COMMUNICATION
CD38 Disruption Impairs Glucose-induced Increases in Cyclic ADP-ribose, [Ca2+]i, and Insulin Secretion*

Ichiro Kato, Yasuhiko YamamotoDagger , Miki Fujimura, Naoya NoguchiDagger , Shin Takasawa, and Hiroshi Okamoto§

From the Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan

    ABSTRACT
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Abstract
Introduction
References

Increases in [Ca2+]i in pancreatic beta cells, resulting from Ca2+ mobilization from intracellular stores as well as Ca2+ influx from extracellular sources, are important in insulin secretion by glucose. Cyclic ADP-ribose (cADPR), accumulated in beta cells by glucose stimulation, has been postulated to serve as a second messenger for intracellular Ca2+ mobilization for insulin secretion, and CD38 is thought to be involved in the cADPR accumulation (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Here we created "knockout" (CD38-/-) mice by homologous recombination. CD38-/- mice developed normally but showed no increase in their glucose-induced production of cADPR in pancreatic islets. The glucose-induced [Ca2+]i rise and insulin secretion were both severely impaired in CD38-/- islets, whereas CD38-/- islets responded normally to the extracellular Ca2+ influx stimulants tolbutamide and KCl. CD38-/- mice showed impaired glucose tolerance, and the serum insulin level was lower than control, and these impaired phenotypes were rescued by beta cell-specific expression of CD38 cDNA. These results indicate that CD38 plays an essential role in intracellular Ca2+ mobilization by cADPR for insulin secretion.

    INTRODUCTION
Top
Abstract
Introduction
References

Glucose stimulates insulin secretion in pancreatic beta cells of the islets of Langerhans, and mobilization of Ca2+ from intracellular stores in the endoplasmic reticulum as well as Ca2+ influx from extracellular sources has an important role in this process (1-3). Concerning the mechanism of Ca2+ influx from extracellular sources, it has been thought that ATP generated in the process of glucose metabolism inhibits the ATP-sensitive K+ channel, causing beta cell membrane depolarization, thereby opening the voltage-dependent Ca2+ channel and resulting in Ca2+-influx from the extracellular space (4). On the other hand, 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)1 hydrolase)-cADPR signal system in pancreatic beta cells (5-8): millimolar concentrations of ATP generated in the process of glucose metabolism induce cADPR accumulation by inhibiting the cADPR hydrolase activity of CD38 (9-12), and cADPR then acts as a second messenger for intracellular Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (5, 13, 14).

In the present study, we produced knockout mice carrying a null mutation in the CD38 gene by homologous recombination and found that CD38 disruption impairs glucose-induced increases in cADPR, intracellular Ca2+ concentration ([Ca2+]i), and insulin secretion.

    EXPERIMENTAL PROCEDURES

Construction of the Targeting Vector-- A 15-kbp mouse genomic DNA (BamHI-BamHI) fragment containing the first exon of the CD38 gene (DDBJ/EBI/GenBankTM accession number AB016868) was cloned from a TT2 embryonic stem (ES) cell (15) genomic library. The loxP sequence (16) and the BamHI site were introduced into the HindIII site located 0.5 kbp 5' upstream of exon 1 containing the translation initiation site (see Fig. 1A). A neomycin resistance gene with a PGK-1 promoter but without a poly(A)+ addition signal (17), which is flanked by two loxP sequences, was inserted into the HindIII site located 0.6 kbp 3' downstream of exon 1. A diphtheria toxin A (DTA) fragment gene with a MC1 promoter (18) was ligated on the 3' terminus for negative selection, and the targeting vector was designated as pCD38-loxP-DTA.

Generation of Knockout Mice-- TT2 embryonic stem cells were transfected with NotI-cleaved pCD38-loxP-DTA. G418-resistant clones were analyzed by Southern blotting (19) with the Probe 1 (see map in Fig. 1A). The correctly targeted ES cells were injected into eight-cell embryos of ICR mice to produce germ-line chimeras. The Cre-containing plasmid (pBS185; Life Technologies) was microinjected at 2 µg/ml into the male pronuclei of fertilized eggs obtained by mating between the male chimera and (C57Bl/6J × DBA) F1 female mice as described (20). Some of the newborn mice (F1) were found to carry the deleted allele (5.3-kbp BamHI-BamHI fragment; see Fig. 1, A and C) that lacks both CD38 exon 1 and neo cassette. Mutant and wild-type mice used were the littermates intercrossed between male and female heterozygotes that had been backcrossed to ICR mice for two to three generations.

RT-PCR and Western Blot Analysis-- Total RNA (2 µg) was isolated from islets and reverse-transcribed as described (21). PCR was performed on each reverse-transcribed sample (1/20, 1 µl) for 30 cycles for CD38 and GAPDH. The sequences of the primers for CD38 cDNA (22) amplification were 5'-ACAGACCTGGCTGCCGCCTCCCTAG-3' and 5'-GGGGCGTAGTCTTCTCTTGTGATGT-3'; for GAPDH cDNA amplification they were as described (21). Western blot analysis was carried out on 50 µg of islet extract as described previously (9) using an anti-CD38 polyclonal antibody raised against a peptide fragment of mouse CD38 (residues 279-301 in Ref. 22).

CD38 Enzymic Assay-- One thousand islets isolated by collagenase digestion (23, 24) were homogenized in 1 ml of 50 mM MES (pH 7.2), 0.25 M sucrose, 1 mM EDTA. ADP-ribosyl cyclase and cADPR hydrolase activities were measured as described (8). NAD+-glycohydrolase activity was measured as described (25, 26).

Measurement of cADPR by Radioimmunoassay-- Four hundred islets were incubated for 10 min at 37 °C in Krebs-Ringer buffer containing 0.2% bovine serum albumin and glucose (2.8 or 20 mM), followed by radioimmunoassay of cADPR (21). As control, an aliquot of every islet extract was heated to 95 °C for 10 min before being neutralized and analyzed by the radioimmunoassay. The heat treatment converted the immunoreactive cADPR to ADP-ribose, resulting in no cross-reaction with the anti-cADPR antibody. The immunoreactivity of all the samples was abolished by the heat treatment.

Measurement of [Ca2+]i-- Intact islets were loaded with fura-2 by a 30-min incubation at 37 °C in Krebs-Ringer buffer containing 25 µM acetoxymethyl ester of fura-2 (Dojindo, Kumamoto, Japan). Islets were then attached to a cover glass using Cell-Tak (Collaborative Biomedical Products, Bedford, MA). The specimen chamber was continuously perfused with Krebs-Ringer buffer at 37 °C at a rate of 2.5 ml/min. Fura-2 dual excitation (340 and 380 nm) and fluorescence detection (510 nm) were accomplished, and the 340/380 nm fluorescent ratio was converted to [Ca2+]i using QuantiCell 900 (Applied Imaging, Sunderland, UK); the interval of recordings was 4 s.

Measurement of Insulin Secretion from Isolated Islets-- Twenty islets were preincubated for 2 h at 37 °C in 1 ml of RPMI 1640 medium containing 10% fetal calf serum and 2.5 mM glucose and then incubated for another 1 h in the same medium containing various concentrations of glucose, tolbutamide (Sigma), or KCl (Sigma). The medium samples were subsequently assayed for radioimmunoassay of insulin (11, 19).

Glucose-tolerance Tests-- Glucose-tolerance tests were carried out (11, 19) by injecting 3 g of glucose per kg of body weight intraperitoneally.

Data Analysis-- Results are expressed as means ± S.E. The significance of differences between groups was evaluated using unpaired Student's t-test.

    RESULTS AND DISCUSSION

We produced chimeric mice composed predominantly of targeted embryonic stem cell-derived cells (Fig. 1, A and B). A DNA fragment flanked by loxP sequences was removed by a transient expression of Cre recombinase in fertilized eggs (20). Newborn mice (F1) were found to carry the mutated allele that deletes both CD38 exon 1 and neo cassette (Fig. 1, A and C). Homozygotes (-/-) were yielded in the F2 generation in a distribution following Mendelian rules. RT-PCR and Western blot analysis (Fig. 1, D and E) showed that there was no detectable CD38 mRNA and protein in pancreatic islets from CD38-/- mice, suggesting that the gene disruption resulted in a null mutation of CD38.


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Fig. 1.   Disruption of the CD38 gene in mice. Panel A, schematic representation of the CD38 targeting vector, mouse CD38 gene, targeted allele, and deleted allele. Neomycin resistance gene (NEO), DTA gene, and the loxP sequences are shown by open box, filled box, and triangles, respectively. Positions of probes used for Southern blot analyses with BamHI are also shown. B, BamHI; H, HindIII; N, NotI; P, PstI; S, SacI; Xb, XbaI; Xh, XhoI. Panel B, Southern blot analysis of ES cell and mouse tail genomic DNA using Probe 1. The 15-kbp band corresponds to the wild-type allele and the 8.2-kbp band to the targeted allele. W, wild-type; T, targeted; C, chimera. Panel C, Southern blot analysis of mouse tail genomic DNA using Probe 2. The 15-kbp band corresponds to the wild-type allele, the 8.2-kbp band to the targeted allele, and the 5.3-kbp band to the deleted allele. Panel D, RT-PCR analysis of pancreatic islet RNA. Panel E, Western blot analysis of islet protein.

CD38 exhibits both ADP-ribosyl cyclase and cADPR hydrolase activities, and the overall reaction is classified as an NAD+-glycohydrolase reaction (6-10, 27). As compared with CD38+/+ islet homogenates (ADP-ribosyl cyclase, 148.6 ± 30.9 pmol/min/mg; cADPR hydrolase, 398.9 ± 56.3 pmol/min/mg; NAD+-glycohydrolase, 10.9 ± 3.3 nmol/min/mg), the enzyme activities of CD38-/- islet homogenates were greatly reduced (2.7 ± 1.7 pmol/min/mg, 27.6 ± 27.6 pmol/min/mg, 0.4 ± 0.3 nmol/min/mg, respectively), indicating that CD38 is mainly responsible for the synthesis and hydrolysis of cADPR in pancreatic beta cells. The remaining activities presumably reflect the minor contribution of other cADPR-metabolizing enzymes (28, 29). The NAD+-glycohydrolase activity was also greatly reduced in other tissues such as spleen, cerebrum, and ileum (data not shown).

The cADPR content in CD38+/+ islets was greatly increased by high glucose stimulation (Fig. 2A). Although some amounts of cADPR were detected in CD38-/- islets when incubated in low glucose, the cADPR content was not at all increased by high glucose stimulation; this suggests the non-redundant role of CD38 in increasing the intracellular cADPR concentration upon glucose stimulation and that the cADPR detected in CD38-/- islets was produced by ADP-ribosyl cyclases other than CD38 (28, 29).


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Fig. 2.   Measurement of cADPR content and glucose-induced [Ca2+]i changes in CD38+/+ and CD38-/- islets. Panel A, cADPR content in isolated islets under 2.8 mM glucose (LG) or 20 mM glucose (HG) concentrations, expressed as fmol per µg of protein of islet homogenate. n = 3-4 for each point; *, p < 0.05. Panel B, digital imaging of [Ca2+]i in the islets. Panel C, changes in [Ca2+]i in the CD38+/+ islets. A representative record from four experiments. Panel D, changes in [Ca2+]i in the CD38-/- islets. A representative record from seven experiments.

cADPR is a Ca2+-mobilizing agent from intracellular Ca2+ stores (5, 30, 31), and we have already shown that cADPR causes Ca2+ release from islet microsomes of normal mice (C57Bl/6J) (21) and rats (Wistar) (5). As shown in Fig. 2, B-D, the glucose-stimulated [Ca2+]i rise in CD38-/- islets was much lower than that in CD38+/+ islets in the digital imaging of fura-2 fluorescence. This attenuated response of [Ca2+]i in CD38-/- islets was reproducible in a series of experiments (increase in [Ca2+]i by glucose (2.8 mM right-arrow 20 mM) was: 243.7 ± 13.9 nM in CD38+/+ islets, n = 20; 132.2 ± 12.9 nM in CD38-/- islets, n = 36, p < 0.001), suggesting that the loss of the cADPR increase in CD38-/- islets in response to glucose stimulation (Fig. 2A) caused this attenuated response.

Although there were no significant differences in insulin secretion between CD38+/+ and CD38-/- islets at 2.5 and 10 mM glucose, insulin secretion from CD38-/- islets at 20 and 30 mM glucose was more than 50% decreased compared with CD38+/+ islets (Fig. 3A). We investigated the effects of tolbutamide (4) and KCl (32) on insulin secretion from CD38+/+ and CD38-/- islets. CD38-/- islets responded normally to tolbutamide and KCl (Fig. 3B), indicating that CD38 is not essential for insulin secretion by extracellular Ca2+ influx stimulants.


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Fig. 3.   Measurement of insulin secretion from CD38+/+ and CD38-/- islets. Panel A, insulin secretion from isolated islets under various glucose concentrations. n = 5-12 for each point. Panel B, insulin secretion from isolated islets by 0.02 mM tolbutamide (Tol(0.02)), 0.2 mM tolbutamide (Tol(0.2)), 12.5 mM KCl (KCl(12.5)), and 20 mM KCl (KCl(20)) in the presence of the low concentration (2.5 mM) of glucose. n = 6 for each point; *, p < 0.05; **, p < 0.01.

Next, 10-h-fasted mice were subjected to glucose-tolerance tests (Fig. 4A). At 30 and 60 min after glucose injection, CD38-/- mice had much higher glucose levels than CD38+/+ mice. In CD38-/- mice, serum insulin levels at 15 min after glucose injection were significantly lower than those of CD38+/+ mice. The blood glucose levels of CD38-/- mice at 15-60 min after insulin injection were essentially similar to those of CD38+/+ mice (data not shown), suggesting that the glucose intolerance in CD38-/- mice is because of an attenuation of glucose-induced insulin secretion rather than an alteration in peripheral insulin resistance. We further tested whether the observed phenotype could be rescued by a pancreatic beta cell-specific expression of CD38 cDNA. Transgenic mice carrying a human CD38 cDNA under the rat insulin promoter (11) were crossed with CD38-/- mice. The human CD38 transgene ameliorated the glucose intolerance and the decreased insulin secretion, suggesting that the observed phenotype was indeed caused by the absence of CD38 in pancreatic beta cells (Fig. 4B).


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Fig. 4.   Serum insulin and blood glucose levels in glucose-tolerance test and amelioration of glucose intolerance by pancreatic beta cell-specific expression of CD38 cDNA. Panel A, blood glucose and serum insulin levels after glucose injection. Open bars and filled bars show serum insulin levels from CD38+/+ and CD38-/- mice, respectively. Panel B, CD38-/- mice and CD38-/- mice carrying the human CD38 transgene (CD38-/- × Ins-CD38Tg) were subjected to glucose-tolerance tests. Filled bars and open bars show serum insulin levels from CD38-/- mice and CD38-/- × Ins-CD38Tg mice, respectively. n = 8 for each point; *, p < 0.05; **, p < 0.01.

We have recently reported that the CD38 mRNA expression is attenuated in islets of ob/ob diabetic mice as well as in RINm5F cells, which synthesize and secrete very little insulin in response to glucose (21). Likewise, the CD38 mRNA expression was attenuated in pancreatic islets of Goto-Kakizaki diabetic rats (33), which show impaired insulin secretion (34). Interestingly, we found that about 14% of the examined human diabetic patients possessed autoantibodies to CD38, which inhibited the ADP-ribosyl cyclase activity of CD38, accumulation of cADPR in islets, and insulin secretion in vitro (35). More recently, a human CD38 gene mutation (Arg 140 right-arrow Trp) that inhibits the ADP-ribosyl cyclase activity of CD38 was found in four diabetic patients but not in control subjects (36). Considered together with the results of the present study using knockout mice, it is reasonable to assume that there is a causal relationship between CD38 dysfunction and diabetes development in at least some patients.

In this paper, we have focused on the role of CD38 in insulin secretion by glucose as one characteristic of CD38 knockout mice, but it would also be possible and important to study the physiological roles of CD38 in cADPR-mediated signaling in tissues other than pancreatic islets using CD38-/- mice. In fact, Cockayne et al. recently reported that they produced another CD38 knockout mouse line by deleting exons 2 and 3 of the CD38 gene and that the mice exhibited altered humoral immune responses (37).

    ACKNOWLEDGEMENT

We thank B. Bell for critical reading of the manuscript.

    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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016868.

Dagger 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 980-8575, Miyagi, Japan. Tel.: 81-22-717-8079; Fax: 81-22-717-8083.

The abbreviations used are: cADPR, cyclic ADP-ribose; [Ca2+]i, intracellular Ca2+ concentration; kbp, kilobase pair(s); ES, embryonic stem; DTA, diphtheria toxin A; RT, reverse transcriptase; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 4-morpholineethanesulfonic acid.
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