From the Department of Biochemistry, Tohoku
University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai
980-77, Miyagi, Japan, and ¶ Department of Physiological
Chemistry, Faculty of Pharmaceutical Sciences, University of
Tokyo, Tokyo 113, Japan
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ABSTRACT |
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Intracellular Ca2+ mobilization
occurs in a variety of cellular processes and is mediated by two major
systems, the inositol 1,4,5-trisphosphate (IP3) and cyclic
ADP-ribose (cADPR) systems. cADPR has been proposed to be a second
messenger for insulin secretion induced by glucose in pancreatic
-cells (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993)
Science 259, 370-373). Here we show that the cADPR signal
system for insulin secretion is replaced by the IP3 system
in diabetic
-cells such as ob/ob mouse islets and RINm5F
cells. We measured the cADPR content in these
-cells by
radioimmunoassay and found that the increase of the cADPR content by
glucose did not occur in ob/ob mouse islets and RINm5F
cells, whereas the increased cADPR level by glucose was observed in
normal rat and mouse islets. Microsomes of these diabetic
-cells
released Ca2+ in response to IP3 but not to
cADPR. In the diabetic
-cells, CD38 (ADP-ribosyl cyclase/cADPR
hydrolase) and type 2 ryanodine receptor mRNAs were scarcely
detected and, in contrast, an increased expression of IP3
receptor mRNAs was observed. The diabetic
-cells secreted
insulin rather by carbamylcholine than by glucose.
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INTRODUCTION |
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Glucose is the primary stimulus of insulin secretion and synthesis
in pancreatic -cells of the islets of Langerhans (1-3). Increases in the intracellular Ca2+ concentration mediate
the biochemical events that couple glucose stimulation to insulin
secretion and mobilization of Ca2+ from intracellular
stores in the endoplasmic reticulum as well as Ca2+ influx
from extracellular sources (4) are important in this process (5). It
has been thought that inositol 1,4,5-trisphosphate (IP3)1 is a
second messenger for Ca2+ mobilization from intracellular
stores (6). We have recently proposed another pathway for the increase
in the intracellular Ca2+ concentration for insulin
secretion by glucose via the CD38 (ADP-ribosyl cyclase/cyclic
ADP-ribose (cADPR) hydrolase)-cADPR signal system in pancreatic
-cells (7-14).
In the present study, we showed that the
cADPR-dependent signal system for intracellular
Ca2+ mobilization for insulin secretion did not work in
diabetic -cells such as ob/ob mouse islets and RINm5F
cells, which in contrast used the IP3-dependent
Ca2+ signal system. Two systems for intracellular
Ca2+ mobilization thus appear to be differentially used
depending on whether the condition is physiological or
pathological.
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EXPERIMENTAL PROCEDURES |
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Measurement of cADPR by Radioimmunoassay--
Animals were
fasted for 24 h before the experiments. 1200-2000 pancreatic
islets were isolated from 6-10 animals of male Wistar rats (250-350
g, SLC, Hamamatsu, Japan), C57BL/6J mice, or ob/ob mice
(8-14 weeks old, Jackson Laboratories) by the collagenase procedure
using Hanks' solution containing 2.8 mM glucose (3, 9,
12). Hand-picked islets (300-500/tube) were immediately incubated for
5-40 min at 37 °C in 2 ml of Krebs-Ringer's bicarbonate buffer
(KRB) containing 0.2% bovine serum albumin (3, 9, 12-14) and glucose
(2.8 or 20 mM) under an atmosphere of 95%
O2/5% CO2. cADPR was extracted and
concentrated as follows. After incubation, the incubation medium was
removed and the islets were sonicated (3 × 10 s) in 370 µl
of perchloric solution (2.5%, v/v). Likewise, cultured rat RINm5F
insulinoma cells (5 × 106 cells) were sonicated in
370 µl of perchloric acid solution. The homogenates were stored at
80 °C and later defrosted and centrifuged for 10 min at
13,000 × g. The supernatant (330-350 µl) of the
homogenates was then mixed with 150 µl of a suspension of Norit A (27 mg/ml in H2O, Nacalai tesque, Kyoto, Japan). After 30 min
incubation at 37 °C, the samples were again centrifuged, and the
supernatant was discarded. The pellet was washed three times with 1.0 ml of H2O, resuspended in a
pyridine/ethanol/H2O mixture (10:50:40, v/v/v), and
incubated for 120 min at 37 °C. After a further centrifugation, the
supernatant was collected and evaporated (Speedvac; Savant Instrument
Inc., Farmingdale, NY). The recovery of cADPR, monitored by the
recovery of [3H]cADPR added in each homogenate, was
67.8 ± 1.49% (n = 47). Correction was introduced
for the recovery of cADPR. The cADPR content of the cell extracts was
measured by a radioimmunoassay (RIA) as described (15). Briefly, the
evaporated materials eluted from Norit A charcoal were resuspended with
50 µl of H2O and then incubated at 25 °C for 2 h
with bovine alkaline phosphatase (Sigma) and venom phosphodiesterase
(Worthington) at final concentrations of 50 and 2 units/ml,
respectively, in 100 mM imidazole HCl (pH 7.5), 2 mM MgCl2, 100 mM NaCl, and 400 mM KCl. The reaction was terminated by adding a solution of
trichloroacetic acid (at a final concentration of 4%, w/v) and kept on
ice for 20 min. A clear supernatant was obtained after centrifugation
at 13,000 × g for 10 min. An aliquot (10-20 µl) of
the supernatant was immediately neutralized with a solution of 2 M Tris base and subjected to the procedure of cADPR
measurement (15). As a control, another aliquot was heated at 95 °C
for 10 min before being neutralized and analyzed by the RIA. The
identity of the immunoreactive cADPR in islets was also confirmed after
its separation by high performance liquid chromatography with an
anion-exchange column, as described previously (15). Immunoreactive
cADPR was eluted as a single peak at the same retention time as the
standard of cADPR. Almost 100% of the total immunoreactive cADPR
applied to the column was recovered at the elution position of cADPR
(data not shown).
Calcium Release Assay--
Microsomes were prepared as described
previously (9, 12-14). In brief, 2000 hand-picked islets from male
mice (C57BL/6J or ob/ob) or RINm5F cells (1 × 107) were homogenized with a pellet mixer (Treff,
Degersheim, Switzerland) in 0.2 ml of acetate intracellular medium
composed of 250 mM potassium acetate, 250 mM
N-methylglucamine, 1 mM MgCl2, and
20 mM Hepes (pH 7.2) supplemented with 0.5 mM
ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, and 0.5 mM
phenylmethylsulfonyl fluoride. After the homogenates had been
centrifuged for 45 s at 13,000 × g, the
microsomes were prepared by Percoll density gradient centrifugation at
20,000 × g for 40 min at 10 °C. Release of
Ca2+ was monitored in 0.6 ml of intracellular medium
composed of 250 mM potassium gluconate, 250 mM
N-methylglucamine, 1 mM MgCl2, and
20 mM Hepes (pH 7.2) supplemented with 1 mM
ATP, 4 mM phosphocreatine, creatine phosphokinase (2 units/ml), 2.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 7 µg/ml bovine brain calmodulin (13,
14), and 3 µM Fluo 3 with the addition of 30 µl of the islet or RINm5F microsome fraction (10 µg of protein) (9, 12-14). The NADH-cytochrome b5 reductase activity of the
microsome fraction was measured as described (16), and the activities
(rat islets, RINm5F cells, C57BL/6J islets and ob/ob islets;
1.21 ± 0.14, 1.45 ± 0.16, 2.08 ± 0.82 and 1.88 ± 0.36 µmol/min/mg of protein, respectively) were not statistically
different, indicating that similar fractions were isolated and studied
in each case. Fluo 3 fluorescence was measured at 490 nm excitation and
535 nm emission with a Jasco CAF-110 intracellular ion analyzer (Tokyo,
Japan) at 37 °C (13, 14). Total accumulated Ca2+ in
islet microsomes was estimated by the increase of Fluo 3 fluorescence caused by the addition of 200 nM ionomycin (Sigma) to the
Ca2+ release medium containing the microsomes, and the
ambient free Ca2+ concentration ([Ca2+]) was
calculated using the following equation, as described
previously (13, 14): [Ca2+] = Kd × (F Fmin)/(Fmax
F), where Kd = 400 nM.
Insulin Secretion from Isolated Islets-- Islets (10/tube) were preincubated at 37 °C for 30 min in 1 ml of KRB buffer containing 2.8 mM glucose under an atmosphere of 95% O2/5% CO2. Following the preincubation, the medium was discarded, and islets were incubated in 0.1 ml of KRB buffer containing glucose (2.8 or 20 mM) or carbamylcholine (0.2 mM). After 30-min incubation, the medium was removed from the islets, and the insulin content was determined by RIA using a rat insulin RIA kit (Amersham Corp.) and rat insulin standard (9, 12, 13).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)-- Total RNA was isolated (8-10, 14) and 1 µg of total RNA was incubated with 500 units of SuperscriptTM (Life Technologies, Inc.) for 1 h at 42 °C in a total reaction volume of 20 µl containing 1 × RT buffer (50 mM Tris-HCl (pH. 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol), a 0.5 mM concentration of each dNTP, 110 units of RNase inhibitor (Takara Shuzo, Otsu, Japan) and 1.5 ng/µl of oligo(dT)12-18 (Pharmacia Biotech Inc., Uppsala, Sweden). The RT sample (1 µl) was used for PCR in a final volume of 50 µl as described (8, 14, 17, 18). The PCR primers correspond to nucleotides 2-23 and 900-921 for mouse CD38 mRNA (19), 6-25 and 934-953 for mouse bone marrow stromal cell antigen-1 (BST-1) mRNA (20, 21), 363-385 and 465-487 for mouse ryanodine receptor (RyR)-1 mRNA (22), 641-663 and 869-891 for mouse RyR-2 mRNA (22), 374-386 and 729-751 for mouse RyR-3 mRNA (22), 7843-7866 and 8386-8409 for mouse IP3 receptor (IP3R)-1 mRNA (23), 1828-1851 and 2003-2026 for mouse IP3R-2 mRNA (24), 29-52 and 174-197 for mouse IP3R-3 mRNA (24), 11-31 and 107-130 for mouse IP3R-4 mRNA (24), 868-890 and 1043-1064 for mouse IP3R-5 mRNA (25), and 151-171 and 967-987 for mouse glyceraldehyde-3-phosphate dehydrogenase mRNA.2 At present, whether BST-1 functions as a cADPR-metabolizing enzyme in islets or not is unclear (18, 27). The nucleotide sequences of the resultant PCR products were confirmed by dideoxy sequencing.
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RESULTS AND DISCUSSION |
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We first incubated rat islets with low and high glucose and
measured the cADPR content by RIA. As shown in Fig.
1, it was found that the cADPR content of
islets incubated with 20 mM glucose was increased within 5 min, whereas the cADPR content of islets incubated with 2.8 mM glucose was not. The results were consistent with our
previous observation based on the Ca2+ releasing activity
of cADPR in islet extracts (12). We also isolated islets from C57BL/6J
mice, incubated them with glucose, and measured the cADPR content. The
cADPR content was significantly increased by glucose stimulation (Table
I). We next isolated islets from mutant
mice of C57BL/6J, ob/ob diabetic mice, and measured the
cADPR content. Some amounts of cADPR in ob/ob islets was
detected when incubated in 2.8 mM glucose, but the cADPR
content was not increased by glucose stimulation (Table I). Because rat insulinoma-derived RINm5F -cells, which synthesize and secrete very
little insulin in response to glucose (28), were reported to be
insensitive to cADPR in Ca2+ mobilization from
intracellular pools (29, 30), the cADPR content of RINm5F cells was
also examined. The content of cADPR in RINm5F cells was scarcely
detectable even by glucose stimulation (Table I). These results suggest
that cADPR acts as a second messenger in normal
-cells by glucose
stimulation but not in diabetic
-cells.
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Microsomes from C57BL/6J islets released Ca2+ in response
to cADPR but scarcely in response to IP3 (Fig.
2). This response to cADPR was completely
attenuated by the prior addition of 100 nM 8-amino
(NH2)-cADPR, an antagonist of cADPR (31, 32) (Fig. 2,
inset). In contrast to normal islet microsomes, microsomes from diabetic -cells showed quite different responses to the two
Ca2+-releasing second messengers: ob/ob islet
microsomes released very little Ca2+ by cADPR but released
much Ca2+ by IP3. RINm5F cell microsomes
responded well to IP3 to release Ca2+ but
scarcely responded to cADPR (Fig. 2). The Ca2+ releases by
IP3 in ob/ob islet and RINm5F microsomes were
attenuated by the prior addition of 100 µg/ml heparin, an inhibitor
of IP3 receptors, (12, 33), and a small Ca2+
release response to cADPR observed in ob/ob islet microsomes was also attenuated by the prior addition of 8-NH2-cADPR.
These results together with the results that ob/ob
-cells
and RINm5F cells released Ca2+ in response to
IP3 but scarcely in response to cADPR by patch clamp
experiments (29, 30, 34) strongly suggest that cADPR acts as a second
messenger for Ca2+ mobilization from intracellular stores
in normal
-cells and that the Ca2+ release machinery by
cADPR may be replaced with that by IP3 in diabetic
-cells.
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As shown in Fig. 3A, the CD38
mRNA level was significantly decreased in ob/ob islets.
The decrease of CD38 mRNA in ob/ob islets may explain
the low response in cADPR content by glucose stimulation because CD38
has both the ADP-ribosyl cyclase and the cADPR hydrolase activity, and
the increase of cADPR by glucose stimulation is achieved by inhibition
of the cADPR hydrolase activity of CD38 (9-11). Decreased CD38
mRNA was also reported in islets of Goto-Kakizaki diabetic rats
(35), which show impaired glucose-induced insulin secretion (36). In
addition, our recent experiments indicated that the cADPR and insulin
secretion levels of CD38 cDNA-introduced RINm5F clones 1 and 3, RINm5F-derived cell lines into which CD38 had been introduced, were
significantly higher3 than
that of RINm5F cells, in which CD38 mRNA expression was not
detected (17, 18). Concerning intracellular Ca2+ release
channels, the mRNA expression of RyR-2, which is postulated to be a
Ca2+ release channel for cADPR (14, 37), was clearly
detected in normal islets but not in ob/ob islets. In
contrast, IP3R mRNAs (IP3R-1,
IP3R-2, IP3R-4, and IP3R-5) were
not detected in normal islets but were clearly detected in
ob/ob islets, and although IP3R-3 mRNA was
slightly detected in normal islets, the mRNA was significantly
increased in ob/ob islets (Fig. 3B), well fitting with the observation that IP3-induced Ca2+
mobilization preferentially worked in ob/ob islet microsomes (Fig. 2). From these results, it is possible that changes in gene expression involved in intracellular Ca2+ mobilization
occurred in diabetic -cells, resulting in the abnormal response to
glucose.
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The above observed phenomena well explain the differences between C57BL/6J and ob/ob islets in insulin secretion by glucose and carbamylcholine (Fig. 4): ob/ob islets secreted far more insulin by carbamylcholine stimulation, which is thought to be mediated by IP3-induced Ca2+ mobilization, than did C57BL/6J islets. Moreover, simultaneous stimulation to ob/ob islets by glucose and carbamylcholine did not induce much more insulin than that by glucose and carbamylcholine independently, whereas the simultaneous stimulation to C57BL/6J islets induced significantly more insulin than that by glucose alone (Fig. 4). These results suggest that, in normal islets, Ca2+ mobilization from intracellular stores by glucose stimulation for insulin secretion is mediated by the cADPR-dependent system and other stimulants for insulin secretion such as acetylcholine mobilize a little Ca2+ via the IP3-dependent system independently. In ob/ob islets, the intracellular Ca2+ mobilization for insulin secretion appears to be mainly mediated by the IP3-dependent system.
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Intracellular Ca2+ mobilization has been reported to be mediated by both IP3 and cADPR in a variety of cells (33, 38, 39). However, some cells are reported to utilize preferentially one of the two second messengers (12, 40). The present results indicate that cells can use one of the messengers, depending on differences in the cellular conditions, physiological, or pathological.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Yasunari Kanda and Kenji Kontani, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo for useful suggestions in the determination of cADPR content in islets and Brent Bell for valuable assistance in preparing the manuscript for publication.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.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.
§ Recipient of a fellowship from the Japan Society for the Promotion of Science.
To whom all correspondence should be addressed: Dept. of
Biochemistry, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-77, Miyagi, Japan. Tel.: 81-22-717-8079; Fax: 81-22-717-8083.
1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; cADPR, cyclic ADP-ribose; KRB, Krebs-Ringer's bicarbonate buffer; RIA, radioimmunoassay; RT, reverse transcriptase; PCR, polymerase chain reaction; BST-1, bone marrow stromal cell antigen-1; RyR, ryanodine receptor; IP3R, IP3 receptor; 8-NH2-cADPR, 8-amino-cADPR.
2 D. E. Sabath, H. E. Broome, and M. B. Prystowsky, EBI accession no. M32599.
3 cADPR levels of RINm5F-CD38 clones 1 and 3 were significantly higher (42.54 ± 5.07 and 43.11 ± 4.51 fmol/µg protein, respectively) than that of RINm5F cells (0.19 ± 0.013 fmol/µg of protein, see also Table I. Insulin secretion from RINm5F-CD38 clones 1 and 3 (0.42 ± 0.11 and 0.48 ± 0.056% of total insulin in cells/h, n = 3, respectively) incubated with 20 mM glucose was also significantly higher than that of RINm5F cells (0.078 ± 0.0078% of total insulin in cells/h, n = 3).
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REFERENCES |
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