Animal models for induction of diabetes and its complications

Abstract

Objectives

Animal models are widely used to develop newer drugs for treatment of diabetes and its complications. We conducted a systematic review to find various animal models to induce diabetes and also the suitable methods in various diabetic complications. With an emphasis on the animal models of diabetes induction, this review provides a basic overview of diabetes and its various types. It focused on the use of rats and mice for chemical, spontaneous, surgical, genetic, viral, and hormonal induction approaches.

Methods

All observations and research conducted on Diabetes and its complications published up to 18 May 2023 in PubMed, Web of Science, Scopus and Conchrane Library databases were included. Main outcome measures were reporting the induction of diabetes in experimental animals, the various animal models for diabetic complications including diabetic nephropathy, diabetic retinopathy, diabetic neuropathy and diabetic osteopathy. The quality of reporting of included articles and risk of bias were assessed.

Results

We reached various articles and found that rats and mice are the most frequently used animals for inducing diabetes. Chemical induction is the most commonly used followed by spontaneous and surgical methods. With slight modification various breeds and species are developed to study and induce specific complications on eyes, kidneys, neurons and bones.

Conclusions

Our review suggested that rats and mice are the most suitable animals. Furthermore, chemical induction is the method frequently used by experimenters. Moreover, high quality studies are required to find the suitable methods for diabetic complications.

Introduction

Approximately 537 million people are living with diabetes. Diabetes affected million people worldwide and it is expected to increase up to 643 million till year 2030 [1]. Diabetes mellitus is a well-known chronic metabolic disorder that causes hyperglycaemia due to a relative or complete lack of insulin. Chronic hyperglycaemia leads to many micro and macro complications like nephropathy, retinopathy, neuropathy, osteopathy, etc. [2]. The effects of diabetes mellitus on people’s lives have fueled research into the disease’s mechanisms as well as efforts to develop better therapies [3]. Animal models are widely used for novel drug development for treatment of diabetes and its complications. Various rodents like mice, rats and hamster have found to be the suitable models for the purpose of research and they also help to find the pathogenesis of the disease [4]. The majority of research on diabetes is conducted on animals because of its complicated aetiology and multi-systemic interactions, and despite advancements in in vitro and in silicon studies, they are unable to fully replicate the knowledge acquired from animal models [5]. Induction of diabetes in experimental animals can be carried out by different ways by using various chemical diabetogens, surgically by partial pancreatectomy, viral induction, hormonal therapy and genetic manipulation by selective inbreeding [6]. The present review focuses on various animal models of diabetes mellitus and its complications.

Animal models

Chemically induced models

Diabetogens are chemicals that have been shown to have an immediate harmful effect on beta cells in the islets of Langerhans, which is followed by a diabetic state. The most used animal model for studying diabetes and its complications is STZ-induced diabetes (Table 1). The term beta cytotoxic substances have also been used to describe these chemicals and are mentioned in Table 2.

Table 1.

Various types of STZ induced models

Types of STZ induced model	Animal model	Age	Weight	Dose	Reference
Repeated low doses of STZ	C57BL/6 or CD-1 male mice	8 to 12 weeks	25 g	40 mg/kg (1.0 ml/100 g) i.p.	[14]
Single high dose of STZ	C57BL/6 or CD-1 male mice	8 to 12 weeks	25 g	200 mg/kg (1.0 ml/100 g) i.p.	[14]
STZ induced type 1 diabetes mellitus	Sprague-Dawley or Wistar male rats	8 to 10 weeks	150 to 200 g	65 mg/kg (2.0 ml/kg) i.v.	[15]
Streptozotocin-Nicotinamide rat model	Sprague-Dawley or Wistar male rats	8 to 10 weeks	150 to 200 g	Nicotinamide dose: 230 mg/kg (1.0 ml/kg) i.p. STZ dose: 65 mg/kg (2.0 ml/kg) i.v.	[16]
Fat fed Streptozotocin model	Sprague-Dawley or Wistar male rats	8 to 10 weeks	150 to 200 g	40 mg/kg (1.0 ml/kg), i.p. or Two lower doses of STZ (30 mg/kg, i.p.) administered at weekly intervals with high fat diet (60% fat by caloric content) for 8 weeks	[17]

Table 2.

Other chemicals for diabetes induction

Compound	Dose	Animal	Reference
Dehydrogluco ascorbic acid	3.5–3.9 g/kg	Rat	[21]
Dehydro ascorbic acid	650 mg/kg three days	Rat	[24]
Dehydroisoascorbic acid	1.5 mg/kg	Rat	[25]
Methyl alloxan	53 mg/kg	Rat	[26]
Ethyl alloxan	50–130 mg/kg	Rat	[27]

Streptozotocin induced models

Streptozotocin is a glucosamine derivative of nitrosourea that is produced naturally by the fungus Streptomycetes achromogenes [7]. Chemically known as N-(methylnitrosocarbamoyl)-alpha-d-glucosamine, it has antibacterial properties. It causes the destruction of beta cells in the pancreas. The processes of methylation, the formation of free radicals, and the production of nitric oxide all contribute to the destruction of beta cells [8]. Due to its selective targeting of pancreatic beta cells, it is the chemical that is most routinely employed to induce diabetes mellitus. It causes necrosis, which kills the cells, when it penetrates pancreatic cells via the glucose transporter GLUT 2 (transmembrane Carrier protein) [9]. While STZ’s alkylating activity drives its impact at larger doses, it also triggers an immunological and inflammatory response at lower doses by releasing glutamic acid decarboxylase. For mice, the optimal dose is between 170 and 200 mg/kg (i.p. or i.v.), while for Wistar rats, it is 60 mg/kg (i.p. or i.v.) [10]. It has been established that golden hamsters administered to a dose of 50 mg/kg i.p. develop diabetes [11]. Recently, a new type 2 diabetes model was developed using STZ and NAD (230 mg/kg, i.p.). The NAD (65 mg/kg, i.v.) is administered 15 min before the STZ and causes moderate hyperglycaemia while protecting the pancreatic beta cells due to its antioxidant properties [12]. The blood sugar level exhibits a triphasic reaction, with the first hyperglycaemia occurring after one hour, followed by a six-hour hypoglycaemic phase, and a 24-hour period of sustained hyperglycaemia, likely as a result of damage to the beta cells that are being depleted [13]. The blood glucose levels normally achieved is between 200 and 250 mg/dl. The mechanism of action of STZ is further given in Fig. 1.

Fig. 1.

Mechanism of action of STZ

Alloxan induced model

Pancreatic beta cells are selectively necrotized by the urea product alloxan. It is chemically 2,4,6-trione of a 5,5-dihydroxyl pyrimidine [4]. Following the administration of alloxan, diabetes can develop in two different ways. There is a sudden rise in insulin levels, which inhibits the islet response to glucose [18]. First, beta cells of the pancreas are destroyed by free radicals produced when alloxan is converted to diuleric acid, which is then reoxidised. the second mechanism that results in cell death is the interaction of alloxan with the SH group, particularly with membrane proteins like glucokinase on the beta cells, ultimately leads in cell necrosis [19]. The most common dose of alloxan administered to rats is 100–175 mg/kg (s.c.) [20]. Diabetogenic dose varies with species. The blood glucose level exhibits a triphasic reaction, with the first hyperglycaemia occurring after one hour, followed by a six-hour hypoglycaemic phase, and then persistent hyperglycaemia at 24 h, most likely as a result of beta cell injury and beta cell depletion. The route of administration has a big impact on the dose [19]. The drawbacks include high rates of mortality in rats, reversible hyperglycaemia, and the induction of ketosis in animals as a result of the production of free fatty acids [21]. Its diabetogenic effects are resistant in some species, such as guinea pigs. Alloxan has been almost entirely replaced by STZ for the induction of diabetes as a result of these shortcomings.

Dithizone induced model

The chemical name for dithizone is 8-(p-toluene-sulfonyl amino) quinoline (8- TSQ). Diabetes was induced by intravenous injection of dithizone. In dithizonized diabetic mice, blood zinc, iron, and potassium levels were found to be higher than average, although serum calcium and sodium levels were lower. Copper and magnesium levels were stable [22]. Zinc can be combined within liposomes by the zinc chelating chemical dithizone, which can also release protons, increasing the diabetogenicity. When injected, these complexing chemicals cause the lipid vesicles’ contents, which contain trapped zinc ions, to become acidic at pH 6. The zinc-containing insulin storage granules in pancreatic beta-cells undergo such proton release, which makes the insulin soluble and produces osmotic stress, which ruptures the granule and results in diabetes [23].

Spontaneously induced model

Studies have shown that both type 1 and type 2 diabetes have a large number of spontaneous models. Non-Obese Diabetic (NOD) mice and Bio Breeding (BB) rats are most likely the best spontaneous models for type 1 diabetes. There are Goto Kakizaki (GK) and Otsuka Long Evans Tokushima Fatty (OLETF) rat models for type 2 diabetes as well [27].

When NOD mice are 4–5 weeks old, insulitis first appears, then the beta cells are destroyed. Young NOD mice develop hyperglycaemia after receiving STZ at a dose as low as 30 mg/kg for five days. In BB rats, pancreatic beta cells are attacked by the immune system, which damages them. In Outbred Wistar rats that have given rise to type 1 Bio Breeding rats (BB), diabetes has been observed to manifest in these animals as a result of a cell-mediated auto immunological process [28]. It consists of the Bio Breeding Diabetic Prone (BBDP) and Bio Breeding Diabetic Resistant (BBDR) strains [29]. It has been found that retinopathy and neuropathy, two complications of diabetes, are more likely to occur in those with BBDP. BB rats are more susceptible to diabetes after puberty, which can result in hyperglycaemia, ketonuria, and hyperinsulinemia[27].

Goto-Kakizaki rats were developed through the repeated mating of Wistar rats at the upper limit of the normal distribution for glucose tolerance. They develop peripheral insulin resistance 56 days after birth and are classed as non-obese models [30]. Early in life, they show signs of mild hyperglycaemia. This strategy induces type 2 diabetes. The other model, OLETF rats develop hyperglycaemia, hyperinsulinemia, and moderate obesity between 12 and 24 weeks [31].

Surgically induced model

The pancreas can be completely or partially removed surgically to induce experimental diabetes. Diabetes that is completely dependent on insulin (Type 1 Diabetes) is the result of pancreas excision. One can also utilise pancreatectomy in combination with chemical agents like STZ and alloxan as it causes a stable form of diabetes in animals. Partial pancreatectomy removes more than 90% of the organ to cause diabetes (Type 2 Diabetes) [32]. The ability of the beta cells to regenerate is investigated [33]. Despite being used, this model has a disadvantage that it causes the loss of beta cells, alpha and delta cells, and extra pancreatic enzymes. Therefore, this model is not the first-choice model and is used under specific conditions.

Genetically induced models

Some of the well-known genetically produced diabetes models include the Akita mouse, Zucker Diabetic Fatty (ZDF) rats, and Obese Spontaneously Hypersensitive Rats (SHR) [24].

Akita mice are used as a model for early-onset diabetes. They were developed using the C57BL/6 inbred strain in Akita, Japan. The pancreatic beta cells in these subjects are destroyed by a mutation in the Ins2+/C96Y gene, and their abnormal protein folding renders the beta cells toxic [24]. Some of the symptoms include polyuria, hyperinsulinemia, hyperglycaemia, and polydipsia. In Akita mice, early-onset diabetes is autosomal dominantly inherited and is not associated with insulitis or obesity. The circulation insulin levels in the Akita diabetic mice are much lower than those in non-diabetic animals.

The Zucker Diabetic Fatty Rats were developed in Indianapolis, USA, from a colony of outbred Zucker rats. In 1991, the genetic model of the ZDF rats was established. A straightforward autosomal recessive leptin receptor gene (fa) mutation on chromosome 5 is responsible for hyperglycaemia, insulin resistance, and obesity [34]. These rats show advanced insulin and glucose resistance by the time they are 4 to 8 weeks old, and by the time they are 8 to 10 weeks old, they are clearly diabetic with typical feeding state glucose levels of 500 mg/dl.

Virus induced model

Viruses are used to cause insulin dependent diabetes for investigational purposes in experimental animals. By infecting and killing beta cells in the pancreas, viruses can cause diabetes. RNA picornoviruses, Coxsackie B4 (CB4), Encephalomylocarditis, Mengo-2T, and other human viruses are among the numerous ones utilised to cause diabetes. First, in the 1960s, Gamble et al. described juvenile onset diabetes, which was caused by a viral infection [35]. The pancreatic acinar cells of mice are damaged by the coxsackie virus. As a result of tissue damage and inflammation, this virus infects the mice. Islet antigen sensitization is an indirect result of the infection that results in the reactivation of dormant auto-reactive T cells [36].

While D-Encephalomyocarditis Virus in some inbred mouse strains damages the pancreatic beta cells. By giving the mice an immunosuppressive drug first, followed by a viral infection, the likelihood of success is increased. Though the viruses are thought to be etiologic agents to produce diabetes mellitus by infecting and destroying beta cells, however, these human pathogenic agents are generally not pancreatotropic or ilytic to beta cells [37].

Hormone induced method

Growth hormone has been shown to cause diabetes and renal complications [38]. The use of corticosteroids can lead to the condition known as steroid diabetes. The two glucocorticoids that lead to steroid diabetes most frequently are prednisolone and dexamethasone [39]. When dexamethasone is administered intraperitoneally twice daily at a dose of 2 to 5 mg/kg, Type 2 Diabetes develops in rats [40].

A brief summary of the experimental rodent models for induction of diabetes has been further given in Table 3.

Table 3.

Experimental rodent models for induction of diabetes

Method of induction	Animal model	Key points	References
Chemically induced model	Streptozotocin induced models	Destruction of beta cells resulting hyperglycaemia	[8]
	Alloxan induced model	Beta cells of the pancreas are destroyed by free radicals	[19]
	Dithizone induced model	It reacts with zinc in the pancreas causing destruction of islets cells	[22]
Spontaneously induced model	NOD (Non-Obese) mouse	Model of type 1 Diabetes	[28]
	Bio Breeding (BB) rats	Develops auto immune diabetes	[28]
	Goto Kakizaki (GK) rats	Diabetes develops due to peripheral insulin resistance	[30]
	Otsuka Long Evans Tokushima Fatty (OLETF) rats	Moderate obesity between 12 and 24 weeks	[31]
Surgically induced model	Pancreatectomy model	Surgically removing all or part of the pancreas	[32]
Genetically induced model	Akita mouse	Model for diabetes with early onset	[24]
	Zucker Diabetic Fatty (ZDF) rats	It develops obesity without diabetes	[34]
Virus induced model	Encephalomyocarditis virus	It harms the beta cells of the pancreas	[35]
	Coxsackie virus	It harms mice’s pancreatic acinar cells	[36]
Hormone induced model	Dexamethasone treated rats	It causes the development of Type 2 diabetes in rats	[39]

Models for various diabetic complications

Diabetes is spreading quickly in practically every nation. Other acute or chronic diseases that damage people’s health and quality of life are emerging as a result of the rise in diabetes. The complications are divided into two: (1) Microvascular complications, when blood vessels are damaged. It includes both retinopathy and neuropathy. (2) Macrovascular complications, such as stroke and coronary artery disease. It also causes liver and bone damage (osteopathy) and it has been linked to cancer. There are numerous models to examine the diabetic complications (Table 4) [41].

Table 4.

Various models of Diabetic complications

Diabetic complications	Models for induction	References
Diabetic Neuropathy	STZ induced Wistar rats	[45, 46]
	BB/Wor rats
	C57BL/Ks (db/db) mouse
Diabetic Nephropathy	Akita, NOD mouse	[51, 54]
	STZ induced Sprague Dawley rats
	Black and Tan Brachyuric (BTBR) mice
Diabetic Retinopathy	Akita mouse	[55, 56]
	NOD (Non-Obese) Mouse
Diabetic Osteopathy	Wistar rats Goto-Kakizaki (GK) rats Zucker Diabetic Fatty (ZDF) rats	[57, 58]

Diabetic neuropathy

One of the most prevalent consequences of diabetes is neuropathy. Peripheral nerve dysfunction is a type of nerve injury that is more common in type 2 diabetes. It happens because of a metabolic cascade that is generated downstream as a result of increased polyol pathway input brought on by persistent hyperglycaemia [42]. Wistar rats with STZ-induced diabetic neuropathy experience anomalies in pain perception that are easily detected by quantitative behavioural testing, similar to neuropathic pain models involving traumatic injury to a peripheral nerve [43].

The model that is most frequently used to understand the motor conduction velocity is the STZ induced model. Here, the peroneal nerve’s myelin sheath axon and smaller fibre size are examined. Another model is the BB/Wor rat, which lacks T cells [44]. These are used to produce type 1 diabetes, which manifests suddenly. After 2 weeks of diabetes, a shift in nerve conduction is apparent, and a loss of sural nerve fibre is seen after 4 months. Streptozotocin-induced diabetic animal models have also been used to study mechanical hyperalgesia and hypoalgesia [45]. The C57BL/Ks (db/db) mouse model is another one that is utilised [46]. The decrease in nerve conduction, lowering of response and decrease in velocity are observed.

Diabetic nephropathy

Diabetic nephropathy usually results from both type 1 and type 2 diabetes. Over time, blood vessel clusters in the kidney can get harmed by poorly controlled diabetes [47]. This could lead to both excessive blood pressure and kidney impairment. The primary direct cause of diabetic nephropathy is assumed to be the diabetes complication of hypertension, or high blood pressure [48]. Hypertension is thought to be both a contributing factor to diabetic nephropathy and a negative outcome of the damage the condition causes[49].

The symptoms of diabetic nephropathy include albuminuria, a decline in glomerular filtration rate (GFR), hypertension, mesangial matrix expansion, thickening of the glomerular basement membrane, and tubule-interstitial fibrosis [50]. Different rodent strains have been produced in order to generate a model that accurately depicts the signs of human diabetic nephropathy[51].

The STZ-induced rat model is frequently employed. 90% of the rats develop hyperglycaemia. Albuminuria increases by 10 times in Wistar rats 1 week after STZ induction and by 400-fold in Sprague Dawley rats 6 weeks after STZ induction. After 4 weeks, albuminuria increases by 4 times in the model of a high-fat diet. At six months of age, albuminuria increases by two folds and mesangial matrix similarly increases in the Akita mouse model of the DBA/2 and C57BL6 strains [52].

The common classic rodent nephropathy models, such as Akita, NOD, or obese type 2 models, only exhibit the early stages of nephropathy. Although the bulk of these conventional models do not display all characteristics, they do simulate a diabetic state. The multigenic and environmental nature of the of diabetic nephropathy makes it difficult to identify and address every risk factor [53].

The Black and Tan Brachyuric (BTBR) mice crossed with C57BL/6 mice, which were described by Clee et al. [51], are a well-known mouse strain. By four weeks of age, these mice exhibit proteinuria, by eight weeks, hypertrophy and mesangial matrix build-up, by twenty weeks, glomerular lesions, and by twenty weeks, an increase in glomerular basement membrane [54].

Diabetic retinopathy

With numerous structural, biochemical, molecular, and functional problems, it is a complicated complication. Along with hyperglycaemia, serum lipids play a significant role in the development of this illness [55]. Mismanagement of lipid metabolism, damage to retinal mitochondria, anomalies in retinal genes and proteins, and DNA damage to mitochondria that increases oxidative stress are all significant contributors to the course of this illness [59]. The most popular models are rodents. The Insulin 2 gene has a missense mutation in the Akita mouse, a model for type 1 diabetes.

The missense mutation results in a structural change in the insulin protein, which causes the protein to accumulate in pancreatic beta cells and ultimately lead to beta-cell death. According to this model, the disease begins 12 weeks after hyperglycaemia and is signalled by reactive gliosis and an increase in retinal vascular permeability [60]. Up until 8 months of age, the condition progresses with diminished dendrites and axons. Another popular type 1 diabetes model is the NOD mouse. By the time the mice are 12 weeks old, the disease has already begun to show symptoms, and they have developed spontaneous hyperglycaemia. In NOD rats, apoptosis is shown in pericytes, endothelial cells, and retinal ganglion cells (RGC), and the retinal capillary basement membrane starts to thicken at 4 weeks. Additionally, focal vessel proliferation was discovered [56].

Db/db (Leprdb) mice were developed to study type 2 diabetes. After six weeks, the number of RGCs decreases and they are concentrated more in the thicker central retina [61]. The OLETF rat, a monogenic diabetic retinopathy model with obesity, hyperglycaemia, and glycosuria, was developed by selectively selecting Long-Evans rats. Six weeks following the initiation of hyperglycaemia, leukocyte entrapment in retinal microcirculation and other micro vessel-related symptoms are observed [62].

Diabetic osteopathy

It is generally known that diabetes mellitus raises the risk of osteopenia, fractures, osteoporosis, and skeletal abnormalities [58]. The development of bone abnormalities in diabetes is thought to be caused by a number of potential pathways. Osteoporosis causes a decline in bone mineral density (BMD), bone quality, and bone strength as well as an increase in skeletal fragility and micro architecture degeneration of bone tissue. Bone has been demonstrated to be impacted by altered insulin levels, insulin resistance, hormonal changes, hyperglycaemia, and peripheral neuropathy [63]. The High Fat Diet- STZ Wistar rat model replicates a close homology to human type 2diabetes and has been used as a model to study the effects of diabetes on bone parameters [64]. Diabetes was induced in male Wistar albino rats by streptozotocin (65 mg/kg, i.v.) after 15 min of nicotinamide (230 mg/kg, i.p.) administration. () In another model a combination of high fat diet fed and low dose of STZ (35 mg/kg i.p.) was used to develop diabetic osteopathy in rats [57].

A lot of people utilise the Goto-Kakizaki (GK) rat model [63]. They have signs of regional osteopathy, lower levels of local IGF-I in diaphyseal bone, and maybe impaired local IGF-I response in trabecular bone. Twelve-month-old GK rats serve as a model for spontaneous type-2 diabetes. Male Zucker Diabetic Fatty (ZDF) rats also serve as a purpose to develop bone fragility [65]. They were fed with high sugar and high fat diets to develop diabetes and later on the osteopathy.

Conclusion

Diabetes is a social and financial hardship. According to studies on the disease, diabetes has significant global direct and indirect financial effects. In research, a variety of anti-diabetic medications are used, and the goal is always to find something more practical, effective, affordable, and simple to use. The frequency of diabetes mellitus is rising, and diabetic rats and mice models are thought to be crucial for understanding the pathophysiology of human diabetes and its consequences. Additionally, the use of diabetic rat models is vital for researching and creating new medications to treat diabetes and its side effects.

Despite all of their advantages, all of the rat and mouse models that have so far been made available have significant drawbacks. The optimal model is still being sought after; it must be sensitive to anti-diabetic medications and suited for studying disease pathophysiology as well. Prolonged diabetes is the cause of the many diabetic complications. These models have the capacity to grow a variety of difficulties. Although a single model can produce all defects, particular breeds and strains are more susceptible to the quick emergence of a given problem and are therefore frequently chosen.

These afflicted animal models have aided scientists and researchers in their efforts to better understand the causes of other diseases in preclinical studies that allowed the screening of medications and pharmacological agents, but their value in foretelling the efficacy of treatment approaches is limited in clinical studies.

Authors’ contributions

The thorough review of articles and literature was done by FQ. SS edited, implemented and modified the contents continuously, MS read the final manuscript and suggested the necessary changes. All the authors read and approved the final manuscript.

Funding

No sources of support provided.

Declarations

Competing interests

The authors declare that they have no competing interests.