Streptozotocin-Induced Diabetic Models in Mice and Rats
Kenneth K. Wu1 and Youming Huan2
1Merck Research Laboratories, Rahway, New Jersey
2Mount 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 . type 1 diabetes mellitus . hyperglycemia .
insulin deficiency . insulitis . mouse . rat

UNIT 5.47

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.

Current Protocols in Pharmacology 5.47.1-5.47.14, March 2008
Published online March 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471141755.ph0547s40
Copyright ⓍC 2008 John Wiley & Sons, Inc.
Animal Models of Disease
5.47.1
Supplement 40

BASIC PROTOCOL 1

Streptozotocin- Induced Diabetic
Models

INDUCTION OF TYPE 1 DIABETES MELLITUS IN MICE USING 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
± ±
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.
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.
On experimental day 1, 4 hr prior to STZ treatment, remove all food from cages for all groups. Provide water as normal.

Treat animals with STZ
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.
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.
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).
Return the mice to their cages. Provide normal food and 10% sucrose water.
On days 2 to 5 (the next 4 consecutive days), repeat steps 3 to 7.
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

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 PROTOCOL

Streptozotocin-
INDUCTION OF TYPE 1 DIABETES MELLITUS IN MICE USING A SINGLE, 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
See Basic Protocol 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.
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.
On experimental day 1, 4 hr prior to STZ treatment, remove all food from cages for all groups. Provide water as normal.
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.
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.
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).
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.
Usually at week 3, most STZ-injected mice develop severe diabetes with blood glucose levels typically >300 to 600 mg/dl.

Animal Models of Disease

Blood glucose (mg/dl)
Water intake (ml)
Body weight (g)

Streptozotocin-
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.

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
∼
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)
± ±
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 rats free access to food and water.

BASIC PROTOCOL 2

Animal Models of Disease

Streptozotocin-
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.
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.
On experimental day 1, fast all rats for 6 to 8 hr prior to STZ treatment. Provide water as normal.
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.
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.
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.
Return the rats to their cages. Provide normal food and 10% sucrose water.
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 Parameters and Troubleshooting).

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
This unit describes methods for using STZ to selectively destroy pancreatic islet β-cells in mice and rats to generate animal mod- els of T1DM. These T1DM models can fur- ther develop diabetic complications, e.g., di- abetic neuropathy (Usuki et al., 2007), dia- betic nephropathy (Breyer et al., 2005), and diabetic atherosclerosis (Wu and Huan, 2007). The models are not only widely used to study the mechanism and pathogenesis of T1DM, but also to assess and evaluate experimental approaches for the treatment of this condition, in particular the hyperglycemia. These models have distinct advantages over others, including the small size of the animals, short induction time period, ease of inducing diabetes, and cost effectiveness (Wu and Huan, 2007).
Multiple, low-dose STZ-induced diabetic
mouse models resemble human T1DM in many aspects. These include the association of hyperglycemia with lymphocyte infiltrates of the pancreatic islets, a marked β-cell necro- sis, insulitis, and insulin deficiency (Like and Rossini, 1976; Bonnevie-Nielsen et al., 1981; Kolb, 1987; Weide and Lacy, 1991). How- ever, because STZ may be toxic to organs and tissues other than the pancreatic islet β-cells these models do not completely mimic the human disease. For this reason, extrapo- lation of the relationships observed with the human condition to the model is not straight- forward. This is particularly true when using a single, high dose of STZ, which is directly toxic to β-cells, destroying them rapidly and completely, therefore resulting in the absence of some T1DM features, such as pancreatic insulitis (Kolb, 1987).
Critical Parameters and Troubleshooting
STZ stability
−
Streptozotocin should be stored at 20◦C to avoid desiccation. After weighing STZ,
cover the microcentrifuge tube with aluminum foil to protect it from light (STZ is light- sensitive). Streptozotocin is unstable in solu- tion, even at an acidic pH, so do not mix STZ into citrate buffer until immediately prior to in- jection. The STZ solution should be prepared fresh each time and injected within 5 min of being dissolved because the drug decomposes in citrate buffer within 15 to 20 min.

Animal gender sensitivity to STZ
There is a strong influence of gender on the development of diabetes. Females appear to be largely resistant to the effects of low-dose STZ, although this can be overcome by in- creasing the dose (Kolb, 1987). Because male pancreatic islet β-cells are more prone to STZ- induced cytotoxicity than females, most inves- tigators employ male subjects for study (Kolb, 1987). The etiology of the observation is not well understood.

Animal strain sensitivity to STZ
Different strains of animals have differ- ent sensitivities to STZ-induced diabetes. For mice, CD-1 and C57BL/6 are reliably sensi- tive to STZ (Like and Rossini, 1976; Rossini et al., 1977), with Sprague-Dawley and Wistar rats being particularly sensitive as well. If blood glucose levels show insufficient hyper- glycemia (fasting <150 mg/dl) at week 3 or 4 after terminating STZ administration, it is possible the animal strain is insensitive to this toxin.

Fatal hypoglycemia after STZ injection
Some animals rapidly die from STZ- treatment due to massive islet β-cells necro- sis with the sudden release of insulin, caus- ing fatal hypoglycemia usually within the first and second days after STZ injection. To pre- vent this it is best to routinely provide animals with 10% sucrose water after STZ treatment (see http://www.AMDCC.org). If the number of animal deaths are >20% using the single,

Animal Models of Disease

high-dose STZ diabetic mouse protocol, treat the animals with 1 ml of 5% glucose solution intraperitoneally 6 hr after STZ injection in- stead of providing 10% sucrose water (Huang and Wu, 2005). To avoid fatal hypoglycemia in multiple, low-dose STZ-treated diabetic mice, provide 10% sucrose water for 6 days, begin- ning on experimental day 1. If mortality is high (>20%) in single dose, STZ-treated diabetic rats, provide 10% sucrose water for 2 days after the STZ injection.

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

Streptozotocin- Induced Diabetic

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

Diabetes incidence (%)
† ††
=
≥ ±
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.

Blood glucose (mmol/liter)
diabetic mice, a second round of STZ injec- tions is required at week 7 to insure mainte- nance of the diabetic state.

Anticipated Results
For diabetic mice, typical time-dependent daily changes of glucose and insulin secre-
tion are summarized in Figure 5.47.1. The fig- ure illustrates glucose and insulin secretory responses to multiple, low doses of STZ in treated mice. Usually, the blood glucose levels in the STZ-injection groups are significantly higher than in the control groups on experi- mental day 10. At experimental weeks 3 to

Animal Models of Disease

= = =
±
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.

Streptozotocin- Induced Diabetic
4, blood glucose levels indicate severe hy- perglycemia (300 to 600 mg/dl) in 50% of STZ-treated animals (it is generally not neces- sary to measure insulin levels to verify hyper- glycemia). Besides glucose and insulin level changes, mice also show typical T1DM fea- tures, i.e., body weight loss and polydipsia at 3 weeks post STZ treatment (Flood et al., 1990). Figure 5.47.2 illustrates typical features of mouse diabetes. In diabetic rats, the hyper- glycemia lasts for months (Arison et al., 1967). Figure 5.47.3 illustrates the typical diabetic condition in rats.
∼
The diabetic models described in this unit can be used to assess diabetic mechanisms, screen compounds, or evaluate therapeutic op- tions. For example, inosine, an immunomodu- lator and antiinflammatory agent, significantly reduced blood glucose levels in the multiple, low-dose STZ-induced diabetic mouse model (Fig. 5.47.4; Mabley et al., 2003). In the single, high-dose STZ-induced mouse model, syn- geneic islet transplantation partially restored
β-cell function and corrected hyperglycemia (Fig. 5.47.5; Yin et al., 2006). The STZ-
induced diabetic rat model has also been used to evaluate antidiabetic drugs, in which val- sartan (an angiotensin II receptor antagonist) showed that the antidiabetic effect is dose- dependent (Fig. 5.47.6; Chan et al., 2003).

Time Considerations
Approximately 1 working day is needed to perform the STZ injections. On the first day of the experiment, mice need to fast for 4 hr and rats need to fast for 6 to 8 hr before the STZ injection. During the fasting period the citrate buffer can be prepared. The STZ doses should be prepared immediately before injection due to rapid STZ decomposition. To prevent early fatal hypoglycemia in single, high-dose STZ- induced diabetic mice, treat the mice with 1 ml of 5% glucose intraperitoneally 6 hr after the STZ injection.
It takes several hours to test the blood glucose levels in all experimental animals to

Plasma glucose (mmol/liter)
±
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- STZ-injection days, depending on the proto- col. The amount of time required over subse- quent days or weeks is dependent on the aim of the study.
∼
If the animals are to be used to study di- abetic complications (e.g., STZ-induced di- abetic atherosclerosis), repeat the STZ treat- ment at experimental week 7, which takes 1 working day, then continue observations for several weeks or months, depending on the aims of the study.

Literature Cited
Arison, R.N., Ciaccio, E.I., Glitzer, M.S., Cassaro, J.A., and Pruss, M.P. 1967. Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin. Diabetes 16:51-56.
Bond, J.S., Failla, M.L., and Unger, D.F. 1983. Ele- vated manganese concentration and arginase ac- tivity in livers of streptozotocin-induced diabetic rats. J. Biol. Chem. 258:8004-8009.
Bonnevie-Nielsen, V., Steffes, M.W., and Lernmark, A. 1981. A major loss in islet mass and B-cell function precedes hyperglycemia in mice given multiple low doses of streptozotocin. Diabetes 30:424-429.
Breyer, M.D., Bottinger, E., Brosius, F.C. 3rd, Coffman, T.M., Harris, R.C., Heilig, C.W., and Sharma, K., AMDCC. 2005. Mouse models of diabetic nephropathy. J. Am. Soc. Nephrol. 16:27-45.
Brondum, E., Nilsson, H., and Aalkjaer, C. 2005. Functional abnormalities in isolated arteries from Goto-Kakizaki and streptozotocin-treated diabetic rat models. Horm. Metab. Res. 37:56- 60.
Chan, P., Wong, K.L., Liu, I.M., Tzeng, T.F., Yang, T.L., and Cheng, J.T. 2003. Antihyperglycemic action of angiotensin II receptor antagonist, val- sartan, in streptozotocin-induced diabetic rats. J. Hypertens. 21:761-769.
Flood, J.F., Mooradian, A.D., and Morley, J.E. 1990. Characteristics of learning and memory in streptozotocin-induced diabetic mice. Diabetes 39:1391-1398.
Ganda, O.P., Rossini, A.A., and Like, A.A. 1976. Studies on streptozotocin diabetes. Diabetes 25:595-603.
Hartnell, J.M., Storrie, M.C., and Mooradian, A.D. 1990. Diabetes-related changes in chromatin structure of brain, liver, and intestinal epithe- lium. Diabetes 39:348-353.
Huang, F. and Wu, W. 2005. Antidiabetic effect of a new peptide from Squalus mitsukurii liver (S-8300) in streptozocin-induced diabetic mice.
J. Pharm. Pharmacol. 57:1575-1580.
Junod, A., Lambert, A.E., Stauffacher, W., and Renold, A.E. 1969. Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J. Clin. Invest. 48:2129- 2139.
Kolb, H. 1987. Mouse models of insulin depen- dent diabetes: Low-dose streptozocin-induced diabetes and nonobese diabetic (NOD) mice. Diabetes Metab. Rev. 3:751-778.
Kolb-Bachofen, V., Epstein, S., Kiesel, U., and Kolb, H. 1988. Low-dose streptozocin-induced diabetes in mice. Electron microscopy re- veals single-cell insulitis before diabetes onset. Diabetes 37:21-27.
Kunjathoor, V.V., Wilson, D.L., and LeBoeuf,
R.C. 1996. Increased atherosclerosis in streptozotocin-induced diabetic mice. J. Clin. Invest. 97:1767-1773.

Animal Models of Disease

Like, A.A. and Rossini, A.A. 1976. Streptozotocin- induced pancreatic insulitis: New model of dia- betes mellitus. Science 193:415-417.
Mabley, J.G., Rabinovitch, A., Suarez-Pinzon, W., Hasko´, G., Pacher, P., Power, R., Southan, G., Salzman, A., and Szabo´, C. 2003. Inosine pro- tects against the development of diabetes in multiple-low-dose streptozotocin and nonobese diabetic mouse models of type 1 diabetes. Mol. Med. 9:96-104.
Rossini, A.A., Appel, M.C., Williams, R.M., and Like, A.A. 1977. Genetic influence of the streptozotocin-induced insulitis and hyper- glycemia. Diabetes 26:916-920.
Usuki, S., Ito, Y., Morikawa, K., Kise, M., Ariga, T., Rivner, M., and Yu, R.K. 2007. Effect of pregerminated brown rice intake on diabetic neuropathy in streptozotocin-induced diabetic rats. Nutr. Metab. (Lond). 4:25.
Weide, L.G. and Lacy, P.E. 1991. Low-dose streptozocin-induced autoimmune diabetes in
islet transplantation model. Diabetes 40:1157- 1162.
Wu, K.K. and Huan, Y. 2007. Diabetic atheroscle- rosis mouse models. Atherosclerosis. 191:241- 249.
Yin, D., Tao, J., Lee, D.D., Shen, J., Hara, M., Lopez, J., Kuznetsov, A., Philipson, L.H., and Chong, A.S. 2006. Recovery of islet beta- cell function in streptozotocin-induced diabetic mice: An indirect role for the spleen. Diabetes 55:3256-3263.

Internet Resource

http://www.AMDCC.org

Web site for Animal Models of Diabetic Compli- cations Consortium (AMDCC), providing new ani- mal models of diabetic complications, with the goal of identifying the most appropriate animal models to study the etiology, prevention, and treatment of diabetic complications.