Resistance to insulin (INS)-induced signaling in tissues has been reported and associated with several high prevalence diseases, such as type 2 diabetes mellitus, non-alcoholic fatty liver disease (NAFLD), and Alzheimer’s disease. Currently, several INS analogs, including Glargine and Detemir, have been approved for treating type 2 diabetes. However, the major mode of action of those INS analogs is prolonging the plasma half-life of INS to extend the glycemic effect in the body, rather than exerting stronger INS-mediated signaling to overcome the resistance. So far, there are very few FDA-approved drugs that can overcome INS resistance safely and effectively. Therefore, it is still a great challenge to develop new therapeutics, including novel INS analogs, for the treatment of INS resistance-associated diseases.
Previously, we have introduced an INS prodrug, proinsulin-transferrin (ProINS-Tf) fusion protein, which can be converted into the active form (irINS-Tf) in the liver to achieve blood glucose lowering effect in type 1 diabetes mouse model 1. Besides, the active form of the fusion protein exhibit higher binding affinity and longer retention to the INS receptor compared to the native INS. In this report, we aim to study whether the stronger binding and longer retention to the INS receptor observed in irINS-Tf could induce an enhanced INS downstream signaling. In addition, we also investigate the potential use of irINS-Tf in overcoming the INS resistance in cultured HepG2 cells, which could be further exploited to treat INS resistance-associated diseases.
Preparation of H4IIE-mediated activation of ProINS-Tf: The production and purification of ProINS-Tf fusion was previously described 1. Serum-free DMEM containing 10 nM of ProINS-Tf was incubated with H4IIE cells to produce the activated form, irINS-Tf. The amount of irINS-Tf in the conditioned medium was quantified using INS-specific radioimmunoassay (RIA) and was estimated as 1 nM (10% conversion) after 24 h incubation with H4IIE cells.
Induction of insulin resistance by palmitate in HepG2 cells: The bovine serum albumin (BSA)-palmitate complex stock was prepared by mixing 8% BSA and 8 mM palmitate with 1:1 ratio and stirred for 1 h at 37. To induce INS resistance in HepG2 cells, cells were treated with 0.25 % BSA/0.25 mM palmitate solution in serum-free DMEM for 16-18 h.
Akt phosphorylation assay in INS resistant cells: Normal or INS resistant HepG2 cells were exposed to INS (10 nM) or irINS-Tf (~ 1 nM, converted from 10 nM of ProINS-Tf) for 10 min to compare their ability in activating INS downstream signaling by detecting the phosphorylation level of Akt on Ser473. For time course study, INS resistant HepG2 cells were treated with INS (1 nM or 10 nM) or irINS-Tf (~ 1 nM) from 10 min to 8 h, and the Akt phosphorylation level were measured in the cell extract with anti-pAkt Western blot.