IA-2 and IA-2 beta were cloned in our laboratory from a beta cell subtraction library in the mid 1990s and found to be enzymatically inactive members of the protein tyrosine phosphatase family. IA-2 and IA-2 beta are widely distributed in neuroendocrine cells throughout the body and belong to an ancient gene family with homologs in Drosophila and C. elegans. Both proteins are integral transmembrane components of dense core secretory vesicles (DCV). Examination of sera from patients with autoimmune diseases revealed that IA-2 and IA-2 beta are major autoantigens in type 1 diabetes. Our studies showed that at diagnosis approximately 70% of patients with type 1 diabetes had autoantibodies to IA-2. More important these autoantibodies appeared as long as 10 years before the development of clinical disease and could be used as predictive markers, especially when combined with the two other major autoantigens (i.e., insulin and glutamic acid decarboxylase) associated with this disease. Studies from a number of laboratories including our own showed that if an individual had one autoantibody the risk of developing clinical disease in 5 years was 10%. In the presence of two autoantibodies the risk increased to 50% and in the presence of three autoantibodies the risk was even higher, between 60 and 80%. As a result of these findings it is now clear that type 1 diabetes is a chronic disease and that it is possible to identify individuals at high disease risk for entry into therapeutic intervention trials long before serious end-organ disease develops and thus determine whether intervention will delay or prevent the disease. Over the last few years we have attempted to determine the function of IA-2 and IA-2 beta. Our approach was to knock out both of these genes. In previous reports we described the knockout of the individual IA-2 and IA-2 beta genes. We have now succeeded in making double knockout mice and have studied the effect of the combined deletion of these two genes on insulin secretion and blood glucose levels. In brief, the knockout mice appeared healthy at birth and showed normal growth and development. Histologic examination and immunostaining for insulin, glucagon, somatostatin and pancreatic polypeptide revealed no difference between the double knockout and wild type mice. Non-fasting blood glucose and insulin levels also were within the normal range. No evidence of insulin resistance was observed nor were there alterations in fasting blood glucose, insulin or leptin levels in the double knockout mice maintained on a high fat diet as compared to the wild type mice maintained on the same diet. However as compared to wild type mice, the double knockout mice showed glucose intolerance and an absent first-phase insulin release curve. Moreover, the glucose tolerance tests were more severely impaired in the double knockout than the single knockout mice. In addition, to determine whether the combined deletion of IA-2 and IA-2 beta played any role in the development of diabetes in NOD mice, the most widely studied animal model for human type 1 diabetes, we generated double knockout mice on the NOD/LtJ background. The incidence of diabetes in these mice was not significantly different than in the wild type mice. Taken together our experiments show that the dense core vesicle proteins IA-2 and IA-2 beta, alone or in combination, are involved in insulin secretion, but neither alone nor in combination are they required for the development of diabetes in NOD mice. Over the last year we also studied in greater depth the mechanism by which IA-2 influences insulin secretion. Insulin homeostasis is a complex process involving a cascade of regulatory factors, and IA-2 is widely expressed in neuroendocrine cells throughout the body. Consequently, it was uncertain whether the impairment of insulin secretion in IA-2 knockout mice was a direct result of the knockout of IA-2 in beta cells or to counter regulatory alterations resulting from IA-2 knockout in other neuroendocrine cells. To define the function of IA-2, we studied the secretion of insulin in a single cell type, MIN-6, by overexpressing and knocking down IA-2. Our experiments showed that overexpression of IA-2 resulted in a 6-fold increase in glucose- or K+-induced insulin secretion and approximately a 3 fold increase in the number of secretory vesicles and the insulin content of cells. In contrast, knockdown of endogenous IA-2 by short interfering RNA resulted in nearly a complete loss of glucose-induced insulin secretion and a 50% decrease in basal insulin release. The half-life of insulin in cells overexpressing IA-2 was nearly twice as great as that in mock-transfected cells, suggesting that IA-2 was stabilizing the insulin-containing vesicles. From these results we conclude that in beta cells, IA-2 is an important regulator of dense core vesicle number and glucose-induced and basal insulin secretion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Intramural Research (Z01)
Project #
1Z01DE000423-20
Application #
7146104
Study Section
(OIIB)
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Cai, Tao; Hirai, Hiroki; Fukushige, Tetsunari et al. (2009) Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2. PLoS Genet 5:e1000447
Kim, Soo Mi; Theilig, Franziska; Qin, Yan et al. (2009) Dense-core vesicle proteins IA-2 and IA-2{beta} affect renin synthesis and secretion through the {beta}-adrenergic pathway. Am J Physiol Renal Physiol 296:F382-9
Khil, L-Y; Jun, H-S; Kwon, H et al. (2007) Human chorionic gonadotropin is an immune modulator and can prevent autoimmune diabetes in NOD mice. Diabetologia 50:2147-55
Notkins, Abner Louis (2007) New predictors of disease. Molecules called predictive autoantibodies appear in the blood years before people show symptoms of various disorders. Tests that detected these molecules could warn of the need to take preventive action. Sci Am 296:72-9
Mett, Vadim; Shamloul, Abdel-Moneim; Hirai, Hiroki et al. (2007) Engineering and expression of the intracellular domain of insulinoma-associated tyrosine phosphatase (IA-2ic), a type 1 diabetes autoantigen, in plants. Transgenic Res 16:77-84
Harashima, S-I; Harashima, C; Nishimura, T et al. (2007) Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction. Clin Exp Immunol 150:49-60
Li, Na; Shigihara, Toshikatsu; Tzioufas, Athanasios G et al. (2007) Human chorionic gonadotropin prevents Sjogren's syndrome-like exocrinopathy in mice. Arthritis Rheum 56:2211-5
Kubosaki, Atsutaka; Nakamura, Shinichiro; Clark, Anne et al. (2006) Disruption of the transmembrane dense core vesicle proteins IA-2 and IA-2beta causes female infertility. Endocrinology 147:811-5
Kubosaki, Atsutaka; Nakamura, Shinichiro; Notkins, Abner Louis (2005) Dense core vesicle proteins IA-2 and IA-2beta: metabolic alterations in double knockout mice. Diabetes 54 Suppl 2:S46-51
Hu, Y F; Zhang, H L; Cai, T et al. (2005) The IA-2 interactome. Diabetologia 48:2576-81

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