An ideal cell-based therapy for type 1 diabetes would be a source of cells that could secrete abundant amounts of insulin in response to physiological cues but that lacks immunogenicity, thereby avoiding the risk of graft rejection. We have discovered that cells in the intermediate lobe (IL) of the pituitary could be engineered with transgenic mouse techniques to produce sufficient amounts of insulin (ins) to cure diabetes when implanted into diabetic NOD mice but, in contrast to Betacells, the ILins cells evaded immune attack. In initial attempts to introduce glucose-sensing properties into these cells, they were engineered to express high levels of the key glucose sensing components, GLUT2 and glucokinase (GK). Although these maneuvers conferred impressive glucose sensing capabilities, the high-level expression of these genes was also associated with cellular toxicity. More recent work with transgenic mice has underscored the need to closely optimize GK gene expression to ensure cell viability. We have recently developed a line of transgenic mice that express physiological levels of GLUT2 and GK in ILins tissues. In parallel, we have created inducible viral vector systems that will allow us to titrate the expression levels of GK (and GLUT2) in ILins cells. Together, these systems will enable us to determine the optimal gene dosage levels for glucose sensing and glucose-stimulated insulin secretion. However, ILins cells do not express KATP channels or GLP-1 receptors that are critical for coupling glucose metabolism to insulin secretion. The goal of this proposal is to: 1. Engineer expression of the B cell KATP channel complex into ILins cells that express GK and GLUT2; 2. Evaluate the ability these cells to sense and secrete insulin. Determine whether the performance of these cells can be further optimized by altering GK expression. Use novel analytical instrumentation to measure insulin secretory dynamics and metabolism in single cells and in real time. 3. Assess whether the introduction of GLP-1 receptors will improve the ability of lLins cells to sense and respond to glucose, in addition to, and independent of the KATP channel pathway 4. Assess the physiological effect of these manipulations on glucose homeostasis in vivo and evaluate the performance of the bioengineered lLins cells in the setting of autoimmune diabetes. This project is a multidisciplinary collaboration between Dr. Lipes, an established type 1 diabetes researcher and a molecular biologist, Dr. Colin Leech, an expert electrophysiologist, and Dr. Robert Kennedy, an analytical chemist who has pioneered innovative technology for evaluating islet function in single cells and in real time. The result of these studies could contribute importantly to the development of a closed-loop cell based sensor in insulin delivery system for the treatment of diabetes.
