We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into β cell–cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their β cell–cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill β cells, we examined the islet antigen–specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of β cell–cytotoxic T cells that cause β cell destruction, resulting in autoimmune diabetes in NOD mice.

Type 1 diabetes results from the destruction of insulin-producing pancreatic beta cells by a beta cell-specific autoimmune process. Beta cell autoantigens, macrophages, dendritic cells, B lymphocytes, and T lymphocytes have been shown to be involved in the pathogenesis of autoimmune diabetes. Beta cell autoantigens are thought to be released from beta 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 beta 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, beta 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 beta cells. In addition, beta 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 beta cell-cytotoxic CD8+ T cells act synergistically to destroy beta cells, resulting in autoimmune type 1 diabetes.

A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of beta cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic beta cells by autoimmune responses specific to beta cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic beta cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

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Because there is a deficiency of beta-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.

A healthy 10-year-old boy was admitted to the hospital in diabetic ketoacidosis within three days of onset of symptoms of a flu-like illness. He died seven days later and post-mortem examination showed lymphocytic infiltration of the islets of Langerhans and necrosis of beta cells. Inoculation of mouse, monkey and human cell cultures with homogenates from the patient's pancreas led to isolation of a virus. Serologic studies revealed a rise in the titer of neutralizing antibody to this virus from less than 4 on the second hospital day to 32 on the day of death. Neutralization data showed that the virus was related to a diabetogenic variant derived from Coxsackievirus B4. Inoculation of mice with the human isolate produced hyperglycemia, inflammatory cells in the islets of Langerhans and beta-cell necrosis. Staining of mouse pancreatic sections with fluorescein-labeled antiviral antibody revealed viral antigens in beta cells. Both the clinical picture and animal studies suggested that the patient's diabetes was virus induced.

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 ␤-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 enhances ␤-cell function; stimulates ␤-cell growth, survival, differentiation, and proliferation; and promotes satiety and delaying gastric emptying (1,2). Furthermore, impaired GLP-1 secretion was observed in patients with type 2 diabetes (3). Therefore, GLP-1 has been proposed as a treatment for type 2 diabetes. Treatment with GLP-1 or its analog, exendin-4, improved insulin sensitivity and glucose tolerance and reduced hyperinsulinemia in animal models of type 2 diabetes (4,5). In type 2 diabetic patients, subcutaneous infusion of GLP-1 for 6 weeks resulted in improved insulin sensitivity and ␤-cell function (6). However, the precise mechanisms by which insulin sensitivity and glucose tolerance are improved are not known.Although subcutaneous injections or intravenous or subcutaneous infusions of GLP-1 showed therapeutic effects on lowering blood glucose levels, the short half-life (ϳ2 min) and rapid clearance of GLP-1 limits the maintenance of therapeutic levels by exogenous administration. GLP-1 is degraded by the enzyme dipeptidyl peptidase IV (7,8); therefore, GLP-1 agonists that are resistant to dipeptidyl peptidase IV degradation and inhibitors of dipeptidyl peptidase IV have been investigated for the treatment of type 2 diabetes (9). We hypothesized that continuous expression of GLP-1 in vivo by a gene therapy strategy may remit hyperglycemia and maintain normoglycemia. In this study, we produced a recombinant adenovirus that expresses and secretes GLP-1 under the control of the cytomegalovirus promoter (recombinant adenovir...

Insulin-dependent diabetes mellitus (IDDM) is caused by the progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the pathogenesis of autoimmune IDDM has been extensively studied, the precise mechanisms involved in the initiation and progression of beta cell destruction remain unclear. Animal models used in the study of IDDM, such as the BioBreeding (BB) rat and the nonobese diabetic (NOD) mouse, have greatly enhanced our understanding of the pathogenic mechanisms involved in this disease. In these animals, macrophages and/or dendritic cells are the first cell types to infiltrate the pancreatic islets. Macrophages must be involved in the pathogenesis of IDDM early on, since inactivation of macrophages results in the near-complete prevention of insulitis and diabetes in both NOD mice and BB rats. The presentation of beta cell-specific autoantigens by macrophages and/or dendritic cells to CD4+ T helper cells, in association with MHC class II molecules, is considered the initial step in the development of autoimmune IDDM. The activated macrophages secrete IL-12, which stimulates Th1 type CD4+ T cells. The CD4+ T cells secrete IFN-gamma and IL-2. IFN-gamma activates other resting macrophages, which, in turn, release cytokines, such as IL-1beta, TNF-alpha, and free radicals, which are toxic to beta cells. During this process, IL-2 and other cytokines induce the migration of CD8+ peripheral T cells to the inflamed islets, perhaps by inducing the expression of a specific homing receptor. The precytotoxic CD8+ T cells that bear beta cell-specific autoantigen receptors differentiate into cytotoxic effector T cells upon recognition of the beta cell-specific peptide bound to MHC class I molecules in the presence of beta cell-specific CD4+ T helper cells. The cytotoxic CD8+ T cells then effect beta cell damage by releasing perforin and granzyme, and by Fas-mediated apoptosis. In this way, macrophages, CD4+ T cells, and CD8+ T cells synergistically destroy beta cells, resulting in the onset of autoimmune IDDM.