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ATP‐sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT2 transporter regulate glucose‐stimulated insulin secretion
Author(s) -
Liu Guo Jun,
Simpson Ann M.,
Swan M. Anne,
Tao Chang,
Tuch Bernard E.,
Crawford Russell M.,
Jovanovic Aleksandar,
Martin Donald K.
Publication year - 2003
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.02-0051fje
Subject(s) - diazoxide , glibenclamide , glut2 , tolbutamide , insulin , medicine , potassium channel , endocrinology , kir6.2 , glucose transporter , sulfonylurea receptor , intracellular , transfection , chemistry , biology , cell culture , microbiology and biotechnology , biochemistry , protein subunit , diabetes mellitus , genetics , gene
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 K ATP was 61pS. K ATP was inhibited by ATP (1 mM) or cAMP (50 µM) applied to the cytosolic side of the membrane. Single K ATP channels and macroscopic whole‐cell currents were inhibited by glucose (20 mM) and glibenclamide (20 µM) and were activated by diazoxide (150 µM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 K atp channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 µM) 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 µM) from 0.4 ± 0.06 to 2.1 ± 0.3 ( n =4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 µM) completely inhibited glucose‐stimulated insulin release. Glucose 20 mM and glibenclamide 100 µM increased intracellular Ca 2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca 2+ 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 µM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 µM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on K atp , similar to a normal pancreatic β cell.