SOURCE OF INSULIN SECRETED BY ISLET-LIKE CELL CLUSTERS (ILCCs) DERIVED FROM MURINE EMBRYONIC STEM (mES) CELLS

This study was undertaken to clarify the source of insulin secreted by ILCCs, derived from mES cells through a five-stage process. Although previous published studies demonstrated glucose-responsive insulin secretion by ILCCs, some investigators later disputed that insulin was not synthesized by ILCCs de novo, but rather exogenous insulin supplement in media was sequestered during culture and released upon glucose stimulation. To resolve this issue, exogenous insulin in media for stages 3, 4 and 5 was (1) eliminated or (2) replaced with insulin-like growth factor-I (IGF-I) to test for insulin secretion in a condition where artifactual sequestration of exogenous insulin was not possible. Insulin is known to bind to IGF-I receptors and able to activate the IGF-receptor mediated pathway. Although IGF-I-derived ILCCs showed positive staining for intracellular insulin, they failed to secrete any detectable quantity of insulin by ELISA, upon glucose stimulation. Control ILCCs cultured with neither insulin nor IGF-I did not stain for intracellular insulin. Based on our results and those of others, we propose the following working hypotheses: Insulin or IGF-I receptor-mediated pathways are necessary to initiate insulin synthesis in ILCCs. Both mechanisms of sequestration from media and de novo synthesis are the sources of insulin secreted by ILCCs while insulin absorbed by ILCCs from the media transiently suppresses de novo insulin synthesis; production resumes when exogenous insulin is depleted.