Anti-Insulin Receptor Autoantibodies Are Not Required for Type 2 Diabetes Pathogenesis in NZL/Lt Mice, a New Zealand Obese (NZO)-Derived Mouse Strain

The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte–enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions ofBlymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh6nullmales gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the Blymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers

Sequence Variation in Promoter of Ica1 Gene, Which Encodes Protein Implicated in Type 1 Diabetes, Causes Transcription Factor Autoimmune Regulator (AIRE) to Increase Its Binding and Down-regulate Expression

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC+) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity

Insulin sensitivity in the setting of islet autoimmunity.

<p>Correlation between insulin sensitivity (GIR) and obesity (BMI, central fat) between all antibody negative (n = 11) and antibody positive on insulin±metformin (n = 7). Open squares indicate antibody negative on metformin only while close squares indicate patients on insulin±metformin.</p

Insulin and glucagon immunostaining.

<p>Immunohistochemical staining for insulin (Panel A and C) and glucagon (Panel B and D) in two different islets from different area of the pancreas in a donor with T2DM and pattern A pancreatic pathology. The first islet (panel A and B) contains both insulin and glucagon positive cells, while the second islet (Panel C and D) contains only glucagon positive cells.</p

Clinical characteristics. All of the blood sampling was performed under fasting conditions.

<p>* = p<0.05 (antibody negative (insulin± metformin) vs antibody positive (insulin± metformin)).</p><p>**Antibody Positive Group: 6 out of 7 patients were positive for GAD65 Ab. Of those 6 GAD65 Ab positives, 2 were also positive for IA-2 and 3 for ZnT8 Ab. One of these subjects was positive for IA-2 Ab alone. Antibody titer: 3 GAD65 Ab positive 3^rd tertile; 2 IA-2 Ab positive 3^rd tertile.</p><p>Clinical characteristics. All of the blood sampling was performed under fasting conditions.</p

Beta cell mass determination.

<p>Beta cell mass (expressed as percentage of mean of donors without diabetes mellitus) in T2DM without autoimmune markers [mean 67.70±SEM 13.58 (n = 11) and T2DM with autoimmune markers (mean 12.80±SEM 4.54 (n = 4)] (p = 0.05) Amongst the donors with markers of autoimmune diabetes 4 out of 4 expressed pattern A pathology while among the donors without autoimmune markers, 3 out of 11 expressed pattern B pathology (p = 0.026).</p

Demographic and immunological characteristics of clinically diagnosed T2DM cadaveric donors captured through the nPOD network.

<p>AA: African American donors, AS: Asian-American donors, CA: Caucasian donors, HI: Hispanic donors.</p><p>Demographic and immunological characteristics of clinically diagnosed T2DM cadaveric donors captured through the nPOD network.</p