Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain α-helix: one neonatal in DM onset (ValB18→Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated α-helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding (ΔΔGu 1.2(±0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

Insulin Fused to Apolipoprotein A-I Reduces Body Weight and Steatosis in DB/DB Mice

Background: Targeting long-lasting insulins to the liver may improve metabolic alterations that are not corrected with current insulin replacement therapies. However, insulin is only able to promote lipogenesis but not to block gluconeogenesis in the insulin-resistant liver, exacerbating liver steatosis associated with diabetes.Methods: In order to overcome this limitation, we fused a single-chain insulin to apolipoprotein A-I, and we evaluated the pharmacokinetics and pharmacodynamics of this novel fusion protein in wild type mice and in db/db mice using both recombinant proteins and recombinant adenoassociated virus (AAV).Results: Here, we report that the fusion protein between single-chain insulin and apolipoprotein A-I prolonged the insulin half-life in circulation, and accumulated in the liver. We analyzed the long-term effect of these insulin fused to apolipoprotein A-I or insulin fused to albumin using AAVs in the db/db mouse model of diabetes, obesity, and liver steatosis. While AAV encoding insulin fused to albumin exacerbated liver steatosis in several mice, AAV encoding insulin fused to apolipoprotein A-I reduced liver steatosis. These results were confirmed upon daily subcutaneous administration of the recombinant insulin-apolipoprotein A-I fusion protein for six weeks. The reduced liver steatosis was associated with reduced body weight in mice treated with insulin fused to apolipoprotein A-I. Recombinant apolipoprotein A-I alone significantly reduces body weight and liver weight, indicating that the apolipoprotein A-I moiety is the main driver of these effects.Conclusion: The fusion protein of insulin and apolipoprotein A-I could be a promising insulin derivative for the treatment of diabetic patients with associated fatty liver disease.