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Department of Metabolism and Clinical Nutrition, Kyoto University School of Medicine, Kyoto; and the Division of Molecular Medicine, Center for Biomedical Science, Chiba University School of Medicine (H.Y., H.K., S.S.), Chiba, Japan
Address all correspondence and requests for reprints to: Nobuhisa Mizuno, M.D., Department of Metabolism and Clinical Nutrition, Kyoto University School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606–01, Japan. E-mail: mizuno{at}metab.kuhp.kyoto-u.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bcl-2 gene has been cloned (16) and has been shown to be expressed within pancreatic islets and acini (17). This gene is known to be an intrinsic factor for apoptosis and to contribute to susceptibility to apoptosis, as its overexpression in the pancreatic ß-cell line, ßTC1 cells, was found to partially protect them from cytokine-induced apoptosis (18). On the other hand, it has been established that the bax gene, a bcl-2 family member, promotes apoptosis (19). The ratio of bcl-2 to bax has been known to determine the susceptibility of the cell to certain apoptotic stimuli (19, 20, 21). Protein in the IgM fraction of IDDM serum has been shown to increase L-type calcium channel activity, which is followed by overload of cytoplasmic Ca2+, and also to contribute indirectly to the destruction of ß-cells in vitro (5). On the other hand, amylin-induced apoptosis in islet cells is not associated with Ca2+ influx from extracellular space via calcium channels (6). Although it had been thought that overloaded intracellular Ca2+ is linked to cell death (22, 23, 24), many studies have shown that a rise in intracellular Ca2+ alone is not sufficient for apoptotic cell death (25, 26, 27, 28).
In the present study, we have investigated whether serum deprivation induces ß-cell apoptosis with the altered expression of bcl-2/bax genes and the cytosolic/nuclear Ca2+ dynamics. To ascertain the hypothesis that intracellular Ca2+ overload can induce apoptosis, we examined the apoptotic effects on ß-cells of various agents, such as high concentrations (30 mM) of KCl (induction of membrane depolarization), BayK (VDCC opener), A23187 (Ca2+ ionophore), and ionomycin to elevate intracellular Ca2+ and also the effect of the Ca2+ chelator, EGTA or BAPTA, to reduce it.
In addition, it has been recently found that the insulin secretory capacity is reduced in ß-cells in apoptotic process, which shows DNA damage (29). Here, we examined the decrease in the insulin secretory response to glucose in ß-cells during the apoptotic process and investigated its reversibility using apoptotic blockers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assay for cell viability
MIN6 and RINm5F cells were plated at a density of 1 x
104/well in 96-well plates and cultured in the DMEM medium,
described above, for 2 days. The culture medium was then replaced by
serum-deprived medium or ordinary medium containing KCl, BayK, A23187,
or ionomycin. The viability of the cells was determined by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay
(Cell Proliferation Kit, Boehringer Mannheim Biochemica, Mannheim,
Germany) at the time intervals indicated in Figs. 1
, 2, and 4 according
to the manufacturer’s instructions. The levels of cell viability at
the starting point (0 h) were used as the basal control (100%), and
the results at each point are presented as a percentage of the basal
control value.
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Assessment of apoptosis by DNA fragmentation
At the various times indicated in Figs. 1
and 4, MIN6 or
RINm5F cells were harvested from the culture dishes and washed twice in
PBS. They were lysed in a solution containing 1% SDS, 0.5% Triton
X-100, 20 mM EDTA, and 5 mM Tris-HCl, pH 8.0,
and incubated overnight with 0.25 mg/ml proteinase K at 37 C. DNA was
extracted in phenol/chloroform, ethanol precipitated, resuspended in 10
mM Tris-HCl and 1 mM EDTA (pH 8.0), and
incubated with deoxyribonuclease-free ribonuclease (80 µg/ml; for
1 h). The DNAs (10 µg/lane) were electrophoresed on 2% agarose
gels and visualized by ethidium bromide staining. Apoptosis was
confirmed further by the in situ modified fluorescein
detection method of DNA fragmentation (ApopTag, Oncor, Gaithersburg,
MD), according to the manufacturer’s instructions. Cellular morphology
and nuclear fluorescein were observed under a differential microscope
and a laser-evoked fluorescence microscope (LSM410, Zeiss).
Northern blot analysis
Total RNA was prepared from cells by the guanidinium
isothiocyanate/cesium chloride procedure at various times after the
addition of KCl, BayK, A23187, ionomycin, or the Ca2+
chelators and also after serum deprivation from the medium. Total RNA
was also extracted from mouse brain and liver, as positive and negative
controls, respectively, using the same method. Ten micrograms of total
RNA were denatured with formaldehyde, electrophoresed on 1% agarose
gel, and transfered to a nylon membrane. The 499-bp fragment of
bcl-2 complementary DNA (nucleotides 37–535 relative to
translation start site) was 32P labeled by nick-translation
and used to hybridize the membrane. The hybridizations and washing
conditions were previously described (26). The membranes were
subsequently stripped and rehybridized with 32P-labeled
bax complementary DNA (nucleotides 100–453). These probes
were prepared by RT-PCR from total RNA of mouse brain. The PCR primers
were 5'-GAGATCGTGA TGAAGTACAT-3' (sense) and 5'-TCAGGTACTCAGTCATCCAC-3'
(antisense) for bcl-2, 5'-ACCAGCTCTGAACAGATCAT-3' (sense)
and 5'-AGATGGTCACTG TCTGCCAT-3' (antisense) for bax, and
5'-ATCCGTAAAGACCTCTATGC-3' (sense) and 5'-AACGCAGCTCAGTAACAGTC-3'
(antisense) for ß-actin. These probes were sequenced to confirm their
identities after subcloning into M13mp18. Relative expression levels of
bcl-2 and bax messenger RNA (mRNA) were
determined by densitometric analysis. The intensity of the
bax mRNA band in each lane was considered to be 100%, and
the ratio of bcl-2 to bax mRNA was expressed as
percentage of bax in each lane.
Measurement of intracellular calcium concentration
([Ca2+]i)
MIN6 cells were loaded with 1 µM
fura-2/acetoxymethylester (Molecular Probes, Eugene, OR) for 30 min in
Krebs-Ringer bicarbonate buffer (KRB) containing 109 mM
NaCl, 3.3 mM glucose, 4.6 mM KCl, 3.2
mM CaCl2, 1.15 mM
Na2HPO4, 0.4 mM
KH2PO4, and 20 mM HEPES (pH 7.4).
After replacing KRB with the DMEM medium, the changes in
[Ca2+]i were monitored by a dual excitation
wavelength method (340 and 380 nm). The absolute value of
[Ca2+]i was determined with a
Ca2+ standard solution (Molecular Probes). Fluorescence
emission at 510 nm was monitored, and the ratio calculation was
digitized by a computerized image processor (Argus-100/CA, Hamamatsu
Photonics, Hamamatsu, Japan).
Confocal
[Ca2+]i measurements and
image analysis
The dissociated cells were loaded with 1 µM
fluo-3/acetoxymethylester (Molecular Probes) for 30 min in KRB. After
replacing KRB with DMEM medium, fluorescence emitted from MIN6 cells
was measured by confocal laser scanning microscope (LSM410, Zeiss). An
argon laser was used to excite the dye at 488 nm, and emission signals
were measured through interference filters (510–540 nm). Single
wavelength images were acquired and stored on an optical memory disk.
In our experiments the laser scan strength was set at 20–30% of the
100-mW output, and a 10% neutral density filter was used. The pinhole
aperture was set at 1.5 µm (thickness of Z slice, <0.5 µm). The
scanning X-Y slice in which cytosolic and nuclear Ca2+ were
studied was set at one half the MIN6 cell thickness, which was about 6
µm (Z slice position = mean of 3 µm). Calibration of single
wavelength fluorescence in terms of the absolute concentration of
calcium is difficult and was not attempted because the relative calcium
change in the intracellular cytosolic and nuclear compartments is the
focus of this study. Transmission and fluo-3 fluorescence images during
stimulation were stored on an optical memory disk. After direct
recording, the nuclei of the measured cells were stained with acridine
orange (1 µg/ml; 20 min) to distinguish between cytosol and nuclear
fluorescence. After indicating the cytosolic and nuclear compartments
on a measured MIN6 cell, the fluorescence of the cytosol and nucleus
was recalculated from the data in the memory disk.
Immunoblotting analysis
Cells were lysed, homogenized, and sonicated in lysis buffer
containing 10 mM Tris-acetate, 2 mM EDTA, 100
mM NaCl, 20% glycerol (pH 7.5), 1 µg/ml antipain, 1
µg/ml leupeptin, 0.1 mM phenylmethylsulfonylfluoride, and
4 µM pepstain A. Aliquots of total protein (10 µg
protein/lane) were subjected to electrophoresis on a 12.5%
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membranes (Immobilon, Millipore Intech, Bedford, MA) by electroblotting
overnight at 4 C and 150 mA. Bcl-2 and Bax proteins were detected on
immunoblots with polyclonal rabbit antimouse/rat Bax antibody
(catalogue no. 13686E, PharMingen, San Diego, CA) and
polyclonal rabbit anti mouse/rat Bcl-2 antibody (catalog no. sc-492,
Santa Cruz Biotechnology, Santa Cruz, CA), respectively. The membrane
filters were blocked for 1 h at room temperature with 5%
nonfat dry milk and 10% donkey serum in PBS with Tween-20 (PBS-T; pH
7.4) consisting of 136.9 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM
KH2PO4, and 0.1% Tween-20. After
washing with PBS-T, the membranes were incubated with 1:2000 diluted
anti-Bax antibody or 1:1000 diluted anti-Bcl-2 antibody. The primary
antibody immunoreactions were then detected with horseradish
peroxidase-conjugated donkey antirabbit Ig using an enhanced
chemiluminescense system according to the manufacturer’s instructions
(ECL, Amersham Life Science, Aylesbury, UK).
Measurements of insulin release
MIN6 cells were plated at a density of 1 x
105/well in 48-well plates and were cultured in the DMEM
described above for 3 or 4 days. The culture medium was then replaced
with the serum-deprived medium or the ordinary medium containing 4
mM BAPTA in the absence or presence of 10 µM
aurintricarboxylic acid (ATA) and 50 µM Zn2+.
The glucose-induced insulin secretory capacity of MIN6 cells was
determined during apoptotic conditions with serum deprivation for
96 h or with 4 mM BAPTA for 36 h. In these
conditions, DNA fragmentation could be detected, but cell viability
assessed by the MTT assay remained at more than half the original
values. The cells at 70
80% confluence in 48-well plates were
preincubated with KRB containing 0.2% BSA plus 3.3 mM
glucose for 10 min at 37 C and then incubated for an additional 1
h in KRB containing 3.3 or 16.7 mM glucose. The insulin
released into the medium was measured by RIA (30, 31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(left panel)
shows the decrease in cell viability of the MIN6 pancreatic ß-cell
line in the serum-deprived medium, assessed by MTT assay. The viability
declined together with the lower concentrations of serum in the culture
medium. In the serum-free medium, MIN6 cells can be cultured for at
least 48 h without a significant loss in the number of living
cells, but cell viability gradually decreases thereafter. The decreased
cell viability under serum-free conditions can be reversed
significantly by an endonuclease inhibitor, ATA (10 µM),
72 h after the serum deprivation and thereafter (Fig. 2A). Actinomycin D and cycloheximide,
considered to be apoptosis blockers via inhibition of mRNA or protein
synthesis, do not reverse the decrease in cell viability (data not
shown). We then investigated whether the serum deprivation-induced cell
death in MIN6 cells was due to apoptosis. One of most important
characteristics of apoptosis, the cleavage of DNA into
oligonucleosomal-sized fragments, can be seen in the intensity of the
ethidium bromide staining. Fragmentations of DNA in MIN6 cells were
first detected at 72 h in the serum-free medium and increased
thereafter (Fig. 1A, right panel). The cell viability of
RINm5F also declined together with the lower serum concentrations and
gradually decreased in serum-free medium. Fragmentations of DNA in
RINm5F cells were slightly detected at 12 and 24 h in the
serum-free medium and increased thereafter (Fig. 1B, right
panel).
Photomicrographs of MIN6 cells stained with acridine orange are shown
in the left panels of Fig. 3.
The nucleus of the untreated (Fig. 3A) and undamaged cells (Fig. 3, C and E) are stained round and red by acridine
orange. On the other hand, condensed nuclei of smaller size
were observed with acridin orange after 120-h incubation in
the serum-free medium (Fig. 3C) and after 18-h incubation with BAPTA
(Fig. 3E). These smaller nuclei mean nuclear chromatin condensation
that supports the characteristic of apoptosis. In addition, end-labeled
fragmented DNA was stained green in the nuclei after 120-h
incubation in the serum-free medium (Fig. 3D) and also after 18 h
with BAPTA (Fig. 3F), but it was not detected in the nuclei of the
untreated (Fig. 3B) or nonapoptotic cells (Fig. 3, D and F).
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, left panel). Under these
conditions no DNA fragmentation was detected (Fig. 4A, right
panel). Whereas ionomycin (50 µM) tended to decrease
the cell viability, it was also insufficient to produce DNA
fragmentations. This unexpected result encourages us to investigate
whether the extracellular Ca2+ chelator, EGTA or BAPTA,
might induce apoptosis in MIN6 cells. Four or more millimolar
concentrations of these extracellular Ca2+ chelators
decreased cell viability in a dose-dependent manner (Fig. 4, B and C).
On the other hand, 1 or 2 mM Ca2+ chelators
could not decrease it. Zn2+ (50 µM) was found
to partially, but significantly, inhibit the decline in cell viability
caused by 4 mM BAPTA (Fig. 2B), whereas ATA (1100
µM), actinomycin D (5 mg/liter), and cycloheximide (10
µM) all failed to block it (data not shown). DNA
fragmentation also was detected with both 4 mM EGTA and 4
mM BAPTA (Fig. 4, B and C, right panels). In
addition, chromatin condensation and in situ end-labeled
fragmented DNA were found in the nuclei of the cells 18 h after
the addition of 4 mM BAPTA (Fig. 3, E and F).
Changes in cytosolic and nuclear calcium concentrations, and
expression of the apoptosis-associated genes, bcl-2 and bax
As serum deprivation and chelation of extracellular
Ca2+ by the Ca2+ chelators evoked apoptosis in
MIN6 cells, we examined the intracellular Ca2+ dynamics and
changes in bcl-2 and bax mRNA expression in MIN6
cells. Serum deprivation did not affect either the cytosolic or nuclear
Ca2+ concentrations detected by computerized image
processor (fura-2/AM) and confocal laser scanning microscope
(fluo-3/AM; data not shown). On the other hand, the expression level of
bcl-2/bax (the ratio of bcl-2 to
bax) mRNA was transiently enhanced and then gradually
decreased during the apoptotic process (Fig. 5). After 96 h of serum deprivation
in MIN6 cells, bcl-2/bax mRNA gradually
decreased. In addition, Bcl-2 protein expression apparently persisted
until 96 h after serum deprivation (Fig. 6A). Although not only cytosolic but also
nuclear Ca2+ was elevated by KCl (30 mM), BayK
(40 µM), A23187 (5 µM), and ionomycin (5
µM), the expression levels of
bcl-2/bax mRNA exhibited no change despite the
addition of these agents (data not shown).
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, A, C, and E, 4
mM BAPTA decreased intracellular, nuclear, and cytosolic
Ca2+ levels from resting Ca2+ levels, although
2 mM BAPTA failed to do so. Under this extracellular
Ca2+ chelation, bcl-2/bax mRNA
gradually decreased (Fig. 8, A and B, right panel). Similar
results were obtained when 4 mM EGTA was used as the
extracellular Ca2+ chelator (Fig. 8, A and B, left panel).
Immunoblotting analysis also showed gradually decreased of Bcl-2/Bax
protein expression under the same condition (4 mM BAPTA;
Fig. 6B).
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and 180.5 ng/well·h
(n = 26) for Fig. 9B] were used as the basal control levels
(100 ± 4.7% and 100 ± 2.7%, respectively).
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, the insulin secretion from MIN6 cells was
significantly increased by 16.7 mM glucose compared with
that caused by 3.3 mM glucose under control conditions
(171 ± 7.3%; P < 0.001). However, 96 h
after serum deprivation, basal (by 3.3 mM glucose) insulin
secretion was significantly decreased to 47.5 ± 3.0% of the
control level (P < 0.001), and there was no
significant increase in insulin secretion induced by 16.7
mM glucose. ATA (10 µM), which reversed the
decreased cell viability 96 h after serum deprivation,
significantly reversed the decreased basal insulin secretion under the
serum-free condition to 74.0 ± 14.0% of the control value
(P < 0.001). Interestingly, the glucose responsiveness
of insulin release was also restored by this apoptotic blocker
(P < 0.001). Other blockers (Zn2+,
actinomycin D, and cycloheximide), however, did not reverse the
decreased insulin secretion under this condition (data not shown).
In another series of experiments (Fig. 9B), basal insulin
secretion was significantly reduced to 30.0 ± 3.0% after 36-h
incubation with 4 mM BAPTA (P < 0.001),
and the glucose responsiveness of the insulin secretion also was lost.
Zn2+ (50 µM) partially, but significantly,
reversed the decreased basal insulin secretion to 55.0 ± 5.0% of
the control value (P < 0.001), and the glucose
responsiveness of insulin release also was restored (P
< 0.001). Other apoptotic blockers (ATA, actinomycin D, and
cycloheximide), however, did not reverse the reduced insulin secretion
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In pancreatic ß-cells, it is still unclear whether intracellular Ca2+ overload induces apoptosis. The present study demonstrates that excessive Ca2+ influx from extracellular space or Ca2+ release from intracellular stores is insufficient to evoke apoptosis in MIN6 cells. It is not clear at present why MIN6 cells are resistant to elevated [Ca2+]i, but it is well known that Bcl-2 is very effective in blocking [Ca2+]i overload-induced apoptosis (33, 34). In addition, using immunoperoxidase staining, Bcl-2 protein has been detected within pancreatic islets and acini (17). These findings suggest that the constitutive expression of bcl-2 mRNA and protein under basal condition may play at least in part a significant role in the mechanism of protection from apoptosis by [Ca2+]i overload in MIN6 cells.
The present study shows that the extracellular Ca2+ chelators, EGTA and BAPTA, promote apoptosis in MIN6 cells. This accords with recent studies showing that PC12 cells, T lymphocytes, erythroid progenitor cells, and thymoma cells can be induced to apoptosis under extracellular Ca2+ deprivation (35, 36, 37). Little is known of the apoptotic pathways under unphysiological Ca2+-chelated conditions. Accordingly, the [Ca2+]i dynamics and its relationship to genes such as bcl-2 and bax were investigated. BAPTA is alkaline, precluding acidification as the cause of the apoptosis induced by the Ca2+ chelators. In addition, no significant leakage of lactate dehydrogenase was observed from apoptotic cells (data not shown), and the decreased cell viability was reversed by an apoptotic blocker, Zn2+, excluding the adverse effect of the agents as the cause of the apoptosis.
One or 2 mM extracellular Ca2+ chelators was
insufficient to affect resting [Ca2+]i
(100 nM), but 4 mM or more of the
extracellular Ca2+ chelators was shown to further decrease
its level to 4050 nM, after which both decreased cell
viability and DNA fragmentation were seen. This indicates that higher
concentrations of Ca2+ chelators can deplete the cytosolic
and nuclear free Ca2+ concentrations, which was further
confirmed by confocal laser scanning microscope. Under 4 mM
or more of the Ca2+ chelators conditions,
bcl-2/bax mRNA and protein were decreased. This
indicates that the Ca2+ chelators cause apoptosis,
accompanied by an alteration of the ratio of bcl-2 to
bax expression and a decrease in the cytosolic/nuclear
Ca2+ concentration.
Our results show that glucose-induced insulin secretion is decreased in ß-cell lines during apoptotic changes induced by serum deprivation (for 96 h) or extracellular Ca2+ chelators (for 36 h), whereas cell viability remains at more than half the basal level and no significant leakage of lactate dehydrogenase from cells was observed. Basal insulin secretion (by 3.3 mM glucose) is significantly decreased, but not completely abolished. We also found that the glucose responsiveness of insulin secretion to a high concentration (16.7 mM) of glucose was remarkably diminished. This is consistent with previous observations that cytokine-induced apoptosis in ß-cells (7, 8) is associated with the impaired insulin secretion stimulated by high glucose (29, 38). Interestingly, the decrease in basal insulin secretion and the disappearance of glucose responsiveness under apoptotic conditions were eliminated by the corresponding apoptotic blockers. It is well known that ATA inhibits the DNA fragmentation formation in PC12 cells induced by serum deprivation (39), and also that Zn2+ prevents the appearance of the DNA fragmentation by glucocorticoids in thymocytes (40, 41). It has been found that glucose-induced insulin release is already reduced in the preclinical stage of IDDM (42). Assuming that apoptosis is involved in the destructive process of pancreatic ß-cells and their functional impairment, the preventive studies on the occurrence of apoptosis would help to reduce the incidence of diabetes mellitus.
In conclusion, at least two distinct pathways for activation of apoptosis are present in pancreatic ß-cell lines, and the response of glucose-induced insulin secretion becomes impaired during the course of apoptosis regardless of its molecular pathway. The susceptibility to apoptosis in ß-cells involving altered expression of apoptosis-related genes such as bcl-2 and bax and changes in the cytosolic/nuclear Ca2+ concentration may play an important role in the mechanism of the deterioration of pancreatic ß-cell function in disease.
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Acknowledgments |
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Footnotes |
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Received July 21, 1997.
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), 50 µM ionophore (A21387;
), and 50 µM ionomycin (
; A, left
panel), and typical internucleosomal DNA fragments from MIN6
cells cultured with each ligand for 72 h were separated by
electrophoresis (A, right panel). Each agent is shown to
be insufficient to markedly decrease the cell viability under the
starting points (100%; A, left panel). DNA extracted
from MIN6 cells is not fragmented at 72 h (A, right
panel). B and C show the effect of the extracellular
Ca2+ chelators, EGTA and BAPTA, on cell viability and DNA
fragmentation in MIN6 cells. EGTA (2 mM; 
