Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse

Abstract

It has been reported that an insulin 2 gene knockout, when bred onto nonobese diabetic (NOD) mice, accelerates diabetes. We produced insulin 1 gene knockout congenic NOD mice. In contrast to insulin 2, diabetes and insulitis were markedly reduced in insulin 1 knockout mice, with decreased and delayed diabetes in heterozygous females and no insulitis and diabetes in most homozygous female mice. Lack of insulitis was found for insulin 1 female homozygous knockout mice at 8, 12, and 37 weeks of age. Despite a lack of insulitis, insulin 1 homozygous knockout mice spontaneously expressed insulin autoantibodies. Administration of insulin peptide B:9-23 of both insulin 1 and 2 to NOD mice induced insulin autoantibodies. Insulin 1 is not the only lymphocytic target of NOD mice. Insulin 1 homozygous knockout islets, when transplanted into recently diabetic wild-type NOD mice, became infiltrated with lymphocytes and only transiently reversed diabetes. These observations indicate that loss of either insulin gene can influence progression to diabetes of NOD mice and suggest that the preproinsulin 1 gene is crucial for the spontaneous development of NOD insulitis and diabetes.

Fig. 1.

Glucose levels and Sialitis of insulin 1 -/- knockout mice. (A) Glucose levels of insulin 1 homozygous knockout female mice (-/-, ▪, n = 7) and wild-type littermates (+/+, •, n = 7) plotted from 8 to 30 weeks of age before diabetes onset. (B) Lymphocyte infiltration in salivary glands was present in insulin 1 -/- mice despite absence of insulitis for this 45-week-old mouse.

Fig. 2.

Life table analysis of insulin knockout (KO) mice. Filled squares (▪) indicate insulin homozygous KO (-/-), filled triangles (▴) indicate heterozygous KO (+/-), and filled circles (•) indicate wild-type insulin genes (+/+). (Upper) Progression to diabetes in insulin 2 KO mice is shown (A: female mice -/-, n = 13; +/-, n = 43; +/+, n = 19; B: male mice -/-, n = 13; +/-, n = 25; +/+, n = 24). (Lower) Progression to diabetes in insulin 1 KO mice is shown (C: female mice, -/-, n = 18; +/-, n = 37; +/+, n = 17; D: male mice -/-, n = 19; +/-, n = 51; +/+, n = 30). Statistical analysis was performed with prism logrank software. The three curves are dramatically different for female insulin 2 (A, P < 0.0001) and insulin 1 KO (D, P < 0.001). For male mice, insulin 1 KO mice differ significantly in progression to diabetes (C, P < 0.01).

Fig. 3.

Severe insulitis in insulin 2 knockout mice and lack of insulitis in insulin 1 knockout (KO) mice. Immunohistochemical staining (anti-insulin, anti-glucagon) of pancreatic islets from insulin KO mice was performed. As early as at 10 weeks of age, beta cells are destroyed in insulin 2 KO mice with extensive insulitis (A and D). In contrast, insulin-containing beta cells remain without insulitis for older insulin 1 +/- male (age = 48 weeks) (B and E) and insulin 1 -/- female mice (age = 37 weeks) (C and F).

Fig. 4.

Peak IAA of different genotypes (-/-, +/-, +/+) of insulin knockout mice. Peak IAA index between 8 and 32 weeks of age is plotted. Statistical analysis was performed with Mann-Whitney U test. Heterozygous and homozygous knockouts versus wild type are significantly different for insulin 2 female (P < 0.05) and male (P < 0.01) mice but not for insulin 1 female and male mice.

Fig. 5.

Restoration of normoglycemia by the transplantation of insulin 1 -/- islets but not by insulin 1 +/- islets. (A and B) Blood glucose levels of transplanted mice with insulin 1 -/- islets graft (A) or with insulin 1 +/- islets graft (B). Insulin 1 -/- islet transplant (C and D) and the native recipient pancreas (E and F) were stained for insulin (C and E) or glucagon (D and F) at the time of diabetes recurrence. The recipient mouse pancreas was devoid of islets with insulin-containing cells, with glucagon cells remaining, whereas the transplanted -/- islets had abundant insulin-containing cells.