Islet Injury Research Articles

Release of Glutamate Decarboxylase-65 into the Circulation by Injured Pancreatic Islet β-Cells

The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet beta-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of beta-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that beta-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. beta-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured beta-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.

Antihyperglycemic activity of kinsenoside, a high yielding constituent from Anoectochilus roxburghii in streptozotocin diabetic rats

Different doses of kinsenoside, a high yielding constituent from Anoectochilus roxburghii, was orally administered to further investigate its biological activity and pharmacological mechanisms that involve in the hypoglycemic effect on streptozotocin (STZ) diabetic rats. Our study showed that this compound exhibited significantly antihyperglycemic activity at the dose of 15 mg/kg body weight, which is speculated to be partially attributed to modulating the activity of enzymatic antioxidants, scavenging free radicals, and reducing the content of factor NO. Much more intact β cells in the islets of Langerhans with denser insulin in kinsenoside-treated groups than the negative control were observed, which greatly supported the morphological and functional elucidation. These results displayed that kinsenoside could be useful for repairing β cells in pancreatic islet injury as well as improving its function. The OGTT evidenced that this compound could promote the glucose tolerance of acute glucose increase in both diabetic and normal healthy rats.

Responses of IL-18- and IL-18 receptor-deficient pancreatic islets with convergence of positive and negative signals for the IL-18 receptor.

Pancreatic islets contain cells that produce IL-18 and cells that express IL-18 receptors. In experimentally induced diabetes, islet failure correlates with IL-18 levels and diabetes is delayed with blockade of endogenous IL-18. We studied islet-derived IL-18 and responses to IL-18 in a mouse model of islet allograft transplantation. In vitro, IL-18-stimulated islets produced nitric oxide, which closely matched islet apoptosis. By neutralizing IL-18 activity with IL-18 binding protein (IL-18BP), we observed that islets produce bioactive IL-18. In vivo, transgenic mice overproducing IL-18BP (IL-18BP-Tg) exhibited delayed hyperglycemia induced by beta cell toxic streptozotocin. Similarly, cultured IL-18BP-Tg islets were protected from streptozotocin-induced apoptosis. In the transplant model, islets grafted from WT to IL-18BP-Tg mice achieved prolonged normoglycemia (P = 0.031). Improved graft function was also observed by using IL-18-deficient islets transplanted into WT recipients, demonstrating that endogenous, islet-derived IL-18 mediates IL-18-driven graft damage. Unexpectedly, islets from mice deficient in IL-18 receptor alpha chain (IL-18R) exhibited rapid graft failure (P = 0.024; IL-18- versus IL-18R-deficient grafts in WT recipients). In related studies, IL-18R-deficient splenocytes and macrophages produced 2- to 3-fold greater amounts of IL-18, TNFalpha, macrophage inflammatory protein 1, macrophage inflammatory protein 2, and IFNgamma upon stimulation with Con A, Toll-like receptor 2 agonist, or anti-CD3 antibodies. These data reveal a role for islet-derived IL-18 activity during inflammation-mediated islet injury. Importantly, discrepancies between IL-18- and IL-18R-deficient cells suggest that IL-18Ralpha chain is used by an inflammation-suppressing signal.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes

EPConDB () is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT–PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Tumor Necrosis Factor-α Production by Human Islets Leads to Postisolation Cell Death

Recent successes in islet transplantation highlight the importance of islet isolation by experienced centers and minimization of cell injury as crucial to the achievement of insulin independence. Islet injury may manifest as cell death by apoptosis, shorter graft survival, and the need for retransplantation. Although an inflammatory cytokine response at the graft site is known to inhibit engraftment, recent evidence indicates that islet cells may contribute to this response. Isolated human islets were cultured for up to one week in serum-free CMRL-1066 with 25 microM of tumor necrosis factor (TNF)alpha inhibitor RDP58. Gene expression was measured by reverse transcriptase polymerase chain reaction, apoptosis and TNFalpha secretion by enzyme-linked immunosorbent assay and enzyme-linked immunospot, and islet function by stimulated insulin secretion. Isolation induced a twofold increase in TNFalpha expression between days one and three (P<0.05), while TNFalpha secretion peaked at day one. RDP58 reduced TNFalpha secretion by 70.6% (P<0.02), though TNFalpha gene expression was unaffected. RDP58 reduced the frequency of TNFalpha-secreting islets by 64.4% (P<0.05) and reduced apoptotic levels by 26.4% within 24 hr postisolation (P<0.05). The reduction in apoptosis was maintained throughout the week (P<0.01), while apoptosis increased in control cultures. Finally, RDP58-treated islets displayed increased insulin secretion in response to both elevated glucose (1915.0+/-396.6 vs. 825.3+/-261.1 mU/L, P<0.01) and secretagogues (2294.3+/-529.5 vs. 939.8+/-333.7 mU/L, P<0.02). These data demonstrate that intraislet cytokine production should be considered as a factor leading to islet cell death postisolation and postengraftment, and strategies aimed at countering islet cytokine production represent a novel target for improving islet viability and function.

Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts

Recent advances in clinical protocols have improved the outcomes of pancreatic islet transplantation (PIT), yet PIT recipients typically require pancreatic islet grafts derived from multiple donors to achieve insulin independence. This along with experimental models of syngeneic PIT, showing that up to 60% of pancreatic islet tissue undergoes apoptosis within the first several days post-transplantation, strongly suggest the involvement of nonalloantigen-specific, inflammatory events in partial destruction of the graft following PIT. Interleukin-1beta appears to be among the most important inflammatory mediators, causing pancreatic islet dysfunction and apoptosis through the up-regulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2. Kupffer cells secrete many molecules, including cytokines, NO, and free radicals, which are known to be directly toxic to the pancreatic islets, and depletion or inhibition of Kupffer cells improves outcomes following experimental PIT. Immediately after transplantation, the pancreatic islets are perfused only by portal vein blood until the process of angiogenesis restores arterial blood flow some 7-10 days later. This delayed vascularization may have implications for the expression of leukocyte adhesion molecules, the effects of free radicals, and the role of ischemia-reperfusion injury. Finally, in the immediate post-transplant period, hepatocytes may contribute to pancreatic islet injury through the production of NO. This paper reviews literature regarding the inflammatory events that follow PIT as well as the pathogenesis of diabetes and the pathophysiology of hepatic ischemia-reperfusion and their relation to the survival and function of intrahepatic pancreatic islet grafts.

Failure of G-CSF Mobilization of Hematopoietic Stem Cells To Prevent or Reverse STZ-Induced Hyperglycemia in Mice.

Background. A series of recent studies implies that circulating bone marrow (BM)-derived cells may contribute to both endo- and exogenous regeneration of non-hematopoietic tissue, at least partly through transdifferentiation into differentiated non-hematopoietic cells. We and others have observed donor-derived pancreatic islet cells after BM-transplantation (BMT) to otherwise healthy mice. The above results suggest that BM cells may be useful for the therapeutic regeneration of insulin-producing pancreatic beta cells in diabetes mellitus (DM). The regenerative properties of BM-derived cells appear to be enhanced in models that include pancreatic islet injury. Therefore, we are exploring the capability of the BM-mobilizing cytokine granulocyte colony stimulating factor (G-CSF) to influence regeneration in streptocotocin (STZ)-induced DM.Methods. Female C57BL/6 mice, 8–10 weeks old, underwent lethal irradiation (11 Gy) followed one day later by BMT (tail vein-injection of 5 million male unfractionated, nucleated BM cells, transgenic for the enhanced green fluorescent protein [EGFP+]). Two months after BMT, the mice were allocated to four experimental protocols (n=10 per group). All experimental animals were treated with STZ (200 mg/kg i.p.) on day 5, to induce DM. Group A received human G-CSF (Amgen, CA) pre- and post-STZ administration (200 μg/kg, i.p., in four daily doses; days 1-4 for pre- and days 22-25 for posttreatment). Group B received human G-CSF only before STZ administration, while Group C received G-CSF only after STZ administration. Group D served as diabetic controls. Group E (n=6) underwent BMT but received no G-CSF or STZ treatment. Blood glucose (Glc) was measured using test strips and a blood glucose meter (Accu-ChekR, Roche, IN). Individual mice from each group were sacrificed on day 31, and organs removed for histological studies.Results. In all experimental groups, 60% of the mice became manifestly diabetic (Glc > 350 mg/dL). The majority of the other mice remained clearly non-diabetic (Glc < 200 mg/dL), i.e. non-responsive to STZ. One mouse in Group B, however, became subdiabetic later in the experiment. The Table shows the development of blood glucose in the STZ-responsive mice (mean blood glucose; n=6 per group (days −2–29), n=5 per group (days 35 and 43)).Failure of G-CSF mobilization to influence STZ-induced hyperglycemiaConclusion. G-CSF administration failed to either prevent or treat the STZ-induced hyperglycemia in this experimental model of DM, implying that the mobilization of BM cells did not promote physiologically significant regeneration of pancreatic islets within the time-frame studied. This failure may imply that BM-based regeneration therapy for DM has serious limitations, or, alternatively, that more favorable timing and dosing of G-CSF may be needed to improve the results. Ongoing work, including the histological examination of these mice, will reveal whether partial, but clinically insufficient, regeneration was indeed induced, which will guide future studies.Day−251218293543Group A143*126380498527393396Group B156122331468514454373Group C147152330389480447427Group D139140327392464406459Group E154144123138125131ND*mean glucose level (mg/dL); ND = Not done

Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury.

Endothelial progenitor cells (EPCs) are detectable in the blood and bone marrow throughout life. These cells contribute to new blood vessel formation (neovascularization) in physiological states such as wound healing and in pathological states such as tumor angiogenesis. We hypothesized that bone marrow-derived EPCs could play a role in the response to pancreatic islet cell injury. We used a murine model of experimentally induced beta-cell injury followed by transplantation with genetically marked bone marrow cells. Bone marrow-derived cells were detectable throughout the pancreas after transplantation. Whereas the total number of bone marrow-derived cells in the pancreas decreased over time, the frequency of endothelial cells (of both donor and recipient origin) increased after transplantation in the animals in which beta-cell injury had been induced. There was no evidence in this model that bone marrow-derived cells differentiated into insulin-expressing cells. This study provides evidence that bone marrow-derived EPCs are recruited to the pancreas in response to islet injury. EPC-mediated neovascularization of the pancreas could in principle be exploited to facilitate the recovery of non-terminally injured beta-cells or to improve the survival and/or function of islet allografts.

Interferon regulatory factor-1 down-regulates cytokine-induced IP-10 expression in pancreatic islets

Background. Interferon (IFN)-γ acts synergistically with interleukin (IL)-1β and tumor necrosis factor (TNF)-α to activate isoform of nitric oxide synthetase (iNOS) gene expression, induce apoptosis, and impair glucose-stimulated insulin release (GSIR) in pancreatic islets. This effect is an important mechanism of islet dysfunction in models of pancreatitis, type I diabetes, and islet allograft rejection. We tested the hypothesis that transcription factor interferon regulatory factor (IRF)-1 plays a regulatory role in both this cytokine-induced islet injury and cytokine-induced gene expression for chemotactic chemokines. Methods. Isolated islets from wild-type (WT) or IRF-1−/− C57BL/6 mice were cultured in a mixture of IL-1β, TNF-α, and IFN-γ ± the iNOS inhibitor L-NMMA. The following end points were assessed: i) GSIR; ii) rates of apoptosis; and iii) gene expression for iNOS, IRF-1 and inducible protein (IP)-10, monocyte chemoattractive protein (MCP)-1, macrophage inflammatory protein-1α, and macrophage inflammatory protein-1β. Results. Cytokine-treated WT islets demonstrated an increase in IRF-1 and iNOS gene expression, inhibition of GSIR, increased rates of apoptosis, and increased gene transcription and protein release for IP-10 and MCP-1. Cytokine-treated IRF-1−/− islets demonstrated relatively less iNOS gene expression, preserved GSIR, reduced rates of apoptosis, and a more marked increase in transcription for IP-10 and MCP-1 and in IP-10 protein release. L-NMMA-cotreated WT islets were completely resistant to cytokine-induced dysfunction and apoptosis but demonstrated the same degree of cytokine-induced chemokine gene expression as cytokine-treated WT without L-NMMA. Conclusion. IFN-γ, IL-1β, and TNF-α in combination induce chemokine gene expression in pancreatic islets. Transcription factor IRF-1 mediates cytokine-induced islet dysfunction, apoptosis, and iNOS gene expression but down-regulates IP-10 gene expression. (Surgery 2003;134:134-41.)

Role of donor MHC class III genes in the destruction of transplanted islets in NOD mice.

MHC class III genes are important in immune regulation and inflammation, and the gene products of this region are well conserved between species. Their role in diabetes is, however, unknown. We used islets from NOD mice that lacked expression of both MHC class I and class II molecules to test the effect of class III differences on the injury of transplanted NOD islets. Loss of islet MHC class I was highly protective, while deletion of MHC class II had no benefit on islet survival. However the combined absence of both MHC class I and class II expression by NOD islets resulted in a delayed form of injury, when islets were transplanted to NOD mice. As neither MHC class I or II molecules were expressed by donor islet tissue, these results suggest a previously unrecognized and important contribution of MHC class III differences on islet injury following transplantation.

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.

Hepatic Sinusoidal Endothelium Upregulates IL-1α, IFN-γ, and iNOS in Response to Discordant Xenogeneic Islets in an in Vitro Model of Xenoislet Transplantation

Background. Data indicate that early islet graft failure is due to nonspecific inflammatory mechanisms that occur prior to T-cell-mediated rejection. The role of the host hepatic endothelium in mediating this immediate islet injury has not been elucidated. The endothelial cell may be important in this process because it is essentially the first cellular barrier encountered by intraportally introduced islets. We have characterized the initial response of hepatic endothelium to xenogeneic islets by measuring the expression of Il-1α, TNF-α, IFN-γ, and iNOS in an in vitro dog-to-pig model of xenoislet transplantation.Materials and methods. Dog islets (500 islet equivalents) were cocultured with either porcine hepatic endothelium or porcine aortic endothelium over a 24-h period in serum-free medium. RNA was extracted at eight time points (0, 1, 2, 4, 6, 8, 12, and 24 h). Reverse-transcriptase polymerase chain reaction was performed on each sample. Polymerase chain reaction was done on the cDNA in order to visualize Il-1α, TNF-α, IFN-γ, and iNOS expression. Bands were semiquantitated by comparison to an external standard (GAPDH) using band densitometry.Results. Hepatic endothelium had early (1 h) expression of IL-1α, IFN-γ, and iNOS. IL-1α peaked at 2 h, IFN-γ at 12 h, and iNOS at 1 and 12 h. Aortic endothelium expressed low levels of IL-1α and TNF-α, but not IFN-γ or iNOS.Conclusions. We have demonstrated that xenogeneic islets are able to activate host endothelial cells without serum or immune cells. The observed endothelial response corresponds with known islet toxic substances. Furthermore, the response differs between hepatic and aortic endothelial cells, suggesting that these differences may be important in choosing suitable implantation sites for islets. Our findings suggest that host endothelium may play an important part in early injury of islet xenotransplants.

Clusterin expression during regeneration of pancreatic islet cells in streptozotocin-induced diabetic rats.

Beta-cell regeneration has been reported after islet injury in an animal model for diabetes. Recently, we showed up-regulation of clusterin after islet injury and suggested that clusterin might be involved in cytoprotection and in the regeneration of islet cells. The aim of this study was to investigate the correlation of clusterin expression with islet regeneration and its effect on islet cell replication. Streptozotocin was administrated to rats to induce various types of diabetes. Islet regeneration and clusterin expression were examined after islet injuries. Clusterin cDNA was transfected to MIN6 cells and their proliferation activity was measured by a [3H]thymidine-incorporation assay. A diabetogenic dose of streptozotocin injected in rats provoked an immediate degeneration of beta cells. In this model, islets showed increased clusterin expression with extensive proliferation of alpha cells but showed poor beta-cell replication. A subdiabetogenic dose of streptozotocin, however, led to the proliferation of beta cells with clusterin up-regulation. In streptozotocin-treated neonatal rats, up-regulation of clusterin was noted during beta-cell proliferation. In all experimental models, clusterin was expressed in alpha cells in close correlation with islet cell proliferation, higher transcription of insulin mRNA and MAPKs activation. Cell replication was increased by 31 % in the MIN6 cells transfected by the clusterin cDNA. Up-regulation of clusterin in alpha cells might induce beta-cell proliferation and thus restore their population after islet injury. We suggest that clusterin could be considered as a growth factor-like molecule stimulating islet-cell proliferation by paracrine action.

Chronological Change in Pancreatic Islet Injury Induced by Streptozotocin

Chronological Change in Pancreatic Islet Injury Induced by Streptozotocin

Islet injury induces neurotrophin expression in pancreatic cells and reactive gliosis of peri-islet Schwann cells.

Pancreatic islets are enveloped by a sheath of Schwann cells, the glial cells of the peripheral nervous system (PNS). The fact that Schwann cells of the PNS become reactive and express nerve growth factor (NGF) and other growth factors following axotomy suggested the possibility that peri-islet Schwann cells could become activated by islet injury. To test this hypothesis, we examined two animal models of islet injury. The first model was mice and rats injected with streptozotocin (SZ), a specific beta-cell toxin. The second model was NOD mice, a strain in which beta cells are deleted by an autoimmune process. We found that peri-islet Schwann cells became reactive following islet injury and began to express increased levels of NGF and the neurotrophin receptor p75. Lesions to the pancreas also markedly induced NGF expression by exocrine and endocrine cells. Neurotrophin expression was not unique to adult tissues since pancreatic cells transiently expressed p75, the NGF receptor Trk A, and NGF during development. These observations suggest that NGF could play an important role in pancreas during embryogenesis and in processes leading to repair following islet injury in adults.

Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets.

The ability of the adult pancreas to generate new insulin (beta) cells has been controversial because of difficulties in unequivocally identifying the precursor population. We recently determined that beta cells were generated during development from precursors that expressed the homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1). To investigate whether PDX-1+ stem cells are present in adult pancreas, we examined two animal models of diabetes. One model was produced by injecting adult mice with streptozotocin (SZ), a toxin that produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing IN+/PDX-1+ cells, a population of somatostatin (SOM)+/PDX-1+ cells, a cell type thought to represent an embryonic islet precursor cell, appeared in islets. The appearance of SOM+/PDX-1+ cells was followed in time by the differentiation to SOM+/IN+/PDX-1+ cells. SOM+/PDX-1+ cells also appeared in islets of nonobese diabetic mice, a strain of mice in which beta cell destruction is immune-mediated. Our findings establish the existence of PDX-1+ beta cell precursors in the adult pancreas and indicate that their differentiation is induced by islet injury.

Induction of β-cell Neogenesis by Islet Injury

Induction of β-cell Neogenesis by Islet Injury

Induction of β-cell Neogenesis by Islet Injury

Induction of β-cell Neogenesis by Islet Injury

Induction of β-cell Neogenesis by Islet Injury

Induction of β-cell Neogenesis by Islet Injury

Cytokines as Mediators of Autoimmune Diabetes and Diabetic Complications

Over the last several years, there has been a considerable and rapid expansion in our understanding of the cytokines. Insights into cytokine functions gained from basic biological studies are currently being applied to the study of autoimmune diabetes. It has become clear that cytokines are critical regulating elements involved in the processes of initiating, promoting, and effecting beta-cell destruction. It is also evident that cytokine functions in IDDM are determined by timing of appearance and local synthesis, as well as the prevailing cytokine and cellular milieu. Elucidating individual cytokine responses in IDDM may offer new insights into mechanisms of disease pathogenesis and may lead to novel cytokine-based therapies for the treatment of IDDM. It may also be possible that the intensity or character of a patient's cytokine response to islet injury or to the metabolic changes inherent to diabetes may influence the ultimate expression of IDDM or its complications. Clearly, this rapidly expanding field of study should have a major impact on our understanding of diabetogenesis, our ability to intervene in our understanding of diabetogenesis, our ability to intervene in the disease process itself, and ultimately our capacity to care for individuals with IDDM.