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Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Woolloongabba, Brisbane 4102, Australia; School of Life Sciences Queensland University of Technology, Brisbane 4001, Australia
Address all correspondence and requests for reprints to: John W. Cardinal, Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane 4102, Australia.
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Abstract |
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C57bl/6J mice were more sensitive to STZ than Balb/c mice as judged by the extent of pancreatic insulin depletion and ß cell death, in vivo and in vitro. The mode of cell death was necrosis. After a 30-min in vitro exposure to the drug the more sensitive C57bl/6J islets contained higher levels of streptozotocin metabolites and less NAD + NADH than the more resistant Balb/c islets. The lack of any strain differences in 3-O-methyl glucose transport, Glut2 levels and medium nitrite accumulation suggested that STZ transport and nitric oxide metabolism were not responsible for differences in STZ sensitivity and metabolite accumulation.
Thus the strain differences in STZ sensitivity appears to be due to intracellular events within the ß cell occurring after STZ transport and before NAD + NADH depletion. STZ metabolite accumulation appears to be associated with STZ sensitivity. Further studies are warranted to determine if differential STZ metabolite accumulation is responsible for STZ sensitivity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred strains of mice show varying susceptibility to the effects STZ injected as a single high dose or as multiple subdiabetogenic doses, indicating the importance of genetic background in these models. Rossini et al. (5) showed that a single high dose of streptozotocin will induce hyperglycaemia of varying degrees depending on the strain of mice. They produced the most marked hyperglycaemia in C57bl/KS mice and very little response from C3H/He mice. They then went on to show that this strain variation did not correlate with the degree of insulitis (lymphocyte infiltration) seen when the mice were administered multiple subdiabetogenic doses of STZ. Thus, there appear to be factors affecting susceptibility to the direct cytotoxic effects of the drug in addition to factors affecting the degree of immune response. Rossini et al. (5) demonstrated no association with the MHC locus and susceptibility and concluded that MHC genes were not the sole regulators of hyperglycaemia and insulitis in the multiple low dose streptozotocin model of diabetes. Their findings have since then been supported by several other investigators (6, 7). The present study investigated the basis for genetic susceptibility to STZ at the ß cell level as the same mechanisms may be important in the resistance to environmental triggering agents in human IDDM.
From what is known of the action of the STZ on the ß cell we postulated that strain variation to STZ may be due to differences in the uptake or metabolism of the drug, the rate of DNA repair, the activity of PARP or damage to NAD generating mechanisms. In this study we have investigated STZ uptake and metabolism as a possible cause of differences in STZ sensitivity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pancreatic insulin and blood glucose measurements
The diabetogenic effect of STZ was measured using total
pancreatic insulin content. The mice were injected at doses of 80 or
120 mg STZ/kg BW. After 4 days, the animals were killed by
decapitation. A blood sample was taken from the decapitation site.
Pancreatic insulin was then extracted overnight at 4 C as previously
described (8). The insulin content of the supernatant was determined by
RIA using rat insulin as the standard. Pancreatic insulin (PI) was
expressed per mg wet wt of pancreas. Blood glucose was determined using
a Yellow Springs Instrument 23AM glucose analyzer (Yellow Springs,
OH).
Histology of mouse pancreata and isolated islets
Initial morphological studies were performed to confirm that the
decreased PI levels were due to ß cell death. As biochemical
measurements were performed at 96 h post dose, initial
morphological studies looked at the same time point as well as 24
h post dose. Groups of 2 or 3 C57bl/6J and Balb/c mice were killed
after a streptozotocin dose of 120 mg/kg BW. To investigate the mode of
cell death involved, mice were killed at 3, 6, 8, and 12 h post
STZ dose. The pancreata were placed in Bouin’s fixative for later
paraffin embedding. The Bouin’s fixed tissues were stained with an
aldehyde fuchsin stain. Histological processing of specimens was
performed by J. Easson of the Queensland University of Technology. The
percentage of affected ß cells was calculated by scoring 500 aldehyde
fuchsin positive ß cells per animal.
Semithin sections were used to determine STZ sensitivity in islets treated with STZ in vitro. Islets were fixed in 3% glutaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 3 h then processed for light and electron microscopy by Mr. C. Winterford of the University of Queensland Medical School. The percentage of affected ß cells was calculated by scoring 500 islet cells per experiment.
Islet isolation
Islets used for the NAD studies were isolated using the method
of Gotoh et al. (9) without the Ficoll purification gradient
step as Ficoll was found to cause islet cell lysis. The islets were
picked clean from the collagenase digest with a sterile disposable
pipette. As large numbers of islets were needed for STZ metabolite
accumulation studies and morphology studies, islets used in these
studies were isolated using the method of Lake et al.
(10).
STZ treatment and processing of islets for in vitro morphological
studies
Islets were cultured immediately after isolation. The islets of
three mice from each strain were pooled and used in groups of 100
islets. Islets were preincubated in Hams F-10 tissue culture medium
supplemented with 10% FCS for 30 min. The medium was bubbled with 5%
CO2 in oxygen gas for 15 min before use. Islets were
cultured in six-well culture dishes (Nunc, Newton, NC) in 2 ml of
medium. The dishes were incubated at 37 C in 5% CO2 in
oxygen. STZ was then added to the medium to a final concentration of
1.1 mM and further incubated for 6 h. The medium was
carefully pipetted off and replaced with 3% glutaldehyde in 0.1
M sodium cacodylate buffer (pH 7.2). After 15 min, the
islets were removed to 1.5-ml microcentrifuge tubes. The islets were
left in the fixative for 3 h and then placed in 0.1 M
sodium cacodylate buffer pending processing for light and electron
microscopy. Semithin sections were stained with 1% toluidine blue in a
1% aqueous borax solution. The sections were examined by light
microscopy and areas of containing dying cells selected for electron
microscopy. Ultra-thin sections (50–70 nm) were stained with lead
citrate and examined using a JEOL EXII electron microscope. Light
microscopy of semithin sections was used to compare the morphology of
the treated islet cells with previous in vivo experiments
and to assess the number of pyknotic nuclei present. Electron
microscopy was used to establish the mode of cell death induced in
these experiments.
NAD + NADH studies
Groups of 20 islets were transferred to tubes containing 1 ml
HBSSH. STZ was dissolved in cold 10 mM citric acid buffer
(pH 4.5) and, within 1 min, 10 µl of the solution was added to the
incubation medium to reach a final STZ concentration of 2.2
mM (10 µl of citric acid buffer was added to the control
groups). The incubations with STZ were performed at 37 C in air for 30
min and terminated by washing the islets four times in HBSSH. The
islets were either incubated at 37 C for 1 h for NAD + NADH
experiments or used immediately to determine the STZ content. NAD +
NADH was measured by the method of Brolin et al. (28). The
samples were corrected for DNA content using a modification of Kissane
and Robins method (11).
Measurement of STZ metabolite accumulation
STZ metabolite accumulation was measured by the method of Forist
(12). Groups of 50 islets were incubated in 2.2 mM STZ in
HBSSH for 0, 5, 10, 20, and 30 min, washed four times in HBBSH, lysed
by the addition of 105 µl 0.6 M perchloric acid and
frozen at -70 C for 1 h. The tubes were then spun in a microfuge
at 12,000 x g at 4 C for 10 min. One hundred
microliters of supernatant was added to 250 µl of a solution
containing 0.5% sulfonilic acid, 0.2%
[N-1-napthyl]ethyldiamine dihydrochloride in 30% acetic
acid and 75 µl 6 M HCl. The reaction mixture was
incubated at 60 C for 45 min and the absorbance read at 550 nm on a
Shimadzu UV-1601 spectrophotometer. A standard curve was produced using
STZ solutions freshly prepared in 0.6 M perchloric acid.
The pellet was resuspended in 0.04 M NaOH and the DNA
content was measured in the cell pellet. The results were then
expressed as µg STZ/ng DNA.
Medium nitrite determination
The study investigated the possibility that differences in STZ
metabolite accumulation might have been explained by a difference in
the formation of nitric oxide. STZ is known to be metabolized to nitric
oxide, which rapidly forms nitrite and diffuses out of the cell
accumulating in the extracellular medium. Groups of 100 islets were
incubated in 250 µl of 2.2 mM STZ for 60 min. The medium
was then removed and assayed for nitrite content by the method of Green
et al. (13). Sodium nitrite standards from 0–5 µg/ml were
made up in HBSSH with 2.2 mM STZ and treated in the same
way as the samples.
3-O-methylglucose uptake assay
3-O-methylglucose transport was measured as an indicator of STZ
transport as it is transported into the ß cell by the same
transporter and its accumulation is determined only by transporter
rates as it is not metabolized by the cell. 3-O-methylglucose uptake
was measured using dispersed islets as per the method of Johnson
et al. (14).
Immunoblotting of Glut 2
Groups of 100 freshly isolated islets were incubated in 500 µl
of 10 mM HEPES, pH 7.9, and 1 mM
phenylmethanesulfonyl fluoride (PMSF) for 20 min at 4 C. The islets
were homogenized by pipetting the islets up and down in solution. The
cell membranes were then pelleted by centrifugation at 12,000 x
g in a microfuge for 10 min. The pellet was then resuspended
in 1 mM PMSF, 5% SDS, 5% ß-mercaptoethanol, 80
mM Tris (pH 6.8), 5 mM EDTA, and 10% glycerol.
Homogenates were separated on a 10% SDS-polyacrylamide gel and
electroblotted to PVDF membrane (Bio-Rad). Membranes were incubated
overnight with anti-Glut-2 (1/2000) at room temperature. The anti-Glut
2 was the generous gift of Dr. Gywn Gould of the University of Glasgow.
The second antibody, antirabbit peroxidase (0.01 U/ml) was incubated at
room temperature for 30 min. Peroxidase activity was detected via
chemiluminescence (Boerhinger Mannheim). The intensity of the signals
was quantified by densitometry.
Statistical analysis
Results from groups from each study were firstly analyzed using
ANOVA. Groups which showed differences were further analyzed with the
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) of STZ
treated Balb/c and C57bl/6J mice. Of the three strains of mice used,
C57bl/6J mice were shown to be the most sensitive strain and Balb/c the
most resistant. C57bl/6J and Balb/c PI levels were significantly
different at both 80 mg STZ (P < 0.001) and 120 mg
STZ/kg BW (P < 0.001). However, C57bl/6J and Balb/c
blood glucose levels were only significantly different at 120 mg STZ/kg
BW (P < 0.05). CBA mice were no longer used in this
study.
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Earlier time points were used to investigate the mode of cell death (necrosis and/or apoptosis) involved. Morphologic changes were evident in the islets 3 h after the STZ dose was given. At this time, clear spaces were seen between the cytoplasm of some of the ß cells and adjoining capillaries, suggesting cell shrinkage. A small percentage of ß cells had small uniformly condensed (pyknotic) nuclei and acidophilic or aldehyde fuchsin positive cytoplasms. Using light microscopy, the most evident cell change seen was nuclear pyknosis.
After 6 h, increased numbers of cells containing pyknotic nuclei were seen. Some of the nuclei appeared to be indented by a clear vacuole. There was no evidence of nuclear chromatin margination characteristic of apoptosis. The clear spaces between the cells became more prominent at this time point. At 8 h, many of the pyknotic nuclei present had begun to fragment into the surrounding tissue, suggesting that both nuclear and cell membranes had ruptured. At 12 h, increased numbers of nuclei had begun to fragment and degrade. There was no immune response seen or evidence of phagocytosis by surrounding cells.
Islet cell morphology using light microscopy of semithin sections
in vitro
The nuclei of normal toluidine blue stained islet cells show fine
evenly dispersed chromatin with evenly stained cytoplasm and darkly
staining organelles (Fig. 1a). The
morphology of the islet cells treated with STZ for 6 h in
vitro resembled the cell morphology of in vivo
experiments at the same time post dose. A large number of the islet
cells retained normal morphology. The nuclei of the dying, STZ affected
cells, were small and condensed (Fig. 1, b and c). The majority of the
cells containing pyknotic nuclei had diffuse lightly staining
cytoplasms with cell membrane rupture consistent with necrosis. A more
careful classification of these cells was done using electron
microscopy.
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) contain fairly evenly
distributed chromatin with some concentration at the nuclear membranes.
STZ affected cells displayed a variety of nuclear changes. Most of the
nuclear changes were consistent with necrosis: clumped chromatin with
ill-defined edges. Necrotic nuclei (Fig. 2b) had random nonuniform
clumping with indistinct borders and often displaying nuclear membrane
breakdown. STZ induced, in the majority of cases, cytoplasmic changes
consistent with necrosis: cell membrane swelling and rupture, swollen
mitochondria with flocculent densities, and large swollen endoplasmic
reticulum. An occasional cell had an apoptotic nucleus (Fig. 2c) with
uniformly condensed chromatin that was sharply delineated and
marginated along the nuclear membrane.
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). There was no difference in the
nature of the cell changes between the two strains, only the number of
affected cells varied. After 8 h, 85% of C57bl/6J ß cells and
4% of the Balb/c ß cells showed pyknosis. At 12 h, it became
difficult to count affected cells in the C57bl/6J islets as the ß
cell nuclei had begun to fragment. (As pyknotic nuclei were smaller
than normal ß cell nuclei, the percentage of pyknotic nuclei scored
from tissue sections may be an underestimate. Therefore, the results
are presented semiquantitatively).
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). Control islets contained a small number of
cells that displayed these changes. This was possibly due to damage
incurred during isolation or anoxia of centrally located islet cells
during culture. (As was the case with in vivo studies,
results are semi-quantitative).
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) (P < 0.01). C57bl/6J
treated islets showed a drop in NAD levels to 49% of control islets
(P < 0.001), whereas Balb/c islet NAD + NADH levels
were not significantly different from control Balb/c levels (Table 3).
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). The
levels were higher in C57bl/6J at 30 min (P <
0.01).
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).
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Medium nitrite levels
There was no significant difference in medium nitrite accumulation
(Fig. 6); however, at the same time there was a significant difference
in islet STZ metabolite accumulation (P < 0.01).
Control islets did not release detectable amounts of nitrite into the
medium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphological studies demonstrated, both in vivo and in vitro, that ß cell death was by necrosis and not apoptosis. Although the classification of STZ-induced cell death in vivo has not been previously reported, several studies (17, 18, 19, 20) have reported similar cell changes to those seen in this study. The lack of reference to the type of cell death in these earlier studies was no doubt because little was known about the types of cell death when the studies were done. In contrast, studies by O’Brien et al. (21) in the multiple low dose streptozotocin model reported apoptosis to be the mode of ß cell death (21). However, without in vitro studies it is impossible to determine if the apoptosis seen in the multiple low dose streptozotocin model is a direct result of STZ on the ß cells or is cytokine-induced. Apoptosis has been induced in rat islet cells in vitro using cytokines and nitric oxide but has not been reported with STZ (22). In vitro STZ-induced ß cell apoptosis has only been reported in cultured insulinoma cell lines (23). As many of the genes involved in apoptosis are also onco-genes, the insulinoma cells are likely to have differences to normal differentiated ß cells in the way they undergo apoptosis. Saini et al. (23) reported that in insulinoma cells STZ induces mainly necrosis at higher doses and apoptosis at lower doses. If this is the case in normal ß cells, then it could be argued that the doses used in this study (1.1 mM STZ) were too high to induce apoptotic cell death. It remains to be seen whether STZ does induce apoptosis in islet cells directly.
The morphological studies confirmed that the strain differences observed in STZ sensitivity are due to necrotic cell death. This finding is important as it excludes any strain differences in the apoptotic cell death pathway as being relevant to strain differences in sensitivity to a single high dose of STZ. Strain differences were shown to be at the level of the islet as morphological differences observed in vivo were also present in vitro.
STZ metabolite accumulation studies showed a higher accumulation of STZ metabolites in the more sensitive C57bl/6J mice. This difference was only demonstrated after a 30-min exposure to STZ when it appeared to reach a plateau. Similar STZ metabolite accumulation kinetics have been reported by Schnedl et al. (1) in Glut2 transfected cell lines. Strain differences in STZ metabolite accumulation have not previously been reported and as differences were found, further studies were performed to determine whether STZ transport rates or metabolism was responsible for accumulation differences.
The lack of any strain differences in 3-O-methylglucose transport as well as immunoreactive Glut2 protein levels provided strong evidence against STZ transport being responsible for differences in STZ metabolite accumulation. This finding pointed to STZ metabolism as being responsible for accumulation differences.
No difference in medium nitrite accumulation was found and it was concluded that nitric oxide formation was not responsible for differences in STZ metabolite accumulation and STZ sensitivity.
It is unlikely that the level of nitric oxide per se contributes significantly to ß cell death in this model. At 8 h post dose, STZ induces only 5% ß cell death in Balb/c islets compared with 85% death in C57bl/6J islets. Assuming at the 8 h time point both strains produced the same amount of nitric oxide, as shown in vitro, then nitric oxide can only be responsible for a maximum of 5% of the ß cell death. Several studies have shown nitric oxide to be a major contributor to STZ-induced ß cell death (22, 24). However, these studies have been in rats, not mice. Rats are known to be much more sensitive to STZ than mice. Thus, it is possible that nitric oxide-induced DNA damage is more important in rats than mice explaining their previously reported increased STZ sensitivity (25).
NAD + NADH depletion is known to be due to PARP activation (2). PARP is activated by STZ-induced DNA damage. STZ is also known to effect NAD levels by inhibiting mitochondrial activity and thereby inhibiting the formation of NAD and ATP (26, 27). Further investigations are warranted to determine whether differences in STZ-induced PARP activation or mitochondrial inhibition can explain the difference in NAD + NADH depletion.
It is tempting to conclude that differences in STZ metabolite accumulation will lead to differences in the amount of DNA damage, DNA strand breaks, PARP activation, NAD depletion, and finally ß cell death. It cannot, however, be assumed that differences in metabolite accumulation will lead to differences in the number of DNA lesions or that the DNA repair process operates at the same rate in both strains. Thus further investigations are needed to quantitate the amount of DNA damage induced in both strains.
The interactions between environmental agents and genetic susceptibility in human IDDM are unclear. Much of what we understand about the mechanisms of IDDM has been from animal models of the disease. This study suggests that, at least in the streptozotocin mouse model, genetic susceptibility to a known environmental agent (streptozotocin) is determined at the level of the ß cell. Further research into the genetic controls on STZ susceptibility may help us identify novel IDDM susceptibility genes at the level of the ß cell.
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Footnotes |
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Received December 2, 1997.
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