Proinsulin-Specific Autoantibodies Are Relatively Infrequent in Young Offspring With Pre–Type 1 Diabetes

Insulin is a β-cell–specific and pivotal autoantigen in type 1 diabetes (1) and is produced after proteolysis of its prohormone proinsulin, which is a potential target of autoimmunity. T-cell studies in the nonobese diabetic (NOD) mouse (2,3) and in prediabetic relatives of patients with type 1 diabetes (4,5) indicate the existence of proinsulin-specific immunodominant epitopes localized in the region between the C-peptide and the A-chain, whereas immune intervention with proinsulin in the NOD mouse can prevent diabetes onset. These studies have led to the proposal of proinsulin as a primary target of autoimmunity associated with type 1 diabetes. At the humoral level, proinsulin-specific autoantibodies have also been reported in humans (6,7). Our own data show, however, that proinsulin autoantibody (PAA) and insulin autoantibody (IAA) levels correlate well in newly diagnosed patients, but IAAs are more sensitive and specific for type 1 diabetes (7). Furthermore, IAA or PAA could be completely displaced with either insulin or proinsulin in the majority of patients, suggesting that the main epitopes recognized by these antibodies at diabetes onset are on the insulin molecule. In the present study, we investigated whether antibodies to proinsulin-specific epitopes either preceded or were a more relevant finding early in the development of diabetes-associated autoimmunity.

We took advantage of the German BABYDIAB Study, in which samples from the offspring of parents with type 1 diabetes were obtained at birth, 9 months, and 2 and 5 years of age and where 62 (4.2%) offspring developed IAA by 2 years of age (8). Samples from 48 offspring (median age 2.6 years) were studied. All had islet autoantibodies that included IAA. Antibodies to insulin and proinsulin were measured by microradiobinding assays, incorporating displacement with unlabelled insulin or proinsulin, respectively, as previously described (7,9). To identify proinsulin-specific antibodies, displacement of binding to radiolabeled proinsulin was also performed with excess cold insulin (52 μmol/l).

There was a strong correlation between IAA and PAA levels (r = 0.88, P < 0.01) (Fig. 1). Only 3 of the 48 IAA-positive offspring showed no binding to proinsulin; 1 of these 3 was positive for another diabetes-associated antibody in the index sample, but all 3 children had further IAA-positive samples after the index sample, and 1 of these subsequently developed islet cell antibody, GAD antibody, and antibody to IA2. Binding to insulin in the remaining 45 sera could be completely inhibited with cold proinsulin. Binding to proinsulin in 2 of the 45 PAA-positive sera could not be completely inhibited with cold insulin, suggesting that PAA in these 2 offspring were directed against both epitopes that are shared by proinsulin and insulin and epitopes found only on the proinsulin molecule. Both had relatively high PAA and IAA (case 1: 14.9 units IAA, 6.9 units PAA; case 2: 14.6 units IAA, 44.1 units PAA). Proinsulin-specific antibodies (not displaceable by insulin) accounted for 49% of the binding to proinsulin in case 1 and for 48% of the binding to proinsulin in case 2. Previous and follow-up samples were analyzed in these two subjects. Neither had PAA in a sample obtained before the first detection of IAA. In case 1, both IAA and PAA declined in follow-up samples, but the proportion of proinsulin-specific binding remained >30%. Case 2 developed type 1 diabetes 1 year after the measurement of IAA and PAA. Insulin antibodies increased (32 units) and PAA decreased (12.3 units) after the commencement of insulin therapy in this subject. The proportion of proinsulin-specific antibodies remaining were markedly reduced (4%), indicating that insulin therapy did not promote the production of proinsulin-specific autoimmunity. Type 1 diabetes genotyping identified HLA DR*11/0401 DQA*05/03 DQB*0301/0302 in case 1 and HLA DR*11/12 DQA*05/0102 DQB*0301/0502 in case 2.

The data indicate that proinsulin-specific humoral autoimmunity is relatively uncommon in the initial stages of diabetes-associated autoimmunity. The relative absence of proinsulin-specific autoantibodies during the disease process does not suggest that screening for PAA rather than IAA would be more effective in early identification of young children at risk and does not provide evidence for proinsulin rather than insulin as a primary target in diabetes-associated autoimmunity.