(Received for publication, August 10, 1995; and in revised form, September 20, 1995)
From the
Cyclic ADP-ribose (cADPR) serves as a second messenger for
Ca mobilization in insulin secretion, and CD38 has
both ADP-ribosyl cyclase and cADPR hydrolase activities (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 produced transgenic mice overexpressing
human CD38 in pancreatic
cells. The enzymatic activity of CD38 in
transgenic islets was greatly increased, and ATP efficiently inhibited
the cADPR hydrolase activity. The Ca
mobilizing
activity of cell extracts from transgenic islets incubated in high
glucose was 3-fold higher than that of the control, suggesting that ATP
produced by glucose metabolism increased cADPR accumulation in
transgenic islets. Glucose- and ketoisocaproate-induced but not
tolbutamide- nor KCl-induced insulin secretions from transgenic islets
were 1.7-2.3-fold higher than that of control. In
glucose-tolerance tests, the transgenic serum insulin level was higher
than that of control. The present study provides the first evidence
that CD38 has a regulatory role in insulin secretion by glucose in
cells, suggesting that the Ca
release from
intracellular cADPR-sensitive Ca
stores as well as
the Ca
influx from extracellular sources play
important roles in insulin secretion.
Cyclic ADP-ribose (cADPR) ()(1) induces the
release of Ca
from microsomes of pancreatic islets (2, 3) and from a variety of other
cells(4, 5, 6, 7, 8, 9, 10) .
Glucose raises the cADPR concentration in islets, and cADPR induces
insulin secretion from digitonin-permeabilized islets in
vitro(3) . We have therefore suggested that cADPR plays a
second messenger role in Ca
mobilization for insulin
secretion(2, 3, 11) . Human lymphocyte
antigen CD38 (12, 13) has been shown to have both
ADP-ribosyl cyclase and cADPR hydrolase activities (11, 14) . CD38 was found to be expressed in a variety
of tissues and cells including pancreatic
islets(11, 15) . We have shown that ATP, which is
generated during glucose metabolism in islets, inhibits the cADPR
hydrolase activity of CD38, thereby increasing the accumulation of
cADPR because of decreased destruction(11) . CD38 is thus
thought to play a central role in glucose-induced insulin secretion in
islets. In the present study, we produced transgenic mice
overexpressing CD38 in islets and analyzed the subcellular localization
of expressed CD38, changes in Ca
mobilizing activity,
and glucose-induced insulin secretion.
Figure 6:
Effect
of exogenous cADPR on Ca mobilization from islet
microsomes. Islet microsomes were prepared from control mice and
transgenic mice (line 18). The release of Ca
from the
islet microsomes (5 µg of protein) in response to various
concentrations (0, 0.2, 0.35, 0.5, and 1.0 µM) of cADPR
was measured as described under ``Experimental
Procedures.''
The rat insulin II promoter/human CD38 hybrid gene (Ins-CD38;
see ``Experimental Procedures'') was designed to direct the
overexpression of CD38 in pancreatic cells of transgenic mice.
The linearized gene fragment was microinjected into the fertilized eggs
of (C57Bl/6J
CBA/J) F
mice. 20 out of 94 newborn
mice were found to carry the Ins-CD38 transgene, as detected by
polymerase chain reaction analyses using primers for the insulin
promoter and human CD38 cDNA. In the present study, the six transgenic
lines, 18, 30, 49, 56, 60, and 72, were maintained on ICR background
and analyzed.
Northern blot analysis using the human CD38 cDNA probe (11) showed that all lines of transgenic mice but not that of the nontransgenic mice expressed human CD38 mRNA in the pancreatic islets (Fig. 1A). Densitometric scanning indicated that the transgenic lines 18, 56, and 60 expressed relatively higher levels of human CD38 mRNA in islets, whereas lines 30, 49, and 72 expressed lower levels of human CD38 mRNA. The human CD38 mRNA expression was not detected in other tissues such as brain, lung, heart, stomach, small intestine, liver, kidney, spleen, and testis in the transgenic mice (Fig. 1, B and C), indicating that the expression of human CD38 is limited to islets. In immunohistochemistry, islets of the transgenic mice were densely stained for human CD38 (Fig. 2, B and C). On the other hand, islets of the control mice showed no immunoreactivity for human CD38 (Fig. 2A). In contrast to islets, the pancreatic exocrine cells showed no detectable staining for human CD38 in any of the transgenic and nontransgenic mice.
Figure 1: Northern blot analyses of RNAs from pancreatic islets (A) and from various tissues of transgenic mouse line 18 (B) and line 56 (C) using human CD38 cDNA. A, total RNAs (1.5 µg/lane) isolated from pancreatic islets of control nontransgenic mice (C), transgenic line 18 (18), line 30 (30), line 49 (49), line 56 (56), line 60 (60), and line 72 (72) were used for RNA blot hybridization. B and C, lanes 1, brain; lanes 2, lung; lanes 3, heart; lanes 4, stomach; lanes 5, small intestine; lanes 6, liver; lanes 7, kidney; lanes 8, spleen; lanes 9, testis (lanes 1-9, 10 µg of RNA/lane). Lane 10, pancreatic islets (1.5 µg of RNA/lane). Positions of mouse 28 S and 18 S rRNAs are presented on the left. The endogenous mouse CD38 mRNA did not hybridize under the conditions used in this study.
Figure 2: Immunohistochemical detection of human CD38 in mouse pancreas. Human CD38 protein was detected in mouse islets of transgenic line 18 (B) and line 56 (C) but not in nontransgenic mouse (A).
Next, the pancreatic islet
homogenates prepared from islets of transgenic and nontransgenic
siblings of lines 18 and 56 were incubated with
[P]NAD
, and the reaction
products were analyzed by HPLC. The formation of cADPR in the
transgenic mice (for line 18, 1.5 nmol/min
mg protein; for line
56, 1.5 nmol/min
mg protein) was indeed much higher than in the
controls (<0.05 nmol/min
mg protein). The formation of ADPR in
the transgenic mice (for line 18, 72.8 nmol/min
mg protein; for
line 56, 174.4 nmol/min
mg protein) was also much higher than in
the controls (4.3 nmol/min
mg protein). CD38 exhibits both
ADP-ribosyl cyclase and cADPR hydrolase activities, and the overall
reaction is classified as an NAD
glycohydrolase
reaction(11, 14) . To determine the subcellular
distribution of expressed CD38, islet proteins were fractionated by
centrifugation (22) and fractionated proteins were assayed for
NAD
glycohydrolase activity (Table 1). The
NAD
glycohydrolase activity in the membrane fraction
was greatly increased in the transgenic mice, indicating that expressed
CD38 was predominantly localized in this fraction. Significant
activities were also detected in the nuclear, microsome and cytosol
fractions of the transgenic islets. The distribution of the percentage
of total activity in fractions of transgenic mice showed a similar
tendency to that of the control.
We isolated islets from transgenic mouse line 18 and their nontransgenic litter mates and measured secreted insulin after incubation in medium containing various concentrations of glucose (Fig. 3A). At 6.8-15.6 mM glucose, the transgenic insulin secretion was 1.7-2.3-fold higher than that of the control. Essentially similar results were obtained using transgenic line 56 (data not shown). Time course experiments at 11.1 mM glucose indicated that at 10 min after the exposure to glucose, the glucose-stimulated insulin secretion was significantly higher in the transgenic islets and progressively increased in a time-dependent manner (Fig. 3B).
Figure 3: Glucose-induced insulin secretion from isolated islets. A, insulin secretion from isolated islets under various glucose concentrations. The results shown are the averages from 10-25 assays performed on five different preparations of transgenic (line 18) and control islets. B, time course of insulin secretion from isolated islets under 11.1 mM glucose. n = 5 for each point. Vertical bars indicate S.E. *, p < 0.05;**, p < 0.01;***, p < 0.001.
We
next investigated the effects of other insulin secretagogues on insulin
secretion from transgenic and control islets. When the islets were
exposed to 10 mM KIC, which, like glucose, generates ATP
during the metabolism(24) , the transgenic insulin secretion
was 1.7-fold higher than that of the control (Fig. 4A).
Tolbutamide blocks ATP-sensitive K channel and
facilitates Ca
influx through voltage-dependent
Ca
channels (24) without increasing the islet
ATP concentration(25) . When the islets were exposed to 0.2
mM tolbutamide, the transgenic insulin secretion was not
altered as compared with the control (Fig. 4B). When
the islets were exposed to 25 mM KCl, which directly induces
cell membrane depolarization resulting in Ca
influx(24) , the transgenic insulin secretion was also
not significantly higher than the control (Fig. 4C).
Figure 4: Insulin secretion from isolated islets by KIC, tolbutamide, and KCl. Insulin secretion from islets of control mice and transgenic mice (line 18) stimulated by 10 mM KIC (A), 0.2 mM tolbutamide (B), or 25 mM KCl (C) was measured as described under ``Experimental Procedures.'' Open bars and shaded bars show levels from control mice and transgenic mice (line 18), respectively. n = 5 for each point. Vertical bars indicate S.E. *, p < 0.01.
We prepared cell extracts from the islets incubated in 2.5 or 11.1
mM glucose. The extracts were assayed for the Ca mobilizing activity from microsomes(3) . The
Ca
mobilizing activity of extracts of transgenic
islets incubated in 11.1 mM glucose was 3-fold higher than
that of the control extracts (Fig. 5). The Ca
mobilization by the islet extracts was abolished when the
microsomes had been desensitized by previously releasing Ca
in response to authentic cADPR(3) , indicating that
Ca
mobilization by the extracts of transgenic islets
after high glucose treatment is cADPR-derived. In contrast, at 2.5
mM glucose, the Ca
mobilizing activities of
the transgenic and control islet extracts were lower, and there was no
significant difference between the two extracts (Fig. 5). The
effect of exogenous cADPR on Ca
mobilization from
control and transgenic islet microsomes was essentially similar (Fig. 6), suggesting that microsome sensitivity to cADPR is not
altered by human CD38 overexpression.
Figure 5:
Ca mobilizing activity
in the transgenic and control islets. Release of Ca
from cerebellar microsomes by the islet extracts prepared after
incubation with 2.5 or 11.1 mM glucose was measured as
described under ``Experimental Procedures.'' Open bars and shaded bars show levels from control mice and
transgenic mice (line 18), respectively. n = 3 for each
mouse. Vertical bars indicate S.E. *, p <
0.05.
We have previously shown that
ATP, generated during glucose metabolism in islets, dose-dependently
inhibits the cADPR hydrolyzing activity of CD38 expressed in COS-7
cells and increases the accumulation of cADPR(11) . In fact,
higher concentrations of ATP efficiently inhibited the cADPR hydrolase
activity of the CD38 expressed in transgenic islets (Fig. 7).
The cellular ATP concentration ()in transgenic islets
increased from 2.3 (at 2.5 mM glucose) to 3.3 mM (at
11.1 mM glucose); this rise in cellular ATP concentrations
would inhibit the cADPR hydrolase activity of CD38 and thereby increase
the cADPR concentration. It is therefore reasonable to assume that the
expressed ADP-ribosyl cyclase/cADPR hydrolase (CD38) in transgenic
islets generates the enhanced cADPR accumulation upon stimulation by
glucose or KIC to increase insulin secretion via the cADPR-mediated
intracellular Ca
elevation.
Figure 7: Effects of ATP on cADPR hydrolase activity of expressed CD38. ADPR synthesis from cADPR by islet cell homogenate of transgenic mice (line 18) in the presence of 0, 2, 4, or 6 mM ATP was measured as described under ``Experimental Procedures.''
Next, 10-h fasted mice were subjected to glucose tolerance tests by an intraperitoneal injection of glucose, and then serum insulin levels at each point after the glucose stimulation were determined. In transgenic mice (lines 18 and 56), glucose-induced insulin increases were 2-fold higher (at 15 min) than those of the controls (Fig. 8, A and B). Determinations of blood glucose levels at each point in the glucose tolerance test showed that the three lines of transgenic mice (lines 18, 56, and 60), which expressed higher levels of human CD38 mRNA in the pancreatic islets (Fig. 1A), had lower glucose levels than the controls (Fig. 8, C-E). However, transgenic lines 30, 49, and 72, which expressed lower levels of human CD38 mRNA in islets, did not have significantly lower glucose levels after glucose administration than the controls. These results indicate that the expressed ADP-ribosyl cyclase/cADPR hydrolase (CD38) reproducibly and dose-dependently facilitates glucose-induced insulin secretion in vivo and, by doing so, reduces the blood glucose levels of the transgenic mice.
Figure 8: Serum insulin (A and B) and blood glucose (C-E) levels in glucose-tolerance tests. A and B, transgenic mice of lines 18 and 56 and their respective nontransgenic siblings (control) were subjected to glucose-tolerance tests, and the serum insulin levels were determined as described under ``Experimental Procedures.'' n = 10 (lines 18 and 56) and 10 (respective control). C-E, in the glucose tolerance tests, blood glucose levels of lines 18, 56, and 60 and their respective nontransgenic siblings (control) were determined using whole blood taken at the indicated times from the tail vein. n = 8 (lines 18, 56, and 60) and 8 (respective control). Vertical bars indicate S.E. *, p < 0.05.
It should be noted that the production of human CD38 in islets did not appear to be deleterious to the health of the transgenic mice; fertility and body weight were indistinguishable from controls. The islets of transgenic mice at 1 year of age appeared morphologically normal and were well stained for insulin (not shown).
The results of the present study suggest that
not only Ca from extracellular sources
(Ca
influx through voltage-dependent Ca
channels evoked by glucose-induced cell membrane depolarization; (24) ) but also Ca
released from
intracellular stores (cADPR-induced Ca
release from
microsomes; (3) ) play important roles in regulating the
glucose-induced insulin secretion. In fact, intracellular
Ca
elevation in the absence of external
Ca
has been reported
recently(26, 27) .
The present results also
indicate that CD38 plays a regulatory role in the glucose-induced
insulin secretion. In non-insulin-dependent diabetes mellitus, the
glucose-induced insulin secretion is impaired (28) even when
pancreatic islets retain significant amounts of insulin(29) .
Thus, it would be important to determine whether there are qualitative
or quantitative differences in the CD38 (ADP-ribosyl cyclase/cADPR
hydrolase) in non-insulin-dependent diabetes mellitus cells.