T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes.

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/Du) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/Du and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vß13a T cell receptor ß chain, completely prevents diabetes. Using the RT1B/Du-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 ß chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vß13a TCR and its class II RT1u ligand suggests a mechanism by which a germline TCR ß chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vß13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity.

The Vbeta13 T Cell Receptor Monoclonal Antibody Reduces Hyaluronan and CD68+, CD3+, and CD8+ Cell Infiltrations to Delay Diabetes in Congenic BB DRLyp/Lyp Rats.

The depleting Vß13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vß16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55-73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59-69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60-92). Three (27%) of the 17D5-treated rats were killed at 101-103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.

Genetic Variation Within the HLA-DRA1 Gene Modulates Susceptibility to Type 1 Diabetes in HLA-DR3 Homozygotes.

Type 1 diabetes (T1D) involves the interaction of multiple gene variants, environmental factors, and immunoregulatory dysfunction. Major T1D genetic risk loci encode HLA-DR and -DQ. Genetic heterogeneity and linkage disequilibrium in the highly polymorphic HLA region confound attempts to identify additional T1D susceptibility loci. To minimize HLA heterogeneity, T1D patients (N = 365) and control subjects (N = 668) homozygous for the HLA-DR3 high-risk haplotype were selected from multiple large T1D studies and examined to identify new T1D susceptibility loci using molecular inversion probe sequencing technology. We report that risk for T1D in HLA-DR3 homozygotes is increased significantly by a previously unreported haplotype of three single nucleotide polymorphisms (SNPs) within the first intron of HLA-DRA1. The homozygous risk haplotype has an odds ratio of 4.65 relative to the protective homozygous haplotype in our sample. Individually, these SNPs reportedly function as "expression quantitative trait loci," modulating HLA-DR and -DQ expression. From our analysis of available data, we conclude that the tri-SNP haplotype within HLA-DRA1 may modulate class II expression, suggesting that increased T1D risk could be attributable to regulated expression of class II genes. These findings could help clarify the role of HLA in T1D susceptibility and improve diabetes risk assessment, particularly in high-risk HLA-DR3 homozygous individuals.

A Critical Role for the Type I Interferon Receptor in Virus-Induced Autoimmune Diabetes in Rats.

The pathogenesis of human type 1 diabetes, characterized by immune-mediated damage of insulin-producing ß-cells of pancreatic islets, may involve viral infection. Essential components of the innate immune antiviral response, including type I interferon (IFN) and IFN receptor-mediated signaling pathways, are candidates for determining susceptibility to human type 1 diabetes. Numerous aspects of human type 1 diabetes pathogenesis are recapitulated in the LEW.1WR1 rat model. Diabetes can be induced in LEW.1WR1 weanling rats challenged with virus or with the viral mimetic polyinosinic:polycytidylic acid (poly I:C). We hypothesized that disrupting the cognate type I IFN receptor (type I IFN α/ß receptor [IFNAR]) to interrupt IFN signaling would prevent or delay the development of virus-induced diabetes. We generated IFNAR1 subunit-deficient LEW.1WR1 rats using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) genome editing and confirmed functional disruption of the Ifnar1 gene. IFNAR1 deficiency significantly delayed the onset and frequency of diabetes and greatly reduced the intensity of insulitis after poly I:C treatment. The occurrence of Kilham rat virus-induced diabetes was also diminished in IFNAR1-deficient animals. These findings firmly establish that alterations in innate immunity influence the course of autoimmune diabetes and support the use of targeted strategies to limit or prevent the development of type 1 diabetes.

Management status and its predictive factors in patients with type 2 diabetes in China: A Nationwide Multicenter Study: A Nationwide Multicenter Study.

BACKGROUND: The prevalence of type 2 diabetes in China is increasing rapidly. Appropriate management of glycemia, blood pressure and dyslipidemia in this population is a major public health concern. OBJECTIVE: The aim of this study was to assess metabolic control including glycated hemoglobin A1c (HbA1c ), blood pressure (BP) and low density lipoprotein cholesterol (LDL-c), in a large sample of patients with type 2 diabetes in China and to identify factors that correlated with the achievement of HbA1c, BP and LDL-c goals (ABCs). METHOD: A nationwide survey was conducted in 50 medical centres across China from April to July of 2010. Baseline information on demographics, medical history, HbA1c , BP and LDL-c levels were measured in 5961 patients with type 2 diabetes. RESULTS: Mean age, body mass index (BMI) and HbA1c were 59.5 ± 1.3 years, 24.5 ± 4.1 kg/m(2) and 8.3 ± 2.2%, respectively. With respect to generally accepted ABC treatment goals, 35.2% of participants had HbA1c <7%; 35.5% had BP < 140/80 mmHg, and 45.1% had LDL-c < 100 mg/dl. The proportion of patients who met all three targets was only 5.4%. Logistic regression revealed that smoking (P=0.000), higher BMI (P=0.001) and insulin use (P=0.000) were statistically significant predictors of failing to meet ABC targets. CONCLUSION: The percentage of Chinese patients with type 2 diabetes who met recommended targets for HbA1c , BP and LDL-c in 2010 was low. Smoking, higher BMI and insulin use were the strongest determinants of failing to meet ABC targets.

Medications for weight loss.

PURPOSE OF REVIEW: Overweight and obesity together with their comorbidities have become increasingly prevalent worldwide. The need for well tolerated, effective interventions has become increasingly urgent. Here we review the pharmacology, benefits, and risks of Western and Chinese medications used for weight loss. RECENT FINDINGS: Lifestyle interventions for weight loss are efficacious, but have had limited long-term durability. Bariatric surgery is very effective for weight loss and reversal of type 2 diabetes mellitus (T2DM), but it is invasive and not consistently durable in all patients. Recent studies show that newer Western pharmaceuticals and some traditional Chinese medications may be effective for appropriate patients in need of weight loss. SUMMARY: New Western medications, notably lorcaserin, phentermine/topiramate, naltrexone/bupropion, and liraglutide, are more effective and possibly safer than older medications but have important side-effects. Chinese herbal medicines may have efficacy similar to that of older Western medications and with few side-effects, but data are limited. We suggest that for appropriate patients, in particular those with or at high risk for T2DM, the judicious use of these medications with lifestyle modification is justified. This may be particularly true in Asia where T2DM develops at a low BMI, though more data are needed to support this concept.

Evaluation, Medical Therapy, and Course of Adult Persistent Hyperinsulinemic Hypoglycemia After Roux-en-Y Gastric Bypass Surgery: A Case Series.

OBJECTIVE: To describe the evaluation and treatment of hyperinsulinemic hypoglycemia in adults who had undergone gastric bypass surgery. A small number of patients who undergo Roux-en-Y bypass surgery develop postprandial hypoglycemia in the absence of dumping. In some cases, such patients have been treated with pancreatectomy. METHODS: We report the demographics, diagnostic results, response to medical therapy, and subsequent course of 6 referral patients with post-Roux-en-Y gastric bypass hypoglycemia. RESULTS: Characteristic clinical and metabolic parameters consistent with hyperinsulinemic hypoglycemia were identified. Parameters were similar for both spontaneous and glucose-challenge-induced hypoglycemia. In the context of exclusively postprandial symptoms, simultaneous glucose ≤55 mg/dL, insulin ≥17 µU/mL, C peptide ≥3.0 ng/mL, and insulin to glucose ratio >0.3 were associated with Roux-en-Y gastric bypass hyperinsulinemic hypoglycemia. Five of 6 patients improved on therapy consisting of dietary modification plus either calcium channel blockade, acarbose, or both. Two patients have remained on therapy for 12 to 15 months. The nonresponder was atypical and had had hypoglycemic events for several decades. Three treated patients were subsequently observed to have undergone partial or complete remission from hypoglycemic episodes after 2 to 37 months of therapy. None of the 6 have undergone pancreatectomy, and none have evidence of insulinoma. Invasive diagnostic procedures were of limited utility. CONCLUSION: In a subset of patients with post-Roux-en-Y gastric bypass hyperinsulinemic hypoglycemia, medical management can be efficacious and an alternative to partial pancreatectomy. In some cases, the disorder remits spontaneously.

Autoantigen-induced focusing of Vß13+ T cells precedes onset of autoimmune diabetes in the LEW.1WR1 rat.

The earliest events leading to autoimmune type 1 diabetes (T1D) are not known in any species. A T-cell receptor (TCR)-variable region, TCR-Vß13, is required for susceptibility to autoimmune diabetes in rats, and selective depletion of Vß13(+) T cells with an allele-specific monoclonal antibody prevents disease in multiple rat strains. To investigate the role of Vß13 early in diabetes, we examined islet T-cell transcripts in susceptible (LEW.1WR1) and resistant (LEW.1W and Wistar Furth) strains induced with polyinosinic:polycytidylic acid. Vß13(+) T cells displayed antigenic focusing in LEW.1WR1 islets 5 days postinduction and were characterized by a substantial decrease in complementarity determining region 3 diversity. This occurred prior to significant islet T-cell accumulation (day 7) or frank diabetes (days 10-14). Vß13(+) transcripts increased in LEW.1WR1 islets during diabetes progression, but not in resistant rats. We also analyzed transcript clonality of rat TCR-Vα5, an ortholog of the dominant TCR-Vα chain found on insulin B:9-23-reactive T cells in nonobese diabetic rat islets. We observed clonal expansion of Vα5(+) transcripts in prediabetic LEW.1WR1 islets, suggesting that rat Vα5 is also an important component of islet autoantigen recognition. These data provide additional evidence that genome-encoded TCR sequences are important determinants of genetic susceptibility to T1D.

The Missing Heritability in T1D and Potential New Targets for Prevention.

Type 1 diabetes (T1D) is a T cell-mediated disease. It is strongly associated with susceptibility haplotypes within the major histocompatibility complex, but this association accounts for an estimated 50% of susceptibility. Other studies have identified as many as 50 additional susceptibility loci, but the effect of most is very modest (odds ratio (OR) <1.5). What accounts for the "missing heritability" is unknown and is often attributed to environmental factors. Here we review new data on the cognate ligand of MHC molecules, the T cell receptor (TCR). In rats, we found that one allele of a TCR variable gene, V ß 13A, is strongly associated with T1D (OR >5) and that deletion of V ß 13+ T cells prevents diabetes. A role for the TCR is also suspected in NOD mice, but TCR regions have not been associated with human T1D. To investigate this disparity, we tested the hypothesis in silico that previous studies of human T1D genetics were underpowered to detect MHC-contingent TCR susceptibility. We show that stratifying by MHC markedly increases statistical power to detect potential TCR susceptibility alleles. We suggest that the TCR regions are viable candidates for T1D susceptibility genes, could account for "missing heritability," and could be targets for prevention.

Prevention of type 1 diabetes in the rat with an allele-specific anti-T-cell receptor antibody: Vß13 as a therapeutic target and biomarker.

In earlier studies of the Iddm14 diabetes susceptibility locus in the rat, we identified an allele of the T-cell receptor (TCR) ß-chain, Tcrb-V13S1A1, as a candidate gene. To establish its importance, we treated susceptible rats with a depleting anti-rat Vß13 monoclonal antibody and then exposed them to either polyinosinic:polycytidylic acid or a diabetogenic virus to induce diabetes. The overall frequency of diabetes in the controls was 74% (n = 50), compared with 17% (n = 30) in the anti-Vß13-treated animals, with minimal islet pathology in nondiabetic treated animals. T cells isolated from islets on day 5 after starting induction showed a greater proportion of Vß13(+) T cells than did peripheral lymph node T cells. Vß13 transcripts recovered from day 5 islets revealed focused Jß usage and less CDR3 diversity than did transcripts from peripheral Vß13(+) T cells. CDR3 usage was not skewed in control Vß16 CDR3 transcripts. Anti-rat Vß13 antibody also prevented spontaneous diabetes in BBDP rats. The Iddm14 gene is likely to be Tcrb-V13, indicating that TCR ß-chain usage is a determinant of susceptibility to autoimmune diabetes in rats. It may be possible to prevent autoimmune diabetes by targeting a limited element of the T-cell repertoire.

Implications of using hemoglobin A1C for diagnosing diabetes mellitus.

Until 2010, the diagnosis of diabetes mellitus was based solely on glucose concentration, but the American Diabetes Association (ADA) recommendations now include a new criterion: hemoglobin A1C ≥6.5%. Because this change may have significant implications for diabetes diagnosis, we conducted a comprehensive literature review including peer-reviewed articles not referenced in the ADA report. We conclude that A1C and plasma glucose tests are frequently discordant for diagnosing diabetes. A1C ≥6.5% identifies fewer individuals as having diabetes than glucose-based criteria. Convenience of A1C test might increase the number of patients diagnosed, but this is unproven. Diagnostic cut-points for both glucose and A1C are based on consensus judgments regarding optimal sensitivity and specificity for the complications of hyperglycemia. A1C may not accurately reflect levels of glycemia in some situations, but in comparison with glucose measurements, it has greater analytic stability and less temporal variability. When choosing a diagnostic test for diabetes, the limitations of each choice must be understood. Clinical judgment and consideration of patient preference are required to appropriately select among the diagnostic alternatives.

Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats.

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

A novel role for the centrosomal protein, pericentrin, in regulation of insulin secretory vesicle docking in mouse pancreatic beta-cells.

The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic beta-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in beta-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in beta-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory beta-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.

Identification of a serum-induced transcriptional signature associated with type 1 diabetes in the BioBreeding rat.

OBJECTIVE: Inflammatory mediators associated with type 1 diabetes are dilute and difficult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing preonset risk, and monitoring therapeutic interventions. RESEARCH DESIGN AND METHODS: We previously utilized a novel bioassay in which human type 1 diabetes sera were used to induce a disease-specific transcriptional signature in unrelated, healthy peripheral blood mononuclear cells (PBMCs). Here, we apply this strategy to investigate the inflammatory state associated with type 1 diabetes in biobreeding (BB) rats. RESULTS: Consistent with their common susceptibility, sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induced transcription of cytokines, immune receptors, and signaling molecules in PBMCs of healthy donor rats compared with control sera. Like the human type 1 diabetes signature, the DRlyp/lyp signature, which is associated with progression to diabetes, was differentiated from that of the DR+/+ by induction of many interleukin (IL)-1-regulated genes. Supplementing cultures with an IL-1 receptor antagonist (IL-1Ra) modulated the DRlyp/lyp signature (P < 10(-6)), while administration of IL-1Ra to DRlyp/lyp rats delayed onset (P = 0.007), and sera of treated animals did not induce the characteristic signature. Consistent with the presence of immunoregulatory cells in DR+/+ rats was induction of a signature possessing negative regulators of transcription and inflammation. CONCLUSIONS: Paralleling our human studies, serum signatures in BB rats reflect processes associated with progression to type 1 diabetes. Furthermore, these studies support the potential utility of this approach to detect changes in the inflammatory state during therapeutic intervention.

Infection with viruses from several families triggers autoimmune diabetes in LEW*1WR1 rats: prevention of diabetes by maternal immunization.

OBJECTIVE: The contribution of antecedent viral infection to the development of type 1 diabetes in humans is controversial. Using a newer rat model of the disease, we sought to 1) identify viruses capable of modulating diabetes penetrance, 2) identify conditions that increase or decrease the diabetogenicity of infection, and 3) determine whether maternal immunization would prevent diabetes. RESEARCH DESIGN AND METHODS: About 2% of LEW*1WR1 rats develop spontaneous autoimmune diabetes, but disease penetrance is much higher if weanling rats are exposed to environmental perturbants including Kilham rat virus (KRV). We compared KRV with other viruses for diabetogenic activity. RESULTS: Both KRV and rat cytomegalovirus (RCMV) induced diabetes in up to 60% of LEW*1WR1 rats, whereas H-1, vaccinia, and Coxsackie B4 viruses did not. Simultaneous inoculation of KRV and RCMV induced diabetes in 100% of animals. Pretreatment of rats with an activator of innate immunity increased the diabetogenicity of KRV but not RCMV and was associated with a moderate rate of diabetes after Coxsackie B4 and vaccinia virus infection. Inoculation of LEW*1WR1 dams with both KRV and RCMV prior to pregnancy protected weanling progeny from virus-induced diabetes in a virus-specific manner. CONCLUSIONS: Exposure to viruses can affect the penetrance of autoimmune diabetes in genetically susceptible animals. The diabetogenicity of infection is virus specific and is modified by immunomodulation prior to inoculation. Maternal immunization protects weanlings from virus-induced diabetes, suggesting that modification of immune responses to infection could provide a means of preventing islet autoimmunity.

Successful treatment of persistent hyperinsulinemic hypoglycemia with nifedipine in an adult patient.

OBJECTIVE: To describe the successful treatment of severe noninsulinoma hyperinsulinemic hypoglycemia with use of a calcium channel blocking agent in an adult patient who had previously undergone a gastric bypass surgical procedure. METHODS: A 65-year-old woman who had undergone a gastric bypass surgical procedure 26 years earlier was hospitalized because of severe postprandial hypoglycemia. During and after hospitalization, the patient underwent assessment with conventional measurements of glucose, insulin, proinsulin, and C-peptide; toxicologic studies; magnetic resonance imaging studies of the pancreas; and determination of hepatic vein insulin concentrations after selective splanchnic artery calcium infusion. RESULTS: Metabolic variables were consistent with the diagnosis of hyperinsulinemic hypoglycemia. Magnetic resonance imaging revealed the presence of a side branch intraductal papillary mucinous tumor that had been stable for more than 1 year. The results of the calcium-stimulated insulin release study were consistent with nonlocalized hypersecretion of insulin. A trial of frequent small feedings failed to prevent hypoglycemia. On the basis of reports of successful treatment of childhood nesidioblastosis, the patient was then prescribed nifedipine, 30 mg daily. She has subsequently remained free of symptomatic hypoglycemia for 20 months. CONCLUSION: A calcium channel blocking agent may be efficacious and a potential alternative to partial pancreatectomy in cases of noninsulinoma hyperinsulinemic hypoglycemia in adults.

Virus-induced autoimmune diabetes in the LEW.1WR1 rat requires Iddm14 and a genetic locus proximal to the major histocompatibility complex.

OBJECTIVE: To identify genes that confer susceptibility to autoimmune diabetes following viral infection in the LEW.1WR1 rat. RESEARCH DESIGN AND METHODS: About 2% of LEW.1WR1 rats develop spontaneous autoimmune diabetes. Immunological perturbants including viral infection increase both the frequency and tempo of diabetes onset. To identify diabetes susceptibility genes (LEW.1WR1 x WF), F2 rats were infected with Kilham rat virus following brief pretreatment with polyinosinic:polycytidylic acid. This treatment induces diabetes in 100% of parental LEW.1WR1 rats and 0% of parental WF rats. Linkage to diabetes was analyzed by genome-wide scanning. RESULTS: Among 182 F2 rats, 57 (31%) developed autoimmune diabetes after a mean latency of 16 days. All diabetic animals and approximately 20% of nondiabetic animals exhibited pancreatic insulitis. Genome-wide scanning revealed a requirement for the Iddm14 locus, long known to be required for diabetes in the BB rat. In addition, a new locus near the RT1 major histocompatibility complex (MHC) was found to be a major determinant of disease susceptibility. Interestingly, one gene linked to autoimmune diabetes in mouse and human, UBD, lies within this region. CONCLUSIONS: The Iddm14 diabetes locus in the rat is a powerful determinant of disease penetrance in the LEW.1WR1 rat following viral infection. In addition, a locus near the MHC (Iddm37) conditions diabetes susceptibility in these animals. Other, as-yet-unidentified genes are required to convert latent susceptibility to overt diabetes. These data provide insight into the polygenic nature of autoimmune diabetes in the rat and the interplay of genetic and environmental factors underlying disease expression.

CHOP mediates endoplasmic reticulum stress-induced apoptosis in Gimap5-deficient T cells.

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5(-/-) BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5(-/-) T cells. Knockdown of CHOP by siRNA protected Gimap5(-/-) T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.

Failure of alpha-galactosylceramide to prevent diabetes in virus-inducible models of type 1 diabetes in the rat.

BACKGROUND: Alpha-galactosylceramide (alpha-GalCer) is an invariant natural killer T (iNKT) cell ligand that prevents type 1 diabetes in NOD mice. However, alpha-GalCer can activate or suppress immune responses, raising concern about its potential use in human diabetes. MATERIALS AND METHODS: To evaluate this therapeutic issue further, BBDR and LEW.1WR1 rats were treated with Kilham rat virus (KRV) plus polyinosinic-polycytidylic acid, with or without alpha-GalCer, and followed for onset of diabetes. RESULTS: alpha-GalCer did not prevent diabetes in inducible rat models. To investigate this discrepancy, we analyzed iNKT cell function. Splenocytes stimulated with alpha-GalCer produced similar levels of IFNgamma in all rat strains, but less than mouse splenocytes. Rat splenocytes stimulated with alpha-GalCer preferentially produced IL-12, whereas mouse splenocytes preferentially produced IL-4. CONCLUSION: alpha-GalCer elicits species-specific cytokine responses in iNKT cells. In humans with type 1 diabetes, differences in iNKT cell responses to stimulation with alpha-GalCer due to age, genetic variability and other factors may influence its therapeutic potential.

Analysis of the rat Iddm14 diabetes susceptibility locus in multiple rat strains: identification of a susceptibility haplotype in the Tcrb-V locus.

Iddm14 (formerly Iddm4) is a non-MHC-linked genetic locus associated with autoimmune diabetes. Its effects have been well-documented in BB-derived rats in which diabetes is either induced by immunologic perturbation or occurs spontaneously. The role of Iddm14 in non-BB rat strains is unknown. Our goal was to extend the analysis of Iddm14 in new diabetes-susceptible strains and to identify candidate genes in the rat Iddm14 diabetes susceptibility locus that are common to these multiple diabetic strains. To determine if Iddm14 is important in strains other than BB, we first genotyped a (LEW.1WR1 x WF)F2 cohort in which diabetes was induced by perturbation with polyinosinic:polycytidylic acid. We found that Iddm14 is a major determinant of diabetes susceptibility in LEW.1WR1 rats. We then used nucleotide sequencing to establish a strain distribution pattern of polymorphisms (insertions, deletions, and single nucleotide polymorphisms [SNPs]) that predicts susceptibility to diabetes in a panel of inbred and congenic rats. Using the positional information from the congenic strains and the new linkage data, we identified a susceptibility haplotype in the T-cell receptor Vbeta chain (Tcrb-V) locus. This haplotype includes Tcrb-V13, which is identical in five susceptible strains but different in resistant WF and F344 rats. We conclude that Iddm14 is a powerful determinant of both spontaneous and induced autoimmune diabetes in multiple rat strains, and that Tcrb-V13 SNPs constitute a haplotype of gene elements that may be critical for autoimmune diabetes in rats.