Glucose-induced toxicity in insulin-producing pituitary cells that coexpress GLUT2 and glucokinase - Implications for metabolic engineering

We have shown that intermediate lobe (IL) pituitary cells can be engineered to produce sufficient amounts of insulin (ins) to cure diabetes in nonobes e diabetic mice but, unlike transplanted islets, ILins cells evade immune a ttack. To confer glucose-sensing capabilities into these cells, they were f urther modified with recombinant adenoviruses to express high levels of GLU T2 and the beta -cell isoform of glucokinase (GK). Although expression of G LUT2 alone had negligible effects on glucose usage and lactate production, expression of GK alone resulted in similar to2-fold increase in glycolytic flux within the physiological (3-20 mM) glucose range. GLUT2/GK coexpressio n further increased glycolytic flux at 20 mM glucose but disproportionately increased flux at 3 mM glucose. Despite enhanced glycolytic fluxes, GLUT2/ GK-coexpressing cells showed glucose dose-dependent accumulation of hexose phosphates, depletion of intracellular ATP, and severe apoptotic cell death . These studies demonstrate that glucose-sensing properties can be introduc ed into non-islet cells by the single expression of GK and that glucose res ponsiveness can be augmented by the coexpression of GLUT2. However, in the metabolic engineering of surrogate beta cells, it is critical that the leve ls of the components be closely optimized to ensure their physiological fun ction and to avoid the deleterious consequences of glucose-induced toxicity .