COMMUNICATION
Cyclic ADP-ribose and Inositol 1,4,5-Trisphosphate as Alternate Second Messengers for Intracellular Ca2+ Mobilization in Normal and Diabetic beta -Cells*

Shin TakasawaDagger , Takako AkiyamaDagger , Koji NataDagger , Michio KurokiDagger , Akira TohgoDagger , Naoya NoguchiDagger §, Seiichi KobayashiDagger , Ichiro KatoDagger , Toshiaki Katada, and Hiroshi OkamotoDagger par

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

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 beta -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 beta -cells such as ob/ob mouse islets and RINm5F cells. We measured the cADPR content in these beta -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 beta -cells released Ca2+ in response to IP3 but not to cADPR. In the diabetic beta -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 beta -cells secreted insulin rather by carbamylcholine than by glucose.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Glucose is the primary stimulus of insulin secretion and synthesis in pancreatic beta -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 beta -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 beta -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

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 beta -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 beta -cells by glucose stimulation but not in diabetic beta -cells.


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Fig. 1.   Time course of cADPR content in response to glucose stimulation in rat islets. cADPR in islets was extracted, concentrated, and measured by RIA using an antibody against cADPR (15). The cADPR content was calculated by accounting for the recovery of cADPR in the extraction and concentration procedures and expressed as fmol/µg islet protein. n = 3-4 for each point. Vertical bars indicate S.E. The statistical analysis was performed using Student's t test. Asterisks indicate significant difference from the value with 2.8 mM glucose at p < 0.05. Most recently, Malaisse et al. (41) measured the cADPR content in rat islets (35.4 ± 1.7 fmol/µg of protein) and reported that it appeared not to be significantly affected by glucose. In the present experiments, fasting of rats before isolation of the islets and the usage of Hanks' solution containing 2.8 mM glucose during the islet isolation as described under "Experimental Procedures" may account for the rapid and significant increase of cADPR content in islets in response to glucose stimulation. Furthermore, we determined the cADPR content by assessing the recovery of cADPR in the extraction and concentration procedures as described under "Experimental Procedures," but they did not. Malaisse et al. (41) incubated islets for a relatively longer period (90 min), whereas we incubated them for 5-40 min. In our experiment, the cADPR content in islets increased significantly within 10-min incubation, but the increase after 40-min incubation was not statistically significant. These differences in the experimental conditions may be responsible for the different results.

                              
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Table I
cADPR content of C57BL/6J, ob/ob islets, and RINm5F beta -cells
Islets and RINm5F cells were incubated at 37 °C for 10 min with KRB containing 2.8 or 20 mM glucose. Values are mean ± S.E. (fmol/µg of protein) of at least triplicate experiments.

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 beta -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 beta -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 beta -cells and that the Ca2+ release machinery by cADPR may be replaced with that by IP3 in diabetic beta -cells.


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Fig. 2.   Calcium mobilization by cADPR and IP3 from microsomes of C57BL/6J, ob/ob islets, and RINm5F beta -cells. Ca2+ release from microsomes (10 µg of protein) was induced by the addition of 100 nM cADPR or 1 µM IP3 in the presence of 7 µg/ml calmodulin as described (13). Values are mean ± S.E. of triplicate experiments. The inset shows a typical result of Ca2+ release from C57BL/6J mouse islet microsomes. The Ca2+ release by cADPR was not affected by the pre-addition of IP3, as seen in rat islet microsomes (12).

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 beta -cells, resulting in the abnormal response to glucose.


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Fig. 3.   RT-PCR analyses of C57BL/6J and ob/ob islets. A, expression of CD38 and BST-1 mRNAs. Expression of CD38 and BST-1 in rat islets has been reported previously (17, 18, 42). B, expression of RyR and IP3R mRNAs. Lane 1, skeletal muscle; lane 2, heart; lane 3, cerebrum; lane 4, cerebellum; lane 5, liver; lane 6, kidney; lane 7, spleen; lane 8, small intestine; lane 9, islets of C57BL/6J mouse; lane 10, islets of ob/ob mouse. Expression of IP3R-3 in normal rat islets and the increased expression of IP3R-3 mRNA in RINm5F cells have also been reported (26). PCR products were of expected size on agarose gel electrophoresis and the nucleotide sequences of the resultant PCR products were confirmed by dideoxy sequencing. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

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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Fig. 4.   Insulin secretion from C57BL/6J and ob/ob islets by glucose and carbamylcholine. Insulin secretion from islets of C58BL/6J (n = 5-12) and ob/ob mice (n = 6-9) was examined. The statistical analysis was performed using Student's t test. #, p < 0.01 when compared with 2.8 mM glucose of C57BL/6J islets. ##, p < 0.01 when compared with 20 mM glucose of C57BL/6J islets. * and **, p < 0.05 and p < 0.01 when compared with 2.8 mM glucose of ob/ob islets, respectively.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

par 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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Abstract
Introduction
Procedures
Results & Discussion
References

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