Potential Involvement of Fructosylated Human Insulin and Serological Evidence in Subclinical Autoimmune Activity in Type 2 Diabetes Mellitus

Introduction: Type 2 Diabetes Mellitus (T2DM) involves chronic inflammation and recurrent hyperglycemia, often linked to excessive fructose intake. Some patients develop autoantibodies, indicating immune dysfunction. Fructose promotes protein glycation and the formation of Advanced Glycation End-products (AGEs), which may expose novel epitopes recognized as foreign. This study examined the antigenicity of fructosylated human insulin and its impact on lymphocytes in diabetic patients. Methods: Fructosylated insulin was characterized using spectroscopic techniques, with aggregation assessed by ThT, ANS, and DLS, and conformational changes analyzed via CD spectroscopy. Biochemical and biophysical methods confirmed the presence of structural alterations. Antigenicity was evaluated using ELISAs in sera from 40 healthy controls and 40 newly diagnosed T2DM patients, with ROC and AUC analyses determining prognostic relevance. Results: Concentration-dependent increase in AGE-specific fluorescence, increased hydrophobicity, and significant loss of lysine (48.6%) and arginine (58.8%) residues, accompanied by conformational changes. Direct binding ELISA revealed statistically significant reactivity (p ≤ 0.05) with diabetic sera. Competition ELISA demonstrated 55% ± 3.7% inhibition for fructosylated insulin compared to 31.4 ± 5.1% for native insulin. IgGs isolated from diabetic sera exhibited 96.67% specificity for fructosylated insulin, with sensitivity and specificity analyses yielding AUC = 1.000 (p < 0.01). Discussion: Fructosylation causes changes in insulin chemistry, which leads to altered structural, conformational, and antigenic potential in T2DM patients. Conclusion: Screening for anti-fructosylated insulin antibodies may help identify T2DM patients with hyperglycemia-associated, subclinical autoimmune activity linked to abnormal insulin responses.