Insulin production by engineered muscle cells

Type 1 diabetic patients depend dramatically on insulin replacement therapy , which involves the administration of intermediate- or long-acting insulin , together with short-acting insulin to mimic physiological insulin profile s. However, the delayed-action preparations available are not generally abl e to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stab ly transfected with a chimeric gene obtained by linking the myosin-light ch ain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm), When differentiat ed, C(2)C(12)Insm myotube cells expressed high levels of insulin mRNA and p rotein, whereas no insulin was detected in myoblast cells. HPLC fractionati on of culture medium and cell extracts from differentiated C(2)C(12)Insm ce lls revealed that about 90% of the proinsulin was processed to mature insul in. In addition, these cells released significant levels (about 100 mu U/10 (6) cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the diffe rentiated C(2)C(12)Insm cells and was able to block the expression of the e ndogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepa toma cells. Furthermore, when C(2)C(12)Insm myoblast cells were transplante d into diabetic mice an increase in insulinemia and a decrease in hyperglyc emia were observed. Thus, our results suggest that the use of engineered my otube cells continuously secreting a defined level of insulin might be a us eful approach to improve the efficacy of insulin injection treatment.