Study on the development of therapy for insulin-dependent diabetes melitus addressing the regulation of pancreatic beta-cells death.

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Development of gene therapy for IDDM addressing regration of pancreatic beta-cell death. We used mouse pancreatic beta-cell lines, Min-6 and beta-TC as materials. We estimated antioxidant activities, insulin secretion, expression of preproinsulin gene, and analyzed the preproinsulin gene promoter. beta-cell damage was estimated as DNA damage induced by the treatment of these cells with TNF-alpha and IL- 1beta. To regulate the antioxidant activity and redox, we cloned the gene of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the glutathione (GSH) synthesis, and analyzed the 5'-flanking region of gamma-GCS gene. To suppress the expression of gamma-GCS,beta-cells were transfected with antisense codon of gamma-GCS constructed with ribozyme.Results1. Levels of GSH and activities of GSH related enzymes decreased by 90% in Min-6 and beta-TC cells compared to other mouse tissues cells, suggesting beta-cells are feasible to oxidative stress and possess less redox activity.2. The insulin secretion was down-regulated by GSH.3. The expression of preproinsulin was not regulated by GSH.4. GSH protected beta-cell damage by cytokines.5. Transfection of anti-gamma-GCS-ribozyme caused of increase in insulin-secretion.6. These data suggest low concentration of GSH in beta-cells is essential for the insulin secretion while sensitive to cellular damage by oxidants.Collectively, new gene therapy targeting the suppression of GSH synthesis thought to be effective to IDDM.