Strain differences in the diabetogenic activity of streptozotocin in mice.

Abstract

We have already reported that slowly progressive non-insulin-dependent diabetes mellitus (NIDDM) is produced by a single i.p. injection of a subdiabetogenic dose (100 mg/kg) of streptozotocin (STZ) to 8-week-old male ICR mice. The aim of the present study was to clarify whether or not the progressive NIDDM is induced in ddY, BALB/c, C57BL/6 and ICR mice by the administration of STZ. Eight-week-old male mice of the 4 different strains were administered a single i.p. injection of STZ at various doses (ICR, ddY and BALB/c: 100-200 mg/kg; C57BL/6: 75-150 mg/kg). Among the ddY, BALB/c and C57BL/6 mice, a time course-related rise in non-fasting serum glucose levels throughout the observation period of 1-12 weeks after STZ administration was only induced in the 125 mg/kg STZ ddY and 100 mg/kg STZ ICR mice. In contrast with serum glucose levels, the area of islets and the percentage of the relative number of insulin-immunoreactive cells (beta-cells) to glucagon-immunoreactive cells (alpha-cells) in the 100 mg/kg STZ ICR and 125 mg/kg STZ ddY mice continued to decrease gradually over time. In addition, in low dose STZ mice of both strains, the insulin response to glucose stimulation was extremely impaired over time, although non-fasting serum insulin levels were maintained near normal levels. The rate of the progression of diabetes was faster in the 125 mg STZ ddY mice than in the 100 mg/kg STZ ICR mice.

Full text

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Diabetes mellitus is classified into two types, insulin-de-
pendent (IDDM, type 1) and non-insulin-dependent diabetes
mellitus (NIDDM, type 2). NIDDM is a disease character-
ized by peripheral insulin resistance and impaired insulin se-
cretion. NIDDM patients are divided into 2 categories, obese
type with hyperinsulinemia and non-obese type with hy-
poinsulinemia.
Streptozotocin (STZ; N-nitroso derivative of glucosamine)
is a broad-spectrum antibiotic extracted from Streptomyces
acromogenes.1)It is a pancreatic b-cell toxin that induces
rapid and irreversible necrosis of b-cells2)and is widely used
in making experimental animal models of IDDM.3—6) Junod
et al.5)reported that the i.v. injection of 25 to 100mg/kg STZ
to rats was able to produce a dose-dependent hyperglycemia.
Recently, we succeeded in producing a new mouse model
of slowly progressive NIDDM by only a single i.p. injection
of a subdiabetogenic dose (100mg/kg) of STZ to 8-week-old
male ICR mice.7,8)However, there is yet no experimental evi-
dence about whether or not the slowly progressive NIDDM is
produced in mice of other strains, except for ICR, by a single
i.p. injection of a subdiabetogenic dose of STZ. Therefore, in
the present study, we examined the dose-dependent diabeto-
genic action of STZ in 8-week-old male ddY, BALB/c, and
C57BL/6 mice and compared it with that in 8-week-old male
ICR mice. We found that slowly progressive NIDDM was
produced in ddY mice as well as ICR mice by a single i.p. in-
jection of a subdiabetogenic dose (125mg/kg) of STZ. In
order to clarify the characteristics of the progressive diabetes
in ddY mice, we also followed-up the changes in islet mor-
phology (the number of a- and b-cells) and serum glucose
and insulin responses to oral glucose load after STZ adminis-
tration.
MATERIALS AND METHODS
Animals
C57BL/6 mice (Nippon SLC, Shizuoka, Japan) were housed
Eight-week-old male ICR, ddY, BALB/c and
in an isolator caging system in air-conditioned animal room
at 23?1°C. All experimental procedures described were ap-
proved by the Experimental Animal Research Committee of
Meijo University, Faculty of Pharmacy.
Induction of Diabetes
Mice were fasted for 20h before
diabetes was induced with STZ. ICR and ddY mice received
a single i.p. injection of 100, 150 or 200mg/kg and 100, 125,
150 or 200mg/kg, respectively, of STZ (Sigma, St. Louis,
MO, U.S.A.) freshly dissolved in 0.05 M citrate buffer, pH
4.5. BALB/c and C57BL/6 mice received a single i.p. in-
jection of 100, 150, 175 or 200mg/kg and 75, 100 or
150mg/kg, respectively, of STZ. Normal mice of each strain
were injected with the equivalent volume of citrate buffer.
Serum Glucose, Insulin and Total Cholesterol Levels,
Body Weight, Urine Volume, and Drinking Water and
Food Consumption
In the first experiment, blood samples
from normal ICR, ddY, BABL/c and C57BL/6 mice and
mice administered various doses of STZ were taken from the
cavernous sinus with a capillary tube under ether anesthesia
at 1, 3, 5, 7, 9 and 12 weeks after STZ administration for the
determination of serum glucose, insulin (100 and 200mg/kg
STZ ICR and 125 and 200mg/kg STZ ddY mice and normal
mice of both strains) and total cholesterol. Body weight was
measured immediately before blood collection. After collec-
tion of blood samples, the animals were kept in individual
metabolic cages for 24h and drinking water (distilled water)
and food consumption per 24h were measured.
In the second experiment, blood samples from the 100 and
200mg/kg STZ ICR and 125 and 200mg/kg STZ ddY mice
were obtained at 20 (non-fasting time) and 0.5h (fasting
time) before and 2, 7, 24, 48 and 72h after STZ administra-
tion in order to measure serum glucose and insulin. Serum
glucose, insulin and total cholesterol were determined using
commercial reagents: Glucose CII-test Wako (Wako Pure
Chemical Industries, Ltd., Tokyo), ELISA Insulin kit (Mori-
naga Seikagaku Industries, Ltd., Tokyo) and Detamina TC-5
(Kyowa Medix Co., Ltd., Tokyo), respectively.
1110Vol. 29, No. 6
Biol. Pharm. Bull. 29(6) 1110—1119 (2006)
Strain Differences in the Diabetogenic Activity of Streptozotocin in Mice
Koji HAYASHI, Rhyoji KOJIMA, and Mikio ITO*
Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University; 150 Yagotoyama, Tenpaku-ku, Nagoya
468–8503, Japan.
Received October 13, 2005; accepted February 17, 2006
We have already reported that slowly progressive non-insulin-dependent diabetes mellitus (NIDDM) is pro-
duced by a single i.p. injection of a subdiabetogenic dose (100mg/kg) of streptozotocin (STZ) to 8-week-old male
ICR mice. The aim of the present study was to clarify whether or not the progressive NIDDM is induced in ddY,
BALB/c, C57BL/6 and ICR mice by the administration of STZ. Eight-week-old male mice of the 4 different
strains were administered a single i.p. injection of STZ at various doses (ICR, ddY and BALB/c: 100—
200mg/kg; C57BL/6: 75—150mg/kg). Among the ddY, BALB/c and C57BL/6 mice, a time course-related rise in
non-fasting serum glucose levels throughout the observation period of 1—12 weeks after STZ administration was
only induced in the 125mg/kg STZ ddY and 100mg/kg STZ ICR mice. In contrast with serum glucose levels, the
area of islets and the percentage of the relative number of insulin-immunoreactive cells (b b-cells) to glucagon-im-
munoreactive cells (a a-cells) in the 100mg/kg STZ ICR and 125mg/kg STZ ddY mice continued to decrease grad-
ually over time. In addition, in low dose STZ mice of both strains, the insulin response to glucose stimulation was
extremely impaired over time, although non-fasting serum insulin levels were maintained near normal levels.
The rate of the progression of diabetes was faster in the 125mg STZ ddY mice than in the 100mg/kg STZ ICR
mice.
Key words
streptozotocin; slowly progressive diabetes; ICR; ddY
© 2006 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed.e-mail: mitoh@ccmfs.meijo-u.ac.jp

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Immunocytochemistry
munostainings, the 100 and 200mg/kg STZ ICR and 125 and
200mg/kg STZ ddY mice and normal mice of both strains
were killed with an overdose of ether and their pancreata
were removed 1d, and 1, 4, 9 and 12 weeks after STZ admin-
istration. The pancreata were fixed in 10% buffered formalin
and then embeded in paraffin. Two consecutive sections (sec-
tion thickness, 1-mm) were cut from the paraffin block.
One of the two sections was stained with anti-glucagon
monoclonal antibody (Histofine SAB-PO kit) (Nichirei,
Tokyo, Japan) to observe glucagon immunoreactive cells (a-
cells). The other section was stained with anti-insulin mono-
clonal antibody (Histofine SAB-PO kit) (Nichirei, Tokyo,
Japan) to observe insulin immunoreactive cells (b-cells).
The glucagon- and insulin-immunoreactive cell popula-
tions in the islets were counted.
Oral Glucose Tolerance Test
test in the 100mg/kg STZ ICR and 125mg/kg STZ ddY
mice and normal mice of both strains was carried out on 3d
and 1, 2, 4, 6 and 8 weeks after STZ administration. These
animals were fasted for 18h before the test and then given 2
g/kg glucose solution orally. Blood samples were taken from
the cavernous sinus with a capillary under ether anesthesia at
0.5h before and at 0.5, 1, 2 and 4h after glucose loading.
Serum glucose and insulin levels (only at 3d and at 1, 2, 6
weeks) were determined as described above.
Statistical Analysis
The results obtained are expressed
as the mean?S.E.M. The data were analyzed using one-way
analysis of variance and Duncan’s multiple range test or non-
parametric statistics. In all cases, p?0.05 was considered
significant.
For glucagon and insulin im-
Oral glucose tolerance
RESULTS
Changes in Serum Glucose, Total Cholesterol and In-
sulin Levels after STZ Administration in ICR, ddY,
BALB/c and C57BL/6 Mice
cose levels of the normal groups of ICR, ddY, BABL/c
and C57BL/6 mice were 176—225mg/dl, 206—252mg/dl,
133—180mg/dl and 196—228mg/dl, respectively, through-
out the observation period of 1—12 weeks (Fig. 1).
In the 150 and 200mg/kg STZ ICR mice, the serum glu-
cose levels rose markedly from 1 week after STZ administra-
tion and the high glucose levels were thereafter maintained
until 12 weeks (termination of the experiment) (150mg/kg
STZ: 585—835mg/dl; 200mg/kg STZ: 644—849mg/dl).
The glucose levels in the 100mg/kg STZ ICR mice were
within a normal range at 1 week, but continued to rise there-
after till 12 weeks when the experiment terminated (1 week:
270?22mg/dl; 12 weeks: 706?38mg/dl). In the 150 and
200mg/kg STZ ddY mice as well as high doses of STZ in
ICR mice, the high glucose levels were maintained through-
out the observation period of 1—15 weeks (150mg/kg STZ:
489—755mg/dl; 200mg/kg STZ: 520—772mg/dl). In the
100mg/kg STZ ddY mice, no apparent rise of the glucose
levels was observed throughout the observation period. How-
ever, in the 125mg/kg STZ ddY mice, the time course-
related rise in serum glucose levels was seen throughout
the observation period (1 week: 305?39mg/dl; 12 weeks:
728?41mg/dl). In the 175 and 200mg/kg STZ BALB/c
mice, the high serum glucose levels were observed through-
out 1—12 weeks (175mg/kg STZ: 406—483mg/dl: 200
mg/kg STZ: 501—611mg/dl). In the 150mg/kg STZ
BALB/c mice, a slight but significant increase in the glucose
levels was seen from 1 week, but a time course-related rise of
the glucose levels thereafter was not seen. The glucose levels
in the 100mg/kg STZ BALB/c mice were within a normal
range even at 12 weeks. In the 150mg/kg STZ C57BL/6
mice, high glucose levels over 950mg/dl were recognized at
1 week but all mice died of severe weakness by 5 weeks. In
the 100mg/kg STZ C57BL/6 mice, high glucose levels were
maintained throughout 1—12 weeks (406—541mg/dl).
The non-fasting serum glu-
June 2006 1111
Fig. 1.
tocin (STZ)
Each plot denotes the mean?S.E.M. for 8 mice. Significantly different from respective normal, ∗p?0.05, ∗∗p?0.01.
Changes in Non-fasting Serum Glucose Levels in ICR, ddY, BALB/c and C57BL/6 Mice after a Single i.p. Injection of Various Doses of Streptozo-

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However, the glucose levels in the 75mg/kg STZ C57BL/6
mice were within the normal range for the observation pe-
riod. Thus, slowly progressive diabetes was induced in the
125mg/kg STZ ddY mice as well as the 100mg/kg STZ ICR
mice.
In only the highest dose STZ ICR (200mg/kg) and
C57BL/6 mice (150mg/kg STZ), non-fasting serum total
cholesterol levels were markedly higher than those in respec-
tive normal mice (Fig. 2). However, the total cholesterol lev-
els of the 100 and 150mg STZ ICR and 125, 150 and 200
mg/kg STZ ddY mice were within the normal range, al-
though hyperglycemia was observed in these mice.
The non-fasting serum insulin levels were determined only
in the 100 and 200mg/kg STZ ICR and 125 and 200mg/kg
STZ ddY mice. The serum insulin levels in the 100mg/kg
STZ ICR mice, which induced the progressive diabetes, were
within a normal range from 1 to 12 weeks when the experi-
ment was terminated after STZ administration (Fig. 3. left).
On the other hand, the insulin levels in the 125mg/kg STZ
ddY mice, which slowly progressive diabetes was induced,
decreased slightly over time after STZ administration and
were significantly lower than the normal levels at 12 weeks
(Fig. 3. right). However, the insulin levels in the 200mg/kg
STZ ICR and ddY mice, in which hyperglycemia was in-
duced from 1 week after STZ administration, were extremely
low.
Changes in Serum Glucose and Insulin Levels within
72h after STZ Administration in ICR and ddY Mice
The serum glucose and insulin levels within 72h after STZ
administration were determined only in the 100 and 200
mg/kg STZ ICR and 125 and 200mg/kg STZ ddY mice. In
the 100mg/kg STZ ICR and 125mg/kg STZ ddY mice, a
1112 Vol. 29, No. 6
Fig. 2.
Streptozotocin (STZ)
Each plot denotes the mean?S.E.M. for 8 mice. Significantly different from respective normal, ∗p?0.05, ∗∗p?0.01.
Changes in Non-fasting Serum Total Cholesterol Levels in ICR, ddY, BALB/c and C57BL/6 Mice after a Single i.p. Injection of Various Doses of
Fig. 3.
mg/kg ddY Mice
Serum insulin levels in 200mg/kg STZ ICR mice were below measurable values. Each plot denotes the mean?S.E.M. for 8 mice. Significantly different from respective normal
at ∗p?0.05, ∗∗p?0.01.
Changes in Non-fasting Serum Insulin Levels after a Single i.p. Injection of Streptozotocin (STZ) in 100 and 200mg/kg STZ ICR and 125 and 200

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transient rise of serum glucose levels was observed at 2h
after STZ administration (Fig. 4. left-top panel). All mice of
both strains given low dose STZ returned to normoglycemia
within 24h. On the other hand, in the 200mg/kg STZ ICR
and ddY mice, a transient rise in the serum glucose levels
was seen at 2h after STZ administration, following which
they returned to near normal levels by 7h (Fig. 4. left-bottom
panel). Thereafter, the glucose levels in the high dose STZ
ICR and ddY mice again rose and stable hyperglycemia was
observed at 24, 48 and 72h. Serum insulin levels in the 100
mg/kg STZ ICR and 125mg/kg STZ ddY mice showed a re-
markable rise at 7h after STZ administration (Fig. 4. right-
top panel). Thereafter, the insulin levels in both strains of
mice given low dose STZ slowly decreased. On the other
hand, in the 200mg/kg STZ ICR and ddY mice, a transient
rise of serum insulin levels was recognized at 7h after STZ
administration (Fig. 4. right-bottom panel). Thereafter, the
insulin levels in both strains of mice given a high dose of
STZ rapidly decreased and were under measurable values by
48h.
Changes in Body Weight, Urine Volume, and Drinking
Water and Food Consumption after STZ Administration
in ICR and ddY Mice
Body weight in the normal groups
of ICR and ddY mice increased gradually during the 12-
week-observation period. Body weight in the 150mg/kg or
200mg/kg STZ ICR and ddY mice with severe hyper-
glycemia from 1 week after STZ administration was signifi-
cantly lower than that in the respective normal mice through-
out the observation period of 1—12 weeks (Fig. 5A). On the
other hand, body weight in the 100mg/kg STZ ICR and 125
mg/kg ddY mice, in which a time course-related rise of
serum glucose levels was induced, was not significantly dif-
ferent from that in the respective normal mice for the obser-
vation period.
Urine volume per 24h in the 150 or 200mg/kg STZ ICR
and ddY mice was markedly greater than that in the respec-
tive normal mice throughout 3—12 weeks after STZ admin-
istration (Fig. 5B). In addition, drinking water (Fig. 5C) and
food consumption (Fig. 5D) per 24h at the same doses in
STZ ICR and ddY mice were also markedly greater than
those in the respective normal mice throughout the same pe-
riod. On the other hand, urine volume and drinking water and
food consumption in the 100mg/kg STZ ICR and 125mg/kg
STZ ddY mice steadily increased for 1—12 weeks, although
the degrees of increases were less than those found in mice
of both strains given 150 or 200mg/kg STZ.
Changes in Pancreatic Islet Morphology after STZ Ad-
ministration in ICR and ddY Mice
involution of pancreatic islets in the 200mg/kg STZ ICR and
ddY mice, and the area of islets in both strains markedly de-
creased from 1 week after STZ administration (ICR: Fig. 6;
ddY: Fig. 7). On the other hand, in the 100mg/kg STZ ICR
and 125mg/kg STZ ddY mice, the area of islets decreased
gradually as time passed after STZ administration. However,
the degree of involution of the islets was greater in the 125
mg/kg STZ ddY mice than in the 100mg/kg STZ ICR mice
at all stages after STZ administration.
The percentage of the number of glucagon (a-cells)- and
insulin-immunoreactive cells (b-cells) in pancreatic islets in
normal ICR and ddY mice was about 20% and 80%, respec-
tively, throughout the observation of 1—12 weeks (ICR:
Figs. 6, 8A; ddY: Figs. 7, 8B). The percentage of the number
of b-cells in 200mg/kg STZ ICR and ddY mice decreased
markedly, by less than 10% and 14%, respectively, from 1
There was a marked
June 2006 1113
Fig. 4.
Each plot denotes the mean?S.E.M. for 8 mice.
Changes in Serum Glucose and Insulin Levels within 72 h after a Single Injection of Streptozotocin (STZ) in ICR and ddY Mice

Page 5

week. On the other hand, the percentage of the number of b-
cells in the 100mg/kg STZ ICR mice continued to decrease
slowly over time, but was still preserved by 64% even at 12
weeks. The number of b-cells in 125mg/kg STZ ddY mice
also decreased slowly over time, but the degree of the de-
crease was greater in mice of this strain (percentage of the
number of b-cells at 12 weeks: 33%).
Serum Glucose and Insulin Responses to Oral Glucose
Loading after STZ Administration in Low Dose STZ ICR
and ddY Mice
Glucose tolerance testing in the 100mg/kg
STZ ICR and 125mg/kg STZ ddY mice in comparison with
the respective normal mice was carried out at various inter-
vals from 3d to 8 weeks after STZ administration. The fast-
ing serum glucose levels in the 100mg/kg STZ ICR and 125
mg/kg STZ ddY mice at 0.5h before oral glucose loading
was already significantly higher than those in the respective
1114Vol. 29, No. 6
Fig. 5.
tion of Various Doses of Streptozotocin (STZ)
Each plot denotes the mean?S.E.M. for 8 mice. Significantly different from respective normal, ∗p?0.05, ∗∗p?0.01.
Changes in Body Weight (A), Urine Volume (B), and Drinking Water (C) and Food Consumption (D) in ICR and ddY Mice after a Single i.p. Injec-