, and osmoregulatory roles of taurine have been well documented. We studied the effect of physiological and supra-physiological concentrations (0.3 and 3.0 mM) of taurine on islet cryopreservation. Islet viability on cryopreservation was significantly improved in both the taurine-treated groups (91.9 +/- 2.3% in 0.3 mM and 94.6 +/- 1.58% in 3.0 mM group, p < 0.05) compared with the controls (85.7 +/- 3.4%). Loss of peripheral islet cells was highly reduced in the taurine group, as examined under phase contrast and quantified by islet morphometric analysis (p < 0.05) using a digital image analysis system. Taurine-treated islets showed significant reduction in lipid peroxidation (0.905 and 0.848 nM MDA/microg protein for 0.3 and 3.0 mM taurine, respectively, p < 0.05) compared with control (1.307 nM MDA/microg protein) islets. In all, 500 islet equivalents (IE) of treated or control group islets were transplanted to BALB/c mice rendered diabetic with STZ. All animals showed a normal glucose clearance following a glucose load. Graft functionality was confirmed by normoglycemia (fasting plasma glucose: fpg < 150 mg/dl) after transplantation and reappearing hyperglycemia (fpg > 200 mg/dl) following removal of the graft. Suboptimal islet transplantation using 250 IE suggests that the grafted islet mass was inadequate for diabetes reversal. In addition, no significant differences were observed in the islet insulin content between the three groups following cryopreservation of the islets at -196 degrees C. Our studies indicate that taurine pretreatment and its continued presence during islet cryopreservation improves the postthawing viable recovery of islets.

Transduction of TAT/PTD Antiapoptotic Fusion Proteins in Pancreatic Islets.

INTRODUCTION. With the development of new strategies to avoid immunological rejection, transplantation of pancreatic islets has become a therapeutic reality to cure diabetes (1-2). However, despite the progress in islet isolation procedures, a single donor transplant does not provide enough islets to attain insulin independence. There is evidence that significant loss of islet cells takes place during isolation due to the triggering of apoptosis (3). There is also substantial evidence that reduction of isolation-induced apoptosis can improve the success rates of islet transplantation. The goal of this proposal is to address the needs for reduced apoptosis and improved viability of islets in conjunction with islet isolation procedures. We present in this study novel transduction methods that allow manipulation of islets to reduce apoptosis. Proteins can be directly transferred to cells when they are linked to protein transduction domains (PTDs), small peptide domains that can freely cross cell membranes. In particular, proteins fused to an 11-amino acid PTD from the human immunodeficiency virus, the TAT protein, readily diffuse across membranes and are efficiently transduced into virtually any cell type (4). The expression as well as the biological function of the TAT fusion protein are temporary without permanent modification of the cell sensitivity to apoptosis, thus avoiding undesirable long-term effects.

Development of pancreatic islets (review).

Recent studies have revealed that islet cells differentiate from the epithelial cells of primitive pancreatic ducts during embryogenesis, and can regenerate in response to the loss of islet cells even in adult pancreas. The ability of islet cells to regenerate raises the possibility that impaired and decreased islets of diabetic patients can be restored. In this review, factors regulating islet development including differentiation factors (Shh, activin, follistatin, and TGF alpha), transcriptional factors (PDX1, Isl1, Pax4, Pax6, Nkx2.2, Nkx6.1, BETA2, and HNF), growth factors (the EGF family, HGF, IGF-I, IGF-II, Reg, INGAP, PDGF, FGF, VEGF, and NGF), hormones (insulin, the GH family, PTHrP, TRH, and gastrin), and cell adhesion molecules (N-CAM and cadherins) are described after a short introduction and an outline of pancreatic development.

Nitric oxide donors decrease the function and survival of human pancreatic islets

Nitric oxide (NO) has been proposed as a possible mediator of β-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human β-cells are affected by NO. In view of species differences in β-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similkar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in β-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

Inactivation of the Poly(ADP-ribose) Polymerase Gene Affects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.

Immunohistochemical Analysis of Spontaneous Pancreatic Islet Amyloid Deposits in Nonhuman Primates

Pancreatic islet amyloidosis has been previously reported in aged diabetic and nondiabetic humans, nonhuman primates, cats, raccoons (Procyon lotor), and degus (Ocrodon degus, a South American rodent).h In nonhuman primates, islet amyloidosis, often accompanied by diabetes mellitus, has been reported in Celebes crested macaques (Macaca nigYU)~ rhesus monkeys (Macaca ~nulatta), cynomolgus monkeys (Macaca fascicularis), a Formosan rock macaque (Macaca cyclopis), and a drill baboon (Mandrillus leucophams). 1.2.7.8 Human and feline islet amyloid deposits are derived from the polymerization of islet amyloid polypeptide (IAPP; amylin), whereas in the degu, they are derived from insulin.69 IAPP is an islet beta-cell product secreted with insulin by humans and many other ~pecies.~ The definitive biological functions of IAPP and the significance of IAPP in the pathogenesis of human type 2 (non-insulin dependent) diabetes mellitus and feline diabetes mellitus are unre~olved.~.~-~ The purpose of this study was to examine the immunoreactivity of pancreatic islet amyloid in captive nonhuman primates. A review of necropsy records from several sources identified four cynomolgus monkeys, three Celebes crested macaques, and one orangutan (Pongo pygmaeus) with islet amyloid deposits. All of the nonhuman primates were adults ranging from 7 to 26 years of age. One of the Celebes crested macaques had diabetes mellitus, but the metabolic status of the other animals was not known. Formalin-fixed, paraffin-embedded tissues were sectioned at 5 lm and stained with either hematoxylin and eosin (HE) or Congo red with or without KMnO, pretreatment. Congo red-stained sections were examined by both bright field and polarized light microscopy. Using a modified avidin-biotin-peroxidase complex (ABC) method,> pancreatic tissues were evaluated for immunoreactivity with guinea pig anti-porcine insulin serum (Dako Corp., Carpinteria, CA) diluted 1 : 800 in phosphate-buffered saline (PBS), pH 7.4; 1 : 400 rabbit anti-human glucagon serum (Dako); 1 : 100 rabbit anti-human somatostatin serum (Dako); 1 : 400 rabbit anti-human pancreatic polypeptide serum (Dako); 1 : 800 rabbit anti-human calcitonin gene-related peptide (CGRP) serum (Peninsula Laboratories, Belmont, CA); and 1 : 200 rabbit anti-human amylin serum (Peninsula). Positive controls included internal controls in the tissue sections containing islet amyloid and application of the primary antisera to tissue sections from normal bonnet macaque (Macaca radiata) pancreas. Negative controls included replacing the primary antisera with the appropriate nonimmune sera and applying the primary antisera to tissue sections from a canine kidney containing glomerular amyloid. Five-micrometer tissue sections were collected on positively charged glass slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, rehydrated, and incubated in 1.5% normal goat serum in PBS for 20 minutes at room temperature in a humidified chamber. Sections were incubated with primary antibody for 1 hour under the same conditions, followed by a 30-minute incubation in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity and 30-minute incubations of biotinylated goat anti-rabbit or anti-guinea pig IgG (Vector Laboratories, Burlingame, CA), and ABC (Vector). The chromagen was 0.0 16% diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO) with NiCI, (Digene Diagnostics, Silver Spring, MD) in 0.024% hydrogen peroxide and PBS. Sections were counterstained with nuclear fast red (Digene). All the animals in this study had either multifocal or diffuse pancreatic islet amyloidosis. The extent and distribution of amyloid ranged from mild multifocal deposition surrounding islet capillaries with little or no loss of islet cells to marked diffuse deposition in enlarged islets with severely reduced

Isolated pancreatic islets of the rat: An immunohistochemical and morphometric study

Although there is a recent increase in the use of the isolated pancreatic islets of the rat in the transplantation and functional studies, there has been no detailed quantitative assessment on the size and cellular constituents of islets after the isolation procedure. The present work was undertaken to study the size classes of the isolated islets and the morphometry of their cellular populations. Islets of the rat pancreas were isolated by using the intraductal collagenase digestion technique, the most commonly used procedure for the isolation of pancreatic islets. Different endocrine cells of the isolated islets were stained by immunoperoxidase staining techniques. The distribution of the cellular constituents of the isolated islets was similar to that of the intact islets of the normal pancreas; A, D, and PP cells were peripherally arranged around the centrally located B cells. However, morphometric quantitative study showed that the percent volume and percent number of A, D, and PP cells of the isolated islets were lower than those of the corresponding intact ones. Further, the mean true diameter of the isolated islets was lower than that of the intact ones. These data indicate loss of islet cells during the process of isolation. Most of the lost cells were from the periphery of islets. This may provide an explanation for the incomplete metabolic control and recurrence of hyperglycemia encountered after isolated islet transplantation in the treatment of diabetes mellitus. It seems that further refinements of the isolation techniques are necessary to obtain islet tissue with total cellular integrity, before a complete success in transplantation could be achieved.

ISLET CELL DESTRUCTION AND GROWTH STUDIED IN TRANSGENIC ANIMAL MODELS

We have produced and studied several transgenic models of insulin dependent diabetes mellitus (IDDM). For understanding the etiology of IDDM, we have investigated the consequences of expression of host defense molecules in the pancreatic islets. IFN-γ is produced in response to infection and has immune-stimulatory and proliferative activities. To investigate the potential role of IFN-γ in inflammatory autoimmune diseases, transgenic mice expressing IFN-γ in pancreatic beta cells were created. These mice suffer from islet cell loss following the appearance of increasing numbers of lymphocytes within and surrounding the islets. We have studied the islet destruction in these mice and have demonstrated that it is mediated by inflammatory cells. Interestingly a proliferative/regenerative response opposes the lymphocyte destruction. We have recently been characterizing this proliferative axis in the transgenic pancreas. We have acquired evidence that it is initiated by duct cell proliferation and with the appearance of more primitive neuroendocrine progenitor cells along the apical regions of the ducts. The regenerative process in the ins-IFN-γ transgenic mice appears similar to the events that occur during embryonic islet cell development. These studies underscore the lymphokine's ability to initiate a complex “transdifferentiation” pathway within a terminally differentiated structure. Another fascinating molecule with potential relevance for treatment of IDDM is the cytokine IL-10. This molecule appears to have immuno-inhibitory effects that include the suppression of antigen presentation by macrophages. We have recently investigated the effects of the cytokine IL-10 on the pancreatic islets. These efforts were designed to test whether the expression of IL-10 could allow the creation of neutral islet tissue for allografting. Our work demonstrates that this molecule has both the ability to attract lymphocytes in vivo as well as to suppress the anti-islet immune response. We have utilized several experimental protocols to study the in vivo effects of this molecule. The results of these studies argue that this cytokine could be relevant for therapeutic intervention in IDDM. Our methodologies should clarify the potentially pathogenic capabilities of host defense molecules when expressed in vivo. Additional IDDM models, produced by ourselves as well as our colleagues will be discussed.

Intrathymic islet cell transplantation reduces beta-cell autoimmunity and prevents diabetes in NOD/Lt mice.

Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for beta-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted beta-cells with < 1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scid/scid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 NOD-scid/scid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-scid/scid recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to beta-cells.

Loss of pancreatic islet tolerance induced by β-cell expression of interferon-γ

Interferon-gamma (IFN-gamma) is produced during the response to infection and participates in immunostimulatory events. We have previously reported the induction of diabetes in transgenic mice (ins-IFN-gamma) in which the expression of the lymphokine IFN-gamma is directed by the insulin promoter. This diabetes is a result of the progressive destruction of pancreatic islets that occurs with the influx of inflammatory cells. Here we demonstrate that this islet cell loss is mediated by lymphocytes, that engrafted histocompatible islets are destroyed, and that lymphocytes from the transgenic mice are cytotoxic to normal islets in vitro. These results indicate that the pancreatic expression of IFN-gamma can result in a loss of tolerance to normal islets, consistent with its role as an inducer of costimulatory activity, which is essential for lymphocyte activation during an immune response.

Heat-shock treatment of mouse pancreatic islets results in a partial loss of islet cells but no remaining functional impairment among the surviving beta cells.

To elucidate the role of thermal stress on the function of pancreatic beta cells, isolated mouse pancreatic islets were incubated for 30 min at 42 degrees C. This resulted in decreased glucose-stimulated insulin secretion, inhibited total protein and pro-insulin synthesis and the induction of heat-shock proteins with molecular weights of 64 and 88 kDa. Six days later, the islets exposed to heat shock showed a lower DNA content, indicating islet cell death. However, the insulin secretory response and rates of oxygen consumption in the presence of glucose were normal. It is suggested that the induction of heat-shock proteins does not permanently impair beta-cell function, but rather protects these cells from lasting damage.

Administration of a 60 kD molecular fraction from pancreatic islets suppresses immune mediated diabetes in mice

Treatment of male C57BL6/J mice with low doses of streptozotocin causes the development of diabetes. Infiltration of pancreatic islets by immune cells and the concomitant loss of beta islet cells can be prevented by immunosuppression. We now report that i.v. administration of islet homogenate 13 d prior to low dose streptozotocin treatment largely protects from development diabetes. Homogenates from liver, kidney or exocrine pancreas were without effect. The suppressive activity was isolated partially from mouse islets as well as from a rat insulinoma cell line. After gelchromatography the suppressive activity was found in the 600 kD fraction. The relevant substance appears negatively charged and does not bind to lentil lectin or concanavalin A.

Longitudinal studies on the development of diabetes in individual Macaca nigra.

Development of spontaneous diabetes has been monitored in individual Macaca nigra. In this study, pancreatic biopsies were taken, islets were assessed morphologically, and results were related to the metabolic/clinical status. A biopsy or autopsy sample was obtained 4 to 10 years later, and the islet morphological state was again related to the metabolic/clinical status. Metabolic deterioration was correlated to the islet lesion, in which there was gradual loss of islet secretory cells and concurrent amyloid deposition. As nondiabetic monkeys with 0 to 3% islet amyloid progressed up to 20 to 40% amyloid, the insulin secretion and glucose clearance were both decreased (p less than or equal to 0.01), and the glucose and glucagon levels increased (p = 0.05). Impaired monkeys progressed to overt diabetes when islet amyloid exceeded 50 to 60%. Diabetic monkeys developed hyperglycaemia, along with impaired insulin secretion and glucose clearance (p less than 0.01). Loss of islet cells results in metabolic deterioration. The lesion precedes development of overt diabetes in Macaca nigra.