Hee-Sook Jun

A new look at viruses in type 1 diabetes.

Type 1 diabetes (T1D) results from the destruction of pancreatic beta cells. Genetic factors are believed to be a major component for the development of T1D, but the concordance rate for the development of diabetes in identical twins is only about 40%, suggesting that nongenetic factors play an important role in the expression of the disease. Viruses are one environmental factor that is implicated in the pathogenesis of T1D. To date, 14 different viruses have been reported to be associated with the development of T1D in humans and animal models. Viruses may be involved in the pathogenesis of T1D in at least two distinct ways: by inducing beta cell‐specific autoimmunity, with or without infection of the beta cells, [e.g. Kilham rat virus (KRV)] and by cytolytic infection and destruction of the beta cells (e.g. encephalomyocarditis virus in mice). With respect to virus‐mediated autoimmunity, retrovirus, reovirus, KRV, bovine viral diarrhoea‐mucosal disease virus, mumps virus, rubella virus, cytomegalovirus and Epstein‐Barr virus (EBV) are discussed. With respect to the destruction of beta cells by cytolytic infection, encephalomyocarditis virus, mengovirus and Coxsackie B viruses are discussed. In addition, a review of transgenic animal models for virus‐induced autoimmune diabetes is included, particularly with regard to lymphocytic choriomeningitis virus, influenza viral proteins and the Epstein‐Barr viral receptor. Finally, the prevention of autoimmune diabetes by infection of viruses such as lymphocytic choriomeningitis virus is discussed. Copyright

Autoimmune destruction of pancreatic beta cells.

Type 1 diabetes results from the destruction of insulin-producing pancreatic β cells by a β cell-specific autoimmune process. β Cell autoantigens, macrophages, dendritic cells, B lymphocytes, and T lymphocytes have been shown to be involved in the pathogenesis of autoimmune diabetes. β Cell autoantigens are thought to be released from β cells by cellular turnover or damage and are processed and presented to T helper cells by antigen-presenting cells. Macrophages and dendritic cells are the first cell types to infiltrate the pancreatic islets. Naive CD4+ T cells that circulate in the blood and lymphoid organs, including the pancreatic lymph nodes, may recognize major histocompatibility complex and β cell peptides presented by dendritic cells and macrophages in the islets. These CD4+ T cells can be activated by interleukin (IL)-12 released from macrophages and dendritic cells. While this process takes place, β cell antigen-specific CD8+ T cells are activated by IL-2 produced by the activated TH1 CD4+ T cells, differentiate into cytotoxic T cells and are recruited into the pancreatic islets. These activated TH1 CD4+ T cells and CD8+ cytotoxic T cells are involved in the destruction of β cells. In addition, β cells can also be damaged by granzymes and perforin released from CD8+ cytotoxic T cells and by soluble mediators such as cytokines and reactive oxygen molecules released from activated macrophages in the islets. Thus, activated macrophages, TH1 CD4+ T cells, and β cell-cytotoxic CD8+ T cells act synergistically to destroy β cells, resulting in autoimmune type 1 diabetes.

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter–based cell sorting. ES cell–derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell–derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell–derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice.

Objective:The objective of this study was to demonstrate that islet neogenesis-associated protein (INGAP) peptide, a pentadecapeptide containing the biologically active portion of native INGAP, increases functional β-cell mass in normal animals and can be used therapeutically to reverse hyperglycemia in streptozotocin-induced diabetes. Summary Background Data:INGAP, a 175 amino acid pancreatic acinar cell protein, has been suggested to be implicated in β-cell mass expansion. Methods:In the first part of this study, normoglycemic hamsters were administered either 500 &mgr;g INGAP peptide (n = 30) or saline (n = 20) intraperitoneally daily and sacrificed after 10 or 30 days of treatment. Blood glucose and insulin levels were measured, and a histologic and morphometric analysis of the pancreas was performed to determine the effect of INGAP peptide on the endocrine pancreas. In the second part of the study, 6- to 8-week-old C57BL/6J mice (n = 8) were administered multiple low doses of the β-cell toxin streptozotocin (STZ) inducing insulitis and hyperglycemia. The mice were then injected with INGAP peptide (n = 4) or saline (n = 4) for 39 days and sacrificed at 48 days. Two additional groups of diabetic mice were administered either a peptide composed of a scrambled sequence of amino acids from INGAP peptide (n = 5) or exendin-4 (n = 5), an incretin that has been associated with amelioration of hyperglycemia. Results:Islet cell neogenesis was stimulated in INGAP-treated hamsters by 10 days. At 30 days, the foci of new endocrine cells had the appearance of mature islets. There was a 75% increase in islet number, with normal circulating levels of blood glucose and insulin. Administration of INGAP peptide to diabetic mice reversed the diabetic state in all animals, and this was associated with increased expression of PDX-1 in duct cells and islet cell neogenesis with a reduction of insulitis in the new islets. Diabetic mice treated with exendin-4 or a scrambled INGAP peptide did not revert from hyperglycemia. Conclusion:Because there is a deficiency of β-cell mass in both type-1 and type-2 diabetes, INGAP peptide stimulation of fully functional neoislet differentiation may provide a novel approach for diabetes therapy.

Protective Role of Autophagy in Palmitate-Induced INS-1 β-Cell Death

Autophagy, a vacuolar degradative pathway, constitutes a stress adaptation that avoids cell death or elicits the alternative cell-death pathway. This study was undertaken to determine whether autophagy is activated in palmitate (PA)-treated beta-cells and, if activated, what the role of autophagy is in the PA-induced beta-cell death. The enhanced formation of autophagosomes and autolysosomes was observed by exposure of INS-1 beta-cells to 400 microm PA in the presence of 25 mm glucose for 12 h. The formation of green fluorescent protein-LC3-labeled structures (green fluorescent protein-LC3 dots), with the conversion from LC3-I to LC3-II, was also distinct in the PA-treated cells. The phospho-mammalian target of rapamycin level, a typical signal pathway that inhibits activation of autophagy, was gradually decreased by PA treatment. Blockage of the mammalian target of rapamycin signaling pathway by treatment with rapamycin augmented the formation of autophagosomes but reduced PA-induced INS-1 cell death. In contrast, reduction of autophagosome formation by knocking down the ATG5, inhibition of fusion between autophagosome and lysosome by treatment with bafilomycin A1, or inhibition of proteolytic degradation by treatment with E64d/pepstatin A, significantly augmented PA-induced INS-1 cell death. These findings showed that the autophagy system could be activated in PA-treated INS-1 beta-cells, and suggested that the induction of autophagy might play an adaptive and protective role in PA-induced cell death.

Cellular and Molecular Pathogenic Mechanisms of Insulin-Dependent Diabetes Mellitus

Abstract: Insulin‐dependent diabetes mellitus (IDDM), also known as type 1 diabetes, is an organ‐specific autoimmune disease resulting from the destruction of insulin‐producing pancreatic β cells. The hypothesis that IDDM is an autoimmune disease has been considerably strengthened by the study of animal models such as the BioBreeding (BB) rat and the nonobese diabetic (NOD) mouse, both of which spontaneously develop a diabetic syndrome similar to human IDDM. β cell autoantigens, macrophages, dendritic cells, B lymphocytes, and T cells have been shown to be involved in the pathogenesis of autoimmune diabetes. Among the β cell autoantigens identified, glutamic acid decarboxylase (GAD) has been extensively studied and is the best characterized. β cell‐specific suppression of GAD expression in NOD mice results in the prevention of IDDM. Macrophages and/or dendritic cells are the first cell types to infiltrate the pancreatic islets. Macrophages play an essential role in the development and activation of β cell‐cytotoxic T cells. B lymphocytes play a role as antigen‐presenting cells, and T cells have been shown to play a critical role as final effectors that kill β cells. Cytokines secreted by immunocytes, including macrophages and T cells, may regulate the direction of the immune response toward Th1 or Th2 as well as cytotoxic effector cell or suppressor cell dominance. β cells are destroyed by apoptosis through Fas‐Fas ligand and TNF‐TNF receptor interactions and by granzymes and perforin released from cytotoxic effector T cells. Therefore, the activated macrophages and T cells, and cytokines secreted from these immunocytes, act synergistically to destroy β cells, resulting in the development of autoimmune IDDM.

A human β-cell line for transplantation therapy to control type 1 diabetes

A human pancreatic β-cell line that is functionally equivalent to primary β-cells has not been available. We established a reversibly immortalized human β-cell clone (NAKT-15) by transfection of primary human β-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed β-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

The role of viruses in Type I diabetes: two distinct cellular and molecular pathogenic mechanisms of virus-induced diabetes in animals

Abstract Type I (insulin-dependent) diabetes mellitus results from the progressive loss of pancreatic beta cells. Environmental factors are believed to play an important part in the development of Type I diabetes by influencing the penetrance of diabetes susceptibility genes. As one environmental factor, the virus has long been considered to play a part in this disease. To date 13 different viruses have been reported to be associated with the development of Type I diabetes in humans and in various animal models. The most clear and unequivocal evidence that a virus induces diabetes in animals comes from studies on the d variant of the encephalomyocarditis (EMC-D) virus in mice and the Kilham rat virus (KRV) in rats. The infection of genetically susceptible strains of mice with a high titre of EMC-D virus results in the development of diabetes within 3 days. This is largely due to the rapid destruction of beta cells by the replication of the virus within the beta cells. In contrast, the infection of mice with a low titre of EMC-D virus results in a limited replication of the virus before the induction of neutralizing anti-virus antibody and the subsequent recruitment of activated macrophages. The Src kinases, particularly hck, play an important part in the activation of macrophages and the subsequent production of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and nitric oxide (NO), leading to the destruction of beta cells which results in the development of diabetes. The Kilham rat virus causes autoimmune diabetes in diabetes resistant (DR)-BB rats without infection of beta cells. The infection of DR-BB rats with KRV results in the disruption of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC-CD4+ T cells, leading to the selective activation of beta-cell-cytotoxic effector T cells. [Diabetologia (2001) 44: 271–285]

Glucagon-Like Peptide-1 Gene Therapy in Obese Diabetic Mice Results in Long-Term Cure of Diabetes by Improving Insulin Sensitivity and Reducing Hepatic Gluconeogenesis

Long-term treatment with glucagon-like peptide (GLP)-1 or its analog can improve insulin sensitivity. However, continuous administration is required due to its short half-life. We hypothesized that continuous production of therapeutic levels of GLP-1 in vivo by a gene therapy strategy may remit hyperglycemia and maintain prolonged normoglycemia. We produced a recombinant adenovirus expressing GLP-1 (rAd-GLP-1) under the cytomegalovirus promoter, intravenously injected it into diabetic ob/ob mice, and investigated the effect of this treatment on remission of diabetes, as well as the mechanisms involved. rAd-GLP-1–treated diabetic ob/ob mice became normoglycemic 4 days after treatment, remained normoglycemic over 60 days, and had reduced body weight gain. Glucose tolerance tests found that exogenous glucose was cleared normally. rAd-GLP-1–treated diabetic ob/ob mice showed improved β-cell function, evidenced by glucose-responsive insulin release, and increased insulin sensitivity, evidenced by improved insulin tolerance and increased insulin-stimulated glucose uptake in adipocytes. rAd-GLP-1 treatment increased basal levels of insulin receptor substrate (IRS)-1 in the liver and activation of IRS-1 and protein kinase C by insulin in liver and muscle; increased Akt activation was only observed in muscle. rAd-GLP-1 treatment reduced hepatic glucose production and hepatic expression of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fatty acid synthase in ob/ob mice. Taken together, these results show that a single administration of rAd-GLP-1 results in the long-term remission of diabetes in ob/ob mice by improving insulin sensitivity through restoration of insulin signaling and reducing hepatic gluconeogenesis.

Recent advances in insulin gene therapy for type 1 diabetes

Type 1 diabetes results from the loss of insulin-producing pancreatic beta cells following the action of beta-cell-specific autoimmune responses. One possible treatment for type 1 diabetes is the development of beta-cell substitutes by introducing an insulin-producing gene into non-beta cells, which would evade the beta-cell-specific autoimmune attack. However, this approach has been hampered by the absence of (1) an appropriate glucose-sensing system to regulate insulin gene transcription; (2) enzymes that process proinsulin to insulin; and (3) glucose-regulatable exocytosis in the target cells. Recent attempts to solve these problems have sought new methods for effective gene transfer and have addressed issues such as the expression and release of insulin in response to the physiological stimulus of glucose, the production of biologically active insulin, and the selection of an ideal target cell for the expression of the insulin gene.