Enhanced protection of Ins-1 beta cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4.

In this study, we developed an expression system of exendin-4, a glucagon-like peptide (GLP-1) analog, using a secretion signal peptide (SP) to facilitate exendin-4 secretion. For delivery of the exendin-4 expression system, high-molecular-weight polyethylenimine (25 kDa, PEI25k), low-molecular-weight polyethylenimine (2 kDa, PEI2k), and polyamidoamine (PAMAM) dendrimers were evaluated as gene carriers to Ins-1 beta cells. As a result, PEI25k showed the highest transfection efficiency. For the construction of the exendin-4 expression vector, DNA coding the SP sequence was inserted upstream of the exendin-4 cDNA, resulting in the construction of pbeta-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the pbeta-SP-Ex-4 transfected cells, compared with the pbeta-Ex-4 transfected cells. To identify the beta-cell protection effect of pbeta-SP-Ex-4 delivery, the Ins-1 beta cells were transfected with pbeta-SP-Ex-4 or pbeta-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the pbeta-SP-Ex-4 transfected cells had higher beta-cell viability than the pbeta-Ex-4 transfected cells under hypoxia. In addition, the pbeta-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the pbeta-Ex-4 transfected cells. Therefore, PEI25k/pbeta-SP-Ex-4 complex may be useful to protect isolated beta cells from apoptosis during transplantation.