Christian Quaade

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.

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]

Analysis of the protein products encoded by variant glucokinase transcripts via expression in bacteria

Five variant transcripts of the single rat glucokinase gene have been described that are naturally expressed in islets of Langerhans, liver and anterior pituitary. Four of these were prepared as cDNA and expressed in bacteria in order to begin to address their physiological roles. Expression of constructs pGKB1 (normal islet/pituitary glucokinase) and pGKL1 (normal liver glucokinase) resulted in glucose‐dependent, glucokinase‐like activity, 7‐fold and 45‐fold, respectively, above background. Expression of pGKB3 (variant islet/pituitary glucokinase) and pGKL2 (variant liver glucokinase) in contrast, did not result in any glucokinase‐like activity.

Molecular Engineering of Glucose-Regulated Insulin Secretion

Rapid advances in the technology of gene transfer into mammalian cells have been made in recent years. These improvements in methodology have allowed investigators to begin to use molecular strategies for testing hypotheses about the role of certain genes in the process of fuel-mediated insulin release (1). To this point, most of the work has focused on genes that regulate the acute insulin secretory response to glucose. Interest in the glucose response is naturally motivated by the fact that its loss is an early event in the etiology of both major forms of diabetes (2,3). In this chapter, recent studies employing molecular approaches for dissecting the relative contributions of glucose transport and glucose phosphorylation in the control of glucose-stimulated insulin release will be reviewed.