title: ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimulated insulin secretion.
abstract: As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a normal pancreatic beta cell.
authors.affiliation: Department of Health Sciences, University of Technology, Sydney, Broadway, NSW, Australia.
authors.forename: Guo Jun
authors.initials: GJ
authors.lastname: Liu
authors.forename: Ann M
authors.initials: AM
authors.lastname: Simpson
authors.forename: M Anne
authors.initials: MA
authors.lastname: Swan
authors.forename: Chang
authors.initials: C
authors.lastname: Tao
authors.forename: Bernard E
authors.initials: BE
authors.lastname: Tuch
authors.forename: Russell M
authors.initials: RM
authors.lastname: Crawford
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Jovanovic
authors.forename: Donald K
authors.initials: DK
authors.lastname: Martin
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005947:Glucose / Q000494:pharmacology*; D051273:Glucose Transporter Type 2; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D008099:Liver / Q000378:metabolism*; D018613:Microscopy, Confocal; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D015221:Potassium Channels; D000255:Adenosine Triphosphate; D005947:Glucose