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Transplantation of isolated pancreatic islets is a promising approach to curative therapy of type 1 diabetes. However, many immunosuppressive drugs, including corticosteroids, cyclosporin, and tacrolimus, are either diabetagenic or toxic [...], OBJECTIVE--The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS--NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS--Although human T-cell engraftment was similar in all groups, ILT3-Fc-treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human [CD8.sup.+] T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc-treated mice and control recipients rejecting the grafts. CONCLUSIONS--The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.

Type 1 diabetes is an autoimmune disease that results in destruction of the insulin-producing β-cells (1). Cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α, and interferon-γ/(IFN-γ), induce β-cell death [...], OBJECTIVE--The transcription factor nuclear factor-κB (NFκB) and the mitogen-activated protein kinases (MAPKs) c-Jun N[H.sub.2]-terminal kinase (JNK) 1/2 are known to play decisive roles in cytokine-induced damage of rodent β-cells. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of the MAPK kinase kinase-1 (MEKK-1) in cytokine-induced signaling. RESEARCH DESIGN AND METHODS--To establish a functional role of MEKK-1, the effects of transient MEKK-1 overexpression in βTC-6 cells, achieved by lipofection and cell sorting, and MEKK-1 downregulation in βTC-6 cells and human islet cells, achieved by diced-small interfering RNA treatment, were studied. RESULTS--We observed that overexpression of wild-type MEKK-1, but not of a kinase dead MEKK-1 mutant, resulted in potentiation of cytokine-induced JNK activation, inhibitor of κB (IκB) degradation, and cell death. Downregulation of MEKK-1 in human islet cells provoked opposite effects, i.e., attenuation of cytokine-induced JNK and MKK4 activation, IκB stability, and a less pronounced NF-κB translocation. βTC-6 cells with a downregulated MEKK-1 expression displayed also a weaker cytokine-induced iNOS expression and lower cell death rates. Also primary mouse islet cells with downregulated MEKK-1 expression were protected against cytokine-induced cell death. CONCLUSIONS--MEKK-1 mediates cytokine-induced JNK- and NF-κB activation, and this event is necessary for iNOS expression and cell death. Diabetes 57:1896-1904, 2008

Despite improvements in immunosuppression protocols over the last years, pancreatic islets from at least two donor pancreata are still needed to reverse type 1 diabetes in clinical islet transplantation (1,2). [...], OBJECTIVE--Insufficient development of a new intra-islet capillary network after transplantation may be one contributing factor to the failure of islet grafts in clinical transplantation. The present study tested the hypothesis that the angiostatic factor thrombospondin-1 (TSP-1), which is normally present in islets, restricts intra-islet vascular expansion posttransplantation. RESEARCH DESIGN AND METHODS--Pancreatic islets of TSP-1-deficient ([TSP-1.sup.-/-]) mice or wild-type islets transfected with siRNA for TSP-1 were transplanted beneath the renal capsule of syngeneic or immunocompromised recipient mice. RESULTS--Both genetically [TSP-1.sup.-/-] islets and TSP-1 siRNA-transfected islet cells demonstrated an increased vascular density when compared with control islets 1 month after transplantation. This was also reflected in a markedly increased blood perfusion and oxygenation of the grafts. The functional importance of the improved vascular engraftment was analyzed by comparing glucose-stimulated insulin release from islet cells transfected with either TSP-1 siRNA or scramble siRNA before implantation. These experiments showed that the increased revascularization of grafts composed of TSP-1 siRNA-transfected islet cells correlated to increments in both their first and second phase of glucose-stimulated insulin secretion. CONCLUSIONS--Our findings demonstrate that inhibition of TSP-1 in islets intended for transplantation may be a feasible strategy to improve islet graft revascularization and function.

Type 2 diabetes is characterized by a chronic elevation of plasma glucose concentration, causing complications such as retinopathy, neuropathy, and nephropathy and increasing the risk of cardiovascular disease and stroke. [...], OBJECTIVE--Fructose-l, 6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic β-cell lines exposed to high fat. However, whether specific β-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet β-cell FBPase can result in reduced glucose-mediated insulin secretion. RESEARCH DESIGN AND METHODS--To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet β-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic β-cell lines (MIN6) stably overexpressing huFBPase. RESULTS--FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels. CONCLUSIONS--Taken together, these results suggest that upregulation of FBPase in pancreatic islet β-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.

Defective negative selection caused by weak interactions between serf-peptides and major histocompatibility complex (MHC) molecules has been proposed as a mechanism that may contribute to autoimmunity. We have been interested [...], OBJECTIVE--Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a β-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes. RESEARCH DESIGN AND METHODS--The binding of a proinsulin epitope, proinsulin-l(47-64) (PI-1[47-64]), to the MHC class II molecules [I-A.sup.g7] and [I-A.sup.k] was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in [I-A.sup.g7+] and [I-A.sup.k+] mice. RESULTS--C-peptide epitopes bound very weakly to [I-A.sup.g7] molecules. However, C-peptide-reactive T-cells were induced after immunization in [I-A.sup.g7]-bearing mice (NOD and B6.g7) but not in [I-A.sup.k]-bearing mice (B10.BR and NOD.h4). T-cells reactive with the PI-1(47-64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated β-cells in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice. CONCLUSIONS--These data demonstrate an inverse relationship between self-peptide-MHC binding and T-cell autoreactivity for the PI-1(47-64) epitope in autoimmune diabetes.

Programmed death 1 (PD-1) is an immunoreceptor of the CD28/CTLA-4 family whose expression is induced in activated T- and B-cells and in macrophages (1,2). PD-1 has two cytoplasmic tyrosine motifs: [...], OBJECTIVE--Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T-cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic p-cells in nonobese diabetic (NOD) mice. RESEARCH DESIGN AND METHODS--We established an insulin promoter-driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells, and regulatory T-cells were analyzed in these transgenic mice; and T-cell proliferation, adoptive transfer, and islet transplantation were performed to evaluate the PD-L1 transgene-mediated immuneprotective mechanisms. RESULTS--The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed, and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets. CONCLUSIONS--Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes and illustrate its role in downregulating diabetogenic T-cells in NOD mice. Diabetes 57:1861-1869, 2008

Recent investigations have revealed a pivotal role for estradiol ([E.sub.2]) in regulating energy metabolism and have opened new insights into the physiological role of estrogen receptors (ERs) (1,2). Mice that [...], OBJECTIVE--Estradiol ([E.sub.2]) is known to modulate insulin sensitivity and, consequently, glucose homeostasis. Resveratrol (RSV), an agonist of estrogen receptor (ER), has exerted anti-hyperglycemic effects in streptozotocin-induced type 1 diabetic rats in our previous study and was also shown to improve insulin resistance in other reports. However, it remains unknown whether activation of ER is involved in the metabolic effects of RSV via insulin-dependent and -independent mechanisms. RESEARCH DESIGN AND METHODS--Male Sprague-Dawley rats were given a high cholesterol-fructose (HCF) diet for 15 weeks and were treated with RSV for either 15 days or 15 weeks. RESULTS--Here, we show that RSV shifts the metabolic characteristics of rats on an HCF diet toward those of rats on a standard diet. RSV treatment increased insulin-stimulated whole-body glucose uptake and steady-state glucose uptake of soleus muscle and liver in HCF-fed rats as well as enhanced membrane trafficking activity of GLUT4 and increased phosphorylation of insulin receptor in insulin-resistant soleus muscles. Interestingly, the phosphorylated ER level in insulin-resistant soleus muscle was significantly elevated in rats with RSV treatment in both basal and euglycemic-hyperinsulinemic conditions. RSV exerted an insulin-like stimulatory effect on isolated soleus muscle, epididymal fat and hepatic tissue, and C2C12 myotubes. The RSV-stimulated glucose uptake in C2C12 myotubes was dependent on extracellular signal-related kinase/p38 (early phase, 1 h) and p38/phosphoinositide 3-kinase (late phase, 14 h) activation. Inhibition of ER abrogated RSV-induced glucose uptake in both early and late phases. CONCLUSIONS--Collectively, these results indicate that ER is a key regulator in RSV-stimulating insulin-dependent and -independent glucose uptake, which might account for the protective effects of RSV on diet-induced insulin resistance syndrome.

The development of new mouse models has allowed investigators to address questions related to glucose metabolism in ways that were not previously possible. Use of inbred mouse strains and proliferation [...], OBJECTIVE--To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains. RESEARCH DESIGN AND METHODS--Hyperinsulinemic-euglycemic (~8.5 mmol/l) and -hypoglycemic (~3.0 mmol/l) clamps were done in catheterized, 5-h-fasted mice to assess insulin action and hypoglycemic counter-regulatory responsiveness. Hyperglycemic clamps (~15 mmol/l) were done to assess insulin secretion and compared with results in perifused islets. RESULTS--Insulin action and hypoglycemic counter-regulatory and insulin secretory phenotypes varied considerably in four inbred mouse strains. In vivo insulin secretion was greatest in 129X1/Sv mice, but the counter-regulatory response to hypoglycemia was blunted. FVB/N mice in vivo showed no increase in glucose-stimulated insulin secretion, relative hepatic insulin resistance, and the highest counter-regulatory response to hypoglycemia. In DBA/2 mice, insulin action was lowest among the strains, and islets isolated had the greatest glucose-stimulated insulin secretion in vitro. In C57BL/6 mice, in vivo physiological responses to hyperinsulinemia at euglycemia and hypoglycemia were intermediate relative to other strains. Insulin secretion by C57BL/6 mice was similar to that in other strains in contrast to the blunted glucose-stimulated insulin secretion from isolated islets. CONCLUSIONS--Strain-dependent differences exist in four inbred mouse strains frequently used for genetic manipulation and study of glucose metabolism. These results are important for selecting inbred mice to study glucose metabolism and for interpreting and designing experiments.

Type 1 diabetes is a disorder of insulin deficiency resulting from the autoimmune destruction of β-cells (1). Monoclonal antibodies directed against a variety of lymphocyte subsets or their products (e.g., [...], OBJECTIVE--Antilymphocyte serum can reverse overt type 1 diabetes in NOD mice; yet, the therapeutic parameters and immunological mechanisms underlying the ability for this agent to modulate autoimmune responses against β-cells are unclear, forming the rationale for this investigation. RESEARCH DESIGN AND METHODS--A form of antilymphocyte serum, rabbit anti-mouse thymocyte globulin (mATG), was utilized in a variety of in Vivo and in vitro settings, each for the purpose of defining the physiological, immunological, and metabolic activities of this agent, with particular focus on actions influencing development of type 1 diabetes. RESULTS--We observed that mATG attenuates type 1 diabetes development in an age-dependent fashion, only proving efficacious at disease onset or in the late pre-diabetic phase (12 weeks of age). When provided at 12 weeks of age, mATG reversed pancreatic insulitis, improved metabolic responses to glucose challenge, and rapidly increased frequency of antigen-presenting cells in spleen and pancreatic lymph nodes. Surprisingly, mATG therapy dramatically increased, in an age-dependent fashion, the frequency and the functional activity of [CD4.sup.+] [CD25.sup.+] regulatory T-cells. Adoptive transfer/cotransfer studies of type 1 diabetes also support the concept that mATG treatment induces a stable and transferable immunomodulatory repertoire in Vivo. CONCLUSIONS--These findings indicate that an induction of immunoregulation, rather than simple lymphocyte depletion, contributes to the therapeutic efficacy of antithymocyte globulin and suggest that time-dependent windows for the ability to delay or reverse type 1 diabetes exist based on the capacity to enhance the functional activity of regulatory T-cells.

Type 1 diabetes is an autoimmune disease characterized by a local inflammatory reaction in and around islets followed by selective destruction of insulin-secreting β-cells (1). The factors leading to the [...], OBJECTIVE--Type 1 diabetes is an autoimmune disease characterized by a local inflammatory reaction in and around islets followed by selective destruction of insulin-secreting p-cells. We tested the hypothesis that chemokines affect different mechanisms responsible for the development of diabetes in NOD mice. RESEARCH DESIGN AND METHODS--We examined chemokine expression in islets of NOD mice and tested their functional relevance to development of diabetes using transgenic mice expressing the mouse herpesvirus 68-encoded chemokine decoy receptor M3 (NOD-M3 mice) in insulin-secreting β-cells. RESULTS--Multiple chemokines were expressed in pancreatic islets of NOD mice before development of diabetes. Islet-specific expression of the pan-chemokine inhibitor M3 dramatically reduced leukocyte infiltration and islet destruction and completely blocked development of diabetes in NOD-M3 mice. M3 blocked diabetes by inhibiting the priming of diabetogenic cells in the pancreatic lymph nodes and their recruitment into the islets. This effect was specific to the pancreatic islets because M3 expression did not affect other ongoing autoimmune processes. CONCLUSIONS--These results demonstrate that chemokines mediate afferent and efferent immunity in type 1 diabetes and suggest that broad chemokine blockade may represent a viable strategy to prevent insulitis and islet destruction.

Over the past 20 years, the molecular underpinnings of energy homeostasis have come to light (1-4) at a rate strikingly similar to that of the rising prevalence of obesity (5, [...]