Experimental diabetes induced by alloxan and streptozotocin: The current
state of the art

Miroslav Radenković, Marko Stojanović, Milica Prostran

PII: S1056-8719(15)30002-2
DOI: doi: 10.1016/j.vascn.2015.11.004
Reference: JPM 6312

To appear in: Journal of Pharmacological and Toxicological Methods

Received date: 28 June 2015

Revised date: 14 November 2015
Accepted date: 15 November 2015

Please cite this article as: Radenković, M., Stojanović, M. & Prostran, M., Experimental
diabetes induced by alloxan and streptozotocin: The current state of the art, Journal of
Pharmacological and Toxicological Methods (2015), doi: 10.1016/j.vascn.2015.11.004

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Title:
Experimental diabetes induced by alloxan and streptozotocin: the current state of the art

Type of article:

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Appraisal of state-of-the-art

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Authors:

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Miroslav Radenković1*, Marko Stojanović1 and Milica Prostran1

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Affiliation:
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Department of Pharmacology, Clinical Pharmacology and Toxicology; Faculty of Medicine;
University of Belgrade; PO Box 38; 11129 Belgrade; Serbia
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E-mail addresses:
MR: mradenkovic@med.bg.ac.rs
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MS: marko.stojanovic@mfub.bg.ac.rs
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MP: mprostran@doctor.com
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*Corresponding Author:
Miroslav Radenković, MD, PhD
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Associate Professor
Clinical Pharmacology Specialist
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Department of Pharmacology, Clinical Pharmacology and Toxicology;

Medical Faculty; University of Belgrade
PO Box 38; 11129 Belgrade; Serbia
Tel.: +381 11 3643 404; Fax: +381 11 3643 397
E-mail: mradenkovic@med.bg.ac.rs

Conflict of Interest:
Authors declare no conflicts of interest.

Contributions:
All authors contributed in the literature search, data extraction, writing of the manuscript and critical
review of the final text.
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Abstract

Diabetes mellitus is a chronic metabolic disorder with a high prevalence worldwide.

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Animal models of diabetes represent an important tool in diabetes investigation that helps us

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to avoid unnecessary and ethically challenging studies in human subjects, as well as to obtain
a comprehensive scientific viewpoint of this disease. Although there are several methods

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through which diabetes can be induced, chemical methods of alloxan- and streptozotocin-

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induced diabetes represent the most important and highly preferable experimental models for
this pathological condition. Therefore, the aim of this article was to review the current

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knowledge related to quoted models of diabetes, including to this point available information
about mechanism of action, particular time- and dose- dependent protocols, frequent
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problems, as well as major limitations linked to laboratory application of alloxan and
sterptozotocin in inducing diabetes. Given that diabetes is known to be closely associated
with serious health consequences it is of fundamental importance that current animal models
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for induction of diabetes should be continuously upgraded in order to improve overall

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prevention, diagnosis and treatment of this pathological condition.

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Keywords: experimental diabetes, alloxan, streptozotocin, methods

Chemical compounds:
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Alloxan (PubChem CID: 5781), streptozotocin (PubChem CID: 29327), phlorizin

(PubChem CID: 6072), nicotinamide (PubChem CID: 936)
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1. Introduction

Diabetes is a chronic metabolic disorder, which appears when there is a defect in

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insulin secretion or/and when the body cannot use insulin in an effective way. This leads to
an increase in blood level of glucose, which is called hyperglycemia. Two major forms of

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diabetes are generally identified, namely diabetes type 1, also known as juvenile-onset

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diabetes, and diabetes type 2, formerly termed as adult-onset diabetes (Cefalu, 2006). Type 1
diabetes represents approximately 10% of all cases of diabetes, and it develops as a result of

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autoimmune process in which B cells destruction is a final consequence of a T-cell –
mediated autoimmune attack (Mathis et al., 2001; Rother, 2007). This type of diabetes results
from the pancreas failure to produce insulin and the person with this type of diabetes requires
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regular insulin injections, which is why this type is also known as an insulin-dependent
diabetes mellitus. The main characteristics of type 1 diabetes are high glucose and low insulin
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levels in blood. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, and
it represents more than 90% of all cases (Cefalu, 2006). This type is known as a progressive
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disorder, and it is associated with gradual diminishing of pancreatic function over a period of
time. Although type 2 diabetes can be sometimes concomitantly found with an absolute
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insulin deficiency, at the beginning, the main problem is usually not insulin secretion but the
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peripheral resistance to insulin. This type can also progress toward insulin dependency during
a certain period of time. This is why type 2 diabetes can be associated with elevated, normal
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or low insulin levels (Cefalu, 2006). Apart from these two types of diabetes there is an
additional type called gestational diabetes which occurs in pregnant women, who never
experienced any problem with high blood glucose level before pregnancy (Radenković,
2011). Additionally, gestational diabetes is considered to be very important since it can
frequently precede development of type 2 diabetes (Kumar et al., 2012; Radenković et al.,
2007).

Over the recent years the prevalence of diabetes throughout the world population has
increased dramatically, unfortunately this trend will continue in the future. This rise in
prevalence of diabetes can be explained with several factors, including overall population
growth, aging, urbanization and an increase of obesity and physical inactivity (Kumar et al.,
2012). In order to better understand the significance of this increase in prevalence of
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diabetes, it is important to note that the number of people affected by diabetes in 1980 was
153 million and that this number significantly increased to 347 million in the year 2008
(Danaei et al., 2011). This is indeed a major public issue because diabetes is a complex,
multifactorial disease and the overall risk of dying among people with diabetes is at least

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double, if comparing to the risk of their peers without diabetes (Roglic et al., 2005). The
economic costs of medical care provided for patients with diabetes, as well as related

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complications are stupefying. For example, in 2002 it was estimated that direct and indirect

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medical expenditures related to diabetes were 132 billion US dollars, whereas in 2007 this
number was even higher and the estimated costs were 174 billion US dollars (Dall et al.,

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2003, American Diabetes Association, 2008). Finally, in 2012 the estimated
total economic cost of diagnosed diabetes was 245 billion US dollars, which is a 41%
increase from the previous estimation reported in 2007 (American Diabetes Association
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2013). Concerning these assessments, it is easy to comprehend the substantial burden that
diabetes imposes on society.
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Considering previously mentioned facts, it is clear that well designed researches in the
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field of diabetes are needed in the future to obtain new knowledge that will be used to reduce
the consequences associated with this disease. Although the epidemiology of diabetes has
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been well described, there is much more about the pathological process itself that we need to
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learn. For example, we need to find out more about complex mechanisms that underlie the
development of diabetes and its complications, to understand the natural history of the
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disease, to identify new targets for therapy, and to re-evaluate already existing interventions
and treatments. Although some of the previously mentioned problems can be resolved by
further studies in human population, unfortunately there are many questions that can be
answered only through invasive manipulations or observations that are not allowed in humans
either for logistical or ethical reasons (Kaplan & Wagner, 2006). A major concern in
investigation of diabetes is its natural history that takes years to be developed, as well as time
needed before all complications would be detectable. Also, it is time consuming and very
expensive to assess the effect of different interventions aimed to modulate development of
diabetes or its complications (Cefalu, 2006). To address this concern, it is also important to
summarize current knowledge about existing animal models of diabetes, and if possible to
develop and utilize new ones in order for planed interventions to be assessed in much shorter
time spans. Accordingly, the aim of this review paper was to collect and present all the
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important facts concerning chemically induced diabetes with streptozotocin and alloxan, and
to point out all positive and negative characteristics of these experimental diabetes models.

2. Ethical issues of animal use in diabetes mellitus research

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Whenever animal research is mentioned, including diabetes research in animals, two
important opinions can be distinguished (Rees & Alcolado, 2005). One of them is that

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animals are unable to provide informed consent and have no direct or indirect benefit from

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the experiments, while the other opinion points out those experiments on animals should be
performed with little or no external influence. The truth is that animal research is essential for

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obtaining many conclusions that can help us in everyday medical practice and also to reduce
human and financial impairments. On the other hand, animal use in research should not be
taken for granted, since this could be a serious ethical issue (Radenković et al., 2012). The
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ethical problem is regarded in the fact that the use of animals in experiments, can also involve
the suffering of the animals, which can be physical and/or psychological (Levy, 2012). This
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means that existing similarities between humans and animals cannot entirely justify the use of
animals for research, since those similarities also raise some ethical issues, given that as
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humans, animals are also able to suffer. Numerous efforts have been made to replace
needless use of animals with alternative in vitro models. This is with the intention of to
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reducing the number of animals used in experiments and to reduce the pain and discomfort
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(Kroeger, 2006). One of the most important regulation concepts of animal welfare is the 3Rs
concept (replacement, reduction and refinement) created by Russell and Burch in 1959
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(Kroeger, 2006). The 3Rs concept allows fair use of animals in research and the correct
application of this model reduces the possibility of compromising overall animal welfare.
Also, a good scientist should never forget that the "best animal welfare" results in the "best
science" (Russell & Burch, 1959).

When ethics of utilizing animal models of diabetes are concerned, it is important to

realize that advances in biomedical sciences usually come as a combination of results
obtained from all levels of research, from molecular to clinical, and that animal use is only
one of the steps in obtaining these results. Also, one should always bear in mind that animal
models of diabetes in particular have provided many benefits to humans including discovery
of insulin. Nevertheless, this does not mean that animal use in diabetes-related investigations
should not be justified at all times. Moreover, before the start of any research one must
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establish if that investigation will provide substantial benefits, such as saving of human lives,
or the considerable contribution to human welfare (Hoff, 1980). The major problem is that
biomedical studies in which animals are used do not always provide an immediate benefit to
humans (Sieber & Traystman, 1993), and this is something that makes the real challenge, not

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only in diabetes research, but in all other fields that include use of laboratory animals.

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3. Animal models of diabetes

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Animal models have been used extensively to obtain different information about
various pathological conditions. Until now, many animal models of diabetes have been

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created. For an animal model of diabetes it is important that it can mirror the pathogenesis
and natural history of diabetes or/and to induce further development of specific complications
connected to it. However, the fact is that we cannot underline any single animal model of
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diabetes to be entirely suitable in terms of mimicking development of human diabetes and its
complications. Since there is no animal model of diabetes that encompasses all of the above
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mentioned, it is desirable to continue with the development of new improved models of

diabetes that will completely, or at least more adequately than current models, mimic
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different aspects of human diabetes.

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In earlier animal studies, a model of pancreatectomized dog was used in order to

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confirm the central role of the pancreas in glucose homeostasis, as well as for discovery and
purification of insulin (Rees & Alcolado, 2005, Lenzen, 1988). In fact, pancreatectomy was
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the first animal model of diabetes. Since that period many new animal models of diabetes
have been created. Nowadays, experiments are rarely performed on dogs for many reasons.
One of them is based on the guiding principles for animal research directing us to utilize the
lowest level of animal species available. Also, the smaller the animal is, the more convenient
and less expensive the experiment will be. This is why the majority of experiments are
performed on rodents. It is important to note that rats are the first choice of use, comprising
over 85% of these models (Wilson & Islam, 2012). Nevertheless, one should bear in mind
that for example according to the Annual Statistics of Scientific Procedures on Living
Animals Great Britain 2013 (2014) rodents are the most commonly used animals for
scientific research, and that at the same time mice are generally far more frequently used than
rats. This can also be the case in diabetes investigations since mice are more appropriate for
developing of transgenic strains. Although rodents are the most commonly used species, they
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are not completely ideal for diabetes research. One of important issue is that they may not
adequately reflect various human homeostatic mechanisms (Rees & Alcolado, 2005). For this
reason some larger animals including cats, dogs, pigs and primates are also used.
Accordingly, domestic cats have been used because this is one of the few outbreed models

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characterized by insulin resistance, defective insulin secretion, islet amyloid formation, and B
cells loss (Henson & O'Brien, 2006). Moreover, amyloid formation in the pancreas is one of

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the main pathological findings in primates (Wagner et al, 2006), which is important since

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islet amyloid is found in approximately 90% of human T2DM cases (Cefalu, 2006).
Additionally, one of the major advantages of using the primate model lays in its compatibility

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in terms of development of atherosclerosis in humans (Cefalu, 2006, Clarkson, 1998). To
substantiate this piece of information it is known that swine model of diabetes is a relevant
animal model for studying metabolic abnormalities that follows diabetes. In that way, these
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animals can be used for investigation of cardiovascular complications of diabetes since they
can develop coronary, aortic, iliac, and carotid atherosclerotic lesions in anatomical locations
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particularly relevant to the appropriate human condition, and these lesions recapitulate the
histopathology seen in humans (Cefalu, 2006).
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Experimental diabetes mellitus in laboratory animals can be induced by using

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chemical, surgical and genetic/immunological manipulation. This can be also regarded as one
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of the classification methods of experimental diabetes, which is in this case based on the
method of induction. In this review paper our aim was to specifically put an accent on
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chemically-induced diabetes. The most important and the most frequently used chemical
compounds for induction of experimental diabetes are alloxan and streptozotocin. Next to
alloxan and streptozotocin there are several other compounds that also possess diabetogenic
activity and these are used for induction of experimental diabetes in animals, as well. Thus,
compounds including vacor (Ahn et al., 1998, Seon, 1999, Kim et sl., 2002), dithizone
(Halim et al., 1977, Hansen et al., 1989), goldthioglucose (De Leeuw et al., 1981, Heydrick et
al., 1995), monosodium glutamate (Nagata et al., 2006, Morrison et al., 2008, Sasaki et al.,
2009) and 8-hydrooxyquinolone (Root & Chen, 1952) can be also used to induce
experimental diabetes. Apart from these compounds, there are several others that can be
administered to animals with already developed diabetes, and they are usually aimed to
mimic specific problem related to this disease. For example phlorizin, a naturally occurring
flavanoid is used as an animal model of glycosuria. Namely, phlorizin blocks Na/glucose
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transporter in the proximal renal tubule. If phlorizin is administered to a rodent with diabetes
it will produce near-normoglycemia, which is actually accomplished due to massive
glycosuria and polyuria (Rees & Alcolado, 2005). This model can help us to separate specific
effects of glucose from the myriad of other metabolic consequences of experimental diabetes,

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and it can be also used for studying the particular role of hyperglycemia in the development
of diabetes complications (Leung et al, 2004).

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Animal models of diabetes can be also classified based on the type of diabetes that

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they might represent (Rees & Alcolado, 2005). Both alloxan and streptozotocin can be used
for induction of type 1 and type 2 diabetes. Still, these chemicals are most commonly used for

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induction of type 1 diabetes because they are unable to directly induce an insulin resistance.
Additionally, some other models have been developed as well, such as nicotinamide-
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streptozotocin model (Masiello et al., 1998), fat-fed streptozotocin-treated rat model (Reed et
al., 2000, Srinivasan et al., 2005) and fructose-fed streptozotocin-injected model (Wilson &
Islam, 2012). Effects of gestational diabetes can be investigated with alloxan and
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stereptozotocin model, too (Van Assche et al., 1991). More information on this topic is
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presented below.

One of the most frequently used classifications regarding experimental diabetes is

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classification related to animal models for screening of anti-diabetic agents (Kumar et al.,
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2012). This classification recognizes three main types, which are further classified into
several subtypes (Table 1).
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4. Basic properties of alloxan and streptozotocin

Alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil) is a pyrimidine derivative that

is synthesized by uric acid oxidation (Lenzen, 1988, Lenzen, 2008). It is a hydrophilic
unstable compound with a structure similar to glucose. These alloxan properties are essential
for the development of diabetes. Namely, its hydrophilicity prevents alloxan from penetrating
the lipid bilayer of the plasma membrane, while glucose-like structure enables an entrance of
alloxan into beta cells (Weaver et al., 1979, Gorus et al., 1982, Lenzen & Munday, 1991).
Additional structural property of alloxan, which is important for development of diabetes, is
its central 5-carbonyl group that reacts with thiol groups of different enzymes, especially
glucokinase (hexokinase IV), which is known to be the most sensitive thiol enzyme in beta
cells (Lenzen, 1988). The half-life of alloxan is very short (Lenzen, 2008). In aqueous
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solution alloxan spontaneously decomposes into non-diabetogenic alloxanic acid, within

minutes (Lenzen & Munday, 1991). At body temperature (37°C) and pH level of 7.4 the half-
life of alloxan equals 1.5 min (Lenzen, 2008). At lower temperatures alloxan‘s half-life is
longer, and since alloxan is a weak acid it is more stable at lower pH (Lenzen, 2008, Lenzen

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et Munday, 1991).

The fungal antibiotic streptozotocin (2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-

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glucopyranose) is synthesized by Streptomycetes achromogenes (Szkudelski, 1998), and it

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has similar chemical properties as alloxan. The similarity between streptozotocin and alloxan
is reflected in the fact that both compounds are hydrophilic, and they can be classified as beta

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cell-toxic glucose analogues (Szkudelski, 1998). Interestingly, streptozotocin possesses
antimicrobial activity and has been also used as a chemotherapeutic alkylating agent (Lenzen,
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2008, Szkudelski, 1998). In regard to chemical structure of streptozotocin, it represents a
nitrosourea analogue in which the N-methyl-N-nitrosourea moiety is linked to the carbon-2 of
a hexose (Lenzen, 2008). Generally speaking nitrosoureas are mostly lipophilic. Despite this
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fact streptozotocin is hydrophilic as a result of the hexose substitution. Owing to its glucose-
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like similar structure, it enters beta cells in a similar way as glucose and alloxan. In contrast
to alloxan, streptozotocin is relatively stable at pH 7.4 and 37°C for at least 1 h (Lenzen,
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2008).
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5. Alloxan and streptozotocin mechanism of action and onset of diabetes

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A more detailed description of mechanisms of alloxan- and streptozotocin-induced

diabetes mellitus has been already presented in the review paper by Lenzen in 2008 (Lenzen,
2008). Therefore, our aim was only to briefly present the most important steps in diabetes
induction process and to point out some possible problems associated with the mechanism of
action of quoted substances. Both compounds have identical mechanisms of action on beta
cells, but their diabetogenic potential is achieved through different cytotoxic pathways
(Figure 1).

5.1. The mechanism of alloxan-induced diabetes

Alloxan is a hydrophilic compound and it is not able to freely pass the cellular
membrane without additional action of specific protein transporters. Namely, it enters B cells
via GLUT2 transporters and this mechanism is tightly connected to its glucose-like structure
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(Lenzen, 2008, Elsner et al., 2000). Considering a similar structure of alloxan and glucose,
and also the fact that they both may enter beta cells through GLUT2 transporters, it is
obvious that glucose can competitively limit alloxan‘s uptake by B cells (Jorns et al., 1997,
Szkudelski, 2001). This means that glucose is able to protect B cells from alloxan-induced

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toxicity. It should be underlined that glucose protection against the toxicity of alloxan is
concentration-dependent (Elsner et al., 2002). This is just one of the reasons why 24h-long

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fasting is performed before alloxan administration (Radenković et al., 2013). Hydrophilic

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nature of alloxan is essential for its use in animal models of diabetes, otherwise alloxan
would be able to penetrate every cell type in animal organism and cause significant damage.

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However, many cells including hepatocytes and kidney tubular cells express GLUT2
transporters, which explain alloxan-induced liver and kidney toxicity. Although alloxan is
able to enter these cell types, it is more selective for pancreatic B cells due to a more rapid
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uptake by those cells, and also because the activity of glutathione peroxidase and the
resistance to exogenous peroxide are around 20 times higher in liver and kidney than in
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pancreas (Malaisse et al., 1982). Despite alloxan‘s selectivity for B cells, it is per se distinctly
a nephrotoxic compound (Evan et al., 1984); thus it is not considered as a first choice model
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of diabetes mellitus type 1 in investigations related to kidney pathology.

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Alloxan induces an insulin-dependent diabetes. Nevertheless, after just two minutes

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upon alloxan application, the rise in the blood insulin level can be spotted (Szkudelski et al.,
1998). This increase in insulin level is considered as one of the metabolic effects of alloxan,
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and it is followed by the reduction of plasma glucose level. One of the possible explanations
for this alloxan-induced metabolic effect is probably connected to alloxan-induced increase in
intracellular calcium. Namely, alloxan is able to depolarize B cells, which can lead to
opening of voltage- dependent calcium channels and increase in cytosolic calcium level
(Dean & Matthews, 1972). The importance of calcium in alloxan-induced insulin increase
was supported by studies in which calcium channel blocker verapamil was administrated
prior an alloxan application (Kim et al., 1994). Also, it has been shown that different calcium
channel blockers such as verapamil, diltiazem or nicardipine have suppressive effects on
development of alloxan-induced diabetes (Kim et al., 1994, Katsumata et al., 1992,
Katsumata et al., 1998, Wei et al., 1992). The effects of calcium channel blockers on
development of alloxan-induced diabetes thus confirm a possible role of calcium in this
model of diabetes. The other explanation for high insulin level at the beginning of alloxan-
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induced diabetes can be linked to an initial reduction of ATP consumption resulting from
blockade of glucose phosphorylation by glucokinase, which produces a transient increase in
ATP in beta cells and triggers a transient release of insulin (Lenzen, 2008).

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After entering beta cells, alloxan produces its pathological effect through two

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independent mechanisms, namely glucokinase inhibition and reactive oxygen species (ROS)
cycle generation. Glucokinase is an isozyme of the hexokinases and it is a major glucose-

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phosphorylating enzyme in the liver, as well as in pancreatic B cells (Lin & Accili, 2011,

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Wilson, 2003, Iynedjian, 2009). In the liver glucokinase is important for the process of
glucose storage in form of glycogen, while in B cells it has a function of glucose sensor and it

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controls insulin secretion (Wilson, 2003, Iynedjian, 2009). As a potent glucokinase inhibitor,
alloxan reduces glucose oxidation and ATP generation, which further suppresses insulin
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secretion (Lenzen, 1988, Lenzen, 2008, Gunnarsson & Hellerström, 1973). Alloxan exhibits a
high affinity for the SH-containing cellular compounds such as glucokinase (Szkudelski,
1998). Inactivation of glucokinase was shown to be the consequence of alloxan reaction with
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two glucokinase -SH groups in the sugar binding side of this enzyme (Szkudelski, 1998).
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Recently, Zhang et al. (2009a) have shown that alloxan is able to substantially reduce both
concentration and activity of glucokinase in liver, and that this alloxan‘s characteristic plays
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an important role in the process of diabetes mellitus type 1 induction. This also means that
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alloxan-induced diabetes is inconsistent with the natural pathogenesis of human diabetes,

which can be one of disadvantages related to this model. The role of liver in alloxan diabetes
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model is most probably in inducing hyperglycemia, since beta 3 - adrenergic agonist

trecadrine was shown to normalize glycemia without affecting insulin levels in alloxan-
induced diabetic rats (Milagro et al., 1999). It has been previously mentioned that glucose
can affect alloxan action and that 24h-long fasting should be performed before alloxan
administration. An additional reason for this procedure is linked with the fact that glucose can
also protect glucokinase against its inactivation by hindering the contact of alloxan with the -
SH groups of an enzyme (Szkudelski, 1998).

Alloxan is able to generate ROS in a redox cycle in which it is chemically reduced to

dialuric acid, and dialuric acid is re-oxidized back to alloxan (Munday, 1988). In these
processes middle products called alloxan radicals and an unidentified "compound 305" are
formed. For the reactions in which alloxan is reduced to dialuric acid, through alloxan
radicals as middle products, a proper thiol is needed as a reducing agent. Most commonly,
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tripeptide glutathione (GSH) is involved in this reaction; nonetheless other thiols such as
cysteine, ascorbic acid and dithiols can also serve as reducing agents (Elsner et al., 2006).
Additionally, thiol groups on proteins, including enzymes or albumin can also be used in this
reaction (Lenzen, 2008). In quoted reactions GSH is converted to thiyl radicals (GS·), which

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further form GSSG (oxidized glutation). Although thiols are necessary for dialuric acid
generation, there have been reports that they (cysteine and GSH) can protect rats against the

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development of alloxan-induced diabetes if they are administrated together with alloxan. This

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paradoxical situation can be explained by assuming that more alloxan is reduced outside of
cells considering higher extracellular concentration of thiols (Lenzen, 2008). Another

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explanation for this situation considers GSH protection against free radicals (Donnini et al.,
1996). Namely, in the alloxan-induced redox cycle hydrogen peroxide is formed, which can
further lead to additional formation of hydroxyl radicals, while GSH can divert this process.
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In vitro generation of hydroxyl radicals in the presence of alloxan is dependent upon the GSH
concentration (Szkudelski, 1998). Low GSH concentration increases the formation of
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hydroxyl radicals, oxygen consumption and auto-oxidation of dialuric acid, while higher
concentrations of GSH decrease these processes. Although high concentration of extracellular
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GSH can protect animals from alloxan-induced effects, while low intracellular concentrations
of GSH increase the formation of hydroxyl radicals, a sufficient concentration of GSH is
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needed in B cells for alloxan to develop diabetogenic effects. This is because alloxan in its
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oxidized form does not generate ROS, which means that it is not toxic. This also indicates
that without GSH alloxan is not cytotoxic, and that thiols from plasma membrane are not
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sufficient to enable the generation of ROS (Elsner et al., 2006). On the other hand dialuric
acid is able to spontaneously auto-oxidize in the presence of O2, which means that ROS are
generated even in the absence of thiols (Lenzen, 2008). In this course of action dialuric acid
is converted to alloxan radicals, which are further transformed back to alloxan, and this is
how the circle ends. In these two reactions O2 is needed and it is transformed to superoxide
radicals. Most probably, superoxide radicals are not responsible for the cytotoxicity of
alloxan and dialuric acid, and several lines of evidence pointed to hydroxyl radicals as the
principal culprit (Lenzen, 2008). The explanation for this presumption can be associated with
the fact that catalasa, a peroxide-inactivating enzyme, provides better protection against
alloxan and dialuric acid toxicity compared to SOD, which is superoxide radical-inactivating
enzyme (Lenzen, 2008). Superoxide radicals are transformed into hydrogen peroxide by a
reaction in which alloxan radicals are transformed to alloxan. Furthermore, hydrogen
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peroxide can be transformed into hydroxyl radicals or it can be transformed into H2O in
reactions mediated by enzymes, such as catalasa or glutathione peroxidase.

5.2. The mechanism of streptozotocin-induced diabetes

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Like alloxan, streptozotocin is also a hydrophilic compound, and it is able to pass

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cellular membrane via GLUT2 transporters (Lenzen, 2008). The verification of GLUT2 role

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in streptozotocin action is consistent with finding that a reduced expression of these particular

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transporters can prevent diabetogenic effects of streptozotocin (Schnedl et al., 1994, Thulesen
et al., 1997). Paradoxically, streptozotocin is also able to reduce the expression of GLUT2

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transporters, if applied in multiple doses, both in in vivo and in in vitro conditions
(Szkudelski, 1998). Although both alloxan and streptozotocin pass cellular membrane with
GLUT2 transporters, overall affinity of these transporters is higher for alloxan in rats (Elsner
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et al., 2003). Unfortunately, GLUT2 transporters are not expressed exclusively in the
pancreas. They are also present in the kidney and the liver, and this is the reason why
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streptozotocin is able to injure these organs, additionally excluding chemically-induced

diabetes in investigations of diabetes-induced effects on kidney and liver (Rerup, 1970;
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Weiss, 1982).
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After streptozotocin administration, insulin-dependent diabetes is developed by means

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of at least three different mechanisms of action (Lenzen, 2008). All these three pathological
mechanisms show DNA destruction as a final result. The first and the most important is DNA
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alkylation, this is also the most likely mechanism of streptozotocin-induced diabetes. The
second proposed mechanism is aftermath of streptozotocin chemical structure. Namely,
streptozotocin contains the nitroso group and it can release nitric oxide in the further course
of action. Still, intracellular nitric oxide release is not considered as the main mechanism of
streptozotocin action, it can be considered more as an alternative or additional mechanism of
its action. Finally, generation of ROS contributes to streptozotocin diabetogenic effects.
Nevertheless, the pathological pathway of ROS production does not play a central role in this
model of experimental diabetes.

As underlined, the first and also the most important mechanism of B cell death
induced by streptozotocin is alkylation of its DNA (Elsner et al., 2000, Delaney et al., 1995).
The alkylating activity of streptozotocin that induces toxic effects in B cells is a result of
alkylating capacity of methylnitrosourea moiety of guanine at the O6 position (Szkudelski,
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1998). Initial transfer of methyl group from streptozotocin to the B cell DNA molecule
initiates a chain of events that will result in the fragmentation and destruction of DNA
(Lenzen, 2008). The destruction of DNA activates an additional mechanism that will further
boost toxic effects of streptozotocin. Namely, DNA damage activates poly(ADP-ribose)

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polymerase-1 (PARP-1), which is in this case overstimulated, and diminishes cellular NAD+

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and ATP stores (Lenzen, 2008). Additional confirmation of PARP-1 involvement in

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streptozotocin mechanism of action is presented in a fact that PARP-1 null mice are resistant

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to the development of diabetes after streptozotocin administration (Burkart et al., 1999;
Pieper et al., 1999). Reduction of B cell‘s ATP results in B cell necrosis. Streptozotocin-

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induced NAD+ depletion results in inhibition of insulin biosynthesis and secretion (Lenzen,
2008).
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With regard to streptozotocin-related signaling cascades it was detected that the nitric
oxide release occurs inside B cells. This is not a spontaneous process. Nitric oxide is released
when streptozotocin is metabolized, and NO-synthase (an enzyme that generates NO) is not
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required for this process (Szkudelski, 1998). Streptozotocin is able to generate a small
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amount of ROS. The most important types of ROS generated by streptozotocin are
superoxide and hydroxyl radicals. These ROS are formed during hypoxanthine metabolism.
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Although streptozotocin-related generation of ROS is generally considered as a negligible

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one, these oxygen radicals can accelerate the process of B cell destruction and development
of insulin-dependent diabetes mellitus.
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Due to its clinical use as chemotherapeutic, streptozotocin mechanism of action has

recently been even more clarified, and more details about its action are known. For example,
c-Jun N-terminal kinase (JNK), which is activated by cellular stress and is involved in
different types of cellular death, is also considered as a very important factor in B cell death
induced by streptozotocin (Cheon et al., 2010). Additionally, it has been shown that this
kinase has also an influence on pathogenesis of naturally occurring type 1 and 2 diabetes
(Hirosumi et al., 2002; Jaeschke et al., 2005). After streptozotocin administration an increase
in activation of JNK in insulinom and also in islet cells is noted. Nevertheless, JNK inhibitors
prevent this streptozotocin effect, additionally marking JNK as an important link in
streptozotocin-induced B cell death (Cheon et al., 2010). Besides JNK inhibitors, PARP
inhibitors, such as nicotinamide or DPQ are capable of inhibiting streptozotocin-induced JNK
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activation, indicating that that JNK activation occurs downstream of PARP-1 activation
(Cheon et al., 2010).

5.3 Onset of disease after alloxan and streptozotocin administration

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The beginning of disease is considered to be the state of permanent hyperglycemia,

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which occurs 48 hours after alloxan and streptozotocin injection (Lenzen, 2008). In the first

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48 hours several important changes in insulin and glucose status, as well as changes in beta

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cells are developed. All of these changes are shown in more detail in the paper by Lenzen
(2008). In short, after alloxan injection a rise of insulin level and consequential decline of
glucose level, which leads to hypoglycemia, is developed. However, this short term

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hypoglycemia is not present after a streptozotocin injection. After an hour following the
chemical induction of diabetes, a significant rise of glycemia can be detected. This rise is
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followed by a drop of glycemic levels, and serious hypoglycemia reaching its maximum 4 to
8 hours after injection. The developed hypoglycemia can last for several hours and can lead
to the death of an animal. That is the reason why this point represents the most critical
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moment in the chemical induction of diabetes. After established hypoglycemia, a constant

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rise in glucose level occurs, but approximately 48 hours post injection a persistent
hypoglycemia will be developed. At this point glucose measurement could be performed in
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order to confirm diagnosis of diabetes. Nevertheless, at approximately 72 hours post injection

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maximal glucose levels are reached, which means that better time for glucose measurement
would be indeed 72 hours post injection, moreover since detection of maximal glucose level
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could help us in determining what type of diabetes is developed. After this point the glucose
levels are usually stable way above normal levels. Nonetheless, in certain situations,
especially after alloxan induction of diabetes, the glucose level can drop and normoglycemia
can occur. The explanation of this problem is discussed in the next section.

6. The Beta cell regeneration and resistance after induction of diabetes

6.1. Beta cell regeneration after chemically induced diabetes

In adult rats or mice regeneration of beta cells does not normally occur after partial
(50-70%) pancreatectomy, or after beta cells destruction with alloxan or streptozotocin
(Bouwens, 2006). Beta cells only have a limited mitotic potential, and neogenesis from
precursor cells cannot be re-activated under these conditions, but on the other hand an acute
hyperglycemic state can actually stimulate beta cells hyperplasia (Bernard et al., 1999). Also,
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if pancreatic tissue is sufficiently damaged or some external stimuli are added, neogenesis
can be re-activated (Bouwens, 2006). This means that sometimes after the chemical induction
of diabetes, although the state of hyperglycemia had developed, hyperglycemia can be
reversed.

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After application of alloxan to experimental animals, this chemical destroys only
pancreatic beta cells, leaving alpha and delta cells intact (Kumar et al., 2012). Although

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alloxan destroys beta cells, hyperglycemia that develops after this procedure is less stable and

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it is a reversible process that can sometimes, after certain period of time, lead to
normalization of blood glucose levels (Kumar et al., 2012). This means that alloxan should be

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primarily used in studies in which short-term effects of diabetes are investigated. For
example, Radenković et al. (Radenković, 2012) used alloxan to determine effects of 4 week-
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long diabetes on adenosine actions in isolated rat common carotid artery. On the other hand
streptozotocin-induced diabetes is a more stable model of experimental diabetes and it can be
used for both shorter and longer experimental studies. These findings suggest that for short-
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term diabetes both substances can be used, while for a long-term diabetes, streptozotocin
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should be a preferable choice.

6.2. Resistance against toxicity of streptozotocin and alloxan

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Beta cells toxicity induced by diabetogenic agents like alloxan and streptozotocin is
dependent upon the animal species. For example, Gorray et al. (1986A) failed to induce
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stable diabetes in guinea pigs with alloxan, although some changes in blood glucose and
insulin levels were found on day 1, but the following parameters returned to control levels by
day 14 after the initial injection. The same research group injected alloxan into guinea pigs in
the dose of 200 mg/kg by i.v. route, which caused a significant decline of serum insulin levels
and a marked inhibition of normal weight gain during the first 96 hrs following the treatment
(Gorray et al., 1986B). Both of these parameters tended to normalize between 96 and 120 hrs
after initial treatment (Gorray et al., 1986B). However, this time they did not detect
hyperglycemia or glycosuria. On the other hand, streptozotocin treatment of guinea pigs
provoked a diabetes-like condition, which was not reversed in the first two weeks after single
injection (Gorray et al., 1986A). Based on these results it can be proposed that guinea pigs
were resistant to alloxan, but not to streptozotocin. Therefore, the resistance to alloxan and
specific changes that alloxan induces in guinea pigs are the reason why the alloxan-treated
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guinea pigs could represent a unique model for studying the restoration of insulinogenic
capacity in insulinopenic diabetes (Gorray et al., 1986B).

Species differences considering the potential of diabetogenicity of streptozotocin and

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alloxan are even more pronounced if someone were to compare rodents which are

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particularly prone to the diabetogenic action of these compounds, and humans who are
considered to be resistant (Elsner et al., 2003). The resistance of human B cells to

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streptozotocin is one of the reasons why diabetes is not typically expected side effect if this

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drug is used as a chemotherapeutic agent, and also why it is not particularly efficient in the
treatment of human insulinomas (Elsner et al., 2003). There are several explanations why

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human beta cells are more resistant to toxic actions of streptozotocin and alloxan. Some
clarifications considered GLUT2 transporters, while others were focused on better
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antioxidative defense status, or the protection by stress response proteins such as hsp70
(Welsh et al., 1995, Ferrer et al., 1995, De Vos et al., 1995). Still, it is unlikely that stress
response proteins have a major role in protection of beta cells against alloxan and
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streptozotocin, and also beta cells‘ antioxidative defense status is not sufficient enough to
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provide an adequate protection, indicating that the main reason for this resistance is linked to
GLUT2 transporters (Elsner et al., 2003). The explanation how GLUT2 glucose transporters
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affect an influence on human resistance can be considered through two assumptions. The first
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one suggests that GLUT2 transporters can be expressed at a very low level, while the second
one indicates that alloxan and streptozotocin are not taken up through the human GLUT2
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transporters into the intracellular compartment. Most probably, the reason for human beta
cells‘ resistance to alloxan and streptozotocin could be in a very low level of constitutive
GLUT2 glucose transporter expression in the human beta cells (Ferrer et al., 1995, De Vos et
al., 1995).

7. Alloxan- and streptozotocin-induced diabetes – animal models

Chemical substances that are currently used for diabetes induction are able to induce
all important forms of diabetes. Nevertheless, alloxan and streptozotocin are the most
commonly used agents for inducing type 1 diabetes. Although streptozotocin in combination
with different feeding regimes or other types of manipulations can be used for inducing type
2 diabetes, several other models are favored for this purpose (Table 2). On the other hand,
application of alloxan induces a state that is particularly similar to human type 1 diabetes
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(Federiuk et al., 2004). That is why alloxan application would be preferred and more correct
if we consider it as a model of type 1 diabetes, although some authors stated that they have
used alloxan for inducing type 2 diabetes (Devaki et al., 2011, Sathya & Siddhuraju, 2013).
No matter what substance would be used or what type of diabetes we would like to induce,

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several problems are always anticipated. The most important issues are related to the route
and interval of administration, the chosen dose, the laboratory manipulation of selected

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substances, the rate of success, the rate of mortality, and many others that will be summarized

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bellow.

7.1. Chemical induction of type 1 diabetes

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Both alloxan and streptozotocin can be used for induction of type 1 diabetes.
Although the most commonly used is streptozotocin, there are several situations in which
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alloxan application is preferable especially since the rate of animal mortality is lower after
alloxan treatment. Detailed protocols for induction of type 1 diabetes are summarized in
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Figure 2.
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7.1.1. Alloxan- induced type 1 diabetes

When alloxan is administrated as a diabetogenic agent, the main purpose of it is to

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destroy B cells. Although we have previously mentioned that there is an existing tolerance in
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some animal species, there are still many other animals showing sensitivity to diabetogenic
effects of alloxan. For example alloxan can be used in rats (Gurusubramanian & Roy, 2014,
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Ahmed & Zahra, 2011), mice (Dey et al., 2015, Favaro et al., 2015), rabbits (Irshad et al.,
2015, Chiang et al., 2014), swine (Sodha et al., 2008) and dogs (Howell et al., 2013, Martínek
et al., 1992, Tasaka et al., 1988). On the other hand guinea pigs, as well as cats (Hatchell et
al., 1986) tend to show resistance to alloxan, although cats are still susceptible to its toxic
side effects (namely kidney damage).

Of all animals sensitive to alloxan diabetogenic effects, rats are most commonly used,
whereas mice and rabbits are the second most frequently utilized type of animals for this
purpose. In rats alloxan can be administrated by intraperitoneal (Radenković et al., 2013),
intravenous (via the tail vein) (Sano et al., 2014, Ahmed & Zaher, 2011) or even by
subcutaneous (Boura et al., 1986, García et al., 2007) route of administration, even though the
last one is the least frequent one. The administered dose of alloxan usually fluctuates between
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150 mg/kg and 200 mg/kg (Radenković et al., 2013, Federiuk et al., 2004). Intravenous
administration of alloxan in quoted dose range is usually toxic, so the preferable route of
administration should be intraperitoneal, which is easier and better tolerated. Additionally, it
should be noted that if necessary, alloxan can be safely administrated intravenously in lower

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doses ranging from 30-65 mg/kg (Al-Salami et al., 2008A; Akhavan et al., 2012; Reis et al.,
2013; Thomaz et al., 2013; Ramakrishnan et al., 2005; Boylan et al., 1992). Still, even though

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unnecessary, in several studies alloxan was intravenously administered in higher doses. This

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should be avoided because the probable toxicity of alloxan, and also due to the fact that
studies in which lower doses of alloxan were used have already demonstrated successful

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induction of diabetes. It should be pointed out that intravenous dose of alloxan needs to be 2
to 3 times lower than intraperitoneal dose in order to avoid toxic effects and to reduce overall
mortality. A comprehensive information on considering dose and route of alloxan
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administration was provided by Federiuk et al. (2004) who performed a study on 41 Sprague-
Dawley rats by utilizing several doses of alloxan (100 - 200 mg/kg) and different routes of
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administration (i.v. and i.p.), as well. The aim was to obtain information about the best way
to use alloxan. The results of this study have shown that a single alloxan dose of 200 mg/kg
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administrated intraperitoneally was the most successful way of diabetes induction by alloxan
with overall 80% successful inductions of diabetes, and 10% of mortality cases. In this study,
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multiple doses of alloxan led to significantly higher rates of mortality compared to a single
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dose administration.
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Even though a literature search showed that alloxan is most commonly used in rats by
intraperitoneal injection in a dose ranging from 150 mg/kg to 200 mg/kg, this is not
something that is fixed, and a variety of papers were published reporting lower doses of
alloxan (Chikezie & Uwakwe, 2014). By using simple logic, the rate of mortality was
probably lower in these studies; nonetheless a question could be asked about the real success
of this approach, especially knowing that Katsumata et al., (1993) demonstrated that any
intraperitoneal dose of alloxan lower than 150 mg/kg would be insufficient for diabetes
induction. And so, if the rate of success with doses below 150 mg/kg is low, an ethical
problem may arise, since this method is stressful for animals involving fear and pain that they
can experience during this procedure.

In mice, which is the second most often used laboratory animal for an alloxan
diabetes model, similar problems regarding inconsistency of route of administration, as well
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as applied dose is present in the literature. This is actually similar to the rat model of alloxan
type 1 diabetes. Diabetes can be induced both intraperitoneally (Raafat & Samy, 2014) or
intravenously (Sirovina et al., 2013), with doses similar to those quoted for the rat model.
Generally, an applied dose of alloxan in the mice model of type 1 diabetes may vary from

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100 mg/kg to 150 mg/kg, as in rats, especially if alloxan is intraperioneally administered
(Leiter & Schile, 2013). However, it is not unusual to find cases for i.p. administration of

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alloxan in doses that are out of this range. For example da Rocha et al., (2013) used a lower

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dose of 80 mg/kg, while Raafat & Samy (2014) utilized a higher dose of 180 mg/kg of body
weight. When used intravenously, administered doses of alloxan are also 2 to 3 times lower

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compared to intraperitoneal doses for induction of the alloxan type 1 diabetes in mice, in the
same way as they reported for rats (Sällström et al., 2014).
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The use of alloxan in rabbits has some similarities and some differences regarding the
utilization of this agent in other experimental animals. The main difference is linked to the
fact that in rabbits, alloxan is mainly administered intravenously, although there are few
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papers stating intraperitoneal use in this model (Kao et al., 2003; Costa e Forti & Fonteles,
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1998). Also, as in other animals, the dose of alloxan may vary significantly. Most commonly
alloxan is used in a dose of 100 mg/kg (Chiang et al., 2014; Roganović et al., 2011; Shafaei et
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al., 2014) and 150 mg/kg (Oh et al., 2014; O'Loughlin et al., 2013). However, there are some
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experimental studies performed with alloxan, which was administered in doses lower than
100 mg/kg, for example 90 mg/kg (Gomes et al., 2007), 80 mg/kg (Díez et al., 2013) and 75
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mg/kg (Nabzdyk et al., 2013).

Regardless of which animal we select for our experiments, there are several
guidelines that we should follow in order to improve chances of adequate diabetes induction,
and reduction of animal mortality, too. First, in order to increase the success rate of type 1
diabetes induction, a proper fasting protocol should be performed. Since alloxan and glucose
are in competition for the same transporters, lower plasma concentration of glucose should
enable alloxan to enter B cells. So, a fasting protocol should be obligatory, and it should be
performed before alloxan injection. Moreover, different durations of fasting periods have
been reported in the literature, with fasting time usually ranging from 12 to 24 hours
(Radenković et al., 2013, Fortes et al., 1983; Preet et al., 2005; de Alencar Mota et al., 2008).
Second, in order to determine whether diabetes is adequately induced, a detection of blood
glucose concentration should be performed not before 48 hours after alloxan administration
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(Manjunath et al., 2009), although a 72 hours period is also quoted in the literature (Al-
Salami et al., 2008B). Third, glucose levels higher than 200 mg/dl (11.1 mmol/l) were
considered as a proper limit for detecting diabetes presence, although in some studies even
higher concentrations were taken as a cut of values (Oh et al., 2014; Perumal et al., 2014). In

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contrast, in some other investigations lower levels of glucose (150 mg/dl or 8.3mmol/l) were
considered as appropriate for characterizing diabetes (Sunarwidhi et al., 2014). Still, although

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lower, previously quoted levels can be used, as well. For example, in rats proposed normal

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glucose values ranged from 50 to 135 mg/dl (2.8 - 7.5 mmol/l) (Parasuraman, 2011).
Nevertheless, it should be mentioned that in the majority of reported investigation values

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higher than 200 mg/dl were considered as a cut of values.

7.1.2. Streptozotocin- induced type 1 diabetes

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Chemical diabetes is generally induced with streptozotocin. This diabetogenic agent is
used to induce diabetes in several animal species, even in those animals that are resistant to
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the effects of alloxan. Streptozotocin has been shown to be an effective diabetogenic agent in
rats, mice, gerbils, rabbits, dogs, guinea pigs, pigs and monkeys. This model is most
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frequently used to induce diabetes in rats.

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Generally, alloxan is administered intraperitoneally and intravenously. In rats

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streptozotocin can also be administrated both intraperitoneally and intravenously in several

different doses described in the literature. Thus, these doses for streptozotocin may vary from
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35 mg/kg to 80 mg/kg (Kim et al., 2006; Azizi et al., 2014; Al-Malki et al., 2015). Normally,
lower doses are used if streptozotocin is administered intravenously (Arcaro et al., 2014). In
mice, which represent the second most commonly used animal species for streptozotocin-
induced diabetes; several connections can be found in relation to the rat model of
streptozotocin diabetes. Namely, both intravenous and intraperitoneal applications of
streptozotocin have been described (Wang et al., 2012; Wang et al., 2015). The dose of
streptozotocin is usually similar to those reported for rats (Basha & Sankaranarayanan, 2014).
Nevertheless, more and more often, in literature one can find several significantly higher
doses like 150 mg/kg (Nojiri et al., 2014), or even 200mg/kg (Chen et al., 2014) for
streptozotocin-related diabetes induction. Though the way of streptozotocin administration
with a single i.p. injection is the predominant one, and it is similar to an alloxan model, one
should bear in mind that significant amount of data propose several i.p. injections with lower
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dose of streptozotocin (Zhou et al., 2015). Although rarely used for streptozotocin-induced
diabetes, experiments performed on gerbils represent a notable source of information where
usually the same route of administration (intraperitoneal and intravenous) and doses from
150-250 mg/kg are used (Nishigaki et al., 1999; Raz et al., 2003). Also, the dose similar to

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that one in rats could be used (Field et al., 1998). Comparable protocols that were applied in
rats are also used to induce streptozotocin type 1 diabetes in rabbits (Javadi et al., 2014), pigs

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(Nalin et al., 2014; Tang et al., 2014) and monkeys (Kavanagh et al., 2011).

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In contrast to alloxan, which is unable to induce diabetes in guinea pigs,
streptozotocin is able to induce type 1 diabetes in these animals. An induction of

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sterptozotocin type 1 diabetes in guinea pigs is similar comparing to the induction in rats,
regarding routes of administration and applied dose (Xie et al., 2013), although in some cases
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higher doses of streptozotocin have been reported (Aslan et al., 2013). Dogs are also used for
investigation of diabetes. In these animals diabetes is usually induced by surgical
intervention, while dogs that were submitted to the chemical induction of diabetes are not
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often used for investigation. On the other hand, several investigators have described a
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chemical model of diabetes in dogs. For example, combination of alloxan (50mg/kg) and
streptozotocin (30 mg/kg or 50 mg/kg) was reported (Matsuo et al., 2009; Alkharfy, 2009).
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7.2. Chemical induction of type 2 diabetes

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Due to a fact that patients with type 2 diabetes represent the majority in the overall
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population of diabetic subjects, an experimental model of this type of diabetes could be

considered as more important from the scientific point of view. The problem with chemical
models of type 2 diabetes is linked with the fact that diabetes can be adequately induced only
after application of streptozotocin with concomitant animal manipulation, which is
additionally reflected in the change of lifestyle habits of experimental animals. The induction
of type 2 diabetes with alloxan is also possible, but is ethically questionable. The setback for
using alloxan in induction of this type of diabetes primarily considers low number of animals
in which diabetes type 2 (12.5%) would be developed, especially if compared with diabetes
type 1 (87.5%) (Federiuk et al., 2004). Therefore, more animals need to be used, and more
animals will be exposed to stress, pain and discomfort, which is unnecessary and ethically
unjustifiable, considering that other animal models exist and should be used in this situation.
However, if one after all decides to use alloxan for induction of diabetes type 2, several
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parameters should be implemented. Namely, the blood glucose level will be significantly
higher in animals with type 1 diabetes. Although this is not the best parameter, and it is not
recommended to be used alone for differentiation of type 1 and type 2 diabetes, it could be
helpful for fast orientation. Much more acceptable for diabetes type determination, would be

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to measure blood keton concentration. Animals with type 1 diabetes would express blood
keton concentration higher then 1.5 mM, while in animals with type 2 diabetes negative or

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minimal blood keton values would be detected (Federiuk et al., 2004). As with alloxan,

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streptozotocin is also capable of inducing type 2 diabetes in a certain percentage, but several
additional manipulations that could be performed after streptozotocin injection, such as

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dietary modification, can influence the development of type 2 diabetes, which is in more
details explained below.
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7.2.1. Streptozotocin- induced type 2 diabetes

Currently, three modified streptozotocin models of type 2 diabetes are recognized in

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the literature. The so called "Fat-fed, streptozotocin-treated" rat model was the first one
described in the literature and was developed by Reed et al. (2000), while "Nicotinamide-
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streptozotocin induced" and "Fructose-fed, streptozotocin-injected" models were recently

developed, although the effects of nicotinamide in streptozotocin-induced diabetes were
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investigated first. Nicotinamide was referred to as a drug that modifies streptozotocin toxic
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effects (Schein et al., 1973), and also as a substance that can prevent (Hu et al., 1996), or
even reverse (Dulin & Wyse, 1969) development of streptozotocin diabetes. Protocols for
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induction of type 2 diabetes are summarized in the Figure 3.

7.2.1.1. Fat-fed, streptozotocin-treated rat model of type 2 diabetes

This is the most recognized and widely used streptozotocin model of diabetes type 2.
It has been developed with an idea to mimic natural progression and changes that gradually
occur in metabolism of humans with type 2 diabetes. The idea for this model was drawn from
studies in which an important influence of fat-enriched diet on insulin resistance was
demonstrated, which was later fallowed by hyperinsulinemia. Nevertheless, due to
hyperinsulinemia, the percentage of hyperglycemic subjects was unsatisfactory. In order to
change this course Reed et al. (2000) administered streptozotocin (50 mg/kg intravenously) to
7 weeks-old Sprague-Dawley rats, previously fed with high-fat diet (40% of calories as fat)
over two weeks. After the streptozotocin injection, this group of animals had continued to
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receive high fat diet. The result of this experimental study showed an increase of glucose,
insulin, free fatty acids and triglyceride in fat-fed, streptozotocin-treated rats if compared
with control (chow-fed, streptozotocin-treated rats). Additionally, in fat-fed, streptozotocin-
treated rats, administration of antidiabetic agents, namely metformin and troglitazone, led to a

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lowering of blood glucose level. These results were sufficient to properly establish the fat-
fed, streptozotocin-treated rat model of type 2 diabetes.

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After this model was introduced, several other modified models have been developed.

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Two most important modifications are reflected in lowering of single streptozotocin dose
(Srinivasan et al., 2005), and in multiple administrations of streptozotocin in lower doses

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(Zhang et al., 2009b). Both of these models are considered to be effective in inducing type 2
diabetes. Namely, Srinivasan et al. (2005) administered a single lower dose of streptozotocin,
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but at the same time the route of administration was changed. Streptozotocin was given in a
dose of 35 mg/kg by intraperitoneal administration. Also, the formula of the high fat diet was
changed. The previous diet that consisted of 40% fat, 41% carbohydrate, and 18% proteins
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was replaced by another consisting of 58% fat, 17% carbohydrate and 25% proteins.
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Alternatively, Zhang et al., (2009b) administered streptozotocin in two lower doses (each 30
mg/kg) that have been applied intraperitoneally. Also, in this study the fat diet formula was
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different and consisted of 22% fat, 48% carbohydrate, and 20% proteins. Male Wistar rats
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were fed with this formula for four weeks before the streptozotocin injections, afterward
experimental animals received the first streptozotocin injection, and one week later the
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second one, with same streptozotocin dose.

Also, it is important to mention that use of basic model or its modifications are
possible in different animal species. For example, this model was also applied in mice,
usually by using high fat diet, with 60% of calories in fat and with 3 (Mali et al., 2014) or 5
(Wang et al., 2014) injections of streptozotocin in a single dose of 40 mg/kg. In guinea pigs
the use of this model was also described; nevertheless, a change in a food formulation was
performed by introducing high-fat, high-carbohydrate diet (Podell et al., 2014).

7.2.1.2. Fructose-fed, streptozotocin-injected rat model of type 2 diabetes

Wilson & Islam (2012) were aimed at developing an alternative non-genetic model of
type 2 diabetes with the idea to mimic in the best way the pathogenesis of disease itself, while
and at the same time making it performable without incurring too much additional cost. The
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idea of using fructose was a logical step since the fructose-fed model of diabetes already
existed, but it required more time to develop, and at the same time fructose was used for
creating other metabolic disorders, such as metabolic syndrome (Panchal & Brown, 2010).

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This model was investigated on a 6 weeks-old Sprague-Dawley rats that were injected

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with low dose of streptozotocin (40 mg/kg) after supplementation with 10, 20, 30 or 40%
drinking fructose solution for two weeks. After streptozotocin injection animals were

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supplied with normal drinking water. Although the used protocol lasted for 11 weeks

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following streptozotocin administration, only the animals that have had 10% of fructose in a
drinking water, managed to remain active until the end of experiment. The rest of

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investigated groups were excluded due to severity of diabetes induced. Many parameters that
were followed in 10% fructose group, including fluid intake, blood glucose, serum lipids,
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liver glycogen, liver enzymes, insulin resistance, body weight, oral glucose tolerance, number
and function of pancreatic B cells, as well as response to anti-diabetic drugs (glibenclamide
and metformin), clearly indicate that this model has a good perspective in research of
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diabetes. The main advantage of this model if compared to the fat-fed, streptozotocin-treated
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rat model of type 2 diabetes can be summarized in two facts, firstly it is less expensive, and
secondly it is easier to induce diabetes by using this method.
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7.2.1.3. Nicotinamide-streptozotocin – induced rat model of type 2 diabetes

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The relationship between nicotinamide and streptozotocin was investigated for a long
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period of time, while the combination of these two compounds was only recently introduced
as a viable model of type 2 diabetes. The protective effect of nicotinamide in diabetes was
initially evaluated in animals with streptozotocin-induced diabetes (Hu et al., 1996). Indeed,
the obtained protection induced by nicotinamide was the reason why this substance was
continued to be used for development of type 2 diabetes. The clarification for this model is
associated with the fact that the percentage of B cells mass reduction may be related to a
particular type of diabetes (Skovsø, 2014). For example, 60-80% reduction of the functional
B cells is already present at the moment of establishing diagnosis of type 1 diabetes. In this
setting, nicotinamide prevents significant destruction of functional B cells and thus introduces
animals into type 2 diabetes, while at the same time they retain the ability to produce insulin.
Protective effect of nicotinamide is achieved through inhibition of PARP-1 activity
(Szkudelski, 1998). In order to repair DNA damage induced by streptozotocin, the activity of
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26

this enzyme is highly increased in B cells. In the further course, an elevated activity of this
enzyme leads to the reduction of intracellular NAD(+) and ATP, thus facilitating necrosis of
B cells.

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In an improved model, nicotinamide is administrated together with streptozotocin, but

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usually at different intervals. Both of these substances can be administered intraperitoneally,
although intravenous administration was also described (Pierre & Gildas, 2012). Several

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different time intervals between the administrations of these compounds can be found in

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literature. For example, streptozotocin is most often administrated 15 min after nicotinamide
(Nayak et al., 2014; Watcho et al., 2014). Also, this interval can vary and can be prolonged,

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like for example 20 minutes in-between applications (Badole et al., 2013), or shorter, being
only 5 minutes (Sheela et al., 2013). Regardless, variations of these intervals do not affect the
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course of type 2 diabetes development and each protocol was proved to be effective. On the
other hand, nicotinamide could be also administered after streptozotocin (Petchi et al., 2014).
As well as for streptozotocin, nicotinamide concentration often varies - usually from 90
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mg/kg to 120 mg/kg (Nayak et al., 2014; Szkudelski et al., 2013; Maheshwari et al., 2014).
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Nevertheless, application of a much higher concentration of nicotinamide (230 mg/kg) was

described as a part of the nicotinamide-streptozotocin model of type 2 diabetes, too (Kumar
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Bhateja & Singh, 2014).

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In contrast to the previous two models of type 2 diabetes that were originally created
and mainly used in rats, nicotinamide-streptozotocin - induced diabetes is also used as a
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model in different other animal species. In mice, this model is used for type 2 diabetes in a
similar fashion as in rats, yet with the same problem regarding inconsistent doses of
nicotinamide and streptozotocin, as well as interval in-between administration of these
substances (Tahara et al., 2012, Ueno et al., 2014; Ahangarpour et al., 2014). In addition, a
combination of nicotinamide (67 mg/kg) and streptozotocin (125 mg/kg) was used in
göttingen minipigs (Larsen et al., 2003; Larsen et al., 2004) and micro-pigs, too (Lee et al.,
2012).

7.3. Chemical induction of diabetes in pregnancy

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One of the most interesting fields of diabetes investigation is its influence on

pregnancy, and resulting problems that may develop during this period. Since available
possibilities for research in pregnant women are limited and often ethically unacceptable, the
use of experimental models is very important for collecting data in this field. Mainly, diabetes

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is induced in order to follow changes that occur in the next generation of animals (Correia-
Santos et al., 2014; Martins et al., 2014). In this case diabetes is induced before mating,

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although induction of diabetes is also possible in already pregnant animals. Additionally, for

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investigation of diabetes in young animals, an injection of alloxan in rat offspring (22 days
old) may be used (Yaghmaei et al., 2013). Nevertheless, by utilizing this procedure we can

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investigate the effects of diabetes in young animals, but we are unable to investigate the
influence of changes that will occur because of diabetes during pregnancy, and afterward on
the next generation of animals.
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For induction of diabetes in pregnancy both alloxan and streptozotocin are used. Still,
researchers are using streptozotocin more often. The application of streptozotocin for
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induction of diabetes in pregnancy is described in rats (Martins et al., 2014), mice (Ge et al.,
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2014), rabbits (Fletcher & Bassett, 1986) and pigs (Ezekwe et al., 1984). Most commonly,
effects of streptozotocin-induced diabetes were investigated in rats. For induction of this type
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diabetes streptozotocin can be used in different doses and routes of administration, similarly
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as in streptozotocin- induced type 1 diabetes model (Martins et al., 2014; Salazar García et
al., 2015). Also, the use of combination of high-fat diet and streptozotocin is described in the
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literature (Correia-Santos et al., 2014). Like streptozotocin, alloxan was also described as an
inducer of gestational diabetes in several animal species. Thus, alloxan was used for
induction of gestational diabetes in rats (Giachini et al., 2008), mice (Favaro et al., 2013),
rabbits (Thieme et al., 2012) and pigs (Emmrich et al., 1985).

7.4. Chemically- induced diabetes: the summary

Currently there are several protocols for chemically-induced diabetes that can be used
for development of animal models regarding type 1, type 2, or gestational diabetes.
Nonetheless, there is no standard protocol that has been unanimously accepted by the
scientific community. The previous subject matter was created to bring together different
protocols and to highlight the positive and negative aspects of each of them. Summary tables
for the induction of type 1, type 2 and gestational diabetes (Table 3) were created and they
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contain all the above mentioned properties of each model with given recommendations that
were proposed based on evidences found in literature. Summary and recommendations will
hopefully provide adequate information that could help scientific personnel to reduce number
of animals needed for their investigations, to shorten the overall time of investigation, to

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increase the quality of their work and to preserve their finance resources.

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8. Animal care after chemically-induced diabetes

The induction of diabetes, especially type 1 diabetes, can be followed by very high

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mortality rate (Federiuk et al., 2004). In order to reduce the animal number needed for
performing certain experiments, several things can be done. The first problem usually occurs
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between 4 and 8 hours after alloxan or streptozotocin injection. At this time point, severe
hypoglycemia that last for several hours is developed, and sometimes glucose administration
is needed to prevent animal death. To overcome this problem 5 % dextrose solution can and
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should be given in drinking bottles for one day after alloxan/streptozotocin injection (Shah &
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Khan, 2014). In some cases, at the beginning of experiment insulin can be administered to
animals in order to stabilize overall animal health condition and to reduce mortality, as well
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(Siddiqui et al., 2005). The application of insulin is only needed in animals with type 1
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diabetes. Insulin should be used in case that at the beginning of experiment (first two days)
blood glucose levels are higher than 200 mg/dl, and keton values are higher than 1.5 mM
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(Federiuk et al., 2004). Both slow and rapid acting insulin could be given depending on the
glucose level. It is recommended that insulin administration should last at least for 7 days
(Siddiqui et al., 2005). More detailed protocol regarding the dose, time of insulin
administration, type of insulin used, and many other facts regarding insulin treatment were
presented in the paper by Federiuk et al., (2004). In some cases, especially if insulin is not
used, severe dehydration can also occur. For most animals fluid requirements are in the range
between 40-80 ml/kg/24h (Waynforth & Flecknell, 1980). Nevertheless, due to high glucose
level polyuria can be, and it is usually is developed, which leads to dehydration. Even though
animals are capable of drinking after these procedures, significant lapse of fluids could still
be developed. Additionally, a fluid can be administrated subcutaneously or intraperitoneally.
For i.p. route of administration, 0.9% saline or dextrose-saline (4% dextrose, 0.18% saline)
solutions should be used (Waynforth & Flecknell, 1980). Rarely, if hypoglycemia is not
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recognized on time, and severe dehydration is developed (judged by loss of skin tone), these
solutions must be administrated intravenously (Waynforth & Flecknell, 1980). Previous
considerations have been summed up in Table 3.

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9. The importance of described diabetes models

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Currently, there are many animal models of diabetes, and none of them is considered

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to be the perfect one. That is why a better knowledge in understanding of these models,

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especially for their appropriate selection and use, is extremely necessary, particularly in order
to prevent unnecessary loss of money, time and animals, and also to avoid dissemination of

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inaccurate, or even false results. The most important reason for implementation of any
available diabetic animal model in a research process is to investigate antidiabetic action of
different compounds. Accordingly, many researchers are using alloxan (Al-Azzawie et al.,
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2006; Kesari et al., 2005) or streptozotocin (Kesari et al., 2007) model for investigation of
those effects. Nevertheless, this can be considered to be a serious issue since some
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investigators have used specific compounds with antioxidant properties, like vitamins or
substances that are obtained from different plants including oleuropein (Umeno et al., 2015),
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Murraya koenigii (Mohd et al., 2009) and many others, which actually stop pro-oxidant effect
of alloxan and streptozotocin. Although the shown antidiabetic effect of investigated
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compounds in these studies is most probably not completely in accordance with obtained
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results, these investigations are far from useless. Namely, for explanation of these results a
different approach is needed. Many of these studies can be used to confirm an antioxidant
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effect of some of investigated compounds, or even in the best possible case these substances
could be probably used in the prevention of diabetes, since they affect antioxidant component
of diabetes before it occurs, but never as pharmacological agents that could be appropriate for
treatment. For investigation of antidiabetic effect of these compounds, some other models are
more suitable and should be used. However, even if the substance of interest does not have
antioxidant properties, several other problems still need to be addressed. Firstly, alloxan-
induced hypoglycemia can be surmounted in experimental animals after several weeks, and it
is difficult at that point to determine if normoglycemia is a result of applied treatment, or just
spomtaneous animal recovery. And secondly, most antidiabetic drugs act by increasing levels
of insulin production, and since in both alloxan and streptozotocin model of diabetes an
extensive destruction of pancreas is present, these models should be avoided, except in case if
proposed antidiabetic effect of used pharmacological agent is not related to increase of
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insulin secretion (Kanigür-Sultuybek et al., 1995). For the purpose of antidiabetic

investigation, a better option is to use modified chemical models like, fat-fed or fructose-fed,
streptozotocin-injected rat model, since a lower dose of streptozotocin leads to less B cell
destruction, and feeding manipulation tends to affect animal metabolism.

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A better way of using these models is for the purpose of investigation of diabetic
complications, and the effects that hyperglycemia induces with regard to physiological

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functioning of organism. Also, a treatment of diabetic complications should be investigated

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through these models. Again, one must be aware of possible antioxidant effect of some
antidiabetic compounds if they are administrated to the living animals, especially if they are

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administrated after alloxan and streptozotocin.

10. Limitations and problems of alloxan and streptozotocin model of diabetes

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Several limitations of described models of diabetes have been already underlined in
previous chapters. Those limitations primarily include toxic effects of alloxan and
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streptozotocin. Secondly, these experimental models are not able to mimic each feature of
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human diabetes. Therefore, a proper understanding of these limitations can provide better
selection of diabetes model, reduce unnecessary use of animals and give an objective
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interpretation of obtained results. Additionally, except for the above limitations, there is also
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a problem connected to the application of these models, and it concerns the selection of
animal species for investigation, which in some cases could be a problem for the translation
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and interpretation of obtained results.

Alloxan and streptozotocin tend to induce some organ toxicity in investigated

animals. Thus, toxic effects of these substances on different organs may preclude their use in
diabetes investigations. In general, toxic effects occur in kidney and liver due to their
expression of GLUT2 transporters. Moreover, streptozotocin is known to have hepatotoxic
effects. Accordingly, in male Sprague-Dawley rats after the streptozotocin injection a
disruption in bile acid levels and bile flow was detected, furthermore, these changes persisted
for 15 days (Watkins & Sherman, 1992). For this reason, studies related to biliary excretion
need to be performed at least two weeks after streptozotocin injection.

One significant piece of information that can be found in the literature is that
antioxidant effect of different compounds can affect alloxan- and streptozotocin-induced
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diabetes. Since the release of ROS represents one of the most important pathological
mechanisms of alloxan- induced diabetes, it is necessary to understand that the investigation
of oxygen radicals is not completely adequate in this model, because they are able to
influence different features of diabetes. Several vitamins like vitamin E and C that are known

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to have antioxidant effects have been used in alloxan model of diabetes. Thus,
supplementation of vitamin E in a diet before the introduction of alloxan, led to prevention of

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insulin decrease in the blood of investigated experimental animals (Kamimura et al., 2013).

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Several other studies with vitamin E, C, or their combination indicated positive effects of
these supplements on diabetes due to their antioxidant activity (Owu et al., 2006; Olanlokun,

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2008; Hamden et al., 2009). If for some reason the use of a chemical model of diabetes is
really necessary, the streptozotocin model is the preferred one. The rationale for this is linked
with the fact that streptozotocin is able to generate only small amounts of ROS, and that ROS
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are not included in the main mechanism of streptozotocin-evoked induction of diabetes.

In animal models of diabetes induced by alloxan and streptozotocin there are notable
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changes in activity of enzymes included in both phase I and phase II of the metabolic
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processes. It was shown that in rats that alloxan and streptozotocin were both able to induce
certain changes in cytochrome P450 isozymes (Lee et al., 2010). It is assumed that those are
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the result of the indirect effect of these compounds due to their short half-life, and are also
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associated to findings that insulin and metformin could reverse these changes (Ogata et al.,
1996; Lee et al., 2008). Although the explanation of this phenomenon is not fully understood,
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it is important to be aware of it, because data from drug investigation in these models of
diabetes could be influenced by cytohrome P450 status at that point and should be
extrapolated with caution.

So far, one of the most important problems with the application of these models is that
in majority of cases, rodents were used as the investigated animal species. Although this is in
accordance with adopted ethical principles, rodents are not the perfect choice for translational
research. Because of that reason, a scientific stimulus should be given in order for animal use
to be shifted towards some other animals, such as pigs or minipigs. This could be really
important, especially since these animals are considered to be exceptionally valuable species
in translational research due to similar anatomic and physiological characteristics in-between
humans and pigs (Swindle et al., 2012). Additionally, it should be mentioned that these
animals should be used in investigation of diabetes effects on cardiovascular, digestive and
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urinary system, except kidneys (due to toxic effects), due to existing organ system similarity
to humans (Swindle et al., 2012).

11. Final Remarks

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Taking into account the prevalence of diabetes, its duration, as well as the
consequences related to this disease, it can be underlined that diabetes represents a serious

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medical, but also an economic problem for every health system. Significant efforts have been

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made to achieve an evident progress in the understanding of causes, symptoms and
complications of this disease, as well as to develop new antidiabetic therapeutic strategies.

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The considerable amount of information related to different aspects of this disease we have
obtained through preclinical investigations. Therefore, it is important to realize how much
different animal models of diabetes are necessary. Existing chemical models, which include
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alloxan- and streptozotocin-induced diabetes models, allow us to investigate different aspects
of this disease. Although they are currently almost irreplaceable, it should be noted that none
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of quoted models can completely mimic all the features of human diabetes. Thus, each of
these animal models is generally used for investigating only one or more points related to
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diabetes. Nevertheless, their use is highly recommended in view of the necessity of their
continuous improvement, as well as further development of new models that will address
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much more of still unresolved issues linked to diabetes.

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Acknowledgement
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This article was supported by the Ministry of Education and Science – Republic of Serbia
with the Grant 175023.

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Table 1. Animal models for experimental diabetes mellitus - General classification

I Methods for Induction of Experimental Diabetes Mellitus

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Ia Pancreatectomy in dogs (Wahoff et al., 1994)

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Ib Alloxan - induced diabetes (Radenković et al.,2013)
Ic Streptozotocin - induced diabetes (Al-Malki et al., 2015)

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Id Hormone induced - diabetes (Campbell et al., 1966)
Ie Virus - induced diabetes (Alkanani et al., 2014)

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If Other diabetogenic compounds (Kim et sl., 2002)
Ig Insulin deficiency due to insulin antibodies (Andreev et al., 1974)

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II Genetically Diabetic Animals
II a Spontaneously diabetic rats (Miao, et al., 2005)
II b Spontaneously diabetic mice (Matsumoto et al., 2010)
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II c Chinese hamsters (Iwashima et al., 1990)
II d Other species with inherited diabetic symptoms (Kramer et al., 1981)
II e Transgenic animals (Streckel et al., 2015)
III Miscellaneous Models
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IIIa Invertebrate animal model (Shirazi et al., 2014)

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IIIb Diet - induced metabolic dysregulation (Spolding et al., 2014)

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Table 2. Specific models for experimental diabetes mellitus

Model of
Animal Model Basic Characteristics
Diabetes

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Ob/Ob mouse Spontaneously Model of obesity with leptin deficiency. In this

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(Matsumoto et al., 2010) diabetic mice model B cells increase insulin production.

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db/db mouse Spontaneously Model of obesity in which B cells are unable to
(Shi et al., 2013) diabetic mice maintain adequate insulin production.

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KK mouse Spontaneously Obese mouse with insulin resistance followed by
(Taylor et al., 1999) diabetic mice hyperinsulinemia.
The Nagoya–Shibata– Model that mainly affect male animals in which

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Spontaneously
Yasuda (NSY) mouse insulin secretion is impaired as a main
diabetic mice
(Ueda et al., 2000) characteristic.
Mild obesity is developed followed by
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CBA/Ca mouse Spontaneously
hyperglycemia, hyperinsulinemia and insulin
(Al Qatari et al.,1996) diabetic mice
resistance.
New Zealand obese
Spontaneously Polygenic model of diabetes characterized with
mouse
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diabetic mice obesity and hyperglycemia.

(Baumeier et al., 2015 )
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Zucker (fa/fa) rat Spontaneously Model of obesity involving leptin resistance with
(Toblli et al., 2015) diabetic rats B cells' increase of insulin production.
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Model with mildly elevated blood glucose as a

Goto Kakizaki rat Spontaneously
result of insulin resistance and impaired
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(Amiri et al., 2015) diabetic rats

secretion
The Otsuka Long-Evans
In this model animals are obese and are
Tokushima fatty (OLETF) Spontaneously
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developing diabetes in adulthood. Males are

rat diabetic rats
more often affected.
(Nagai et al., 2015)
In this non-obese model hyperglycemia and
Diabetic Torri rat Spontaneously
hypoinsulinemia are developed mainly in mail
(Miao, et al., 2005) diabetic rats
animals.
Psammomys obesus Diet - induced With laboratory diet animals develop obesity
(the Israeli sand rat) metabolic and due to insulin resistance hyperglycemia is
(Spolding et al., 2014) dysregulation developed.
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Table 3. Technical details and recommended approach for induction of different diabetes types

DIABETES MODELS TECHNICAL DETAILS AND RECOMMENDED APROACH SPECIAL POINT OF ATTENTION
ANIMALS RECOMMENDED SHORT-
RECOMMEN- RECOMMENDED TIME OF
TYPE OF MOST FASTING ROUTE OF TIME OF PARAMETERS TO /LONG- ADVANTAGE OF
MODEL DED FASTING ROUTE OF DOSE RECOMMENDED DOSE GLUCOSE ANIMAL CARE WHEN NOT TO USE
DIABETES COMMONLY TIME ADMINISTRATION GLUCOSE FOLLOW TERM EACH MODEL

PT
TIME ADMINISTRATION MEASUREMENT
USED MEASUREMENT DIABETES
I.P. 100-150 mg/kg; I.V. In cats and guinea pigs (do not
MICE 100 mg/kg (I.P.)
2-3 x less then I.P. Blood glucose 5% DEXTROSE SOLUTION (on a first develop), investigation of diabetes
I.P.
I.P. 150-200 mg/kg; I.V. (> 200 mg/dl or SHORT- day); INSULIN (glucose > 200mg/dl) effect on kidney, liver and biliary Lower mortality rate

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RAT 150 mg/kg (I.P.)
ALLOXAN 30-65 mg/kg 11.1 mmol/l) and TERM and/or LIQUID (0.9%SALINE or excretion, and in investigations of and less expensive
keton values DIABETES dextrose-saline (4% dextrose, calcium channel blockers (block model of diabetes
RABBIT I.V. I.P.; I.V. 100 -150 mg/kg 100 mg/kg (I.V.) (> 1.5 mM) 0.18% saline)solutions alloxan effects), and beta-3 adrenergic

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agonists (normalize glycemia )
TYPE 1 60 mg/kg (single I.P.
DIABETES injection); Applicable in guinea
MICE I.P. 35-80 mg/kg;
I.P. 40 mg/kg (I.P. for 5 Blood glucose 5% DEXTROSE SOLUTION (on a first pig and cat, can be
I.V. lower then 35 mg/kg days) SHORT-
(> 200 mg/dl or day); INSULIN (glucose > 200mg/dl) (but not the best

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AND LONG- Investigation of kidney, liver and
STREPTOZOTOCIN RAT 60 mg/kg (I.P.) 11.1 mmol/l) and and/or LIQUID (0.9%SALINE or choice) used for
TERM biliary excretion
I.P. 250 mg/kg; keton values dextrose-saline(4% dextrose, investigation of
GERBIL 150 mg/kg (I.V.) DIABETES
I.V. 150 mg (> 1.5 mM) 0.18% saline)solutions chemicals with
I.V. antioxidant effect
I.P. 35-80 mg/kg;
RABBIT 50 mg/kg (I.V.)

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I.V. lower then 35 mg/kg
I.P.; I.V. and S.C. I.P. 100-150 mg/kg; I.V.2- In cats and guinea pigs (do not
MICE 100 mg/kg (I.P.) Blood glucose
3 x less then I.P. develop), investigation of diabetes
(> 200 mg/dl or 5% DEXTROSE SOLUTION
I.P. 150-200 mg/kg; I.V. SHORT- effect on kidney, liver and biliary Lower mortality rate
RAT 150 mg/kg (I.P.) 8.3 mmol/l) and
ALLOXAN I.P. 30-65 mg/kg TERM (on a first day) excretion, and in investigation of and less expensive
keton values
DIABETES calcium channel blocker (block alloxan model of diabetes
(negative or
effects), and beta-3 adrenergic

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RABBIT I.P.; I.V. 100 -150 mg/kg 100 mg/kg (I.V.)
minimal)
agonists (normalize glycemia )
60 mg/kg (single I.P. Applicable in guinea
I.P. 35-80 mg/kg; pig and cat, can be
MICE I.P. injection); 40 mg/kg
I.V. lower then 35 mg/kg (but not the best
(I.P. for 5 days) Blood glucose

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choice) used for
I.P. 35-80 mg/kg; (> 200 mg/dl or
STREPTO- RAT I.P. 60 mg/kg (I.P.) 5% DEXTROSE SOLUTION investigation of
I.V. lower then 35 mg/kg 11.1 mmol/l) and
ZOTOCIN (on a first day) chemicals with
I.P. 250 mg/kg; keton values
GERBIL I.V. 150 mg/kg (I.V.) antioxidant effect,
I.V. 150 mg (> 1.5 mM)
more chance to
TYPE 2
DIABETES
STREPTO-
RABBIT
12 - 24h 24h
I.V. CE
I.P. 35-80 mg/kg;
I.V. lower then 35 mg/kg
50 mg/kg (I.V.)
2 week fed with high-
48-72h
72 h
develop type 2
diabetes
SHORT-
ZOTOCIN + FAT- RAT I.V. 30-65mg/kg fat diet; then 50 mg/kg
AND LONG- Investigation of kidney, liver and
AC
FAD (I.V.) + high-fat diet
TERM biliary excretion
STREPTO- 2 week fed with high-
I.V. 40 mg/kg DIABETES
ZOTOCIN + FAT- MICE fat diet; then 50 mg/kg
3-5 times Blood glucose Higher rate of
FAD I.V. (I.V.) + high-fat diet
(> 150 mg/dl or success in developing
2 week drinking
8.3 mmol/l) and type 2 diabetes when
STREPTO- I.P.; I.V. fructose solution; then NO SPECIAL CARE NEEDED
keton values compared to alloxan
ZOTOCIN + RAT I.V. 40 mg/kg 40 mg/kg (I.V.) +
(negative or and streptozotocin
FRUCTOSE-FAD drinking fructose
minimal) alone application
solution
Nicotinamide
STREPTO-
I.P. 35-80 mg/kg; 90 mg/kg +
ZOTOCIN + RAT I.P.
I.V. lower then 35 mg/kg 60 mg/kg (I.P.) 15 min
NICOTIN-AMIDE
after
MICE I.V. 30-65mg/kg 40mg/kg (I.V.) In cats and guinea pigs (do not
Blood glucose
develop) and in investigation of
(> 200 mg/dl or SHORT-
calcium channel blockers (block Lower mortality rate
ALLOXAN RAT I.V. 30-65mg/kg 40mg/kg (I.V.) 11.1 mmol/l) and TERM
alloxan effects) and beta- 3 adrenergic and less expensive
keton values DIABETES 5% DEXTROSE SOLUTION (on a first agonists (normalize glycemia) in model of diabetes
GESTATIONAL RABBIT I.V. 100-150 mg/kg 120 mg/kg (I.V.) (> 1.5 mM) day); INSULIN (glucose > 200mg/dl) pregnancy
DIABETES I.V. and S.C. I.V. and/or LIQUID (0.9%SALINE or
MELLITUS I.P. 35-80 mg/kg; I.V. 60 mg/kg (single I.P. Blood glucose dextrose-saline(4% dextrose,
Applicable in guinea
MICE lower then injection); 40 mg/kg (> 200 mg/dl or 0.18% saline)solutions pig and cat, can be
STREPTO- 35 mg/kg (I.P. for 5 days) 11.1 mmol/l) and SHORT-
(but not the best
AND LONG- Investigation of kidney, liver and
ZOTOCIN RAT I.P. 35 mg/kg 60 mg/kg (I.V.) keton values choice) used for
TERM biliary excretion in pregnancy
I.P. 35-80 mg/kg; I.V. (> 1.5 mM) investigation of
DIABETES
RABBIT lower then 50 mg/kg (I.V.) chemicals with
35 mg/kg antioxidant effect
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Figure 1. Alloxan and streptozotocin mechanisms of action

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Figure 2. Experimental protocols for induction of type 1 diabetes

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Figure 3. Experimental protocols for induction of type 2 diabetes

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Figure 1
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Figure 2
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Figure 3