Metformin Protects Human Insulin from Fructosylation: An in Vitro Biochemical Study.

Introduction: Fructose, like other sugars and sugar metabolites, is capable of glycating protein. Insulin's fructosylation leads to the generation of Advanced Glycation End Products (AGEs). Reducing sugars reaction with proteins to form Schiff's bases, which are characterized by the presence of an imine (C=N) bond. The Schiff bases then undergo irreversible rearrangements, followed by the production of much more stable compounds called Amadori products. These Amadori products can further undergo oxidation, dehydration, cyclization, and condensation to form highly toxic advanced glycation end-products (AGEs). These processes are accompanied by oxidative stress, secondary structural perturbations, and altered morphology, progressing toward amyloidogenesis. Metformin, a biguanide, is the most common drug used to treat type 2 Diabetes Mellitus (T2DM).

Aim: The aim of this study was to evaluate the protective effect of metformin against fructosylation-induced cross-β structures and amyloid aggregations of human insulin.

Methods: UV-absorbance and fluorescence spectroscopy, determination of carbonyl content, free lysine and arginine residues, determination of fructosamine content, SDS-PAGE, circular dichroism (CD) spectroscopy, dynamic light scattering, and scanning and transmission electron microscopy.

Results: Physicochemical studies in the presence or absence of metformin revealed a concentration-dependent structural restoration of fructosylated insulin. Results from the thioflavin-T fluorescence assay suggested that metformin limited the transition of insulin from native to fibrillar state, which was validated by scanning and transmission electron microscopy. Metformin lowered the ThT fluorescence intensity in a concentration- dependent manner. The ThT-specific fluorescence intensity was reduced to 114 and 112.5%. The fluorescence intensity at 2.5 mM metformin was close to native insulin. Electron microscopy revealed that insulin fructosylated by 25 mM fructose in the presence of 2.5 mM metformin suppressed the formation of fibrillar structures. Dynamic light scattering data revealed the potential of metformin to conserve and reinstate the increased hydrodynamic radii (Rh) of fructosylated insulin close to the native conformer. The Rh values of native, fructosylated insulin and insulin incubated with fructose and metformin were found to be 2.65 ± 0.28, 307.6 ± 24.19 nm, and 110.1 ± 4.08 nm, respectively. This study also identified metformin as an antioxidant by protecting critical amino acid residues of the insulin domain.

Discussion: The study reports the protective effects of metformin on insulin structure, conformation, and function. The findings suggest a potential role for metformin in improving the risk profile associated with insulin resistance due to altered structure or the accumulation of protein aggregates. Interaction studies between insulin and metformin presented here are due to the chemical effect; hence, further in-depth studies are required to identify the molecular mechanism of insulin sensitivity and changes in cellular processes and pathways.

Conclusion: The results suggest that metformin safeguards against fructosylation-induced structural, conformational, morphological, and amyloidogenic aggregating tendencies of insulin. Protein aggregation has been linked to several neurological and metabolic diseases. Hence, metformin may be crucial in preserving the biological activity of insulin by maintaining and protecting its structural integrity and minimizing the associated comorbidities. The study may further be extended to identify the role of metformin in controlling the gradual insulin resistance in T2DM at the molecular level.