Dose optimization and evaluation of nanoparticle-mediated E4orf1 delivery for improving hyperglycemia

Background: Transgenic or vector-mediated expression of the adenoviral gene E4orf1 (E4) improves glycemic control in a high-fat diet (HFD)-induced mouse model of type 2 diabetes. The attractive feature of E4 is improving glycemic control in response to glucose bolus, even in the presence of HFD and independent of insulin. However, a clinically applicable delivery system is needed for the therapeutic application of E4 in humans. In response, we created nanoparticles (NP) that delivered E4 to 3T3-L1 preadipocytes and promoted cellular glucose uptake. As a next step, the present study determined if subcutaneous (s.c.) administration of E4 NP could improve glucose disposal in HFD-fed mice and if E4 NP increase glucose uptake in hepatocytes and myocytes. We also determined the effect of E4 NP on cell viability in vitro. Methods: C2C12 and HepG2 were treated with E4 NP or empty NP (void NP), and seventy-two hours later, glucose uptake assay was performed. To check the toxicity of E4 NP, 3T3-L1 cells were treated with E4 NP, and seventy-two hours later, the MTT assay was performed. In the in vivo experiment, HFD-fed C57BL/6J mice received s.c. injections of either void or E4 NP. The glucose tolerance test was performed, and blood glucose and serum insulin were measured. Results: In C2C12 and HepG2, the glucose uptake significantly increased in the E4 group compared to the void group, and seventy-two hours treatment with E4 NP was not toxic to the cells. In mice, s.c. injection of E4 NP significantly improved glucose disposal and glucose area under the curve, thereby enhancing overall glycemic control. Conclusions: We report the successful NP-mediated delivery of E4 in-vivo and the predicted effect of E4 on glucose uptake in-vitro. Increased glucose uptake in pre-adipocytes, myocytes, and hepatocytes without having any toxic effect is crucial since adipose tissue, skeletal muscle, and liver are major influencers of glycemic control. Moreover, we show that s.c. injection of E4 NP, a clinically relevant delivery method, can improve glycemic control even in presence of HFD. Thus, this study provides the first proof of concept towards initiating the studies for its clinical use of NP-based E4 delivery system in treating T1D or T2D.

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