Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells.

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes, we differentiated human induced pluripotent stem cells into pancreatic endocrine cells, including ß cells. Here, we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived ß cells, along with a reduced effect on α cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response.

Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

Modifying Enzymes Are Elicited by ER Stress, Generating Epitopes That Are Selectively Recognized by CD4+ T Cells in Patients With Type 1 Diabetes.

In spite of tolerance mechanisms, some individuals develop T-cell-mediated autoimmunity. Posttranslational modifications that increase the affinity of epitope presentation and/or recognition represent one means through which self-tolerance mechanisms can be circumvented. We investigated T-cell recognition of peptides that correspond to modified ß-cell antigens in subjects with type 1 diabetes. Modified peptides elicited enhanced proliferation by autoreactive T-cell clones. Endoplasmic reticulum (ER) stress in insulinoma cells increased cytosolic calcium and the activity of tissue transglutaminase 2 (tTG2). Furthermore, stressed human islets and insulinomas elicited effector responses from T cells specific for modified peptides, suggesting that ER stress-derived tTG2 activity generated deamidated neoepitopes that autoreactive T cells recognized. Patients with type 1 diabetes had large numbers of T cells specific for these epitopes in their peripheral blood. T cells with these specificities were also isolated from the pancreatic draining lymph nodes of cadaveric donors with established diabetes. Together, these results suggest that self-antigens are enzymatically modified in ß-cells during ER stress, giving rise to modified epitopes that could serve to initiate autoimmunity or to further broaden the antigenic repertoire, activating potentially pathogenic CD4+ T cells that may not be effectively eliminated by negative selection.

Surprising Heterogeneity of Pancreatic Islet Cell Subsets.

Two studies clearly demonstrate that pancreatic islets and, more specifically, their cellular constituents, display a much greater complexity than previously appreciated.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion.

T cell-mediated destruction of insulin-producing ß cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in ß cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in ß cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in ß cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates.

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

Exenatide treatment for 6 months improves insulin sensitivity in adults with type 1 diabetes.

OBJECTIVE: Exenatide treatment improves glycemia in adults with type 2 diabetes and has been shown to reduce postprandial hyperglycemia in adolescents with type 1 diabetes. We studied the effects of exenatide on glucose homeostasis in adults with long-standing type 1 diabetes. RESEARCH DESIGN AND METHODS: Fourteen patients with type 1 diabetes participated in a crossover study of 6 months' duration on exenatide (10 µg four times a day) and 6 months off exenatide. We assessed changes in fasting and postprandial blood glucose and changes in insulin sensitivity before and after each study period. RESULTS: High-dose exenatide therapy reduced postprandial blood glucose but was associated with higher fasting glucose concentrations without net changes in hemoglobin A1c. Exenatide increased insulin sensitivity beyond the effects expected as a result of weight reduction. CONCLUSIONS: Exenatide is a promising adjunctive agent to insulin therapy because of its beneficial effects on postprandial blood glucose and insulin sensitivity in patients with type 1 diabetes.

Diabetes: 8 strategies to put into practice.

In search of ways to improve the way you approach diabetes care in your practice? Consider these strategies, which have worked for us-- and have evidence to support them.

Extrapancreatic autoantibody profiles in type I diabetes.

Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing ß-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-ß. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-ß (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.

Humanized mice for the study of type 1 and type 2 diabetes.

The availability of immunodeficient mice engrafted with functional human immune systems and islets permits in vivo study of human diabetes without putting patients at risk.

Diabetes and cancer: a consensus report.

Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and diabetes treatments. This consensus statement of experts assembled jointly by the American Diabetes Association and the American Cancer Society reviews the state of science concerning 1) the association between diabetes and cancer incidence or prognosis, 2) risk factors common to both diabetes and cancer, 3) possible biologic links between diabetes and cancer risk, and 4) whether diabetes treatments influence risk of cancer or cancer prognosis. In addition, key unanswered questions for future research are posed.

Diabetes and cancer: a consensus report.

Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and treatments. This consensus statement of experts assembled jointly by the American Diabetes Association and the American Cancer Society reviews the state of science concerning 1) the association between diabetes and cancer incidence or prognosis; 2) risk factors common to both diabetes and cancer; 3) possible biologic links between diabetes and cancer risk; and 4) whether diabetes treatments influence the risk of cancer or cancer prognosis. In addition, key unanswered questions for future research are posed.

Effects of exenatide alone and in combination with daclizumab on beta-cell function in long-standing type 1 diabetes.

OBJECTIVE: In patients with long-standing type 1 diabetes, we investigated whether improved beta-cell function can be achieved by combining intensive insulin therapy with agents that may 1) promote beta-cell growth and/or limit beta-cell apoptosis and 2) weaken the anti-beta-cell autoimmunity. RESEARCH DESIGN AND METHODS: For this study, 20 individuals (mean age 39.5 +/- 11.1 years) with long-standing type 1 diabetes (21.3 +/- 10.7 years) were enrolled in this prospective open-label crossover trial. After achieving optimal blood glucose control, 16 subjects were randomized to exenatide with or without daclizumab. Endogenous insulin production was determined by repeatedly measuring serum C-peptide. RESULTS: In 85% of individuals with long-standing type 1 diabetes who were screened for participation in this trial, C-peptide levels >or=0.05 ng/ml (0.02 nmol/l) were found. Residual beta-cells responded to physiological (mixed-meal) and pharmacological (arginine) stimuli. During exenatide treatment, patients lost 4.1 +/- 2.9 kg body wt and insulin requirements declined significantly (total daily dose on exenatide 0.48 +/- 0.11 vs. 0.55 +/- 0.13 units x kg(-1) x day(-1) without exenatide; P = 0.0062). No signs of further activation of the underlying autoimmune disease were observed. Exenatide delayed gastric emptying, suppressed endogenous incretin levels, but did not increase C-peptide secretion. CONCLUSIONS: In long-standing type 1 diabetes, which remains an active autoimmune disease even decades after its onset, surviving beta-cells secrete insulin in a physiologically regulated manner. However, the combination of intensified insulin therapy, exenatide, and daclizumab did not induce improved function of these remaining beta-cells.

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.

BACKGROUND: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. RESULTS: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. CONCLUSION: AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Deciphering von Hippel-Lindau (VHL/Vhl)-associated pancreatic manifestations by inactivating Vhl in specific pancreatic cell populations.

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in alpha-cells or an insulin promoter in beta-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated alpha-cells or beta-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function.

Pancreatic islet beta-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate beta-cell function is of immense clinical relevance. Transforming growth factor (TGF)-beta signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-beta pathway in regulation of insulin gene transcription and beta-cell function. We identify insulin as a TGF-beta target gene and show that the TGF-beta signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucose-stimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-beta signaling regulates expression of genes involved in beta-cell function. Together, these studies emphasize TGF-beta/Smad3 signaling as an important regulator of insulin gene transcription and beta-cell function and suggest that components of the TGF-beta signaling pathway may be dysregulated in diabetes.

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Assessment of islet function following islet and pancreas transplantation.

Pancreas and islet transplant recipients are monitored using various metabolic and imaging methods. The inaccessibility of the transplanted whole pancreas and of the isolated islets poses specific problems (eg, all assessment techniques are indirect). Although successful pancreas transplantation typically restores normal glucose homeostasis, islet transplantation into the liver does not completely normalize islet hormone secretion and glucose metabolism. Development of better testing strategies, such as direct islet imaging, will significantly advance the field.