Samuel A. Clark

Novel Insulinoma Cell Lines Produced by Iterative Engineering of GLUT2, Glucokinase, and Human Insulin Expression

Cellular engineering studies in our group are directed at creating insulin-secreting cell lines that simulate the performance of the normal islet β-cell. The strategy described in this article involves the stepwise stable introduction of genes relevant to β-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term “iterative engineering.” RIN cells stably engineered to contain multiple copies of the human insulin gene exhibit a large increase in insulin content, such that they approach the content of human islets assayed in parallel. Analysis by high-performance liquid chromatography demonstrates that these engineered cell lines process human proinsulin to mature insulin with high efficiency. Cell lines that are further engineered to express the GLUT2 and glucokinase genes demonstrate stable expression of the three transgenes for the full lifetime of the lines produced to date (6 months to 1 year in continuous culture). Transplantation of the engineered cell lines into nude rats reveals that stably integrated genes are expressed at constant levels in the in vivo environment over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal β-cell, including rat insulin, amylin, sulfonyhirea receptor, and glucokinase, are stably expressed in the insulinoma lines during these in vivo studies. Endogenous GLUT2 expression, in contrast, is rapidly extinguished during in vivo passage. The loss of GLUT2 is overcome in engineered cell lines in which transporter expression is provided by a stably transfected trans gene. These results suggest that a potential advantage of the iterative engineering approach may be to preserve stability of function and phenotype, particularly in the in vivo setting.

Regulation of Insulin Secretion From Novel Engineered Insulinoma Cell Lines

In the accompanying article, we describe the creation of novel cell lines derived from RIN 1046-38 rat insulinoma cells by stable transfection with combinations of genes encoding human insulin, GLUT2, and glucokinase. Herein we describe the regulation of insulin secretion and glucose metabolism in these new cell lines. A cell line (βG I/17) expressing only the human proinsulin transgene exhibits a clear increase in basal insulin production (measured in the absence of secretagogues) relative to parental RIN 1046-38 cells. βG I/17 cells engineered for high levels of GLUT2 expression and a twofold increase in glucokinase activity ([βG 49/206) or engineered for a 10-fold increase in glucokinase activity alone (βG 40/110) exhibit a 66% and 80% suppression in basal insulin secretion relative to βG I/17 cells, respectively. As a result, βG 49/206 and βG 40/110 cells exhibit potent insulin-secretory responses to glucose alone (6.1- and 7.6-fold, respectively) or to glucose plus isobutylmethylxanthine (10.8- and 15.1-fold, respectively) that are clearly larger than the corresponding responses of βG I/17 or parental RIN 1046-38 cells. βG 49/206 and βG 40/110 cells also exhibit a rapid and sustained response to glucose plus isobutylmethylxanthine in perifusion studies that is clearly larger in magnitude than that of the two control lines. Glucose dose-response studies show that both engineered and non-engineered lines respond maximally to submillimolar concentrations of glucose and that βG 49/206 cells are the most sensitive to low concentrations of the hexose, consistent with their clearly elevated rate of ]5-3H]glucose usage. Finally, 5-thioglucose, a potent inhibitor of low-Km hexokinases, most effectively normalizes glucose concentration dependence for insulin secretion in the cell line with highest glucokinase expression (βG 40/110). We conclude that GLUT2 and/or glucokinase expression imposes tight regulation of basal insulin secretion in cell lines that overexpress human proinsulin, allowing a marked improvement in the range of secretagogue responsiveness in such cells.

Engineered cell lines for insulin replacement in diabetes: current status and future prospects

Summary The recently completed diabetes complications and control trial has highlighted the need for improvement of insulin delivery systems for treatment of insulin-dependent diabetes mellitus. Despite steady improvement in methods for islet and whole pancreas transplantation over the past three decades, the broad-scale applicability of these approaches remains uncertain due in part to the difficulty and expense associated with procurement of functional tissue. To address this concern, we and others have been using the tools of molecular biology to develop cell lines with regulated insulin secretion that might serve as a surrogate for primary islets or pancreas tissue in transplantation therapy. This article seeks to provide a brief summary of the current status of this growing field, with a particular emphasis on progress in producing cell lines with appropriate glucose-stimulated insulin secretion. [Diabetologia (1997) 40: S 42–S 47]