title: Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion.
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.
authors.affiliation: SAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine BT52 1SA, Northern Ireland, United Kingdom. jt.mccluskey@ulster.ac.uk
authors.forename: Jane T
authors.initials: JT
authors.lastname: McCluskey
authors.forename: Muhajir
authors.initials: M
authors.lastname: Hamid
authors.forename: Hong
authors.initials: H
authors.lastname: Guo-Parke
authors.forename: Neville H
authors.initials: NH
authors.lastname: McClenaghan
authors.forename: Ramon
authors.initials: R
authors.lastname: Gomis
authors.forename: Peter R
authors.initials: PR
authors.lastname: Flatt
mesh_terms: D000328:Adult; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000150:complications; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D051246:Glucose Transport Proteins, Facilitative / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000150:complications / Q000473:pathology / Q000628:therapy; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D054086:KATP Channels / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D021381:Protein Transport / Q000187:drug effects
chemical_list: D051246:Glucose Transport Proteins, Facilitative; D007328:Insulin; D054086:KATP Channels; D005947:Glucose; D002118:Calcium