A Comprehensive Review of Preclinical Animal Models Used in Diabetic Research

Streptozotocin (STZ)-induced models of diabetes mellitus

A broad-spectrum antibiotic called STZ is derived from Streptomyces achromogenes and is useful in inducing T1DM and T2DM.^[4,5] Multiple low doses and a single high dose are utilised to elicit diabetes by destroying β-cells of the pancreas.^[9] STZ metabolite acts as an alkylating agent, causes methylation of DNA, and thereby destroys β-cells.^[10,11] Wistar and Sprague-Dawley rats are the typically utilised strains, while some strains, including Wistar-Kyoto, are less sensitive to STZ. Males are more susceptible to STZ than females.^[12,13] Generally, intravenous (i.v.) and intraperitoneal (i.p.) administration methods are used. Intravenous injection produces more stable hyperglycemia. STZ produces dose-dependent β-cells damage, thus making it possible to produce subclinical conditions of diabetes.^[14] Prior fasting of animals is not essential before the STZ challenge. Compared to other diabetogenic agents, STZ develops a more stable model. Mortality due to hyperglycemia and hypoglycemia is the main disadvantage of this method; appropriate measures should be chosen to minimise or avoid mortality. The use of STZ destroys cells other than pancreatic β-cells, which is the main drawback. It was reported that the liver, kidney, lung, gut, testis, and brain all had altered P450 isozymes. STZ also causes lymphopenia.^[10,15]

Alloxan (ALX)-induced diabetes mellitus

Similar to STZ, ALX is a harmful glucose analog that builds up in β-cells through the GLUT-2 transporter. When ALX is combined with glutathione (intracellular thiols), it generates reactive oxygen species (ROS) and forms dialuric acid in a cyclic redox reaction.^[14,16] The radicals produced by dialuric acid, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, cause β-cell destruction and glucose-induced insulin secretion.^[16,17] Compared to STZ, ALX is more widely available and less costly.^[17] The ALX-induced diabetes model is less stable. ALX-induced hyperglycemia is auto-reversible. Inconsistent increases and decreases in glucose concentration cause a multiphasic glucose response. T2D induced by ALX does not mimic the clinical condition. Like STZ, ALX also causes damage to cells other than pancreatic β-cells.^[16,18] ALX also causes renal toxicity.^[19,20]

Diphenylthiocarbazone (Dithizone)-induced diabetes mellitus

Dithizone is a chelator of zinc, which can be administered by both i.v. route and i.p. route for diabetes induction. The dose administered through the i.v. route ranges between 125 mg/kg and 250 mg/kg. The i.p. route needs a dose of 50 mg/kg.^[21-23] The dithizone-induced diabetic rats show higher levels of zinc, iron, and potassium and lower levels of sodium and calcium. Only those drugs that show a hypoglycemic effect by the release of insulin can be screened using this model.^[21,23]

8-hydroxyquinoline induced diabetes mellitus

8-hydroxyquinoline, when administered intravenously, induced diabetes by chelating zinc in pancreatic β-cells.^[24] This causes defective production of insulin. This method induces diabetes in rabbits and is not efficient for diabetes in rats, mice, cats, and dogs.^[24]

Surgery-mediated diabetes mellitus

Pancreatectomy of dogs and pigs develops a diabetes mellitus model in higher animals. One of the major benefits of this approach is that it is free from exposure to cytotoxic chemicals, compared to methods that use STZ and ALX.^[6] During surgical procedures, digestive problems may ensue on account of the surgical excision of the exocrine portion of the pancreas.^[6] Surgical procedures require technical expertise, with risks of damaging other organs.^[25] Mortality is comparatively higher with this method and involves a lot of pre-and postoperative procedures. This model can be less costly and quicker than chemical methods of β-cell destruction. Uncertain efficacy and regeneration of β-cells, and side effects of chemical diabetogenic agents are the drawbacks of the chemical method when used in larger animals.^[26] This can be overcome by pancreatectomy. Pancreatectomy together with splenectomy reduces the chance of infection in an extrapolatory study, and this is relevant for transplantation studies.^[26]

Spontaneous autoimmune models of diabetes mellitus

Non-obese diabetic (NOD) mice, biobreeding (BB) rats, long evans tokushima lean (LETL) rats, LEW-iddm, and Komeda diabetes-prone (KDP) rats are spontaneous animal models of diabetes mellitus.^[14,27-30] High blood sugar and leukocyte infiltration into the islet of Langerhans are traits observed in NOD mice.^[27,28] Clinical signs of diabetes mellitus, such as hyperglycemia and ketoacidosis, occur spontaneously in BB rats. LETL rats spontaneously develop diabetes mellitus without lymphopenia. Pancreatic β-cell apoptosis causes spontaneous development of autoimmune diabetes in LEW-iddm rats.^[25] The hyperglycaemia persists for several days. As the lipid content increases, the spontaneously developed mouse model is useful for hyperlipidemia studies. Both polyphagia and polyuria occur in these animals.^[25]

Virus-induced diabetes mellitus model

In this model, β-cell destruction is caused by viruses, which include the Kilham rat virus and the encephalomyocarditis virus. Virus infection with β-cell autoimmunity destroys β-cells.^[25,31-35] This model describes the potential role of viruses in developing T1DM. The induction using viruses creates a stable model. Since the virus permanently damages beta cells in the pancreas, the model is irreversible. The handling of viruses requires a technical expert, and this method of producing type 1 diabetes is costlier; these are the main disadvantages of this method.^[25,31,32]

Humanised mouse model for T1DM immunology

Humanised mice (hu-mice) models are created through genetic manipulation and engraftment techniques, incorporating human genes, cells, tissues, or organs into mice to replicate human-specific biological processes for research purposes.^[36] For conditions such as T1DM, traditional mouse models often do not reflect the complexity of human disease, making hu-mice a more suitable option.^[37] In studying T1DM immunology, advanced methods like CRISPR allow researchers to insert pivotal human genes linked to diabetes into the mouse genome and remove corresponding murine genes. Specifically, human HLA class I and II alleles, those associated with T1DM susceptibility, are introduced, enhancing the relevance of antigen presentation and immune response within the model.^[37] Deletion of MHC class I and II prevents the presentation of cross-reactive mouse antigens. CRISPR enables the modification of mouse pancreatic β-cells to express human autoantigens relevant to T1DM. The advantage of this method is that it allows experiments that are unethical to be performed on humans. However, this approach is costly.^[36-38]

Gold thioglucose-induced diabetes mellitus

This drug was initially used for the treatment of rheumatism and arthritis. Later, toxicological studies found that it causes a syndrome of hyperphagia and obesity in mice.^[39] The Gold thioglucose (GTG) causes bilateral necrosis of the ventromedial hypothalamus (VMH) region of the brain. This causes damage to the ventromedial nuclei, arcuate nuclei, supraoptic nuclei, and medial eminence.^[40] This impairs the regulation of water and food intake, and thus it causes obesity. These animals develop insulin resistance gradually and show characteristics of T2DM.^[41] GTG can be used in genetically normal mouse strains resistant to HFD-induced obesity. GTG treatment induces diabetes in BKs, KK, DBA, and BDF mice.^[42,43] The success rate of achieving hyperphagia and obesity in this model will vary with the mouse strains used, a limitation. Another limitation is that the effective dose is close to the lethal dose of GTG in some strains. Hence, choosing the optimal dose for each strain is critical.^[42,43]

Monosodium glutamate (MSG)-induced diabetes mellitus

MSG-induced diabetic mice model shows features such as hyperglycemia, hyperinsulinemia, obesity, decreased glucose tolerance, and decreased insulin sensitivity.^[44] A high dose of MSG of ≥4 mg/g body weight and follow-up of >6 months is required to produce T2DM.^[45-47]

Ferric nitrilotriacetate-induced diabetes mellitus

ROS are formed by fenton-type reactions catalysed by the soluble iron complex ferric nitrilotriacetate (Fe-NTA).^[48,49] Iron buildup in the liver, kidney, and pancreas of rats after multiple treatments results in lipid peroxidation, DNA damage, and protein modifications. Animals develop hyperglycemia, glucose intolerance, glycosuria, and decreased insulin responsiveness in a matter of weeks.^[48]

Neonatal streptozotocin (n-STZ) model of diabetes mellitus

Many research papers report the neonatal n-STZ model of diabetes mellitus with STZ injections to neonatal pups, on post-natal day 0 (n0-STZ), day 2 (n2-STZ), and day 5 (n5-STZ), eliciting polyphagia, polyuria, polydipsia, glycosuria, impaired glucose tolerance, and reduction in insulin receptor number.^[49,50] In the n0-STZ rat model, insulin-deficient acute diabetes develops within 3-5 days after birth. The characteristics of this model are high plasma glucose levels, elevated plasma glucagon, and a decrease in plasma insulin of about 93%. By 8 weeks of age, rats begin to display mild hyperglycemia. The n-STZ model exhibits slightly lowered plasma insulin levels, hyperglycemia, and lowered pancreatic insulin levels.^[50] This model is well-established and frequently employed.^[49,50]

Nicotinamide and streptozotocin model

STZ is a nitrosourea compound that enters via the GLUT2 glucose transporter in to pancreatic β-cells selectively. This causes DNA alkylation, activation of poly ADP-ribose polymerase (PARP), NAD⁺/ATP depletion, oxidative stress, and results in β-cell necrosis. STZ, when administered alone, produces almost complete destruction of β-cell and produces T1DM. NA is a pancreatic β-cell protectant. It is a precursor of NAD⁺ and a PARP inhibitor. When administered before or shortly after injecting STZ, it produces a partial pancreatic β-cell protection. Thus, STZ and NA together produce only partial β-cell destruction and thus resemble T2DM.^[51,52] It produces stable and reproducible hyperglycemia useful for long-term studies.^[53,54]

High-fat-induced diabetes mellitus model

T2DM can be induced in experimental animals like mice, rats, guinea pigs, monkeys, dogs, Drosophila, and zebrafish.^[55,56] HFD-induced T2DM shows features like human T2DM. The HFD-fed mice show initial hyperinsulinemia and later show hypoinsulinemia. The HFD diabetic mice model also exhibits symptoms such as impaired glucose tolerance, hyperglycemia, and obesity.^[56] Hyperinsulinemia, abdominal adiposity, insulin resistance, impaired glucose clearance, and high blood glucose are seen in the HFD-fed diabetes rat model.^[57] Drosophila fed with a diet containing 20% coconut oil exhibit insulin resistance, high triglyceride and circulating glucose, and increased expression of TGF-β.^[58] The limitation of this method is that it requires feeding with HFD for several months to bring about all features like human T2DM, making it expensive.^[59]

High-fat diet and streptozotocin model for diabetes mellitus

Feeding a HFD with administration of low-dose STZ, and glucocorticoids, together or HFD diet and STZ alone, induces T2DM, manifested as β-cell destruction and insulin resistance. This model can be used to research T2DM mechanisms and investigate treatments.^[60-62] The advantages of an induced model over spontaneous models are low cost, stable effect, and easy acceptance.^[55,60-63]

Low-protein diet-induced diabetes mellitus model

Animals fed with a low-protein diet (LPD) for a long time exhibited hyperglycemia.^[64-66] Offspring of mothers fed an LPD during pregnancy exhibited elevated blood glucose levels along with a reduced number of pancreatic islet cells. Maternal malnutrition impairs the development of fetal pancreatic islet cells and causes diminished insulin-secreting capacity.^[67] The elevation of blood glucose and reduced insulin level during the undernutrition period is due to the changes occurring in the beta cells, mitochondria, and protein channels.^[68] Feeding the animals for long-term use of LPD itself causes hyperglycemia. Challenge of low-dose STZ to 13-week-old Wistar rats after feeding them with LPD produces a stable T2DM model. These animals exhibited all the cardinal signs of diabetes mellitus: polyuria, polyphagia, and polydipsia. The patent application of this model has been published. This model can be useful for understanding the pathophysiology of T2DM caused by protein malnutrition (PMN) and is effective in screening drug targets for the treatment. (BK A, presented at the MPCON 2025).

Obese models

T2DM involves insulin resistance and obesity. Genetic modification, along with a HFD, can cause obesity. The diabetes induced in Lep^ob/ob mice does not completely represent diabetes in humans. The diabetes in C57BL/KS is more severe and associated with early mortality. Polygenic models represent diabetes similar to humans and are suitable for studying disease complications.^[17,51]

Transgenic animals as models of diabetes mellitus

Transgenic animals, created by genetic manipulation, help study diabetes by determining the functions of specific genes. Further, it helps to study the onset and course of the disease development. Additionally, it helps in recognising the role of inflammation in the development of diabetes and abnormal glucose metabolism.^[6,14] Knockout mice, fed with HFD, can determine whether genes are capable of compensating for insulin resistance.^[69] This method of model development is costlier. Human Islet Amyloid Polypeptide (HIP) rat develops T2DM between 5-10 months of age due to ꞵ-cell apoptosis. Nearly 60% of islet cell apoptosis.^[70]

Zebrafish model of diabetes mellitus

This is an excellent model to explore novel targets for the management of obesity and diabetes mellitus.^[71,72] Because of the functional similarities in pancreatic anatomy, lipid and glucose metabolism, this model serves well for studying metabolic diseases.^[73] Zebra fish can be altered as a T1DM or a T2DM, gestational diabetes, and glomerular cystic kidney disease. Zebra fish have 85% of disease-related genes similar to humans.^[74] The use of diabetogenic substances, Nitroreductase (NTR)-mediated cell destruction, and the expression of activated Bid during apoptosis develop insulin-dependent diabetes mellitus.^[74-76] T2DM is induced by glucose immersion, overnutrition, expression of IGF-IR, and knockdown of insulin receptors in the liver. The pancreatectomy technique has the drawback of technical difficulty. Diabetes induced by diabetogenic compounds such as ALX and STZ, with quick recovery, is a drawback.^[75,77] Glucose immersion to induce T2DM, hyperglycemia, impaired response to insulin, and diabetic nephropathy are the phenotypic features of this method. This model can be used to study diabetic complications such as nephropathy and retinopathy.^[71,78] However, it requires frequent exchange of glucose solutions.^[71,78] Overnutrition induces T2DM and obesity, achieved by overfeeding zebra fish with commercial food, although overfeeding increases mortality.^[71,72,78]

Drosophila melanogaster model

T1DM and T2DM may both be mimicked in the “fruitfly” Drosophila melanogaster. The following represent a few of the benefits of implementing Drosophila as a model of diabetes. Easy genetic manipulation, high fertility, quick generation times, and homogenous genetic backgrounds make Drosophila a good model for investigating the insulin pathway and metabolic processes.^[79]

Caenorhabditis elegans model

The organism Caenorhabditis elegans helps investigate the mechanisms of metabolic disorders in diabetes and the complications of antidiabetic drugs. The use of C. elegans on microarray devices helps high-throughput screening of antidiabetic agents.^[80]

Employing invertebrates as a diabetes mellitus model method requires less time. Higher animals can be spared by using invertebrate animals for developing diabetes mellitus models. However, this method has disadvantages due to the differences in anatomy and physiology compared with humans.^[6]