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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-0051fje. |
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,2
* Department of Health Sciences, University of Technology Sydney, Broadway, N.S.W. 2007, Australia;
Department of Cell and Molecular Biology, University of Technology Sydney, Broadway, N.S.W. 2007, Australia;
Department of Anatomy and Histology and Institute for Biomedical Research, University of Sydney, N.S.W. 2006, Australia;
Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Randwick, NSW, 2031;
|| Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK; and
Institute for Nanoscale Technology, University of Technology Sydney
2Correspondence: Department of Health Sciences, University of Technology, Sydney, P.O. Box 123, Broadway, N.S.W. 2007 Australia. E-mail: donm{at}uts.edu.au
SPECIFIC AIMS
As part of our research into the liver-directed gene therapy of type I diabetes, we have engineered a human hepatoma cell line (HEP G2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to confirm our hypothesis that HEP G2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP) using the techniques of patch-clamp electrophysiology, immunoprecipitation and Western blot, confocal laser scanning microscopy, and biochemistry.
PRINCIPAL FINDINGS
1. Patch-clamp electrophysiology reveals expression of functional KATP channels
Previous studies of ours have shown that the insertion of insulin cDNA into a human hepatoma cell line (HEP G2) that lacked native GLUT 2 expression to produce the cell line HEP G2ins resulted in synthesis, storage, and release of insulin to ß cell secretagogues, but not to glucose. A second insertion of the glucose transporter GLUT 2 resulted in near physiological release of insulin to glucose and other stimuli in the doubly transfected HEP G2ins/g cells. In this study we used patch-clamp electrophysiology to characterize KATP and identify pharmacological inhibitors and activators of KATP.
The membrane potential of the HEP G2ins/g cells, in which cDNA for insulin and GLUT 2 genes had been inserted, displayed greater membrane potentials (–68.9±7.1 mV, F=17.933, 
=0.05) than either the untransfected HEP G2 cells (–18.2±5.8 mV) or the HEP G2 cells transfected with empty vectors alone. The HEP G2ins cells had only the insulin gene inserted and had a membrane potential (–45.3±4.4 mV) between that of the HEP G2 and HEP G2ins/g cells. We also recorded macroscopic ionic currents from the HEP G2 and HEP G2ins/g cells. The currents in the HEP G2ins/g cells were larger than the parent HEP G2 cells. The I-V curve for a group of HEP G2ins/g cells indicated that this current was K+ selective, since the reversal potential (balance of electrical and concentration potential) (ER
–50 mV) for the currents approached the Nernst potential for potassium. The currents in the HEP G2 cells appeared to be nonselective, since the ER was closer to 0 mV. We found no differences in the macroscopic ionic currents between the HEP G2 and HEP G2 cells transfected with empty vectors alone.
The sensitivity of these potassium channels to ATP was measured using single-channel recordings from inside-out patches taken from the HEP G2ins/g cells. ATP (1 mM) inhibited the potassium channels by reducing the frequency of the channel openings, as shown by a reduced number of upward reflections in the recordings (Fig. 1
a). We also found that cAMP (50 µM) inhibited potassium channel activity when applied to the cytosolic face of inside-out membrane patches detached from the HEP G2ins/g cell.
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The sensitivity of the K channels to high glucose concentrations was measured using whole-cell patch clamping of the HEP G2ins/g cells (Fig. 1b
). Glucose (20 mM) added to the superfusing solution inhibited the ATP-sensitive potassium currents. If the channels had a role in stimulating insulin secretion, extracellular application of glucose (20 mM) would be expected to inhibit the channels.
We used single-channel techniques (outside-out patches), which demonstrated that glibenclamide (20 µM) inhibited the ATP-sensitive potassium channels and that diazoxide (150 µM) activated the channels.
2. Western blot and biochemical and confocal scanning laser microscopy (CSLM) studies confirm the presence of functional KATP channels
Western blot with the specific anti-Kir6.2 antibody revealed signals in MIN 6 (positive control), HEPG2ins/g, and HEPG2ins cells at the expected size of 
35 kDa. The intensity of the signal is much higher in MIN6 and HEPG2ins/g cells than in HEPG2ins cells, with little if any expression in HEPG2 cells.
Glucose-stimulated insulin release in HEP G2ins/g cells was examined by perfusing the cells with glucose and the KATP activator diazoxide. HEP G2ins/g cells were grown on cytodex beads and cultured in spinner flasks in normal culture medium for 4 days. Beads with cells (108) attached were transferred to a column, connected to a peristaltic pump and fraction collector, and basal medium (phosphate-buffered saline containing 1 mM CaCl2 and supplemented with 20 mM HEPES, 2 mg/mL BSA, 1.0 mM glucose) was pumped through the system for 30 min. This was followed by 15 min exposures to 20 mM glucose ± 150 µM diazoxide. As seen in Fig. 2
, opening the ATP-sensitive potassium channels with diazoxide reversed the stimulating effects of glucose on insulin secretion. By comparison, in separate experiments the channel inhibitors tolbutamide (100 µM) and glibenclamide (20 µM) were seen to significantly (P<0.001) stimulate insulin secretion similar to what is seen upon glucose (20 mM) stimulation.
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We confirmed these results using CSLM and the fluorescent probe for Zn2+ Zinquin-E (ethyl [2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate), an intracellular zinc-specific fluorophore concentrated in insulin-storing secretion granules due to their relatively high content of labile zinc. A fresh preparation of Zinquin-E was prepared before each experiment by diluting the stock in Dulbecco's modification of Eagle's medium to 25 µM. Cells were incubated in Zinquin-E for 30 min at 37°C. Excess was washed out with three changes of DMEM medium containing 5 mM glucose before imaging with an inverted microscope using a 100x1.4 NA UV-corrected Planapo oil immersion objective, UV illumination, BP 490/440 and a Leica TCSNT confocal laser scanning microscope (CLSM). We visualized secretion granules in live cells as Zinquin-E labeled punctuate fluorescence and were able to follow the effect of glucose and channel openers and closers on insulin secretion of HEP G2ins/g cells. Zinquin-E staining was lost (secretion occurred) upon addition of either 20 mM glucose alone or 20 µM glibenclamide alone. Conversely, adding 150 µM diazoxide blocked secretion; even subsequent washing with 20 mM glucose did not cause insulin-containing granules to be released from Hep G2ins/g cells). Taken together, these data indicate that depolarizing the HEP G2ins/g cells by inhibition of the KATP channels, either by glucose or directly with glibenclamide, stimulated secretion from insulin-containing granules.
In the HEP G2ins/g cells, both glucose 20 mM and glibenclamide 100 µM stimulated an increase in intracellular Ca2+, measured using fluo3 and CLSM. Glucose 20 mM failed to stimulate an increase in intracellular Ca2+ in the untransfected HEP G2 cells.
CONCLUSIONS
The results presented in the present study confirm that the HEP G2ins/g liver cell line that has been engineered to store and secrete insulin to glucose does so via signaling pathways similar to those seen in a normal pancreatic ß cell. Although ATP-sensitive potassium channels are found in the parent cell line, HEP G2, the opening of these channels in resting HEP G2 cells requires pharmacological stimulation with agents such as pinacidil. The novelty of the HEP G2ins/g cells is that dual expression of the glucose transporter GLUT 2 and the insulin gene has induced functional KATP channels in the resting cells. We report that the HEP G2ins/g cells have a resting membrane potential similar to that of pancreatic ß cells, which is in contrast to the relatively depolarized potential of the parent HEP G2 liver cell line. Moreover, the HEP G2ins/g cells developed a macroscopic K+-selective current that was inhibited by glucose (20 mM) and glibenclamide (20 µM) and activated by diazoxide (150 µM). These results were further strengthened by a biochemical and CSLM study of the cells, which revealed that insulin secretion from the secretory granules was increased by exposure to the channel inhibitors tolubutamide and glibenclamide and blocked by the channel activator diazoxide. Thus the single-channels underlying the macroscopic K+-current had the characteristics of ATP-sensitive channels reported earlier in pancreatic ß cells.
While the precise signaling pathway for acute-stimulated insulin secretion needs to be elucidated further, the most likely scenario is that the expression of functional KATP channels in the resting HEP G2ins/g cells provides a mechanism to explain the rapid triggering of insulin secretion that we recorded in response to stimulation of the cells by glucose (Fig. 3
). Glucose in the extracellular environment is sensed and transported across the membrane by the facilitative glucose transporter GLUT 2 and converted to glucose-6-phosphate by glucokinase; there is a rise in the ATP:ADP ratio leading to the closure of ATP-sensitive KATP channels (described in this study) that leads to cell depolarization, influx of extracellular Ca2+, induced rise in [Ca2+]I from intracellular stores, exocytosis, and secretion of insulin. It seems likely that as in a normal pancreatic ß cell, this increase in Ca2+ would have a wide range of effects such as the activation of glycerol phosphate dehydrogenase and other mitochondrial enzymes and activation of protein kinases and phospholipase C; however, such intracellular events further down the cascade of insulin secretion are yet to be confirmed in the HEP G2ins/g cells.
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The subunits that comprise the KATP channel in pancreatic ß cells are SUR1 and KIR 6.2; the subunits comprising the KATP channel in the parent HEP G2 cells were reported to be SUR2 and Kir 6.2, whose mRNA expression was unaltered by glibenclamide. Our results indicate that following overexpression of the insulin and GLUT 2 cDNA, the HEP G2ins/g cells express channels that are blocked by both tolbutamide and glibenclamide and activated by diazoxide. This pharmacology suggests that the expression of the insulin and GLUT 2 genes in these cells possibly modified the SUR2 subunit of the existing channels to enable the KATP channels to be functional. Recent evidence that may support this hypothesis is that the sensitivity of the KATP channel to nicorandil (channel opener) and sulfonylureas can depend on sequence variations in the COOH-terminal group of certain transmembrane loops of SUR2 and the last 42 amino acids of SUR2. While the mechanism remains to be completely elucidated, the results from this study are encouraging with regard to the possibility of engineering liver cells to mimic the physiology of pancreatic ß cells and the eventual use for treatment of type I diabetes.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0051fje; doi: 10.1096/fj.02-0051fje 
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