Glibenclamide-induced apoptosis is specifically enhanced ...jpet.aspetjournals.org/content/jpet/early/2005/11/23/jpet.105.097501... · glibenclamide on recombinant HEK293 cells expressing

JPET#97501 1

Glibenclamide-induced apoptosis is specifically enhanced by expression of

the sulfonylurea receptor isoform SUR1 but not by expression of SUR2B or

the mutant SUR1(M1289T)

Annette Hambrock, Claudia Bernardo de Oliveira Franz, Sabrina Hiller, and Hartmut

Osswald

Department of Pharmacology and Toxicology,

Medical Faculty, University of Tübingen,

Wilhelmstraße 56, D-72074 Tübingen, Germany (A.H., C.B.F., S.H., H.O.)

JPET Fast Forward. Published on November 23, 2005 as DOI:10.1124/jpet.105.097501

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on November 23, 2005 as DOI: 10.1124/jpet.105.097501

at ASPE

T Journals on M

ay 15, 2020jpet.aspetjournals.org

Dow

nloaded from

http://jpet.aspetjournals.org/

JPET#97501 2

Running title:

Expression of SUR1 enhances glibenclamide-induced apoptosis

Author for correspondence:

Annette Hambrock

Department of Pharmacology and Toxicology,

Medical Faculty, University of Tübingen, Wilhelmstraße 56, D-72074 Tübingen, Germany

telephone number: ++49-7071-29 78261

fax number: ++49-7071-29 4942

e-mail address: [email protected]

Number of text pages: 29

Number of tables: 0

Number of figures: 5

Number of references: 40

Number of words (Abstract): 250

Number of words (Introduction): 724

Number of words (Discussion): 1494

Abbreviations:

ABC-protein, ATP binding cassette protein; AFC, 7-amino-4-trifluoromethyl coumarin;

CFTR, cystic fibrosis transmembrane conductance regulator; HEK cells, human embryonic

kidney cells; KATP channel, ATP-sensitive potassium channel; Kir, inwardly rectifying K+

channel; NBF, nucleotide binding fold; PBS, phosphate buffered saline; SUR, sulfonylurea

receptor; TM, transmembrane helix

Recommended section assignment:

Cellular and molecular


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 3

Abstract

Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive K+

channel (KATP channel) which is essential for triggering insulin secretion via membrane

depolarization. Sulfonylureas such as glibenclamide and tolbutamide act as KATP channel

blockers and are widely used in diabetes treatment. These antidiabetic substances are known

to induce apoptosis in pancreatic β-cells or β-cell lines under certain conditions. The precise

molecular mechanisms of this sulfonylurea-induced apoptosis, however, are still unidentified.

In order to investigate the role of SUR in apoptosis induction, we tested the effect of

glibenclamide on recombinant HEK293 cells expressing either SUR1, the smooth muscular

isoform SUR2B, or the mutant SUR1(M1289T) at which a single amino acid in

transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2.

By analyzing cell detachment, nuclear condensation, DNA fragmentation and caspase-3-like

activity, we observed a SUR1-specific enhancement of glibenclamide-induced apoptosis that

was not seen in SUR2B-, SUR1(M1289T)-, or control cells. Coexpression with the pore-

forming Kir6.2 subunit did not significantly alter the apoptotic effect of glibenclamide on

SUR1-cells. In conclusion, expression of SUR1, but not of SUR2B or SUR1(M1289T),

renders cells more susceptible to glibenclamide-induced apoptosis. SUR1 as a pancreatic

protein could therefore be involved in specific variation of β-cell mass and might also

contribute to regulation of insulin secretion at this level. According to our results, TM17 is

essentially involved in SUR1-mediated apoptosis. This effect does not require the presence of

functional Kir6.2-containing KATP channels, which points to additional, so far unknown

functions of SUR.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 4

Introduction

Sulfonylurea receptors (SURs) are members of the ABC- (ATP-binding cassette) protein

family and form important regulatory subunits of ATP-sensitive K+ channels (KATP channels).

These channels are heterotetrameric complexes formed by SUR and the pore-forming Kir6.x

subunit. Different combinations of these subunits (SUR1, SUR2A, or SUR2B and Kir6.1 or

Kir6.2) form channels in various tissues with distinct pharmacological and

electrophysiological properties. SUR2 is encoded by a different gene than SUR1. Alternative

splicing of the SUR2 gene leads to expression of either SUR2A, predominantly found in heart

and skeletal muscle, or SUR2B, typically occurring in smooth vascular muscle (Gribble and

Reimann, 2003). To some extent, SUR1 and SUR2 show inverse pharmacological profiles:

SUR1 exhibits high affinity for several sulfonylureas but low affinity for most KATP channel

openers, whereas SUR2 shows lower affinity for sulfonylureas and high affinity for openers

(Schwanstecher et al., 1998; Hambrock et al., 2002).

In the pancreatic β-cell, KATP channels (constituted by SUR1 and Kir6.2) are essential

for triggering insulin secretion via membrane depolarisation. High blood glucose

concentrations lead to an elevated ATP/ADP ratio and result in closure of KATP channels,

opening of voltage-dependent Ca2+ channels and release of insulin-containing vesicles

(Ashcroft, 2000; Gribble and Reimann, 2003; Bryan et al., 2004). Several sulfonylureas and

glinides act as synthetic KATP channel blockers and are widely used in the treatment of

diabetes type 2 due to their insulinotropic effects (Doyle and Egan, 2003; Henquin et al.,

2004).

Insulin secretion is also modulated and amplified by additional pathways that are not

completely understood so far. KATP channel-independent mechanisms involve e.g. elevated

Ca2+ concentrations, regulation via incretin hormones or certain neurotransmitters or

activation of the adenylyl cyclase second messenger system or of other kinases. There is also


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 5

some evidence for a special role of SUR in controlling cAMP-dependent insulin secretion

without necessarily involving the Kir6.2 subunit (Nakazaki et al., 2002; Eliasson et al., 2003;

Shibazaki et al., 2004). These results are in contrast to other reports that doubt such a function

of SUR (Nenquin et al., 2004).

According to recent studies, one further mechanism regulating the extent of insulin

secretion on a different level is based on adaptive variation of β-cell mass by changes in cell

proliferation or apoptosis. For example, an increasing demand for insulin can be compensated

by a rise in β-cell mass in obese humans while β-cell mass in patients suffering from diabetes

(type 1 or 2) is often reduced in the course of the disease. The precise molecular mechanisms

underlying β-cell specific apoptosis, however, are still unknown (Efanova et al., 1998;

Mandrup-Poulsen, 2001; Donath and Halban, 2004; Ahren, 2005). In addition to glucotoxic

effects, it is known that sulfonylureas such as glibenclamide or tolbutamide are able to induce

apoptosis in β-cells or respective cell lines (Efanova et al., 1998; Iwakura et al., 2000;

Rustenbeck et al., 2004; Maedler et al., 2004, 2005) under certain conditions. Therefore, some

concern is expressed about long-term treatment with sulfonylureas (Mandrup-Poulsen, 2001;

Donath and Halban, 2004; Maedler et al., 2005) although a mechanistic explanation of the

apoptotic effect of these drugs is lacking so far.

The aim of this study was to evaluate whether SUR is involved in the induction of

apoptotic processes by sulfonylurea compounds. As a model system, we employed

recombinant HEK293 cells which are widely used in the study of KATP channels or their

subunits. HEK293 cells expressing either the SUR1 or the SUR2B isoform were analyzed

after glibenclamide-treatment. Cells expressing the mutant SUR1(M1289T) at which a single

amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino

acid of SUR2, were also investigated. It had been shown previously that a mutation at this

position markedly influences some pharmacological properties of SUR (Moreau et al., 2000;

Hambrock et al., 2004). For HEK293 cells stably or transiently expressing the different SUR


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 6

forms, typical apoptotic parameters like cell detachment, nuclear condensation, DNA

fragmentation and enhanced caspase-3-like activity were determined after glibenclamide

treatment. To assess the extent of the glibenclamide-effect, comparable experiments were

performed in which etoposide, a classical inducer of apoptosis widely used in chemotherapy,

was supplemented. Additionally, cells were analyzed after coexpression of SUR1 and Kir6.2

in order to prove if the apoptotic effect of glibenclamide requires the presence of intact KATP

channels (Maedler et al., 2004, 2005) or if it is also mediated by SUR in the absence of the

Kir6.x subunit.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 7

Materials and methods

Mutagenesis and generation of stably transfected cell lines. HEK293 cells were cultured at

37°C in a humidified atmosphere of 95% air and 5% CO2 in minimal essential medium

(MEM) supplemented with glutamine, 10% fetal bovine serum, and 20 µg/ml gentamycin as

described previously (Hambrock et al., 2002). Cells were transfected with pcDNA3.1

expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequences of SUR1

(GenBank X97279), SUR1(M1289T), or SUR2B (GenBank D86038). The mutant

SUR1(M1289T) had been constructed from SUR1 using the QuikChange Mutagenesis

System (Stratagene, Amsterdam, The Netherlands). Control cells were obtained by sham-

transfection with pcDNA3.1. Stably transfected cell lines were isolated as described before

(Hambrock et al., 2002) and cultured in the presence of 300 µg/ml geneticin.

Transient cotransfection of SUR1 and Kir6.2. HEK293 cells at 60-80% confluence were

transiently transfected either with SUR1- and Kir6.2- (GenBank D50581) plasmids or with

SUR1- and pcDNA-plasmids. Lipofectamine was applied as transfection reagent as described

in Hambrock et al. (2002) and the plasmids were added in equimolar amounts.

Treatment with glibenclamide or etoposide. Cells were cultured in the absence of geneticin

for at least 3 days. At 60-80% confluence (14-17 x 106 cells / 9.5 cm diameter culture dish),

glibenclamide or etoposide were supplemented by adding 10 or 20 µl, respectively, of a stock

solution (100 mmol/l glibenclamide in ethanol/DMSO (50:50, v/v) or 50 mmol/l etoposide in

DMSO) to 10 ml of culture medium. In control experiments, 10/20 µl of solvent were added.

In case of the transient transfections, glibenclamide was added 24 h after starting the

transfection. Experiments with differently transfected cells were performed in parallel and


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 8

repeated 3-7 times. In each experiment, drug-exposure was performed in two culture dishes

and solvent treatment in one dish.

Quantification of cell detachment. The numbers of adherent and of detached cells were

determined separately with a CASY TT Analyser System (Schärfe System GmbH,

Reutlingen, Germany) according to the manufacturer’s instructions. After calibration with

dead and vital HEK293 cells, cursor positions were set to 12.75 – 50.00 µm (evaluation

cursor) and 7.63 – 50.00 µm (standardization cursor). From the supernatant of each culture

dish three 100 µl aliquots were withdrawn for measurement. The adherent cells were then

rinsed off with CASY electrolyte solution and diluted if necessary. Again, 100 µl aliquots

were analyzed in triplicate.

Determination of apoptotic nuclei by Hoechst 33258 (bisbenzimide) staining. After drug

or solvent treatment, adherent cells were fixed with paraformaldehyde (3% in PBS) and

stained with Hoechst 33258 (10 µg/ml in PBS) for 10 min. The cells were washed with PBS

buffer (5 min), rinsed with distilled water, air-dried and embedded in gelatine. Cells were

evaluated using a Laborlox D fluorescence microscope (Leitz, Oberkochen, Germany) and an

UV-2A filter system (excitation: 330-380 nm; emission: 420 nm). Microscopic images were

captured with a digital CF20 DXC air CCD camera (Kappa-Meßtechnik, Gleichen, Germany).

Analysis of DNA-fragmentation. Cells from the different treatment groups were collected by

combining adherent and detached cells of each culture dish. For isolation of cytoplasmic

DNA, cells from each culture dish (9.4 cm diameter) were rinsed off separately with culture

medium after different periods of treatment. Cells were pelleted by centrifugation (500 g, 6

min) and resuspended in 1 ml PBS buffer. An aliquot of 300 µl was then withdrawn for

determination of caspase activity. The remaining 700 µl were centrifugated again (200 g, 5


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 9

min) and cells were lysed in 300 µl of lysis buffer (0.5% Triton X-100, 5 mmol/l TRIS, 20

mmol/l EDTA, pH 7.4) on ice for 20 min. Cell debris and nuclei were removed by

centrifugation (14000 g, 15 min, 4°C). Cytoplasmic DNA was extracted from the supernatants

by phenol/chloroform extraction, precipitated with ethanol and incubated with proteinase K

and RNAse A (for 60 and 30 min, respectively, at 37°C). Separation of the isolated DNA

fragments or of a peqgold 100 bp DNA ladder Plus marker (peqlab, Erlangen, Germany) was

performed in 1.2% agarose gels. UV fluorescence of ethidium bromide-stained DNA was

registered with a CCD video camera system (Raytest, Straubenhardt, Germany).

Determination of caspase-3-like (DEVD-caspase) activity. Cells were prepared as

described under “analysis of DNA fragmentation”. The aliquots withdrawn for caspase

measurements were then processed according to the method of Wanke et al. (2004) using a

Wallac 1420 Victor2 Multilabel Reader (Perkin Elmer, Rodgau-Jügesheim, Germany).

Cleavage of synthetic caspase-3-like substrate (DEVD-AFC, AFC: 7-amino-4-trifluoromethyl

coumarin) was monitored by fluorescence emission at 510 nm (excitation at 390 nm) in 20

min-intervals over a total period of 160 min. Caspase activities were calculated as increase in

fluorescence per minute and normalized to the protein content of each sample.

Statistical analysis. Data are presented as means from single experiments ± SEM. Statistical

analyses were performed using GraphPadPrism software (GraphPad Software, San Diego,

USA). For statistical comparisons, two-tailed Student’s paired t-test or repeated measures

one-way ANOVA analysis in combination with Tukey’s post-hoc test were employed as

appropriate.

Materials. All reagents used for cell culture were purchased from Invitrogen (Karlsruhe,

Germany). Glibenclamide, Hoechst 33258, and AFC-standards were from Sigma


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 10

(Deisenhofen, Germany), DEVD-AFC caspase substrate was from Biomol (Hamburg,

Germany) and etoposide from Calbiochem (Bad Soden, Germany).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 11

Results

Cell morphology after glibenclamide treatment of different cell lines.

HEK293 cells stably expressing either SUR1, SUR2B, or the mutant SUR1(M1289T) were

incubated with glibenclamide concentrations (5-100 µmol/l) over a seven-day culture period

and compared to cells that were only treated with the same amount (0.1%) of solvent. In

further controls, cells that were only sham-transfected with pcDNA3.1 expression vector (=

pcDNA-cells) were exposed to either glibenclamide or solvent. At the beginning of the

experiments, all cells showed a vital appearance, exhibited a fibroblast-like morphology and

formed coherent monolayers with 60-80% confluence (Fig. 1). Normal cell growth within the

next seven days was not visibly influenced by solvent treatment. After continuous exposure to

glibenclamide, however, many cells became roundish, detached themselves from the bottom

of the culture dish, and went up into the supernatant. The extent of glibenclamide-induced

detachment of SUR1-cells was much more pronounced than that of pcDNA-cells. Cells

expressing the mutant SUR1(M1289T) resembled pcDNA-cells after glibenclamide

treatment. Glibenclamide-treated SUR2B-cells often showed an even smaller amount of

detached cells than equally treated pcDNA-controls.

Quantification of cell detachment after treatment with glibenclamide or etoposide.

Measurements of the number of adherent cells and of cells in the supernatant revealed an

obvious correlation between concentration of glibenclamide, time of treatment, and degree of

detachment: Exposure of SUR1-cells to 100 µmol/l glibenclamide, for example, led to

intensive cell detachment after 2 days; a similar effect was achieved by incubating the same

cell line with 50 µmol/l glibenclamide for 4 days. Differences between SUR1- and pcDNA-

cells concerning cell detachment were already seen at concentrations higher than 10 µmol/l

after 4 days of glibenclamide treatment (data not shown).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 12

In further experiments, however, we applied 100 µmol/l glibenclamide in order to see

clear effects after a shorter incubation period that were not covered up by side effects like e.g.

cell aging, depletion of nutrients or accumulation of toxic metabolites. Cell counting

confirmed the results from microscopic investigation (Fig. 2): Glibenclamide treatment led to

an enhanced number of cells in the supernatant compared to solvent-treated cells in case of all

cell lines. After glibenclamide-treatment, the number of detached SUR1-cells was

significantly (2.0-2.3fold) higher than that of detached SUR2B- or pcDNA-cells, while there

was no significant difference between SUR2B- and pcDNA-cells (Fig. 2A). For cells

expressing SUR1(M1289T), the number of detached cells was reduced compared to SUR1-

cells, but slightly higher than the amount of detached pcDNA-cells (Fig. 2B). However, these

differences between SUR1(M1289T)-cells and SUR1- or pcDNA-cells were not significant.

In contrast to glibenclamide-treatment, all solvent-treated cell lines did not exhibit any

significant differences.

Comparable experiments in which the anticancer drug etoposide was applied (in 4 series

of parallel experiments) caused cell detachment with no significant differences between either

all treated groups (P > 0.05) or all untreated groups (P > 0.05) (data not shown). Just as

glibenclamide, etoposide was supplemented in the concentration of 100 µmol/l which is

commonly used for apoptosis induction in cell culture. In order to directly compare the effects

of glibenclamide and etoposide, the number of detached cells after drug treatment was

expressed as percent of the number of detached cells after solvent treatment. Again, the

degree of detached SUR1-cells after glibenclamide treatment (462 ± 82% of solvent control)

was considerably higher than that of the other cells (pcDNA: 320 ± 66%; SUR1(M1289T):

262 ± 71%; SUR2B: 254 ± 30%). The amount of glibenclamide-induced detachment of

SUR1-cells was nearly the same as that of etoposide-induced cell detachment (pcDNA: 434 ±

75%; SUR1: 475 ± 127%; SUR1(M1289T): 488 ± 78%; SUR2B: 389 ± 85%).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 13

Effect of coexpression with Kir6.2 on detachment of SUR1-expressing cells.

In order to examine whether the effect of glibenclamide on SUR1-expressing cells was

modified by additional expression of Kir6.2, HEK293 cells were transiently transfected with

equimolar amounts of SUR1- and Kir6.2-plasmids or of SUR1- and pcDNA- plasmids (Fig.

3). 24 hours after transfection, SUR1 and Kir6.2 were already well expressed as previous

experiments had confirmed (data not shown). The cells were then treated with either 100

µmol/l glibenclamide or solvent for up to 3 days. In all cases, the number of cells in the

supernatant was higher for glibenclamide-treated cells than for the respective solvent-controls.

A direct comparison between cells expressing SUR1 alone and cells coexpressing SUR1 and

Kir6.2 was difficult because already after solvent treatment cells expressing SUR1 and Kir6.2

showed a higher degree of detachment than SUR1-cells without Kir6.2. In order to assess the

effect of glibenclamide in these experiments, the background of cell detachment due to

solvent treatment was therefore either subtracted from the total number of detached cells after

glibenclamide treatment (Fig. 3B) or considered in its proportional contribution to the total

amount (Fig. 3C). According to both evaluations, coexpression with Kir6.2 did not

significantly alter the results obtained for cells that were transfected with SUR1 alone.

Nuclear condensation and DNA fragmentation after glibenclamide treatment.

After glibenclamide treatment (100 µmol/l glibenclamide, 48 h), staining with the DNA-

binding dye Hoechst 33258 demonstrated the appearance of brightly stained, condensed and

fragmented nuclei, which is characteristic for apoptotic cells (Fink and Cookson, 2005). In

SUR1-cells, apoptotic nuclei occurred to a much larger extent and were much more

condensed and fragmented than in SUR2B- (data not shown), SUR1(M1289T)-, or pcDNA-

cells (Fig. 4). After solvent treatment, however, all cell lines exhibited almost exclusively

normal intact nuclei with no visible differences between the cell lines. DNA fragmentation

after glibenclamide treatment was also confirmed by a visible DNA laddering pattern typical


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 14

of apoptotic cell death (fragment length: 400-1500 bp). As this method does not allow any

reliable quantification of isolated fragmented DNA, no direct comparison between the

different glibenclamide-treated cell lines was possible. Nevertheless, all solvent-treated cells

exhibited only very slight or no fragmentation.

Determination of caspase-3–like activity after glibenclamide treatment.

Initiation of apoptotic cell death is associated with sequential activation of a cascade of

different caspases. Caspase-3 plays a key role within this signaling pathway in mammalian

cells. Caspase-3 is able to specifically recognize the peptide sequence DEVD, but cleavage of

DEVD-substrates by other enzymes with lower affinity cannot be excluded. Therefore,

turnover of DEVD-substrates strictly speaking is due to caspase-3-like enzymes (Wanke et

al., 2004). We quantified the activity of these enzymes by use of the synthetic fluorescent

substrate DEVD-AFC after different intervals of glibenclamide or solvent treatment (12, 16,

24 and 48 h). Solvent-treated cells showed a basal amount of caspase-3-like activity that,

however, was lower than that of cells supplemented with 100 µmol/l glibenclamide. The latter

was slightly increased after 12 and 24 h of glibenclamide treatment but showed a marked

increase after 48 h (about 2.5 to 3-times higher compared to the beginning of the experiments,

data not shown). After 48 h, caspase-3-like activity in SUR1- and pcDNA-cells was

significantly higher in the glibenclamide-treated groups than in the solvent-treated groups, but

this difference was not observed in SUR1(M1289T)-cells (Fig. 5). Enzyme activity after

glibenclamide exposure was significantly enhanced in SUR1-cells compared to the other cell

lines. SUR1(M1289T)-cells showed even slightly lower activity than pcDNA-controls after

glibenclamide treatment, but this difference was not significant. Small differences were also

detected between the solvent-treated groups: Caspase-3-like activity was a little higher in

SUR1-cells than in pcDNA- and SUR1(M1289T)-cells in most experiments and pcDNA-cells

for their part showed slightly, but not significantly higher activity than SUR1(M1289T)-cells.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 15

Yet, if total activity after glibenclamide treatment was referred to the respective solvent-

dependent activity, the increase in caspase-3-like activity due to glibenclamide was still

higher in SUR1-cells (323 ± 40%) in comparison with pcDNA-cells (269 ± 37%) or

SUR1(M1289T)-cells (200 ± 41%).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 16

Discussion

In this study, we were able to show that expression of SUR1 in HEK293 cells renders these

cells more susceptible towards induction of apoptosis after exposure to glibenclamide. This

was seen by an increase in typical apoptotic parameters like cell detachment, nuclear

condensation, DNA fragmentation and caspase-3-like activity (Mandrup-Poulsen, 2001; Fink

and Cookson, 2005), which rules out the involvement of necrotic cell death. A basal level of

apoptosis was also present in sham-transfected control cells as well as in cells expressing the

SUR2B isoform or the mutant SUR1(M1289T). In SUR1-cells, however, apoptotic processes

were clearly enhanced after glibenclamide-treatment.

These results are in accordance with previous studies reporting a negative impact of

prolonged exposure to some sulfonylureas on islet cell function (Rustenbeck et al., 2004; Del

Guerra et al., 2005). Additionally, the sulfonylureas glibenclamide and tolbutamide are

known to elicit apoptosis in β-cells or pancreatic cell lines (Efanova et al., 1998; Iwakura et

al., 2000; Rustenbeck et al., 2004; Maedler et al., 2004, 2005). A precise mechanistic

explanation, however, is still lacking. Mostly, it is assumed that the apoptotic effect of these

secretagogues is mediated by blockade of functional KATP channels and depolarization-

induced Ca2+ influx into the cell. According to Miki et al. (2001), KATP channels are important

for β-cell survival, as apoptosis occurs more frequently in Kir6.2 knock-out or Kir6.2G132S

transgenic mice. Kim et al. (1999) on the other hand suggest a KATP-channel-independent

mechanism for glibenclamide-induced apoptosis in hepatoblastoma cells that involves

inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) channels.

In contrast to this, our data for the first time give evidence that expression of the SUR1

isoform can be specifically responsible for enhanced induction of apoptosis by glibenclamide:

First, enhanced apoptosis induction is restricted to HEK293 cells expressing SUR1 and does

not occur in SUR2B- or control cells. Artifacts concerning the stably transfected SUR1-cell


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 17

line can be excluded here, because similar results were obtained after transient transfections

(data not shown). Second, a single mutation of SUR1 is able to abolish the SUR1-specific

effect. Third, this effect does not require the presence of Kir6.2 and, furthermore, is not

significantly enhanced by Kir6.2. At first glance, these results are surprising because

assemblage of SUR and Kir6.2 is thought to be a prerequisite for correct trafficking of both

subunits and formation of functional plasmalemmal KATP channels (Neagoe and Schwappach,

2005). On the other hand, trafficking control can be dependent on factors such as temperature,

it might differ between certain cell lines (Giblin et al. 2002) and could be overcome by

recombinant overexpression (Mikhailov et al., 1998). There even might be diverse trafficking

routes for KATP channel subunits within the same cell (Neagoe and Schwappach, 2005), as

there are several reports that SUR or high-affinity sulfonylurea binding sites in the β-cell are

also located intracellularly, mainly in the insulin secretory granules (Carpentier et al., 1986;

Ozanne et al., 1995; Barg et al., 1999; Geng et al., 2003). Even if Kir6.2 is required for

correct targeting of SUR1 in the β-cell, glibenclamide-induced apoptosis would not require

complete functional SUR1/Kir6.2 channels according to our data. Therefore, the results point

to an additional function of SUR beyond being a regulatory subunit of KATP channels. There

are already some considerations that, in the β-cell, SUR1 plays a special role in cAMP-

dependent exocytosis of insulin vesicles (Nakazaki et al., 2002; Eliasson et al., 2003;

Shibasaki et al., 2004). Future work using SUR1-knockout mice might be suitable to confirm

the involvement of the SUR1 isoform in β-cell apoptosis.

Like in other studies (Efanova et al., 1998; Kim et al., 1999; Iwakura et al., 2000;

Rustenbeck et al., 2004; Maedler et al., 2004, 2005), we applied the sulfonylurea in rather

high concentrations of 5-100 µmol/l in our cell culture system in order to see clear rapid

effects (see Results). The glibenclamide-effect, though, is not only dependent on drug-

concentration, but also on cell pretreatment, cell density, and time of drug exposure. Perhaps

because of these reasons Del Guerra et al. (2005) did not notice a change in the apoptotic rate


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 18

of human islets after 24 h of incubation with 10 µmol/l glibenclamide. Maedler et al. (2005),

however, observed that even low glibenclamide-doses of 1 and 10 nmol/l elicited clear effects

(2-2.5fold increase in apoptosis) in cultured human islets after 4 days. According to these

observations it has to be proven if the apoptotic effects of glibenclamide could constitute

problems in long-term treatment with therapeutic sulfonylurea concentrations and contribute

to the phenomenon of secondary sulfonylurea failure.

Comparative experiments with equal concentrations of the chemotherapeutic agent

etoposide demonstrate that the extent of apoptosis induced by glibenclamide in SUR1-cells is

nearly the same as that induced by etoposide. In contrast to glibenclamide treatment,

etoposide exposure does not lead to such pronounced differences among the cell lines.

Apoptosis induction by specific substances depending on the SUR-isoform and thereby on the

type of cell or tissue might in this context provide a new perspective in cancer research. In

that respect, the detection of other compounds that might be more potent than glibenclamide

could be of interest.

The precise signal transduction pathway connected with the SUR1-specific apoptotic

effect of glibenclamide remains to be elucidated. The intensive detachment of glibenclamide-

treated SUR1-cells points to the involvement of “anoikis”, a special form of apoptosis

characterized by extensive impairment of cell adhesion (Valentijn et al., 2004). Interestingly,

an anoikis-like mechanism has been shown to be involved in early isolated islet apoptosis

(Thomas et al., 2001). According to our results, caspase-3-like enzymes are activated after

glibenclamide treatment. Still it has to be noted that caspase-3-like activity in solvent-treated

controls is slightly higher in SUR1-cells compared to pcDNA-cells, but lower in

SUR1(M1289T)-cells. This, however, does not result in further damage of SUR1-cells in the

absence of glibenclamide, because the other apoptotic parameters do not show any differences

between all solvent-treated cell lines. Maybe expression of SUR1 per se (but not of


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 19

SUR1(M1289T)) results in slightly higher caspase-3-like activity, which could be due to e.g.

interaction of SUR1 with endogenous compounds or supplements in the culture medium.

The clear SUR1-specific glibenclamide-induced apoptotic effect is completely abolished

by exchanging a single amino acid in the carboxy-terminal transmembrane helix, TM17, by

the corresponding amino acid of SUR2. In several transporters from the ABC-protein family,

the last carboxy-terminal transmembrane helix plays an important role in binding of ligands

and transport substrates (Oleschuk et al., 2003). SUR1(M1289T) is able to form functional

plasmalemmal channels with Kir6.2 according to electrophysiological experiments (Moreau

et al., 2000) and thus possesses essential properties of an intact SUR protein. In radioligand

binding studies using 3H-glibenclamide, we were able to show that neither glibenclamide

affinity (Hambrock et al., 2004) nor expression rate (unpublished results) of the mutant are

different from SUR1 wild-type. However, the maximal amount of glibenclamide binding sites

is reduced at SUR1(M1289T) by about 30-50% at physiological MgATP concentrations

(unpublished results), which might explain the lower sensitivity of SUR1(M1289T)-cells to

glibenclamide-induced apoptosis. On the other hand, the maximum amount of binding sites (>

2000 fmol/mg membrane protein) is still quite high at the mutant. In contrast to this,

glibenclamide-induced apoptosis in SUR1(M1289T)-cells is at the same level as that of

control cells (or is even lower for SUR1(M1289T)-cells in case of caspase-3-like activity).

For this reason, it is more plausible to assume that the specific integration of SUR1 in the

relevant signaling pathway, maybe via interaction with specific kinases, is disturbed by

conformational changes due to the mutation in TM17. In this respect, SUR1(M1289T) is

probably similar to the SUR2B isoform, which is also less effective in triggering

glibenclamide-induced apoptosis. It has been shown previously that binding and effect of

several KATP channel openers are clearly enhanced in SUR1(M1289T)-cells (Moreau et al.,

2000; Hambrock et al., 2004). Taking into account that negative allosteric interactions exist

between opener and glibenclamide binding (Schwanstecher et al., 1998; Hambrock et al.,


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 20

2004) and that several openers exhibit anti-apoptotic effects (Maedler et al., 2004; Teshima et

al., 2003; Skak et al., 2004), TM17 is obviously involved in SUR1-specific apoptosis

induction. It will, therefore, be an interesting task to investigate whether openers exert

different protective effects in cells expressing SUR1, SUR1(M1289T), or SUR2.

In conclusion, glibenclamide induces apoptotic processes in HEK293 cells that are

markedly enhanced by expression of SUR1, but not of SUR2B or the mutant

SUR1(M1289T). Accordingly, the SUR1 isoform can render cells more susceptible to

glibenclamide and possibly also to other specific agents. SUR1 as a typical pancreatic protein

for this reason might be involved in pancreatic β-cell apoptosis. Adaptive variation of β-cell

mass is known to provide one mechanism in the regulation of insulin secretion. In this

context, SUR1 could well influence insulin secretion by a second mechanism apart from

regulating electrical activity as a KATP channel subunit. These results could contribute to

knowledge of so far unknown SUR functions which may be important in diabetes and/or

cancer research. Nevertheless, future work will have to concentrate on further exploitation of

the signaling pathways involved in SUR1-mediated apoptosis.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 21

Acknowledgments

Dr. C. Derst (Jena, Germany) is acknowledged for kindly providing cDNA of SUR1 and Drs

Y. Kurachi and Y. Horio (Osaka, Japan) for the generous gift of the SUR2B and Kir6.2

clones. We thank Dr. A. Buchmann, J. Mahr und E. Zabinsky for friendly help with some of

the apoptosis assays and C. Müller for excellent technical assistance in cell culture.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 22

References

Ahren, B (2005) Type 2 Diabetes, insulin secretion and β-cell mass. Curr Mol Med 5:275-

286.

Ashcroft SJH (2000) The β-cell KATP channel. J Membrane Biol 176:187-206.

Barg S, Renström E, Berggren P-O, Bertorello A, Bokvist K, Braun M, Eliasson L, Holmes

WE, Köhler M, Rorsman P and Thévenod F (1999) The stimulatory action of

tolbutamide on Ca2+-dependent exocytosis in pancreatic ß cells is mediated by a 65-kDa

mdr-like P-glycoprotein. Proc Natl Acad Sci U S A 96:5539-5544.

Bryan J, Vila-Carriles WH, Zhao G, Babenko AP and Aguilar-Bryan L (2004) Toward

linking structure with function in ATP-sensitive K+ channels. Diabetes 53:S104-S112.

Carpentier JL, Sawano F, Ravazzola M and Malaisse, WJ (1986) Internalization of 3H-

glibenclamide in pancreatic islet cells. Diabetologia 29:259-261.

Del Guerra S, Marselli L, Lupi R, Boggi U, Mosca F, Benzi L, Del Prato S and Marchetti P

(2005) Effects of prolonged in vitro exposure to sulphonylureas on the function and

survival of human islets. J Diabetes Complications 19:60-64.

Donath MY and Halban PA (2004) Decreased β-cell mass in diabetes: significance,

mechanisms and therapeutic implications. Diabetologia 47:581-589.

Doyle ME and Egan JM (2003) Pharmacological agents that directly modulate insulin

secretion. Pharmacol Rev 55:105-131.

Efanova IB, Zaitsev SV, Zhivotovsky B, Kohler M, Efendic S, Orrenius S and Berggren PO

(1998) Glucose and tolbutamide induce apoptosis in pancreatic β-cells - A process

dependent on intracellular Ca2+ concentration. J Biol Chem 273:33501-33507.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 23

Eliasson L, Ma XS, Renstrom E, Barg S, Berggren O, Galvanovskis J, Gromada J, Jing XJ,

Lundquist I, Salehi A, Sewing S and Rorsman P (2003) SUR1 regulates PKA-

independent cAMP-induced granule priming in mouse pancreatic B-cells. J Gen Physiol

121:181-197.

Fink SL and Cookson, BT (2005) Apoptosis, pyroptosis, and necrosis: Mechanistic

description of dead and dying eukaryotic cells. Infect Immun 73:1907-1916.

Geng XH, Li LH, Watkins S, Robbins PD and Drain P (2003) The insulin secretory granule is

the major site of KATP channels of the endocrine pancreas. Diabetes 52:767-776.

Giblin JP, Quinn K and Tinker A (2002) The cytoplasmic C-terminus of the sulfonylurea

receptor is important for KATP channel function but is not key for complex assembly or

trafficking. Eur J Biochem 269:5303-5313.

Gribble FM and Reimann F (2003) Sulphonylurea action revisited - the post-cloning era.

Diabetologia 46:875-891.

Hambrock A, Kayar T, Stumpp D and Osswald H (2004) Effect of two amino acids in TM17

of sulfonylurea receptor SUR1 on the binding of ATP-sensitive K+ channel modulators.

Diabetes 53:S128-S134.

Hambrock A, Löffler-Walz C and Quast U (2002) Glibenclamide binding to sulphonylurea

receptor subtypes: dependence on adenine nucleotides. Br J Pharmacol 136:995-1004.

Henquin JC (2004) Pathways in β-cell stimulus-secretion coupling as targets for therapeutic

insulin secretagogues. Diabetes 53:S48-S58.

Iwakura T, Fujimoto S, Kagimoto S, Inada A, Kubota A, Someya Y, Ihara Y, Yamada Y and

Seino Y (2000) Sustained enhancement of Ca2+ influx by glibenclamide induces

apoptosis in RINm5F cells. Biochem Biophys Res Commun 271:422-428.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 24

Kim JA, Kang YS, Lee SH, Lee EH, Yoo BH and Lee YS (1999) Glibenclamide induces

apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator

(CFTR) Cl- channels and intracellular Ca2+ release in HepG2 human hepatoblastoma

cells. Biochem Biophys Res Commun 261:682-688.

Maedler K, Carr RD, Bosco D, Zuellig RA, Berney T and Donath MY (2005) Sulphonylurea

induced b-cell apoptosis in cultured human islets. J Clin Endocrinol Metab 90:501-506.

Maedler K, Størling J, Sturis J, Zuellig RA, Spinas GA, Arkhammar POG, Mandrup-Poulsen

T and Donath MY (2004) Glucose- and interleukin-1 β-induced β-cell apoptosis

requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and

is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2

(SUR/Kir6.2) selective potassium channel opener in human islets. Diabetes 53:1706-

1713.

Mandrup-Poulsen, T (2001) β-cell apoptosis - Stimuli and signaling. Diabetes 50:S58-S63.

Mikhailov MV, Proks P, Ashcroft FM and Ashcroft, SJH (1998) Expression of functionally

active ATP-sensitive K-channels in insect cells using baculovirus. FEBS Lett 429:390-

394.

Miki T, Iwanaga T, Nagashima K, Ihara Y and Seino S (2001) Roles of ATP-sensitive K+

channels in cell survival and differentiation in the endocrine pancreas. Diabetes 50:S48-

S51.

Moreau C, Jacquet H, Prost A-L, D'hahan N and Vivaudou M (2000): The molecular basis of

the specificity of action of KATP channel openers. EMBO J 19:6644-6651.

Nakazaki M, Crane A, Hu M, Seghers V, Ullrich S, Aguilar-Bryan L and Bryan J (2002)

cAMP-activated protein kinase-independent potentiation of insulin secretion by cAMP

is impaired in SUR1 null islets. Diabetes 51:3440-3449.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 25

Neagoe I and Schwappach B (2005) Pas de deux in groups of four - the biogenesis of KATP

channels. J Mol Cell Cardiol 38:887-894.

Nenquin M, Szollosi A, Aguilar-Bryan L, Bryan J and Henquin JC (2004) Both triggering and

amplifying pathways contribute to fuel-induced insulin secretion in the absence of

sulfonylurea receptor-1 in pancreatic β-cells. J Biol Chem 279:32316-32324.

Oleschuk CJ, Deeley RG and Cole SPC (2003) Substitution of Trp1242 of TM17 alters

substrate specificity of human multidrug resistance protein 3. Am J Physiol Gastrointest

Liver Physiol 284: G280-G289.

Ozanne SE, Guest PC, Hutton JC and Hales, CN (1995) Intracellular localization and

molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells.

Diabetologia 38:277-282.

Rustenbeck I, Krautheim A, Jörns A and Steinfelder HJ (2004) β-Cell toxicity of ATP-

sensitive K+ channel-blocking insulin secretagogues. Biochem Pharmacol 67:1733-

1741.

Rustenbeck I, Wienbergen A, Bleck C and Jorns A (2004) Desensitization of insulin secretion

by depolarizing insulin secretagogues. Diabetes 53:S140-S150.

Schwanstecher M, Sieverding C, Dörschner H, Gross I, Aguilar-Bryan L, Schwanstecher C

and Bryan J (1998) Potassium channel openers require ATP to bind to and act through

sulfonylurea receptors. EMBO J 17:5529-5535.

Seino S and Miki, T (2003) Physiological and pathophysiological roles of ATP-sensitive K+

channels. Prog Biophys Mol Biol 81:133-176.

Shibasaki T, Sunaga Y and Seino S (2004) Integration of ATP, cAMP, and Ca2+ signals in

insulin granule exocytosis. Diabetes 53:S59-S62.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 26

Skak K, Gotfredsen CF, Lundsgaard D, Hansen JB, Sturis J and Markholst H (2004)

Improved b-cell survival and reduced insulitis in a type 1 diabetic rat model after

treatment with a b-cell-selective KATP channel opener. Diabetes 53:1089-1095.

Teshima Y, Akao M, Baumgartner WA and Marban E (2003) Nicorandil prevents oxidative

stress-induced apoptosis in neurons by activating mitochondrial ATP-sensitive

potassium channels. Brain Res 990:45-50.

Thomas F, Wu JG, Contreras JL, Smyth C, Bilbao G, He J and Thomas J (2001) A tripartite

anoikis-like mechanism causes early isolated islet apoptosis. Surgery 130:333-338.

Valentijn AJ, Zouq N and Gilmore,AP (2004) Anoikis. Biochem Soc Trans 32:421-425.

Wanke I, Schwarz M and Buchmann A (2004) Insulin and dexamethasone inhibit TGF-β-

induced apoptosis of hepatoma cells upstream of the caspase activation cascade.

Toxicology 204:141-154.


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 27

Footnotes

This study was supported by the Deutsche Forschungsgemeinschaft (HA 2711/2-1).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 28

Legends for figures

Fig. 1. Effect of glibenclamide treatment on the morphology of SUR1-, SUR1(M1289T)-,

SUR2B-, or pcDNA-cells. Cells were observed within 3 days of treatment with 100 µmol/l

glibenclamide (GBC) or ethanol/DMSO (solv.). Solvent-controls were shown for pcDNA-

cells as an example. Using phase contrast microscopy, adherent cells showed a dark

appearance while detached cells were roundish and more refractive.

Fig. 2. Total number of detached cells after exposing the different cell lines (60-80%

confluence before treatment) to 100 µmol/l glibenclamide for 48 hours. A: The effect of

glibenclamide (GBC) or solvent (solv.) on detachment of pcDNA-, SUR1-, and SUR2B-cells

was determined in parallel in 3 series of experiments (A). pcDNA-, SUR1-, and

SUR1(M1289T)-cells were compared in parallel experiments with n=4 (B). *, P < 0.05.

Fig. 3. Influence of coexpression of SUR1 with Kir6.2 on glibenclamide-induced cell

detachment. HEK293 cells transiently transfected either with SUR1-cDNA + pcDNA vector

or with SUR1-cDNA + Kir6.2-cDNA were incubated with 100 µmol/l glibenclamide (GBC)

or solvent (solv.) for 3 days (n=4). Data are presented as total number of cells in the

supernatant (A), as number of cells obtained after subtracting the number of detached cells

due to solvent treatment from the total number of cells in the supernatant (B) or as amount of

detached cells in percent of the respective controls (C). No significant differences were

detected comparing the following data: SUR1 + pcDNA + glibenclamide vs. SUR1 + pcDNA

+ solvent: P > 0.05, SUR1 + Kir6.2 + glibenclamide vs. SUR1 + Kir6.2 + solvent: P > 0.05,

SUR1 + pcDNA + glibenclamide vs. SUR1 + Kir6.2 + glibenclamide: P > 0.05 (A); SUR1+

pcDNA vs. SUR1 + Kir6.2: P > 0.05 (B, C).


at ASPE

T Journals on M


Dow

nloaded from


JPET#97501 29

Fig. 4. Hoechst 33258 staining after 48 hours of treatment with 100 µmol/l glibenclamide

(GBC) or ethanol/DMSO treatment of pcDNA-, SUR1-, and SUR1(M1289T)-cells. Normal

nuclei are large and faintly stained, apoptotic nuclei are brightly stained, condensed, and

fragmented. Results for SUR2B-cells (not shown) were the same as for pcDNA- or

SUR1(M1289T)-cells. Scale bar: 10 µm.

Fig. 5. Determination of caspase-3-like activity in pcDNA-, SUR1-, and SUR1(M1289T)-

cells after treatment with 100 µmol/l glibenclamide (GBC) or solvent for 48 hours. Data from

7 experimental series are given as arbitrary units (increase in fluorescence per minute

normalized to the protein content of each sample). *, P < 0.05; **, P< 0.01.


at ASPE

T Journals on M


Dow

nloaded from



at ASPE

T Journals on M


Dow

nloaded from



at ASPE

T Journals on M


Dow

nloaded from



at ASPE

T Journals on M


Dow

nloaded from



at ASPE

T Journals on M


Dow

nloaded from



at ASPE

T Journals on M


Dow

nloaded from


Documents

Glibenclamide-induced apoptosis is specifically enhanced ...jpet.aspetjournals.org/content/jpet/early/2005/11/23/jpet.105.097501... · glibenclamide on recombinant HEK293 cells expressing