Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Jane T Mccluskey,Muhajir Hamid,Hong Guo-Parke,Neville H Mcclenaghan,Ramon Gomis,Peter R Flatt

doi:10.1074/jbc.m111.226795

Abstract

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Highlights

The restricted supply of viable human islets greatly limits the opportunity for studies of beta cell function as well as restricting the success of worldwide clinical islet transplantation programs
The INS-1 cell line [8], derived from the original radiation tumor, has high insulin content and is able to maintain insulin secretory responsiveness over a prolonged period in culture [6]. Studies of these rodent pancreatic beta cell lines have provided the foundation for many advances in beta cell research, but generation of stable human insulin-producing cell lines would provide a fundamental progression toward testing on human-derived tissue, assisting drug development and possible clinical diabetes therapy
We report here the characteristics and functional properties of three novel human pancreatic beta cell clones produced by electrofusion of human beta cells with human PANC-1 epithelial cells [20]

Summary

Introduction

The restricted supply of viable human islets greatly limits the opportunity for studies of beta cell function as well as restricting the success of worldwide clinical islet transplantation programs. One of these is the glucose-responsive insulin-secreting BRIN-BD11 cell line generated by electrofusion of the RINm5F clone and normal rat beta cells [7]. HIN D8 cloned from a human insulinoma, showed significant insulin secretory response to glucose and other secretagogues, but no studies have since been reported using this cell line [12, 13].

Results

Conclusion