From the Department of Biochemistry and the
¶ Department of Pathology, Tohoku University School of Medicine,
Sendai 980-77, Miyagi, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We generated transgenic mice carrying the mouse
type 2 nitric-oxide synthase (NOS2) cDNA under the control of the
insulin promoter. Western and immunohistochemical analyses revealed
that NOS2 was expressed abundantly in transgenic islets but not in control islets. When islets were isolated and cultured, high levels of
nitrite were released from the transgenic islets. In transgenic mice,
the cell mass was markedly reduced without the infiltration of
macrophages or lymphocytes, and extensive DNA strand breaks were
detected in the islets by in situ nick translation. All the transgenic mice developed hypoinsulinemic diabetes by 4 weeks of age,
and treatment with an inhibitor of NOS2, aminoguanidine (200 mg/kg body
weight every 12 h), prevented or delayed the development of
diabetes. The present study shows that the production of nitric oxide
by
cell NOS2 plays an essential role in the
cell
degeneration.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Insulin-dependent diabetes mellitus
(IDDM)1 is caused by the
degeneration of insulin-producing cells in pancreatic islets (1-4). Nitric oxide (NO·), first identified as a physiological
signaling molecule, has been also shown to be a cytotoxic effector
molecule when generated in high concentrations by type 2 NO·
synthase (NOS2) (5). In the process of IDDM, activated macrophages produce NO·, which is thought to be cytotoxic to
cells, and
NO·, which is produced by
cell NOS2 induced by
macrophage-derived cytokines such as interleukin-1
(IL-1
), is
also thought to be involved in
cell degeneration (6). Although many
in vitro studies (7-13) suggest that NO· produced by
cytokine-induced NOS2 can cause the degeneration of
cells, no
in vivo study has clearly demonstrated the pathological significance of NO· produced within
cells in the development
of IDDM, because an infiltration of macrophages in islets always
occurred in animal models of IDDM (6, 14). In this study, we produced
transgenic mice expressing NOS2 constitutively in pancreatic
cells
and found that the transgenic mice developed severe IDDM without
macrophage or lymphocyte infiltration in and around islets.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Rat Insulin II Promoter/Mouse NOS2 Hybrid
Gene--
Islets were isolated from ICR mice by the collagenase
digestion method (15) and cultured for 12 h in the presence of 150 units/ml IL-1 (Sigma). A mouse NOS2 cDNA was cloned by
polymerase chain reaction (PCR) of reverse-transcribed RNA from
IL-1
-stimulated islets. Primers used in the PCR reaction were
5
-TTCCCGGGAGCAGAAGTGCAAAGTCTCAGAC-3
and
5
-AAAGATCTGGGCTGTCAGAGCCTCGTGGCTTT-3
; these
sequences correspond to the nucleotides
25 to
1 and 3418 to 3444 of
mouse NOS2 cDNA (16) and contain XmaI and
BglII sites (underlined sequences), respectively. The cloned
cDNA sequence was determined and was found to be exactly the same
as the reported NOS2 sequence (16). To express NOS2 in
cells, the
0.7-kbp BamHI-XmaI fragment of the rat insulin II
promoter (17), the 3.5-kbp XmaI-BglII fragment of
mouse NOS2 cDNA containing the entire coding region, and the 1.6-kbp BglII-EcoRI fragment of the SV40
intron/polyadenylation signal (18) were ligated at the XmaI
and BglII sites in the correct orientation. The resultant
hybrid gene (5.8 kbp) separated from the pBlueScript SK (
)
(Stratagene) by KpnI and NotI was used for
microinjection.
Generation of Transgenic Mice-- To generate transgenic mice, a DNA solution (2 µg/ml) was microinjected into the male pronuclei of fertilized eggs from BDF1 females as described (17, 19). Identification of transgenic mice was performed by PCR on genomic DNA. In the present study, the two diabetic transgenic lines, 31 and 40, were established, maintained on CD-1 mouse background, and analyzed. Because the diabetes occurred in two independent lines of transgenic mice, the pathology was assumed to have resulted from the transgene expression rather than from the positional or insertional effects of the transgene.
Western Blot and Immunohistochemical Analyses-- Pancreatic islets were isolated from 4-8-week-old mice by collagenase digestion (15) and homogenized (19). Protein concentrations were determined using BCA Protein Assay Reagent (Pierce). Western blot and immunhistochemical analyses of NOS2 were carried out as described (17, 20) using the diluted (1:2000) rabbit anti-mouse NOS2 antibody (Wako, Osaka, Japan) (21). Insulin staining was carried out as described (17) using anti-porcine insulin antibody (DAKO, Carpinteria, CA).
Measurement of Nitrite Release from Isolated Islets--
50-200
islets, isolated from transgenic and nontransgenic mice, were incubated
in 150 µl of RPMI 1640 medium without phenol red (Sigma) containing
10% fetal calf serum (JRH Biosciences, Lenexa, KS) and 11.1 mM glucose. Control islets were also incubated in the
presence of IL-1 (150 units/ml; Sigma). Medium samples (50 µl)
were removed at 12 and 24 h. Nitrite levels in the medium were
analyzed by using Griess reagent (22).
Measurements of Glucose and Insulin-- Blood and urinary glucose levels were determined by using Advantage equipment (Boehringer Manheim) and Tes-tape assay (Lilly, Indianapolis, IN), respectively. Pancreatic extracts were obtained from the entire pancreas by acid-ethanol extraction (23). The insulin levels in the serum and pancreatic extracts were determined by using an insulin radioimmunoassay kit (Amersham Corp.) and rat insulin standards.
Treatment with Aminoguanidine-- Aminoguanidine hemisulfate salt (Sigma) at 12 mg/ml in 0.9% NaCl solution was administered intraperitoneally at a dose of 200 mg/kg body weight every 12 h, beginning at 1 day of age.
Analyses of DNA Strand Breaks by Nick Translation--
Pancreata
from 4-week-old mice were fixed in 4% paraformaldehyde and embedded in
paraffin. In situ nick translation reaction was performed as
described (9, 24), and the incorporated biotin-dUTP was visualized by
peroxidase reaction. For analysis of DNA strand breaks by nondenaturing
gel electrophoresis (9), genomic DNA was extracted from isolated islets
by phenol-chloroform extraction. The DNA (25 ng) labeled by nick
translation (25) using [-32P]dCTP (Amersham Corp.) was
electrophoresed on 1.2% agarose gel and autoradiographed. The
incorporation of [
-32P]dCTP into the DNA was
determined as described (25).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blot analysis using an NOS2-specific antibody showed that the transgenic mice (lines 31 and 40), but not the nontransgenic control mice, expressed NOS2 in the pancreatic islets (Fig. 1A). The NOS2 expression was not detected in other tissues such as brain, liver, kidney, and small intestine of the transgenic mice (Fig. 1B). NOS2 mRNA was detected only in the transgenic islets by Northern blot analysis (data not shown). In immunohistochemistry, islets of transgenic mice were stained for NOS2 (Fig. 2, B and C). The NOS2 expression in transgenic islets was observed from 1 week of age (data not shown). The proportion of insulin-producing cell mass to total pancreatic cell mass was markedly reduced in the transgenic mice (Fig. 2, D-G). On the other hand, islets of the control mice showed no immunoreactivity for NOS2, and the pancreatic exocrine cells showed no detectable staining for NOS2 in the transgenic and nontransgenic mice (Fig. 2, A-C).
|
|
We next incubated islets from transgenic and control mice and measured
the nitrite content in the incubation medium, which is indicative of
NO· release (16). As shown in Fig.
3, high levels of nitrite were detected
in a time-dependent manner in the medium containing
transgenic islets, whereas the nitrite content in the control islet
medium was under the limit of detection. The amount of nitrite in the medium of transgenic line 31 was almost equivalent to that of the
medium containing IL-1-stimulated control islets.
|
In the transgenic lines 31 and 40, the mice developed hyperglycemia from 1 week of age (Fig. 4A) and exhibited profound polydipsia/polyuria by 4 weeks of age. The blood glucose levels of both lines of transgenic mice were over 400 mg/dl, and the urine tested strongly positive (250-500 mg/dl) for glucose. The nontransgenic control mice had normal glucose levels (less than 200 mg/dl) and never showed glycosuria. The serum insulin levels in the transgenic lines 31 (0.69 ± 0.07 ng/ml, n = 7) and 40 (0.53 ± 0.07 ng/ml, n = 7) were significantly (p < 0.05) lower than those in the control mice (3.68 ± 1.10 ng/ml, n = 7). Urine from both lines of transgenic mice often tested positive for ketones; the presence of ketonuria indicated the severity of the diabetes. We followed the diabetes development every week from the first week of age by monitoring the blood glucose levels and pancreatic insulin contents. As shown in Fig. 4, the transgenic mice developed diabetes from as early as 1 week of age. The diabetes was clearly insulin-dependent, because the blood glucose level of diabetic mice injected with 4 units of exogenous insulin (Humulin U, Lilly) was reduced to below 200 mg/dl (data not shown). These results indicated that the diabetes observed in the transgenic mice was characteristic of IDDM (type 1 diabetes, ketosis prone) (27). At 1 week of age, NOS2 was immunohistochemically detected in the islets of the transgenic mice, suggesting that NOS2 expression was correlated with the development of diabetes. There was no evidence of transient insulitis such as infiltration of macrophages or lymphocytes at any time from the first to the eighth week of age (Fig. 2).
|
It has been reported that aminoguanidine, an inhibitor of NOS2 (28),
was effective in reducing NO· produced by IL-1-treated islets
in vitro (29). To demonstrate that the diabetes in the
transgenic mice was dependent on NO· production, we undertook a
prophylactic intervention with aminoguanidine. The elevation of blood
glucose levels as well as the reduction of pancreatic insulin contents
in the transgenic mice were prevented (line 31) or delayed (line 40) by
aminoguanidine treatment (Fig. 4). In control mice, the intraperitoneal
administration of aminoguanidine did not affect the body weight, blood
glucose levels, or serum insulin levels during the 8 weeks.
Furthermore, the reduction of the insulin-producing cell mass was also
prevented or improved by aminoguanidine (Fig. 2G),
indicating that the transgenic model represents the effect of
NO· produced by NOS2 in
cells. Immunohistochemical staining
of NOS2 showed that the transgenic mice treated with aminoguanidine did
express NOS2 (data not shown), indicating that the effects of
aminoguanidine were not due to the loss of expression of specific transgenes under the control of the insulin promoter. It has been reported that aminoguanidine treatment has no effect on the appearance of insulitis or the incidence of diabetes in animal models of immune-mediated IDDM (14, 30, 31). It is reasonable to assume that not
only NO· but also other
cytotoxic factors such as hydroxyl
radicals and direct cytotoxic actions by cytotoxic T lymphocytes
can be involved in the development of the immune-mediated model (3, 4).
Because high concentrations of exogenous NO· have been shown to
cause islet DNA strand breaks in vitro (8, 9), we examined DNA strand breaks in transgenic and control islets. DNA was labeled by
nick translation and analyzed by gel electrophoresis. As shown in Fig.
5A, [32P]dCTP
was incorporated into transgenic islet DNA but not into control islet
DNA. The estimated incorporation of [32P]dCTP into
transgenic islet DNA (line 31, 27.2 ± 0.98 cpm/pg DNA; line 40, 45.1 ± 1.58 cpm/pg DNA) was much higher than that into control
DNA (0.66 ± 0.06 cpm/pg DNA). We further confirmed the presence
of DNA strand breaks by in situ nick translation. Transgenic
islet cells showed extensive DNA strand breaks (Fig. 5, C
and D), but nontransgenic islet cells did not (Fig.
5B). We and others have reported that when NO· is
produced in cells upon cytokine stimulation (4, 6-12), extensive
DNA strand breaks can occur to initiate a "suicidal" response; once
cell DNA strand breaks occur, nuclear poly(ADP-ribose) synthetase
is activated, causing the depletion of intracellular NAD+.
The depletion of NAD+ severely impairs
cell functions
and ultimately evokes
cell death (3, 4, 15, 32-35).
|
We have already proposed that although IDDM can be caused by many
different agents such as immunologic abnormalities, inflammatory tissue
damage, and cytotoxic chemical substances, the final pathway for
the toxic agents is the same (3, 4, 12, 15, 32-35). This pathway
involves DNA damage by free radicals such as NO·and hydroxyl
radicals, poly(ADP-ribose) synthetase activation, and NAD+
depletion. Therefore, IDDM is theoretically preventable by suppressing immune reactions, scavenging free radicals, and inhibiting
poly(ADP-ribose) synthetase by its inhibitors. The mechanism of
cell death and its prevention was confirmed by using poly(ADP-ribose)
synthetase gene disrupted mice (13). In the present study, we showed
that NOS2 transgenic mice developed IDDM with
cell DNA damages by NO· produced in the cells. Moreover, it was recently reported
that NO·-mediated poly(ADP-ribose) synthetase activation plays
an essential role in ischemic neuronal cell death (36). It is thus
reasonable to assume that not only
cell death in IDDM but also many
other cell deaths such as ischemic brain injury can be explained by the
pathway described above.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. O. Tanaka, H. Abe, A. Akabane, Y. Narushima, and M. Fujimura for kind help, F. Date and A. Yumoto for technical assistance, and 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.
§ Recipient of a fellowship from the Japan Society for the Promotion of Science.
Present address: Dept. of Biochemistry, Kanazawa University
School of Medicine, 13-1 Takara-machi, Kanazawa 920, Ishikawa, Japan.
** To whom 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: IDDM,
insulin-dependent diabetes mellitus; IL-1,
interleukin-1
; NOS2, type 2 nitric-oxide synthase; NO·,
nitric oxide; PCR, polymerase chain reaction; kbp, kilobase pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|