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The First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan
Address all correspondence and requests for reprints to: Dr. Ken-Ichi Ohtani, M.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-22 Showa-machi, Maebashi Gunma 371, Japan. E-mail: kohtani{at}sb.gunma-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thiazolidinedione derivatives have been reported to modify the
glucose transporter (GLUT) system, especially GLUT1 and GLUT4, in both
muscles and liver (3, 5). The effect of these derivatives on peripheral
insulin resistance is supposed to be mediated by the modification of
peroxisome proliferator-activated receptor 
(PPAR) (6). It is
proposed that these derivatives may directly modulate cell function
through the modification of the glucose transporter system or DNA
binding protein such as PPAR in pancreatic ß-cells.
Hyperinsulinemia, observed in noninsulin-dependent diabetic patients,
is subsequently attenuated by the reduction of peripheral insulin
resistance by these agents (1, 2, 5). However, CS-045 does not change
plasma insulin levels in normal fed rats (7). In contrast, an increased
pancreatic insulin content and ß-cell granulation after 12 h or
24 h of the treatment with ciglitazone, a thiazolidinedione
analog, was observed in control-fed ob/ob mice. This
observation suggests the possibility that ciglitazone may have an
additional, perhaps a direct, action on the pancreatic islets (8).
Malaisse and Conget first demonstrated that CS-045 inhibits insulin
release evoked by glucose (9). Recently, Matsuda and his associates
demonstrated that troglitazone has dual effect on insulin
secretory capacity, mediated through the modulation of glucose
transport activity (10). However, the exact mechanism by which CS-045
modulates insulin secretion in pancreatic ß-cells still remains to be
determined and the long-term, direct effect of this drug has not been
established.
HIT-T 15 cells, a Syrian hamster clonal ß-cell line (11), retain glucose-stimulated insulin release (12) and responds to glucose with increased rates of insulin biosynthesis and preproinsulin messenger RNA (mRNA) levels (13, 14). These cells allow for the study of the long-term effects of drugs on insulin secretion and ß-cell proliferation. The present studies were undertaken to examine whether CS-045 modulates ß-cell function in HIT-T 15 cells (11). We investigated whether the changes in intracellular Ca2+ levels may influence the effect of CS-045 on insulin secretion in HIT-T 15 cells. Finally, we determined the long-term effect of CS-045 on HIT-T 15 cell proliferation rate and insulin secretory capacity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental protocols
CS-045 was kindly provided from Sankyo Company, Ltd. (Tokyo,
Japan). In the first experiment, the direct effects of troglitazone
(CS-045: from 10-6 M to 10-4
M) on insulin secretion in HIT-T 15 cells were examined by
static incubation. CS-045 was dissolved in dimethyl sulfoxide (DMSO)
with a final concentration of DMSO below 0.05% in the culture medium
and at the same concentration among all groups to avoid the influence
of increased osmolarity in insulin secretion. The range of
concentrations of CS-045 used in these experiments was chosen because
it has been shown to be clinically relevant in studies of peripheral
insulin resistance (10-6 M to
10-4 M) (15).
Once confluent, the cells were washed three times with fresh medium and then incubated for 10–60 min in 1 ml of experimental media. The medium was then aspirated and stored at -20 C for insulin assay.
In the second experiment, we determined the effect of removal of extracellular Ca2+ by the addition of 1.5 mM EGTA on the 10-4 M CS-045-induced insulin secretion. A total of 1.5 mM EGTA was added to the medium containing 7 mM glucose, 0.9 mM Ca2+. The medium was collected at 10 min following the addition of CS-045, and the aspirated medium was frozen at -20 C for assay.
In the third experiment, the cells were incubated with various concentrations of CS-045 (10-6 M to 10-4 M) for 1, 3, and 5 days. Following the start of the experiment, the culture medium was changed on day 2 (3-day incubation group), or on days 2 and 4 (5-day incubation group). Total insulin secretion for the final 24 h of the experiment was measured. After aspiration of the medium, cells were used for the measurement of cell proliferation rate by MTT assay, as described below.
In the fourth experiment, the cells were incubated with 10-4 M CS-045 in the presence and absence of 50 nM nifedipine for 5 days with culture protocol similar to that of the third experiment. Insulin secretion for the last 24 h and cell proliferation rate were measured.
Measurement of cytoplasmic free Ca2+ concentrations
([Ca2+]i)
The [Ca2+]i was measured according to
the method described previously (16). Briefly, cells were plated on
round glass coverslips (diameter, 20 mm) 3 days before the experiment.
Before the start of an experiment, the cells were incubated with
Hanks’ (Nippon Suisan Co., Tokyo, Japan) buffer containing 7
mM glucose and 4 mM Fura-2/AM (Molecular
Probes, Eugene, OR) for 10 min in a dark box at room temperature. The
cells were then carefully washed twice with Hanks’ buffer containing 7
mM glucose without Fura-2/AM, and a slide glass was placed
in a 1 ml superfusion chamber on the stage of a fluorescence
microscope. A superfusion chamber was filled with 1 ml of 7
mM glucose containing Hanks’ buffer without Fura-2. Fura-2
fluorescence was measured by using Model FC-300 (Mitsubishi Kasei,
Inc., Tokyo, Japan), allowing fluorometry using two excitation
wavelengths (340 nm and 360 nm). Photon counts were amplified every
seconds. A single cell was selected and the fluorescence from a single
cell was monitored before and after the addition of CS-045 (final
concentration: 10-5 M). Changes of
[Ca2+]i was expressed as the F340/F360
ratios, and the peak value after the addition of CS-045 was compared
with basal value just before drug addition.
Preproinsulin mRNA expression determination by RT-PCR method
Cells were incubated with 1 ml of the F-12 K medium containing
CS-045 for 24 h at 37 C. Following 24-h incubation, the medium was
completely removed and cells were washed three times with fresh F-12 K
medium. The cells were then sonicated in 0.8 ml of ISOGENE (Nippon
Gene, Tokyo, Japan) and centrifuged at 12,000 rpm for 10 min.
Total RNA was extracted from supernatants. Hamster preproinsulin mRNA
and ß-actin mRNA were measured by RT-PCR method using GeneAmp EZ rTth
RNA PCR kit (Perkin Elmer, Foster City, CA). The synthetic
preproinsulin primer sequences used in the present study were as
follows: forward primer: 5'-AGCGTGGCTTCTTCTACACACC-3'; backward primer:
5'-GGTGCAGCACTGATCCACAATG-3', according to the preproinsulin
complementary DNA sequence obtained by Bell and Sanchez-Pescador
(17).
According to the results from our preliminary experiment confirming the linear increase of PCR products, following reverse transcription step at 60 C for 30 min, PCR was performed for 32 cycles using a 1-min denaturation step at 94 C and 1-min annealing-extension step at 60 C. An additional 7-min extension step at 60 C was added after the 32 cycles. The PCR product was loaded on an 8% acrylamide gel, and the intensity of fluorescence of the band stained by ethidium bromide was calculated using the National Institutes of Health Image 1.56 program. The relative expression of preproinsulin mRNA to ß-actin mRNA was calculated in each sample.
Cell proliferation rate assay using MTT
Cell proliferation rate was assayed by the method of Mosmann
(18). Incubation medium was completely aspirated after the incubation
with CS-045 for 1, 3, or 5 days. The MTT-formazon product was dissolved
with a phosphate buffer solution. Following the addition of 10% MTT
containing F-12 K medium, cells were incubated at 37 C for 4 h,
the medium was aspirated, and the cells were lysed by the addition of
100 µl DMSO. Ten microliters of DMSO was collected from each sample
and diluted in 90 µl of fresh DMSO. After mixing with a mechanical
plate mixer, the optical density of each sample was measured by Kinetic
Microplate-Reader (Molecular Devices Co., Menlo Park, CA) using test
and reference wavelengths of 490 nm and 650 nm.
DNA fragmentation assay
According to the method previously reported (19), a Cellular DNA
Fragmentation ELISA Kit (Boehringer Mannheim GmbH, Germany) was used
for the determination of DNA fragmentation by CS-045. HIT-T 15 cells
were incubated with 10 µM BrdU overnight at 37 C,
centrifuged at 250 x g for 10 min adjusted to 1
x 105 cells/ml in F-12 K medium and plated in a
96-multiwell plate. The cells were incubated with 10-6
M to 10-4 M CS-045 at 37 C for
72 h, and the supernatant was completely removed from each well.
The cells were lysed by the addition of an incubation buffer enclosed
in the kit for 30 min at room temperature. The multiplate was
centrifuged at 250 x g for 10 min, and the supernatant
was transferred directly to the well of a multiplate precoated with
anti-DNA antibody. Then, samples were incubated for 90 min at room
temperature. After washing, the samples were denatured and fixed by
microwave (500 W) for 5 min and frozen at -20 C for 10 min.
Peroxidase-conjugated anti-BrdU solution was added and incubated for an
additional 90 min at room temperature. Substrate solution was added,
and it was incubated at room temperature in the dark on a plate shaker
at 250 rpm for 10 min. The reaction mixture was stopped by adding
5.6% H2SO4 to each well and incubating the
plate for 1 min on the shaker at 250 rpm. The absorbance was measured
at 450 nm (reference wavelength: 690 nm) against substrate solution as
a blank.
Measurement of ß-cell insulin content
After HIT-T 15 cells were incubated for 24 h with
10-6–10-4 M CS-045 at 37 C, the
supernatant was removed. The cell insulin content was measured by the
method of Grodsky and Frosham (20). The cells were solubilized in 75%
acid-ethanol solution. Ten microliters of the cell extract was frozen
at -20 C and used for insulin assay.
RIA
Insulin concentrations in the medium and cell extract were
determined by commercially available RIA kits (Phadeceph Insulin,
Pharmacia Japan, Tokyo, Japan).
Statistical analysis
All data represent mean ± SE. The statistical
analysis of the means was performed by ANOVA, followed by Duncan’s
multiple range test for the individual comparisons of the means. The
F340/F360 ratios were analyzed by Student’s paired t
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Both the 10-min and the 60-min
incubation in CS-045 caused a dose-dependent increase in insulin
secretion. At the maximal concentration (10-4
M), a 10-min incubation in CS-045 caused a significant
increase in insulin secretion (Fig. 1, left panel). In the
60-min incubation, the effects of 10-5 and
10-4 M CS-045 were statistically significant.
The addition of 10-4 M CS-045 increased
insulin secretion for 60 min by 182.4 ± 11.9% of controls (Fig. 1, right panel).
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shows a typical change of
[Ca2+]i after the addition of
10-5 M CS-045 in HIT-T 15 cells. As shown in
Fig. 2, the addition of 10-5 M CS-045 caused
an immediate increase of [Ca2+]i. The
increase of the F340/F360 ratio by the 10-5 M
CS-045 addition is statistically significant (P <
0.001), compared with the value before the addition of
10-5 M CS-045 (1.520 ± 0.107
vs. 0.940 ± 0.075, P < 0.001).
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shows the effect of removal of
extracellular Ca2+ by the addition of 1.5 mM
EGTA on the stimulatory effect of CS-045. The addition of EGTA
completely abolished the stimulatory effect of 10-4
M CS-045 (10-min) on insulin secretion. These results
suggest that Ca2+ influx from extracellular space may be
important in the short-term stimulation of insulin secretion by
CS-045.
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). Similarly, the incubation with
CS-045 for 5 days dose dependently decreased cell proliferation rate in
HIT-T 15 cells. The most pronounced effect was seen following exposure
to 10-4 M CS-045 for 5 days. However, an
inhibitory effect on insulin secretion was already apparent as early as
24 h at the highest concentration of CS-045 (10-4
M) (Fig. 4B). At 3-day incubation, an inhibitory effect on
insulin secretion was significant over 10-5 M
and at 5-day incubation, CS-045 dose dependently inhibited insulin
secretion for the final 24 h.
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Using ELISA, we determined changes in the amount of DNA fragmentation
of HIT-T 15 cells after 3-day incubation with CS-045 (Fig. 5). The amount of DNA fragmentation dose
dependently increased in HIT-T 15 cells treated with CS-045 for 3 days,
indicating that the addition of CS-045 may induce apoptosis after 3-day
incubation. Apoptosis may contribute to the reduction of ß-cell
number caused by CS-045.
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shows the effects of 50
nM nifedipine on the reduction of ß-cell proliferation
rate, insulin secretion for 24 h, and the amount of DNA
fragmentation caused by 3-day incubation with 10-4
M CS-045. The addition of nifedipine failed to attenuate
the reduction of ß-cell proliferation rate and insulin secretion by
10-4 M CS-045 (Fig. 6, A and B). In contrast,
the addition of nifedipine completely abolished an increase in the
amount of DNA fragmentation by 10-4 M CS-045
(Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CS-045 binds to the sulfonylurea receptor in noncompetitive manner in both clonal HIT-T 15 cells and isolated pancreatic islet cells (10). Thiazolidinedione analogs have been reported to increase the glucose transporter expression in the peripheral tissues such as liver and muscles (3, 5). It has been recently reported that CS-045 stimulated insulin secretion and increased glucose uptake, suggesting that glucose transport activity may be increased by CS-045 (10). It has been demonstrated that ATP-sensitive K+ channel are not closed by the addition of CS-045 (10), distinct from sulfonylurea itself (22). The present data raised the possibility that the effect of CS-045 may not be mediated only by metabolic changes such as the changes in glucose transporter system or intracellular ATP/ADP ratio, because an immediate within seconds increase in [Ca2+]i was observed after the addition of CS-045.
From our present and previously reported observations (21), we supposed
that CS-045 acts by immediately opening the voltage-dependent
Ca2+ channels through an unknown mechanism after binding to
specific binding sites. CS-045 has been recently reported to modify the
protein kinase C activity in Rat-1 fibroblasts (4). This finding raised
the possibility that CS-045 may affect intracellular phosphatidyl
inositide metabolism. In pancreatic ß-cells, activation of
phosphoinositide (PI) turnover increases inositol 1,4,5-triphosphate
that releases Ca2+ from the endoplasmic reticulum, which
can trigger a peak of insulin secretion (23). It is possible that in
ß-cells thiazolidinedione analogs may cause an increase in PI
turnover leading to an increase of [Ca2+]i,
and thus resulting in the stimulation of insulin secretion in HIT-T 15
cells. In addition, recent studies reported that PPAR may involve an
improvement of peripheral insulin resistance by thiazolidinedione
derivatives (24). Further studies are needed to determine which
intracellular mechanism should involve the stimulation of insulin
secretion by CS-045, and Ca2+ influx from extracellular
space.
The present study uniquely demonstrated that long-term incubation with CS-045 inhibits insulin secretion and cell proliferation rate and increases the amount of DNA fragmentation, indicating the induction of apoptosis in ß-cell. A maximal physiological concentration of CS-045 is about 10-5 M in the serum after oral administration in clinical usage (25). The present data raised the possibility that the continuation of higher concentration (over 10-5 M) of CS-045 may induce ß-cell hypofunction and cause apoptosis in pancreatic ß-cells in vivo. Recent studies about apoptotic regulatory proteins demonstrated that bcl-2 was found in fetal islets (26) and that bcl-xL, bax, and bag-1 are expressed in adults islets (27). It is possible that these apoptotic proteins may be involved in the apoptotic response to CS-045 in ß-cells. Further investigations about the changes in apoptotic proteins by CS-045 are in progress now.
Nifedipine abolished the CS-045 effect on DNA fragmentation, but the
addition of nifedipine failed to attenuate the reduction of cell
proliferation rate and long-term insulin secretion by CS-045. These
observations suggest that the mechanism by which CS-045 induces
apoptosis could be different from the one by which it reduces ß-cell
proliferation rate and long-term insulin secretory capacity.
Ca2+ influx has been reported to induce apoptosis in rat
liver (28). It has also been shown by Juntti-Berggren et al.
that an Ig M-mediated increase in the Ca2+ influx into
RINm5FA insulin producing cells that are blocked by a voltage-dependent
L-type Ca2+ channel antagonist, verapamil, contributes to
pancreatic ß-cell destruction by apoptosis (29). In the present
studies, the finding that nifedipine, a voltage-dependent L-type
Ca2+ channel antagonist (30), abolished the CS-045 effect
on the amount of DNA fragmentation may indicate that the induction of
apoptosis by CS-045 may be in part mediated by the activation of
voltage-dependent L-type Ca2+ channel. In addition, it has
been shown that continuous exposure to PPAR activators led to adipose
conversion in 3T3-L1 preadipocytes, during which PPAR and its
heterodimerization partner (retinoid X receptor) were induced and
treatment of cells cultured in delipidated serum with retinoic acid
caused death of the cells by apoptosis (31). The mode of the cell death
of the Reuber hepatoma cell line FaO, in which PPAR expression is
highest, was characterized as apoptosis (32). These previous
observations may support a possibility that the long-term incubation
with CS-045, PPAR activator, may induce apoptosis in HIT-T 15 cells.
However, to explain the exact mechanism of CS-045-induced reduction of
cell proliferation rate, cell hypofunction, and apoptosis in pancreatic
ß-cells, further studies should be necessary.
The present data provide evidence that thiazolidinedione analog, CS-045, can acutely stimulate in vitro insulin secretion from HIT-T 15 cells. The insulin secretory action of this agent is proposed to be in part mediated by the activation of Ca2+ influx through voltage-dependent Ca2+ channel. In addition, this study uniquely demonstrates that long-term incubation with CS-045 inhibits ß-cell proliferation rate in vitro and may induce apoptosis, which could cause the reduction of ß-cell mass by CS-045.
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Acknowledgments |
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Received June 23, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is activated by inducers
of adipogenesis, including thiazolidinedione drugs. Eur J
Biochem 239:1–7[Medline]
(PPAR). J Biol Chem 270:12953–12956This article has been cited by other articles:
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