From the a Third Department of Internal Medicine, Faculty of
Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan,
d Omiya Medical Center, Jichi Medical School, Omiya, Saitama
330, Japan, f The Institute for Diabetes Care and Research, Recently, a mitochondrial mutation was found to
be associated with maternally inherited diabetes mellitus (Kadowaki,
T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., Tanabe,
Y, Sakura, H., Awata, T., Goto, Y., Hayakawa, T., Matsuoka, K.,
Kawamori, R., Kamada, T., Horai, S., Nonaka, I., Hagura, R., Akanuma,
Y., and Yazaki, Y. (1994) N. Engl. J. Med. 330, 962-968). In order to elucidate its etiology, we have investigated the
involvement of mitochondrial function in insulin secretion. Culture of
the pancreatic The mitochondrial genome of mammalian cells encodes 13 polypeptides, two ribosomal RNAs, and 22 transfer RNAs (for a review, see Ref. 1). The mitochondrion is believed to be an organelle derived
from a genetic component(s) of microorganisms, and thus its
replication, transcription, and translation system has been developed
on its own basis, although several nuclear genome-encoded proteins are
also essential for these systems. Mitochondrial proteins involved in
oxidative phosphorylation are composed of enzyme complexes (I-IV) and
cooperate with a number of nuclear genome-encoded proteins for ATP
production.
Currently, the following hypothesis is widely accepted as a major part
of the glucose-signaling pathways for insulin secretion in pancreatic
To examine the relationship between mitochondrial function and the
stimulation of insulin release, we employed a newly developed insulin-secreting cell line, It has been reported that EB, a reagent that inhibits DNA/RNA
synthesis, affects transcription/replication of extrachromosomal genetic materials more specifically than those of chromosomal genes
(19-21). Recently, human cell lines lacking mitochondrial DNA
( In this report, we investigated the effects of EB treatment and the
virtual elimination of mitochondrial transcription on glucose-stimulated insulin secretion and on
[Ca2+]i. EB treatment blocked the effect of
glucose to increase [Ca2+]i and to stimulate
insulin secretion. The effect of EB was completely reversed after
removal of the EB. These results provide strong evidence that
mitochondrial function is crucial for the stimulation of insulin
release by glucose.
Materials--
Restriction enzymes and DNA-modifying enzymes
were purchased from Takara Shuzo Co. (Kyoto, Japan). EB, bovine serum
albumin (BSA), nifedipine, and fura-2 were obtained from Sigma, and
[ Cell Culture--
DNA Analysis--
The cells were harvested by trypsinization and
suspended in 10 mM Tris-HCl and 100 mM EDTA, pH
8.0, containing 100 µg/ml proteinase K and 1% SDS, following a 2-h
incubation at 37 °C. Total DNA was purified, and 2 µg of the DNA
was digested with BglII, which cleaves the mouse
mitochondrial DNA at nucleotide position 15,329 (25). The resultant DNA
fragments were subjected to agarose (0.7%) gel electrophoresis,
transferred onto Hybond N+ membrane (Amersham Pharmacia Biotech), and
hybridized with the [ RNA Analysis--
Cells were lysed by adding RNAzolB (Biotech
Laboratories, Inc., Houston, TX) to the culture plate, and total RNA
was prepared following the manufacturer's instructions. Ten µg of
the total RNA was denatured and subjected to formaldehyde-containing
agarose (1.2%) gel electrophoresis and transferred onto the Hybond N+ membrane. The membrane was hybridized with the
[ Determination of Glucose-phosphorylating
Activity--
Glucose-phosphorylating activities by hexokinase (HK)
and glucokinase (GK) of either control or EB-treated cells on day 4 were determined fluorometrically as described previously (29, 30).
Electron Microscopic Analysis--
The control and EB-treated
cells were fixed overnight with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3). They were postfixed
with 2% OsO4 in the same buffer, followed by en block staining with
1% uranyl acetate. After dehydration with a graded series of ethanol,
they were substituted by propylene oxide and embedded in Spurr's low
viscosity resin. Silver to gold sections were cut and examined with a
JEOL 1010 electron microscope (JEOL, Tokyo, Japan.).
Insulin Secretion and Content--
The control and EB-treated
cells were cultured in 12-well plates. Each well was washed twice with
1 ml of phosphate-buffered saline and then incubated with 1 ml of
Hanks'-BSA medium composed of Hanks' buffered saline containing 0.2%
BSA and 10 mM HEPES, pH 7.5, plus 0.1 mM
glucose in 5% CO2 at 37 °C. After incubation for 2 h, the medium was replaced with 1 ml of the Hanks'-BSA medium containing various secretagogues. For the experiment shown in Fig. 6,
Krebs-Ringer bicarbonate buffer was used instead of Hanks' buffer.
After incubation for 2 h, the medium was collected and centrifuged
at 6000 rpm for 2 min, and the supernatant was collected and stored at
Measurement of [Ca2+]i--
Control
and EB-treated cells were cultured on a glass coverslip in a 6-cm
diameter dish. Each dish was washed twice with 2 ml of
phosphate-buffered saline and incubated with 4 ml of Hanks'-BSA medium
for 40 min. The cells were then loaded with fura-2 for 40 min in 4 ml
of the Hanks'-BSA medium containing 4 µM fura-2 acetoxymethylester (added from a 1 mM stock solution in
dimethyl sulfoxide) in 5% CO2 at 37 °C. The glass
coverslips were washed three times with 2 ml of the Hanks'-BSA medium
before measurement of [Ca2+]i. The
[Ca2+]i was measured before and then 15 and 30 min after stimulation by replacement with the Hanks'-BSA medium
containing 20 mM glucose or 1 µM
glibenclamide. All measurements of [Ca2+]i were
carried out at 37 °C. A dual excitation digital imaging system
(Argus 100 image analysis system; Hamamatsu Photonics Inc., Japan) was
used for the measurements. Epi-illumination at 340- or 360-nm
wavelength from a mercury lamp was used to excite fura-2. Emitted
fluorescence was magnified through a fluorescence objective lens (× 10; Nikon Inc., Japan) taken by a silicon-intensified tube camera, and
then converted into a digitized value of 256 gray levels at each pixel.
Paired fluorescence images by 360-nm wavelength excitation were taken
before and after a single measurement and interpolated each time. The
signal fluorescence by 340-nm wavelength was converted into a ratio
divided by the value of interpolated 360 nm at each pixel to yield a
pseudocolor image of [Ca2+]i (32). Conversion
from the ratio to [Ca2+]i was performed using a
Ca2+-EGTA standard (Molecular Probes, Inc.). Data were
expressed as two-dimensional mean values of
[Ca2+]i, which were calculated by averaging the
values of all of the pixels over the cellular area.
Statistical Analysis--
Data shown are means ± S.D. of
triplicate observations in a single representative experiment unless
otherwise indicated.
Characteristics of
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-cell line,
HC9, with low dose ethidium bromide
(EB) (0.4 µg/ml) for 2-6 days resulted in a substantial decrease in the transcription level of mitochondrial DNA (to 10-20% of the control cells) without changing its copy number, whereas the
transcription of nuclear genes was grossly unaffected. Electron
microscopic analysis revealed that treatment by EB caused morphological
changes only in mitochondria and not in other organelles such as
nuclei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mM)
could no longer stimulate insulin secretion, while glibenclamide (1 µM) still did. When EB was removed after 3- or 6-day
treatment, mitochondrial gene transcription recovered within 2 days,
and the profiles of insulin secretion returned to normal within 7 days.
Studies with fura-2 indicated that in EB-treated cells, glucose (20 mM) failed to increase intracellular Ca2+,
while the effect of glibenclamide (1 µM) was maintained.
Our system provides a unique way to investigate the relationship
between mitochondrial function and insulin secretion.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-cells. First, glucose is transported into the pancreatic
-cells
and metabolized through the glycolytic pathway and Krebs cycle. ATP is
produced by oxidative phosphorylation within the mitochondria. The
increased ATP and decreased ADP concentrations cause depolarization of
the plasma membrane via closure of ATP-sensitive K+
channels. Depolarization activates the voltage-dependent
calcium channels and increases [Ca2+]i. This
increase in [Ca2+]i stimulates exocytosis of
insulin granules from the pancreatic
-cells (for a review, see Ref.
2). Several lines of evidence suggest an important role for the
mitochondria in this pathway. First, ATP-sensitive K+
channels are believed to "sense" ATP produced by mitochondria and
to convert the information into depolarization of membrane potential.
The presence of this type of channels in pancreatic
-cells was first
demonstrated by electrophysiological approaches (3, 4) and recently
established on a molecular basis (5, 6). Second, 2-ketoisocaproate,
which is metabolized intramitochondrially, exerts the same stimulatory
effects on insulin secretion as glucose does (7, 8). Third, it has been
shown that mutations of mitochondrial DNA are associated with diabetes
mellitus (9-11). We have recently pointed out that diabetic patients
with an A to G mutation at position 3243 in mitochondrial DNA (base
pair 3243 mutation) have reduced insulin secretory capacity rather than
peripheral insulin resistance (12). In addition, Hess et al.
(13) and Chomyn et al. (14) showed by in vitro
study that this mutation results in mitochondrial transcriptional and
translational defects. However, there are few reports that have
demonstrated a correlation between mitochondrial (dys)function and
insulin secretion directly.
HC9 (15-17), and cultured the cells with ethidium bromide (EB),1
an inhibitor of the synthesis of DNA and RNA. To date, few detailed studies on the role(s) of mitochondrial function in glucose signaling have been performed. In one of these, in which
bis-4-piperidyle-dichloride was employed to establish a
0 cell line in the MIN6 insulin-secreting cell, it was
shown that the presence of mitochondria was essential for
glucose-stimulated insulin release (18), as discussed below.
0 cells) were established by long term treatment with
low concentrations of EB (22, 23). Hayashi et al. (24) also
showed that a specific inhibition of replication, transcription, and
translation of genes takes place with mitochondrial DNA when a mouse
fibroblast cell line was treated with EB for up to 7 days.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]dCTP was from Amersham Pharmacia Biotech.
Glibenclamide was provided by Hoechst Co., Ltd.
HC9 was a kind gift from Dr. D. Hanahan
(University of California).
HC9 cells were maintained in Dulbecco's
modified Eagle's medium containing 25 mM glucose, 0.11 mg/ml pyruvate, and 0.05 mg/ml uridine supplemented with 15% horse
serum, 2.5% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml
streptomycin in 5% CO2. Cells reaching 70-90% confluency
were divided to a density of 3 × 104/cm2.
For the treated cells, EB was added to the culture medium 18-24 h
after plating. Cells of passages near passage 20 were used for experiments, except for the experiments for Fig. 6 and Table III, for
which passages 31 and 29 were used, respectively.
-32P]dCTP-labeled mouse
mitochondrial DNA fragment probe (nucleotide positions 643-9047)
containing 12 and 16 S ribosomal RNAs, 10 transfer RNAs, and three
peptides corresponding to cytochrome oxidase subunits 1, 2, and 3, and
ATPase 6 (25). Southern blot analysis was carried out according to the
standard protocol (26). Radioactivity of hybridizing bands was measured
by BAS2000 image analyzer (Fujix Co., Japan), and the relative amount
of mitochondrial DNA was determined.
-32P]dCTP-labeled mouse mitochondrial DNA fragment,
rat insulin I (27), or human
-actin cDNA (28) probe according to
the standard protocol (26). Radioactivity of hybridizing bands was
measured by BAS2000 image analyzer, and the relative amount of each
transcript was determined.
20 °C until radioimmunoassay for insulin concentration. For
determination of insulin content in the cells, each well was washed
twice with 1 ml of phosphate-buffered saline and suspended in 1 ml of a
solution containing 74% ethanol and 1.4% HCl and kept at
20 °C
for 18-24 h. Then the supernatant was collected and stored at
20 °C until assayed. Radioimmunoassay kits (Shionoria; Shionogi & Co., Ltd., Japan) were used for the determination of insulin levels
using mouse insulin as standard. Total cellular proteins were extracted
by boiling the trypsinized cells in 1% SDS, and protein concentration
was determined by the method described by Lowry (31) with BSA as a
standard.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
HC9 Cells upon Insulin Secretion--
Table
I summarizes the characteristics of
insulin secretion and insulin content in
HC9 cells. Analyses were
carried out when cells reached 70-90% confluency. Twenty
mM glucose induced a substantial increase in insulin
secretion from the basal level (0.1 mM glucose alone).
Half-maximal effect of glucose on insulin secretion was obtained at
10-15 mM (shown later), consistent with the results of
Radvanyi et al. (15) and Noda et al. (17). Glibenclamide (1 µM), KCl (20 mM), and
2-ketoisocaproate (10 mM) also stimulated insulin
secretion. In other experiments, it was shown that nifedipine (100 nM), an inhibitor of L-type voltage-dependent calcium channels, inhibited the stimulation of insulin secretion by
glucose (20 mM) or glibenclamide (1 µM)
(percentage of inhibition: 89.3 ± 6.9% and 81.7 ± 11.9%,
respectively), and it was also shown that these secretagogues did not
increase the insulin content during 2-h incubations (total insulin
content after 2-h incubation was 100 ± 6, 105 ± 9, and
99 ± 12% for 0.1 mM glucose, the addition of 20 mM glucose, and 1 µM glibenclamide,
respectively).
Insulin secretion from HC9 cells by glucose and other secretagogues
HC9 cells by various secretagogues were evaluated as
described under "Experimental Procedures." Means ± S.D. of
triplicate observations in a single representative experiment are shown
here.
Effect of EB on Mitochondrial Replication and
Transcription--
HC9 cells were incubated for 4 days with various
concentrations of EB in Dulbecco's modified Eagle's medium
supplemented with pyruvate and uridine, which was previously shown to
be essential for the growth of
0 cells (cells lacking
mitochondrial DNA) (22). Fig.
1A shows changes in gene
transcription in EB-treated cells measured by Northern blot analyses.
The mitochondrial DNA probe used for these analyses detected several
transcripts (two rRNAs, 10 tRNAs, and four peptides), including two
major hybridized bands. The relative quantity of the mitochondrial
transcripts was calculated in comparison with the radioactivity of 16 S
transcript(s). These analyses showed that the reduction of
mitochondrial transcription by EB treatment occurred in a
concentration-dependent manner, and the mitochondrial transcription level of EB-treated (0.4 µg/ml) cells was reduced to
10-20% of the control cells, whereas the transcription of the insulin
gene was not affected. Transcription of
-actin was also unaffected
as described below.
|
Time Course Analysis of Transcription of Nuclear and Mitochondrial
Genes in EB-treated Cells--
We examined the temporal profiles of
mitochondrial transcription and insulin secretion 1-6 days after the
addition of EB. On days 4-6, the growth of EB-treated cells was
significantly retarded (see below). On days 3-6, mitochondrial
transcription levels of the control cells were enhanced, whereas those
of EB-treated cells were inhibited by 80-90% compared with control
cells (Fig. 2). In contrast,
transcription of the insulin gene showed little change between treated
and untreated cells (Fig. 2). Expression of "housekeeping" genes
also seemed to be unaffected by EB treatment, when the messenger RNA of
-actin was assessed on day 0, 2, 4, and 6 by reverse
transcription-polymerase chain reaction using the Mouse
-Actin
Control Amplimer Set (CLONTECH, Palo Alto, CA) (data not shown). Analysis by reverse transcription-polymerase chain
reaction of cells treated with EB for 6 days also showed that
transcription of major glucose-sensing enzymes, GK and HK-I, was not
altered significantly (data not shown). In accord with this,
glucose-phosphorylating activities by HK and GK were not changed
between EB-treated and control cells on day 4 (Fig.
3).
|
|
Electron Microscopic Analysis--
On electron microscopic
analysis (Fig. 4), HC9 cells contained
electron-dense secretory granules in their cytoplasm, which probably
represented insulin-containing granules, because fluorescence microscopy of anti-insulin antibody to these cells revealed fine granular staining in the cytoplasm (data not shown). Although dead or
damaged cells were occasionally seen after EB treatment, most of the
cells looked morphologically intact, as also shown by phase-contrast
microscopy (data not shown). The only prominent finding observed in
these cells was the frequent appearance of ring- or cup- shaped
mitochondria (Fig. 4A). No changes were found in other
organelles including Golgi apparatus or dense core granules.
|
Time Course Analysis of Insulin Secretion in EB-treated Cells-- Basal insulin secretion (0.1 mM glucose) of the control cells maintained a constant low level during cell proliferation. The stimulatory effect of glucose (20 mM) on insulin secretion was gradually increased, and the maximal effect was observed on day 6 (confluency had reached 70-90% at this time). A similar temporal profile to that of glucose-induced insulin secretion was observed with glibenclamide (1 µM)-stimulated insulin release (Fig. 5A). In contrast, in EB-treated cells, glucose-stimulated insulin release was completely abolished by day 5-6, although the stimulatory effect of glibenclamide was still observed (Fig. 5B). The insulin content of EB-treated cells was higher than those of the control cells (tested on days 4 and 6; Table II). In addition, glucose-phosphorylating activity by either HK or GK was unchanged between EB-treated and control cells (on day 4; Fig. 3). Reverse transcription-polymerase chain reaction analysis of these two enzymes of the cells treated with EB for 6 days showed similar expression levels to those of control cells (data not shown).
|
|
|
Reversible Change of Mitochondrial Transcription and Properties of
Insulin Secretion--
After 3 days of treatment with EB, cells were
returned to EB-free culture medium, and mitochondrial transcription and
insulin secretion were examined. As described earlier, mitochondrial
transcription of cells treated with EB for 3 days was only 10-20% of
that of the control cells. After removal of EB from the medium,
mitochondrial transcription was slightly raised after 1 day and
restored at 2 days (Fig. 7). The
transcription levels of the insulin and -actin genes were increased
2-3-fold on days 2-5. Importantly, the effects on insulin secretion
by glucose started to recover 3 days after the removal of EB and were
completely reversed in 5-7 days (at 70-90% confluency),
i.e. both the basal level of insulin secretion and the
stimulatory effect of glucose (20 mM) were normal,
i.e. similar to control cells on days 5 and 7 (Figs.
5A and 8). This 3-day delay in recovery could be
attributable to the time course of restoration of mitochondrial
transcription (see "Discussion"). Mitochondrial transcription and
insulin secretion by glucose were also restored when EB was removed
after 6 days of EB treatment (Fig. 8;
data for transcription not shown). This reversible change in insulin
secretion was also observed with a later passage (passage 29) as shown
in Table III.
|
|
|
Changes in [Ca2+]i-- Changes in the [Ca2+]i in response to 20 mM glucose or 1 µM glibenclamide were examined in control and EB-treated cells using fura-2 as the indicator (Fig. 9). As shown in Fig. 9, the growth of EB-treated cells was significantly retarded compared with the control cells on day 6.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the importance of ATP for glucose signaling in the
stimulation of insulin secretion has been proved by
electrophysiological techniques, only a few reports, including one
report (18) from one of our laboratories, have clearly shown the role
of the mitochondrion, the ATP-producing organelle, in this pathway. In
the present study, we have modulated (reduced) mitochondrial
transcription and hence mitochondrial function by EB treatment and have
investigated the effect of this on glucose-regulated insulin secretion
in the -cell line,
HC9. This cell line, established from the
hyperplastic stage of pancreatic islets of transgenic mice expressing
SV40 large T antigen in their
-cells (15), synthesized and secreted insulin in response to glucose and other stimuli. Furthermore, the
half-maximal glucose concentration for insulin secretion was 10-15
mM, similar to that of normal islets, indicating that this cell line retains the innate glucose-signaling pathway of normal
-cells (17).
Inhibitors of oxidative phosphorylation such as sodium azide, antimycin A, rotenone, cyanide, and iodoacetamide have been used to study the role of mitochondrial function in insulin secretion and intracellular Ca2+ handling (33-35). These studies suggest the importance of mitochondrial function both for the stimulation of insulin secretion and for the increase in [Ca2+]i. Unfortunately, the inhibitors possess strong cytotoxicity, and thus the inhibitory effects are irreversible. Therefore, inhibition of insulin secretion by these compounds provides only limited evidence for the role of mitochondrial function for insulin secretion.
One of our laboratories established a 0 system
introduced into another insulin-secreting cell line, MIN6, and
demonstrated that mitochondrial function is indispensable for
glucose-induced insulin release (18). In the current report, we not
only showed that glucose-induced insulin release and
[Ca2+]i rise were decreased in EB-treated cells
but also demonstrated that neither of them was decreased when
glibenclamide was used as the stimulus. Additionally, we showed by
electron microscopy that morphological changes were specifically
located in the mitochondria; i.e. electron microscopic
analyses demonstrated the presence of secretory granules and normal
mitochondria in the control
HC9 cells; changes in the EB-treated
cells were only found in mitochondria, not in other cellular components
such as nuclei, endoplasmic reticula, Golgi bodies, or secretory
granules. These results are consistent with an action of EB
specifically on the mitochondria. We also demonstrated that
reversibility of changes in mitochondrial gene transcription, insulin
secretion, and [Ca2+]i rise upon removal of the
EB.
EB has been established as a useful tool for the study of
extrachromosomal genetic components because it specifically inhibits their transcription and subsequently replication by deleting RNA primers required for initiating replication (19-21, 24). In this report, we have demonstrated that in HC9 cells, EB is also able to
inhibit the transcription of mitochondrial genes without suppressing that of chromosomal genes, e.g. insulin (Fig. 2),
glucokinase, and
-actin genes; we also showed that glucokinase and
hexokinase activities were unchanged by EB treatment. In addition, the
results in Table II show that synthesis of insulin is even enhanced in the EB-treated cells compared with the control cells. One possible explanation for this phenomenon is an enhanced posttranslational mechanism(s), since long term exposure to high glucose has been reported to elevate insulin biosynthesis, which outgrows the increase in mRNA content of insulin (36). In EB-treated cells, possible elevated glucose 6-phosphate levels, as a result of unchanged GK and HK
activities and suppressed mitochondrial function, might well affect
this level of regulation of insulin biosynthesis, since similar
phenomena were reported in other cell types (37, 38). The inhibitory
effect of EB on mitochondrial transcription was preceded by its effect
on mitochondrial replication (Fig. 2), suggesting that the remaining
transcription level (10-20%) was enough to maintain mitochondrial
replication.
When EB was removed, all of the changes induced by the treatment (cell growth, insulin secretory profiles, and Ca2+ dynamics) were reversed, along with the restoration of mitochondrial transcription. This suggests that the changes observed during this treatment were due to mitochondrial dysfunction and that the treatment caused no irreversible effects on chromosomal genes. In fact, recovery of the insulin secretory profile was in good accord with the restoration of mitochondrial transcription. As shown in Fig. 7, it needed 2 days for the mitochondrial transcription to recover fully, and the secretory response to glucose started to recover 1 day later (Fig. 8). This 1-day delay might well be due to the time required for protein synthesis following the recovery of mitochondrial transcription.
During treatment with 0.4 µg/ml EB for 6 days, the growth of HC9
cells was retarded. This contrasts with the previous report by King and
Attardi (22) that
0 cells lacking mitochondrial DNA,
derived from the human osteosarcoma cell line 143B, did not show growth
inhibition compared with the parent cells when pyruvate and uridine
were supplemented in the medium. Although the reason for this
difference is unclear, one possible explanation could be that
HC9
cells (and possibly normal
-cells) are more "fragile" and
sensitive to ATP depletion than cells of other types like osteosarcoma
cells. In fact, we recently found that pancreata from patients with a
mutation of mitochondrial DNA at the position of base pair 3243 showed
a lower number of islet cells and exhibited their atrophy
(predominantly
-cells), which may contribute to the pathogenesis of
diabetes mellitus of such
subjects.2
In the current study, the [Ca2+]i was increased
in the control cells by 20 mM glucose. This increase was
lost in EB-treated cells and was restored after removal of EB. In this
series of experiments, glucose-induced increase in
[Ca2+]i was well associated with glucose
responsiveness in insulin secretion. In contrast, 1 µM
glibenclamide increased the [Ca2+]i in EB-treated
cells to a similar extent as in control cells. These results indicate
the involvement of mitochondrial function in the glucose-stimulated
Ca2+ increase and subsequent insulin release, compatible
with the current model that ATP generated from glucose in mitochondria is the key component in glucose-stimulated insulin release, acting upon
ATP-sensitive K+ channels and subsequent activation of
voltage-dependent calcium channels in -cells (2, 39,
40).
The lack of response to glucose in EB-treated cells appears not to be
due to the decreased expression of GK, which has been suggested to
underlie the left shift of the dose-responsive curve of
glucose-regulated insulin secretion in several -cell lines (15, 41)
and also in pancreatic islets (42). In EB-treated
HC9 cells,
however, glucose-phosphorylating activities by HK or GK as well as
expression levels of these enzymes were unchanged (Fig. 3). Regarding
the glucokinase activity in this cell line, Liang et al.
(16) reported that the Vmax value of its
activity of
HC9 cells was 10 times higher than that of hexokinase
(in supernatant of homogenates), and the present study showed equal activities of these two enzymes (in sonicates). In this regard, it
should be mentioned that the same laboratory (43) reported similar
activities of glucokinase and hexokinase in sonicates of rat islets,
whereas the former activity was less than half of the latter in the
supernatant of the homogenates. This could be ascribed to the
difference in the distribution between supernatant and sonicates of the
two enzymes. In addition,
3-O-methyl-N-acetylglucosamine used to stabilize
the assay by suppressing the perturbation by N-acetylglucosamine kinase (29, 43) might have affected
glucokinase activity. In our hands,
HC9 cell glucokinase activity
increases by 25% when measured without this
compound.3
On day 6 when mitochondrial transcription was decreased to ~10%, the basal level of insulin secretion was enhanced (Fig. 5). This could be explained in part by the increase in insulin content as described earlier. It may also be due to decreased intercellular contact as a result of occasional loss of cells, for a decrease in cell-to-cell interaction has been proved in islets to cause elevation of the basal level of insulin secretion (44). This would agree well with the gradually decreasing basal secretion in the control cells along with the cell proliferation (Fig. 5A).
Other possibilities to be considered include the possibility that the
basal level of [Ca2+]i might be increased (due to
impaired activity of the Ca-ATPase pump, for example), which could
contribute to the accelerated insulin secretion at low glucose levels;
but, as shown in Fig. 9 and as described under "Results," the basal
[Ca2+]i level was lower in EB-treated cells than
in control cells. Next, we considered that activation of the
"immature" or constitutive secretory pathway with relative
suppression of the regulatory pathway might be associated with the high
basal secretion. Therefore, we measured the proinsulin/insulin ratio in
the medium of the EB-treated and control cells on day 6 by high
performance liquid chromatography analysis; the elevation of this ratio
could be attributable to relative activation of constitutive secretion. The results showed no significant increase in the ratio for the cells
deficient in mitochondrial function (data not shown), suggesting that
impairment of the regulatory pathway is little or none in EB-treated
cells. Third, this increase in basal secretion might also be due to
defective energy production resulting from low (0.1 mM)
glucose incubation; however, fractional secretions at glucose
concentrations of 2.5 and 5.0 mM, known to maintain
cellular energy production, were also raised in EB-treated cells
compared with control cells (Fig. 6). Finally, the decreased
mitochondrial volume in EB-treated cells (Fig. 4) may be associated
with some changes in intracellular distribution of substances related
to insulin secretion such as long-chain acyl-CoA (45), which is believed to be stored for the most part in mitochondria (46). In any
case, both an enhanced level of insulin secretion and a loss of glucose
responsiveness in EB-treated cells are correlated to mitochondrial
dysfunction, since these characteristics were completely restored after
EB removal. Thus, mitochondrial function might have some additional,
unknown correlation to insulin secretion in -cells other than its
effects on ATP-sensitive K+ channels (47).
In conclusion, we have developed a unique system to study the role of
mitochondrial function in insulin secretion. In our experience, EB
rarely failed to exert an effect on insulin secretion. In this regard,
this approach is both reproducible and reversible. These
characteristics make the method useful for studying the mechanisms of
insulin secretion, especially in relation to mitochondrial
(dys)function. Our results presented here as well as the previous
report (18) with a 0 system in MIN6 cells demonstrate
the necessity of mitochondrial function for glucose-stimulated insulin
release. Our data clearly show that mitochondrial function is essential
for glucose signaling for insulin secretion through generating an
increase in [Ca2+]i with possible interplay with
some other unknown mechanisms. Moreover, the present report is the
first to demonstrate a reversible change, upon removal of the agent
suppressing mitochondrial transcription. Our results show that 80-90%
inhibition of transcription of mitochondrial DNA is enough to cause a
similar change to
0 cells in terms of glucose-stimulated
insulin release and [Ca2+]i rise, which provides
practical convenience and usefulness as an experimental system for
mitochondrial dysfunction studies. It negates the need to maintain the
0 cell line or to verify the complete absence of
mitochondrial gene products. In addition, a variety of degrees of
mitochondrial dysfunction can be induced by changing the duration of EB
treatment. Finally, it should be pointed out that the system described
here should also serve as a useful tool for investigating the role of
mitochondrial function and dysfunction in cell lines of other origin.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. D. Hanahan (University
of California, San Francisco) for kindly providing HC9 cells. We
also thank Dr. Keiji Iwamoto (University of Tokyo) for critical reading
of this manuscript and helpful discussions.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Present address: Pharmaceutical Research Laboratories II, Takeda Chemical Industries, Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532, Japan.
c These authors contributed equally to this work.
g To whom correspondence should be addressed: Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3815-5411 (ext. 3111); Fax: 81-3-5689-7209.
The abbreviations used are: EB, ethidium bromide; BSA, bovine serum albumin; GK, glucokinase; HK, hexokinase.
2 S. Otabe and T. Kadowaki, unpublished observation.
3 I. Miwa, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|