title: Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose.
abstract: We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.
authors.affiliation: Department of Metabolic Medicine, Kumamoto University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Motoyoshi
authors.forename: T
authors.initials: T
authors.lastname: Shirotani
authors.forename: E
authors.initials: E
authors.lastname: Araki
authors.forename: K
authors.initials: K
authors.lastname: Sakai
authors.forename: K
authors.initials: K
authors.lastname: Kaneko
authors.forename: H
authors.initials: H
authors.lastname: Motoshima
authors.forename: K
authors.initials: K
authors.lastname: Yoshizato
authors.forename: A
authors.initials: A
authors.lastname: Shirakami
authors.forename: H
authors.initials: H
authors.lastname: Kishikawa
authors.forename: M
authors.initials: M
authors.lastname: Shichiri
mesh_terms: D000236:Adenoma / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D015870:Gene Expression; D005941:Glucokinase / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology; D051273:Glucose Transporter Type 2; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D007004:Hypoglycemic Agents; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009543:Nifedipine / Q000494:pharmacology; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002121:Calcium Channel Blockers; D051273:Glucose Transporter Type 2; D007004:Hypoglycemic Agents; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D011189:Potassium Chloride; D003346:Corticotropin-Releasing Hormone; D005941:Glucokinase; D009543:Nifedipine; D005947:Glucose; D003981:Diazoxide; D005905:Glyburide