From the Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-8575, Miyagi, Japan
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ABSTRACT |
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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 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.
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.
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 (/
) 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
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
/
) 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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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
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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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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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 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).
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ACKNOWLEDGEMENT |
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We thank B. Bell for critical reading of the manuscript.
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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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016868.
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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REFERENCES |
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