Engineering of a glucose-responsive surrogate cell for insulin replacementtherapy of experimental insulin-dependent diabetes

Glucose responsiveness in the millimolar concentration range is a crucial r equirement of a surrogate pancreatic beta cell for insulin replacement ther apy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones w ith millimolar glucose responsiveness were generated from an early-passage glucose-unresponsive RINm5F cell line. This line expressed constitutively b oth the K-ATP channel and the GLUT2 glucose transporter; but it had a relat ive lack of glucokinase, Through overexpression of glucokinase, however, it was possible to generate glucose-responsive clones with a glucokinase-to-h exokinase ratio comparable to that of a normal pancreatic beta cell. This a im, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transpor ter into the range of rat liver, without correcting the glucokinase-to-hexo kinase enzyme ratio, did not render the cells glucose responsive. These glu cokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into str eptozotocin diabetic immunodeficient rats, glucokinase-overexpressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo conditions, These cells represent a notable step toward the future bioengineering of a surrogate beta cell for insulin replacement ther apy in insulin-dependent diabetes mellitus.