Streptozotocin-Induced Diabetic Models UNIT 5.

47
in Mice and Rats
Kenneth K. Wu1 and Youming Huan2
1
Merck Research Laboratories, Rahway, New Jersey
2
Mount Sinai School of Medicine, New York, New York

ABSTRACT
Streptozotocin (STZ) is an antibiotic that can cause pancreatic β-cell destruction, so it is
widely used experimentally as an agent capable of inducing insulin-dependent diabetes
mellitus (IDDM), also known as type 1 diabetes mellitus (T1DM). This unit describes
protocols for the production of insulin deficiency and hyperglycemia in mice and rats,
using STZ. These models for diabetes can be employed for assessing the mechanisms of
T1DM, screening potential therapies for the treatment of this condition, and evaluation of
therapeutic options. Curr. Protoc. Pharmacol. 40:5.47.1-5.47.14.
C 2008 by John Wiley

& Sons, Inc.

Keywords: streptozotocin r type 1 diabetes mellitus r hyperglycemia r
insulin deficiency r insulitis r mouse r rat

INTRODUCTION
Streptozotocin (STZ) was initially isolated from Streptomyces achromogenes in 1960,
after which it was shown to be a broad-spectrum antibiotic possessing antitumor, onco-
genic, and diabetogenic properties (Like and Rossini, 1976). Its diabetogenic property
is characterized by selective destruction of pancreatic islet β-cells, causing insulin de-
ficiency, hyperglycemia, polydipsia, and polyuria, all of which mimic human type 1
diabetes mellitus (T1DM; Kolb, 1987).

Several species, including the mouse, rat, rabbit, and monkey, are sensitive to the pan-
creatic β-cell cytotoxic effects of STZ. Currently, STZ is often used to induce diabetes
in all of those animals and is routinely used for that purpose in the mouse. This unit
describes two protocols that create STZ-induced diabetes in mice (Basic Protocol 1) and
rats (Basic Protocol 2). Basic Protocol 1 employs multiple administrations of low-dose
STZ to produce diabetic mice, and it is the most widely used STZ-induced diabetic
model. This model has two major advantages: (1) its close resemblance to human T1DM
with chronic pancreatic islet inflammation, insulitis, and insulin deficiency and (2) the
cost effectiveness related to the small size of the animal. Similarly, in Basic Protocol 2
STZ is used to induce diabetes in rats. Despite the fact that the STZ-induced diabetic rat
model is not used as commonly as the mouse model, it was one of earliest STZ-induced
animal models, and many investigators still use this model.

NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) and must comply with the
guidelines as established by the IACUC regarding the care and use of laboratory animals
in scientific experiments.

Animal Models
of Disease

Current Protocols in Pharmacology 5.47.1-5.47.14, March 2008 5.47.1

Published online March 2008 in Wiley Interscience ([Link]).
DOI: 10.1002/0471141755.ph0547s40 Supplement 40
Copyright
C 2008 John Wiley & Sons, Inc.
 BASIC INDUCTION OF TYPE 1 DIABETES MELLITUS IN MICE USING
PROTOCOL 1 REPEATED LOW DOSES OF STREPTOZOTOCIN
Streptozotocin (STZ) is a highly selective pancreatic islet β-cells cytotoxic agent, so it
was conventionally administrated as a single high dose to cause complete β-cell necrosis
and diabetes within 48 hr (Kolb, 1987). However, in 1976 Like and Rossini (1976)
administered multiple, low-dose STZ to mice on 5 consecutive days and observed a
delayed onset of hyperglycemia which, for kinetic reasons, could not be due to the direct,
rapid, toxic activity of the drug. This multiple, low-dose STZ approach partially damaged
pancreatic islets, triggering an inflammatory process that caused the further loss of
β-cell activity and resulted in insulin deficiency and hyperglycemia, which more closely
resembled type 1 diabetes mellitus (T1DM) in pathogenesis and morphologic changes
than the method employing a single high dose of STZ (Like and Rossini, 1976; Kolb,
1987; Kolb-Bachofen et al., 1988; Weide and Lacy, 1991). The multiple, low-dose STZ
approach is now the most widely used in T1DM research animals (Wu and Huan, 2007).

This protocol involves intraperitoneal administration of multiple, low doses of STZ

(40 mg/kg) to mice on 5 consecutive days to cause diabetes. The diabetic mouse model
can be used for testing the effectiveness of potential antidiabetic compounds. The com-
pounds can be initiated prior to and/or following induction of diabetes, depending on the
experimental goals.

Materials
C57BL/6 or CD-1 male mice: ∼25 g, 8 to 12 weeks old (The Jackson Laboratory
or Taconic); 12 to 20 per treatment group, recommended
Standard rodent chow diet (Harlan)
50 mM sodium citrate (enzyme grade; Fisher) buffer, pH 4.5: prepared just before
use
Streptozotocin (STZ; Sigma)
10% (w/v) sucrose (Sigma): prepared just before use
Test compound(s)
Rodent cages
Temperature-, humidity-, and light-controlled housing
1.5-ml microcentrifuge tubes
Aluminum foil
1-ml syringes
25-G needles
One Touch Basic blood glucose monitoring system (Lifescan)
Prepare animals
1. At least 5 days prior to the start of the experiment, house two to five male mice per
cage at 24◦ C ± 1◦ C and 55% ± 5% humidity, with a 12-hr light-dark cycle (light on
at 8:00 and light off at 20:00). Allow the mice free access to food and water.
Female mice are less sensitive to this islet-cell toxin; therefore, most STZ-induced diabetic
mouse studies have used males (Kolb, 1987).
The protocol described here minimizes variability, but group sizes of 12 to 20 are rec-
ommended. These high numbers per group will allow for the anticipated morbidity and
variance.

2. Weigh all mice accurately to 1 g. Randomly divide the mice into control groups and
study groups.

Streptozotocin-
The number of mice should be equal for each group.
Induced Diabetic
Models 3. On experimental day 1, 4 hr prior to STZ treatment, remove all food from cages for
all groups. Provide water as normal.
5.47.2
Supplement 40 Current Protocols in Pharmacology
Treat animals with STZ
4. Prepare the 50 mM sodium citrate buffer (pH 5.4). Place 1 ml of the buffer into
each 1.5-ml microcentrifuge tube (about one tube for five animals) and cover the
tubes with aluminum foil.
5. Immediately prior to injection, dissolve the STZ in the 50 mM sodium citrate buffer
(pH 4.5) to a final concentration of 6 mg/ml.
The STZ solution should be prepared fresh for each use and injected within 5 min of
being dissolved because STZ is not stable and degrades after 15 to 20 min in the citrate
buffer.

6. Using 1-ml syringes and 25-G needles, inject the STZ solution intraperitoneally at
40 mg/kg for the study groups. Inject an equal volume of citrate buffer (pH 4.5)
intraperitoneally for the control groups.
The results of intravenous and intraperitoneal injections are essentially equivalent (Like
and Rossini, 1976).

7. Return the mice to their cages. Provide normal food and 10% sucrose water.
8. On days 2 to 5 (the next 4 consecutive days), repeat steps 3 to 7.
9. On experimental day 6, switch the 10% sucrose water back to regular water.
Induce diabetic state
10a. For studies involving early-stage T1DM: On experimental day 14 (9 days after the
last STZ injection), fast all mice for 6 hr (e.g., from 7 a.m. to 1 p.m.). Test the blood
glucose level from the tail vein using a One Touch Basic blood glucose monitoring
system to check for STZ injection-induced hyperglycemia.
If the diabetic animals are for studying early-stage mechanisms of T1DM or for screening
compounds for early treatment of diabetes, the models are validated for further study
when the hyperglycemia is established in the STZ-injected mice (i.e., blood glucose levels
are >150 mg/dl and/or statistically higher in STZ-injected mice than in control mice).
Step 10b can be skipped if injected mice meet this criteria prior to experimental day 28.
If <40% of mice in the STZ-injected group attain a diabetic state by day 14, retest blood
glucose levels as described in step 10b.
Usually, determination of blood glucose levels is sufficient to diagnose diabetes, so it is
unnecessary to measure insulin levels.

10b. For retest of groups failing the first test for diabetes and for studies involving later-
stage T1DM: On experimental day 28, fast all mice for 6 hr (e.g., from 7 a.m. to 1
p.m.). Test the blood glucose level from the tail vein using a One Touch Basic blood
glucose monitoring system to confirm STZ injection-induced hyperglycemia.
Fasting glucose levels for mild hyperglycemia should be >150 mg/dl and/or exhibit
statistically significant increases in the STZ-injected mice compared to control mice.
Usually, severe diabetes develops in ∼50% of mice ∼3 weeks after STZ injection, with
blood glucose levels typically >300 to 600 mg/dl.
If >60% of STZ-injection mice still do not exhibit mild hyperglycemia at week 4, a
second round of STZ-injection is required at week 7 (repeat steps 3 to 8, plus step 10a,
except do not provide 10% sucrose water because in the second round of STZ injection,
the incidence of fatal hypoglycemia is much lower), or check whether there are any
errors or problems in the experiment (see Critical Parameters and Troubleshooting).
Usually, determination of blood glucose levels is sufficient to diagnose diabetes, so it is
unnecessary to measure insulin levels.
A blood glucose level of 18 mg/dl = 1 mM (Hartnell et al., 1990). Animal Models
of Disease

5.47.3
Current Protocols in Pharmacology Supplement 40
 Figure 5.47.1 Schematic representation of the time course of multiple, low-dose STZ-induced
diabetes in mice. Mice were treated with STZ (40 mg/kg) or without STZ (vehicle control) for
5 consecutive days. Typical changes in insulin secretion and blood glucose levels are illustrated.

11a. To test an agent or compound for ability to correct diabetes or affect hyperglycemia:
Once the diabetic state is established as described in step 10, treat the animals with
the potential restorative agent or compound. Include groups that receive appropri-
ate vehicle injections as a control. Extend the protocol longer, depending on the
experimental needs.
11b. To study a chronic condition or diabetic complications (e.g., STZ-induced diabetic
atherosclerosis): Repeat steps 3 to 8 (except do not provide 10% sucrose water
because in the second round of STZ injection, the incidence of fatal hypoglycemia
is much lower) at week 7 to maintain hyperglycemia in the STZ treatment group
(Kunjathoor et al., 1996).
The length of the experiment depends on the investigator’s purposes, e.g., from several
days to weeks for diabetic nephropathy or atherosclerosis studies.
Figure 5.47.1 illustrates the typical insulin secretion and blood glucose levels following
administration of multiple, low doses of STZ.

ALTERNATE INDUCTION OF TYPE 1 DIABETES MELLITUS IN MICE USING A SINGLE,

PROTOCOL HIGH DOSE OF STREPTOZOTOCIN
Administration of a single, high dose of STZ (200 mg/kg) was commonly used to cause
complete β-cell necrosis and diabetes before Like and Rossini (1976) applied multiple,
low doses of STZ to cause hyperglycemia. A single, high dose of STZ causes direct
toxicity to the β-cell, rapidly causing diabetes, and blood glucose levels can reach
>500 mg/dl within 48 hr (Like and Rossini, 1976). Although multiple, low doses of STZ
have minimal toxic effects and have replaced a single, high dose of STZ as the major
regimen in STZ-induced diabetic mice, some investigators still use the single high-dose
STZ method.

Materials
Streptozotocin- See Basic Protocol 1
Induced Diabetic
Models

5.47.4
Supplement 40 Current Protocols in Pharmacology
 1. At least 5 days prior to the start of the experiment, house two to five male mice per
cage at 24◦ C ± 1◦ C and 55% ± 5% humidity, with a 12-hr light-dark cycle (light
on at 8:00 and light off at 20:00). Allow the mice free access to food and water.
Female mice are less sensitive to this islet-cell toxin; therefore, most STZ-induced
diabetic mouse studies have used males (Kolb, 1987).
The protocol described here minimizes variability, but group sizes of 12 to 20 are
recommended. These high numbers per group will allow for the anticipated morbidity
and variance.
2. Weigh all mice accurately to 1 g. Randomly divide the mice into control groups
and study groups.
The number of mice should be equal for each group.
3. On experimental day 1, 4 hr prior to STZ treatment, remove all food from cages for
all groups. Provide water as normal.
4. Immediately prior to injection, dissolve the STZ in sodium citrate buffer (pH 4.5)
to a final concentration of 10 mg/ml.
The STZ solution should be prepared fresh for each injection and injected within 5 min
of being dissolved. Note the higher dose compared to Basic Protocol 1.
5. Inject the STZ intraperitoneally at 200 mg/kg for the study groups. Inject an equal
volume of citrate buffer (pH 4.5) intraperitoneally for the control group.
6. Return the mice to their cages. Provide normal food and 10% sucrose water. Closely
monitor the mice.
Some mice will die early from a high dose of STZ due to rapid massive β-cell necrosis,
which releases large amounts of insulin and causes fatal hypoglycemia (mostly within the
first day of STZ injection). If the number of early deaths is >20%, treat the remaining
mice with 1 ml of 5% glucose solution intraperitoneally (instead of providing 10%
sucrose water for drinking) 6 hr after STZ injection, to prevent fatal hypoglycemia
(Huang and Wu, 2005).
7. On experimental day 3, switch the 10% sucrose water back to regular water.
8a. For studies involving early-stage T1DM: On experimental day 10, fast all mice for
6 hr (e.g., from 7 a.m. to 1 p.m.). Then test the blood glucose level from the tail vein
with a One Touch Basic blood glucose monitoring system to check hyperglycemia.
If the diabetic animals are to be used for assessing early stage mechanisms of T1DM
or for screening potential compounds for early treatment of diabetes, the models are
validated for further study once hyperglycemia is established in STZ-injected mice
(glucose levels statistically higher than in control mice). In this case, skip step 8b. If
the diabetic animals are not for early stage assessment of T1DM mechanisms or for
screening potential compounds not for early treatment of diabetes, skip step 8a.
If <40% of the STZ-injected mice attain a diabetic state, retest blood glucose levels as
described in step 8b.
Usually, determination of blood glucose levels is sufficient to diagnose diabetes, so it is
unnecessary to measure insulin levels.
8b. For retest of groups failing the first test for diabetes or for studies involving later-
stage T1DM: On experimental day 21, fast all mice for 6 hr (e.g., from 7 a.m. to 1
p.m.). Test the blood glucose level from the tail vein using a One Touch Basic blood
glucose monitoring system to confirm STZ injection-induced hyperglycemia.
Fasting glucose levels for mild hyperglycemia should be >150 mg/dl and/or exhibit
statistically significant increases in the STZ-injected mice compared to control mice.
Animal Models
Usually at week 3, most STZ-injected mice develop severe diabetes with blood glucose of Disease
levels typically >300 to 600 mg/dl.
5.47.5
Current Protocols in Pharmacology Supplement 40
 A
*
600

500

Blood glucose
400

(mg/dl)
300

200

100

0
Vehicle STZ-treatment

B
40

30
Body weight (g)

20

10

0
Vehicle STZ-treatment

C *
40

30
Water intake (ml)

20

10

0
Vehicle STZ-treatment

Figure 5.47.2 A single, high dose of STZ causes diabetes in mice (n = 20). Mice were treated
with 200 mg/kg STZ or sodium citrate buffer vehicle (control). The effect of STZ on (A) nonfasting
blood glucose level, (B) body weight, and (C) daily water intake at 3 weeks after the STZ injection.
Data represent the mean ± SEM. *p < 0.001 versus control.

Streptozotocin-
Induced Diabetic
Models

5.47.6
Supplement 40 Current Protocols in Pharmacology
 If >60% of the STZ-injected mice still do not exhibit mild hyperglycemia, check whether
there are any problems with the experiment (see Critical Parameters and Troubleshoot-
ing), or use the multiple low-dose STZ approach (see Basic Protocol 1).
Usually, determination of blood glucose levels is sufficient to diagnose diabetes, so it is
unnecessary to measure insulin levels.
A blood glucose level of 18 mg/dl = 1 mM (Hartnell et al., 1990).

9a. To test an agent or compound for ability to correct diabetes or affect hyperglycemia:
Once the diabetic state is established as described in step 8, treat the animals with
the potential restorative agent or compound. Include groups that receive appropri-
ate vehicle injections as a control. Extend the protocol longer, depending on the
experimental needs.
9b. To study a chronic condition or diabetic complications (e.g., STZ-induced diabetic
atherosclerosis): Extend the protocol longer, depending on the experimental needs.
The length of the experiment depends on the investigator’s purposes, e.g., from several
days to weeks for diabetic nephropathy or atherosclerosis studies.
Figure 5.47.2 illustrates some characteristics of a typical mouse diabetic state 3 weeks
after injection of single high dose of STZ.

STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES MELLITUS IN RATS BASIC

PROTOCOL 2
The rat is commonly used as an STZ-induced diabetic model. An STZ-induced diabetic
state in rats is (as in the mouse) also dose-dependent (Arison et al., 1967; Junod et al.,
1969; Ganda et al., 1976). The most frequently used procedure is to administer one dose
of STZ (40 to 70 mg/kg) to rats aged 8 to 10 weeks (Brondum et al., 2005). Many
investigators use a single dose of ∼65 mg/kg to establish diabetes using the procedure
described in this protocol.

This protocol describes administration of a single intraperitoneal dose of STZ (65 mg/kg)
to rats to generate a T1DM state. The diabetic rats can be used to study the pathogenesis
of T1DM, as well as to evaluate antidiabetic agents or compounds (Bond et al., 1983).
Materials
Sprague-Dawley or Wistar male rats: ∼150 to 200 g, 8 to 10 weeks old (Charles
River Breeding Laboratories); 10 to 16 per treatment group, recommended
Standard rodent chow diet (Harlan)
50 mM sodium citrate (enzyme grade; Fisher) buffer, pH 4.5: prepared just before
use
Streptozotocin (STZ; Sigma)
10% (w/v) sucrose (Sigma): prepared just before use
50 mM sodium citrate (enzyme grade; Fisher) buffer, pH 4.5: prepared just before
use
Test compound(s)
Rodent cages
Temperature-, humidity-, and light-controlled housing
1.5-ml microcentrifuge tubes
Aluminum foil
3-ml syringes
23-G needles
One Touch Basic blood glucose monitoring system (Lifescan)
1. At least 5 days prior to the start of the experiment, house two to five male mice per
cage at 24◦ C ± 1◦ C and 55% ± 5% humidity, with a 12-hr light-dark cycle (light on Animal Models
at 8:00 and light off at 20:00) Allow the rats free access to food and water. of Disease

5.47.7
Current Protocols in Pharmacology Supplement 40
 Because female rats are less sensitive to STZ, most investigators use only males.
The protocol described here minimizes variability, but it is recommended that group sizes
of 10 to 16 be used. These high numbers per group will allow for the anticipated morbidity
and variance. Usually >80% of STZ-injected rats develop diabetes in an experiment.

2. Weigh all rats accurately to 1 g. Randomly divide the animals into control groups
and study groups.
The number of rats should be equal for each group.

3. On experimental day 1, fast all rats for 6 to 8 hr prior to STZ treatment. Provide
water as normal.
4. Prepare the 50 mM sodium citrate buffer (pH 4.5). Place 1 ml of the buffer into each
1.5-ml microcentrifuge tube and cover the tubes with aluminum foil.
5. Immediately prior to injection, dissolve STZ in the 50 mM sodium citrate buffer
(pH 4.5) to a final concentration of 10 mg/ml.
The STZ solution should be prepared fresh for each injection and injected within 5 min
of being dissolved.

6. Using a 3-ml syringe and 23-G needle, inject the STZ solution intraperitoneally at
65 mg/kg for the study group. Inject an equal volume of citrate buffer (pH 4.5)
intraperitoneally for the control group.
7. Return the rats to their cages. Provide normal food and 10% sucrose water.
8. On experimental day 2, switch the 10% sucrose water to regular water.
9a. For studies involving early-stage T1DM: On experimental day 10, fast all rats for
6 to 8 hr (between 7 a.m. and 1 to 3 p.m.). Test the blood glucose level from a tail
vein sample using a One Touch Basic blood glucose monitoring system to check
hyperglycemia.
If the diabetic animals are for assessing early-stage mechanisms of T1DM or for screening
compounds for treatment of early-stage diabetes, the models are validated for further study
when hyperglycemia is established in the STZ-injected rats (i.e., blood glucose levels are
>150 mg/dl and/or statistically higher compared to control rats). In this case, skip step
9b. If the diabetic animals are not for assessment of early-stage T1DM mechanisms, or
are for screening potential compounds not intended for early treatment of diabetes, skip
step 9a.
If <40% of the STZ-injected rats attain a diabetic state, retest blood glucose levels as
described in step 9b.
Usually, determination of blood glucose levels is sufficient to diagnose diabetes, so it is
unnecessary to measure insulin levels.

9b. For retest of groups failing the first test for diabetes or for studies involving later-
stage T1DM: On experimental day 21, fast all mice for 6 to 8 hr (e.g., from 7 a.m.
to 1 or 3 p.m.). Test the blood glucose level from a tail vein sample using a One
Touch Basic blood glucose monitoring system to confirm STZ injection-induced
hyperglycemia.
Fasting glucose levels for mild hyperglycemia should be >150 mg/dl and/or exhibit
statistically significant increases in the STZ-injected mice compared to control mice.
Usually at week 3, most STZ-injected rats develop severe diabetes with blood glucose
levels typically >250 to 600 mg/dl. If >60% of STZ-injected rats still do not exhibit mild
hyperglycemia, check whether there are any problems in the experiment (see Critical
Streptozotocin- Parameters and Troubleshooting).
Induced Diabetic
Models

5.47.8
Supplement 40 Current Protocols in Pharmacology
10. If a test agent or compound is being assessed for its ability to correct hyperglycemia,
extend the protocol longer, depending on the experimental needs. Treat groups of
animals as described in steps 3 to 9 to establish a diabetic state and then treat the
animals with the potential restorative therapy. Include groups that receive appropriate
vehicle injections as controls.
The length of the experiment depends on the investigator’s purposes, e.g., from several
days to weeks for diabetic nephropathy or atherosclerosis studies, although most investi-
gators use mouse rather than rat models for atherosclerosis studies.
Figure 5.47.3 illustrates rat blood glucose changes after STZ (65 mg/kg) injections on
different days.

COMMENTARY
Background Information cover the microcentrifuge tube with aluminum
This unit describes methods for using STZ foil to protect it from light (STZ is light-
to selectively destroy pancreatic islet β-cells sensitive). Streptozotocin is unstable in solu-
in mice and rats to generate animal mod- tion, even at an acidic pH, so do not mix STZ
els of T1DM. These T1DM models can fur- into citrate buffer until immediately prior to in-
ther develop diabetic complications, e.g., di- jection. The STZ solution should be prepared
abetic neuropathy (Usuki et al., 2007), dia- fresh each time and injected within 5 min of
betic nephropathy (Breyer et al., 2005), and being dissolved because the drug decomposes
diabetic atherosclerosis (Wu and Huan, 2007). in citrate buffer within 15 to 20 min.
The models are not only widely used to study
the mechanism and pathogenesis of T1DM, Animal gender sensitivity to STZ
but also to assess and evaluate experimental There is a strong influence of gender on the
approaches for the treatment of this condition, development of diabetes. Females appear to
in particular the hyperglycemia. These models be largely resistant to the effects of low-dose
have distinct advantages over others, including STZ, although this can be overcome by in-
the small size of the animals, short induction creasing the dose (Kolb, 1987). Because male
time period, ease of inducing diabetes, and cost pancreatic islet β-cells are more prone to STZ-
effectiveness (Wu and Huan, 2007). induced cytotoxicity than females, most inves-
Multiple, low-dose STZ-induced diabetic tigators employ male subjects for study (Kolb,
mouse models resemble human T1DM in 1987). The etiology of the observation is not
many aspects. These include the association well understood.
of hyperglycemia with lymphocyte infiltrates
Animal strain sensitivity to STZ
of the pancreatic islets, a marked β-cell necro-
Different strains of animals have differ-
sis, insulitis, and insulin deficiency (Like and
ent sensitivities to STZ-induced diabetes. For
Rossini, 1976; Bonnevie-Nielsen et al., 1981;
mice, CD-1 and C57BL/6 are reliably sensi-
Kolb, 1987; Weide and Lacy, 1991). How-
tive to STZ (Like and Rossini, 1976; Rossini
ever, because STZ may be toxic to organs
et al., 1977), with Sprague-Dawley and Wistar
and tissues other than the pancreatic islet
rats being particularly sensitive as well. If
β-cells these models do not completely mimic
blood glucose levels show insufficient hyper-
the human disease. For this reason, extrapo-
glycemia (fasting <150 mg/dl) at week 3 or
lation of the relationships observed with the
4 after terminating STZ administration, it is
human condition to the model is not straight-
possible the animal strain is insensitive to this
forward. This is particularly true when using
toxin.
a single, high dose of STZ, which is directly
toxic to β-cells, destroying them rapidly and Fatal hypoglycemia after STZ injection
completely, therefore resulting in the absence Some animals rapidly die from STZ-
of some T1DM features, such as pancreatic treatment due to massive islet β-cells necro-
insulitis (Kolb, 1987). sis with the sudden release of insulin, caus-
ing fatal hypoglycemia usually within the first
Critical Parameters and and second days after STZ injection. To pre-
Troubleshooting vent this it is best to routinely provide animals
STZ stability with 10% sucrose water after STZ treatment
Streptozotocin should be stored at −20◦ C (see [Link] If the number Animal Models
to avoid desiccation. After weighing STZ, of animal deaths are >20% using the single, of Disease

5.47.9
Current Protocols in Pharmacology Supplement 40
 high-dose STZ diabetic mouse protocol, treat ing state are lower and less variable than in
the animals with 1 ml of 5% glucose solution a nonfasting state. There is no standardized
intraperitoneally 6 hr after STZ injection in- hyperglycemia level for mice or rats because
stead of providing 10% sucrose water (Huang different institutes and investigators use dif-
and Wu, 2005). To avoid fatal hypoglycemia in ferent, nonstandardized fasting and nonfasting
multiple, low-dose STZ-treated diabetic mice, methods. However, there are three key points
provide 10% sucrose water for 6 days, begin- to remember: (1) use the same approach to
ning on experimental day 1. If mortality is high test blood glucose levels for both control and
(>20%) in single dose, STZ-treated diabetic STZ-treatment groups in the same experiment,
rats, provide 10% sucrose water for 2 days e.g., either a nonfasting state or a fasting state
after the STZ injection. for both control and STZ-treatment groups;
(2) never use fasting and nonfasting states in
Fasting and nonfasting blood glucose levels the same experiment; and (3) hyperglycemia
Mice and rats are nocturnal feeders and an means the blood glucose level in the STZ-
overnight fast before measuring blood glu- treatment groups is statistically higher than
cose levels usually translates into a more that found in the control groups.
prolonged fast of ∼24 hr. This 24-hr fast can Generally speaking, the blood glucose level
activate several physiologic responses that ob- in the STZ-treatment groups for nonfasting hy-
scure the reliability of glucose readings. Be- perglycemia should be >200 mg/dl, whereas
cause of this, fasting should be started on for fasting diabetes the blood glucose level
the morning of blood sampling. The National should be >150 mg/dl (glucose of 18 mg/dl =
Institutes of Health (NIH) and the Animal 1 mM). The most important point is that
Models of Diabetic Complications Consor- there should be a statistically significant dif-
tium (AMDCC) have established a protocol ference between the STZ-treatment and con-
of fasting mice from 7 a.m. to 1 p.m. with trol groups. Usually 3 weeks after STZ in-
blood drawn at 1 p.m. (Breyer et al., 2005; jection, >50% of animals will develop severe
[Link] Similarly, the ac- hyperglycemia and blood glucose levels will
cepted rat fasting time is 6 to 8 hr, between reach 300 to 600 mg/dl (see Animal Mod-
7 a.m. and 1 to 3 p.m., after which the blood els of Diabetic Complications Consortium,
sample is taken for analysis. [Link] If the study involves
Blood glucose levels between fasting and a chronic condition or diabetic complica-
nonfasting animals are quite different. The tion (e.g., STZ-induced diabetic atheroscle-
absolute levels of blood glucose in a fast- rosis) using multiple, low-dose STZ-induced

350

300

250
Blood glucose

200
(mg/dl)

150

100

50

0
day 0 day 2 day 8 day 36 day 120
Experiment time

Figure 5.47.3 STZ-induced hyperglycemia in rats. A single 65 mg/kg dose of STZ causes hyper-
Streptozotocin- glycemia in rats. Fasting blood glucose levels were monitored before and after the STZ injection
Induced Diabetic on the indicated days.
Models

5.47.10
Supplement 40 Current Protocols in Pharmacology
 A
16

14
**

Blood glucose (mmol/liter)

12

10
†
8 †

4
1 7 14 21
Days of treatment

B
100

80 **
Diabetes incidence (%)

60

40

†
20
††

0
1 7 14 21
Days of treatment

Figure 5.47.4 Effect of daily treatment with inosine on STZ-induced diabetes in mice. Daily
treatment with 100 or 200 mg/kg inosine for 21 days decreased hyperglycemia (A) and incidence
of diabetes (B) following multiple, low-dose STZ (MLDS) treatment of the mice. Mice were either
untreated (open circles); given daily doses of 200 mg/kg inosine alone (open squares); or treated
with STZ (on days 1 to 5) in combination with vehicle (filled circles), 100 mg/kg inosine (filled
triangles), or 200 mg/kg inosine (filled squares) starting on day 1. Diabetes incidence is expressed
as a cumulative percentage of mice with a blood glucose ≥11 mmol/liter. Results are means ±
SE for n = 20 mice in two separate experiments with 10 mice per experimental group. **p < 0.01
compared with vehicle-treated mice; †p < 0.05; ††p < 0.01 compared with MLDS-treated mice.
Reproduced from Mabley et al., 2003 with permission from The Feinstein Institute for Medical
Research.

diabetic mice, a second round of STZ injec- tion are summarized in Figure 5.47.1. The fig-
tions is required at week 7 to insure mainte- ure illustrates glucose and insulin secretory
nance of the diabetic state. responses to multiple, low doses of STZ in
treated mice. Usually, the blood glucose levels
Anticipated Results in the STZ-injection groups are significantly
For diabetic mice, typical time-dependent higher than in the control groups on experi- Animal Models
of Disease
daily changes of glucose and insulin secre- mental day 10. At experimental weeks 3 to
5.47.11
Current Protocols in Pharmacology Supplement 40
 Figure 5.47.5 Restoration of ß-cell function in STZ-induced diabetic mice. (A) Blood glucose
(BG) levels of single high-dose STZ-induced diabetic (open circles) or restored (filled squares)
mice. (B) Mean BG levels (±SEM) were calculated from each group. * and ** indicate statistical
differences (ANOVA) between the restored (n = 14) and the normal (n = 10) or diabetic (n = 34)
groups, respectively. Reproduced from Yin et al., 2006 with permission from the American Diabetes
Association.

4, blood glucose levels indicate severe hy- β-cell function and corrected hyperglycemia
perglycemia (300 to 600 mg/dl) in ∼50% of (Fig. 5.47.5; Yin et al., 2006). The STZ-
STZ-treated animals (it is generally not neces- induced diabetic rat model has also been used
sary to measure insulin levels to verify hyper- to evaluate antidiabetic drugs, in which val-
glycemia). Besides glucose and insulin level sartan (an angiotensin II receptor antagonist)
changes, mice also show typical T1DM fea- showed that the antidiabetic effect is dose-
tures, i.e., body weight loss and polydipsia dependent (Fig. 5.47.6; Chan et al., 2003).
at 3 weeks post STZ treatment (Flood et al.,
1990). Figure 5.47.2 illustrates typical features Time Considerations
of mouse diabetes. In diabetic rats, the hyper- Approximately 1 working day is needed to
glycemia lasts for months (Arison et al., 1967). perform the STZ injections. On the first day of
Figure 5.47.3 illustrates the typical diabetic the experiment, mice need to fast for 4 hr and
condition in rats. rats need to fast for 6 to 8 hr before the STZ
The diabetic models described in this unit injection. During the fasting period the citrate
can be used to assess diabetic mechanisms, buffer can be prepared. The STZ doses should
screen compounds, or evaluate therapeutic op- be prepared immediately before injection due
tions. For example, inosine, an immunomodu- to rapid STZ decomposition. To prevent early
lator and antiinflammatory agent, significantly fatal hypoglycemia in single, high-dose STZ-
reduced blood glucose levels in the multiple, induced diabetic mice, treat the mice with 1
low-dose STZ-induced diabetic mouse model ml of 5% glucose intraperitoneally 6 hr after
(Fig. 5.47.4; Mabley et al., 2003). In the single, the STZ injection.
Streptozotocin- It takes several hours to test the blood
Induced Diabetic high-dose STZ-induced mouse model, syn-
Models geneic islet transplantation partially restored glucose levels in all experimental animals to

5.47.12
Supplement 40 Current Protocols in Pharmacology
 28 *

Plasma glucose (mmol/liter)

26

*
24

**
22 **
**

20
0 0.05 0.1 0.2 0.4 0.8
Valsartan (mg/kg)

Figure 5.47.6 Effect of valsartan on plasma glucose concentration in STZ-induced diabetic rats.
The diabetic rats were treated with valsartan 2 weeks after STZ-injection. Values of mean ± SE
were obtained from each group of eight animals. *p < 0.05 and **p < 0.01 versus data from animals
treated with vehicle (0 mg/kg valsartan). Reproduced from Chan et al., 2003 with permission from
Lippincott, Williams, & Wilkins.

confirm the diabetic state on different post- Chan, P., Wong, K.L., Liu, I.M., Tzeng, T.F., Yang,
STZ-injection days, depending on the proto- T.L., and Cheng, J.T. 2003. Antihyperglycemic
action of angiotensin II receptor antagonist, val-
col. The amount of time required over subse-
sartan, in streptozotocin-induced diabetic rats.
quent days or weeks is dependent on the aim J. Hypertens. 21:761-769.
of the study.
Flood, J.F., Mooradian, A.D., and Morley, J.E.
If the animals are to be used to study di- 1990. Characteristics of learning and memory in
abetic complications (e.g., STZ-induced di- streptozotocin-induced diabetic mice. Diabetes
abetic atherosclerosis), repeat the STZ treat- 39:1391-1398.
ment at experimental week 7, which takes ∼1 Ganda, O.P., Rossini, A.A., and Like, A.A. 1976.
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several weeks or months, depending on the 25:595-603.
aims of the study. Hartnell, J.M., Storrie, M.C., and Mooradian, A.D.
1990. Diabetes-related changes in chromatin
structure of brain, liver, and intestinal epithe-
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T., Rivner, M., and Yu, R.K. 2007. Effect of Web site for Animal Models of Diabetic Compli-
pregerminated brown rice intake on diabetic cations Consortium (AMDCC), providing new ani-
neuropathy in streptozotocin-induced diabetic mal models of diabetic complications, with the goal
rats. Nutr. Metab. (Lond). 4:25. of identifying the most appropriate animal models
Weide, L.G. and Lacy, P.E. 1991. Low-dose to study the etiology, prevention, and treatment of
streptozocin-induced autoimmune diabetes in diabetic complications.

Streptozotocin-
Induced Diabetic
Models

5.47.14
Supplement 40 Current Protocols in Pharmacology