Development of Proinsulin-secreting Non-endocrine Cell

Yoon, Do Jun; Kim, Seok Hyun; Kim, Jae Woo; Kim, Yu Kyong; Song, Young Duk; Ahn, Yong Ho

Abstract

BACKGROUND: Recently, the advent of genetic engineering technics enabled. us to transfer foreign genes of interests into various cells and establish an "artificial b-cells" capable of secreting insulin in response to plasma glucose level. In this study, we have designed a study to establish an "artificial b-cells" by transfecting liver/pancreatic b-cell type glucose transporter 2(GLUT2) cDNA and genomic DNA of proinsulin into non-endocrine cell. Because GLUT2 molecules on the plasma membranes act as a sensor of glucose outside the cell and promote the secretion of proinsulin from the cells, cotransfection of GLUT2 cDNA along with insulin gene will translate the GLUT2 molecules necessary for glucose transport into the cells and hence leading to insulin secretion.
METHODS
We have subcloned GLUT2 cDNA and proinsulin gene into separate eukaryotic expression vectors and transfected them to Chinese hamster ovary cells. The stable cell lines harboring GLUT2 cDNA and proinsulin gene were selected by G418, neomycin analogue. The surviving clones were harvested and subjected to Southem blot analysis by digesting the chromosomal DNA either with BamHI for insulin gene detection or Xho I/Sma I double digestion for GLUT2 gene detection. The amount of proinsulin secretion into the medium was measured by the insulin radioimmunoassay(DPC, Coat-A-Count insulin, LA, USA) which detected proinsulin with 40% cross-reactivity.
RESULTS
1) We were able to find out 3 clones positive for both GLUT2 gene and insulin gene. 2) Of these clones, clone 5 cells secreted proinsulin 3 times as much as that of the control CHO cells.
CONCLUSION
There was some increase of proinsulin secretion in artificial g-cells compared to control cells. But this increased proinsulin secretion was not enough to be used as therapeutics. We need more expriments to find out more efficient way of proinsulin secretion and to identify the glucose-regulated insulin secretion in these artificial b-cells.