Analysis of human insulin promoter activity in primary human endocrine pancreatic beta-cells

Insulin biosynthesis in pancreatic beta-cells is regulated at the transcriptional level by the activity of the insulin promoter. Due to the lack of human pancreatic beta-cell lines, detailed analysis of the regulation of the human insulin promoter was restricted in the past to heterologous rat, mouse and hamster pancreatic beta-cell lines, although it is known, that substantial differences exist in transcriptional regulation of genes in different species. Here we describe the development of a method for specific investigation of the regulation of the human insulin promoter in primary human pancreatic beta cells in isolated pancreatic islets from human donor organs. Adenoviral vectors were constructed, that express secreted alkaline phosphatase (SEAP) as a reporter gene under the control of -336 base pairs of the human insulin promoter in transcriptional connection with an expression cassette for the luciferase gene under the control of the CMV promoter in a single vector. These vectors allow transduction of primary human pancreatic beta-cells. 72 hours after transduction SEAP activity in the culture supernatant is monitored by a fluorescent assay and normalized to constitutively expressed luciferase activity. In preliminary experiments were were able to demonstrate glucose dependent activation of the human insulin promoter in primary human pancreatic beta-cells, when the glucose concentration was raised from 2,5 mM to 11,1 mM. Further analysis using 5’-deletion mutants and block mutations within key transcriptional response elements for PDX-1, BETA2 and CREB will yield detailed insights into glucose and hormone-dependent regulation of the human insulin promoter in primary human pancreatic beta-cells. In conclusion we have developed a method to analyze for the first time regulation of human insulin promoter activity in primary human pancreatic beta-cells. The results of these experiments will help to elucidate the molecular mechanisms of insulin biosynthesis in the human endocrine pancreas in physiology and diabetes mellitus.