Progress in experimental models to investigate the in vivo and in vitro antidiabetic activity of drugs

Abstract Diabetes mellitus is one of the world's most prevalent and complex metabolic disorders, and it is a rapidly growing global public health issue. It is characterized by hyperglycemia, a condition involving a high blood glucose level brought on by deficiencies in insulin secretion, decreased activity of insulin, or both. Prolonged effects of diabetes include cardiovascular problems, retinopathy, neuropathy, nephropathy, and vascular alterations in both macro‐ and micro‐blood vessels. In vivo and in vitro models have always been important for investigating and characterizing disease pathogenesis, identifying targets, and reviewing novel treatment options and medications. Fully understanding these models is crucial for the researchers so this review summarizes the different experimental in vivo and in vitro model options used to study diabetes and its consequences. The most popular in vivo studies involves the small animal models, such as rodent models, chemically induced diabetogens like streptozotocin and alloxan, and the possibility of deleting or overexpressing a specific gene by knockout and transgenic technologies on these animals. Other models include virally induced models, diet/nutrition induced diabetic animals, surgically induced models or pancreatectomy models, and non‐obese models. Large animals or non‐rodent models like porcine (pig), canine (dog), nonhuman primate, and Zebrafish models are also outlined. The in vitro models discussed are murine and human beta‐cell lines and pancreatic islets, human stem cells, and organoid cultures. The other enzymatic in vitro tests to assess diabetes include assay of amylase inhibition and inhibition of α‐glucosidase activity.

pancreatic diabetes and classified as type 3 diabetes. 3The longterm effects of DM include cardiovascular problems, retinopathy, neuropathy, nephropathy, and vascular alterations in both macro and microblood vessels. 4The International Diabetic Federation has estimated that 1 in 10 adults, or 537 million people globally, have diabetes.According to their projections, there will be 643 million adults worldwide who have diabetes by 2030, and 784 million, that is, 1 in 8 individuals, by 2045.In 2021, diabetes contributed to 6.7 million fatalities, or 1 every five seconds.Over 240 million patients with diabetes are believed to go undiagnosed.
[7] The two countries with the greatest prevalence of the disease are China with 141 million people 8 and India with 77 million people. 9abetic people are more likely to contract the virus COVID-19 and are likely to experience more significant complications.
Patients with comorbid conditions like diabetes and heart disease are more likely to experience problems arising from the recent world-wide COVID-19 epidemic. 10abetes poses an important threat to people's health and burdens society financially, 6 and one of the most popular areas of research currently is the management and treatment of DM.In particular, appropriate animal and advanced in vitro research is crucial for the establishment of innovative, efficient methods of treating conditions like diabetes. 11More generally, use of animal models helps researchers create more effective treatments for many disorders and diseases.
Humans and other mammals share many biologically related organs, including the heart, lungs, kidneys, liver, and other organs.They are genetically quite similar as well.For instance, the genes of mice and humans are almost identical. 12All new medications must first pass legal testing on rodents (often mice or rats) and a bigger nonrodent mammal (typically a dog, pig, or monkey) before being administered to humans.This is done because unfavorable effects in either species frequently point to comparable reactions in people, and if a dose is toxic in both rodent and nonrodent species, it is probably also going to be toxic in people. 13However, recently the FDA changed the legislation originally passed in 1938 on animal studies to state that they "no longer require drugs to be tested on animals". 14

| ME THODOLOGY
This review article is based on the databases PubMed, Cochrane, Virtual Health Library, High Wire, Science Direct, Web of Science, Elsevier, Wiley, and academic Google, etc.The databases were systematically searched for articles published in English from 1922 to 2023 with keywords like diabetes animal models, genetically modified rodent models, chemically induced models, surgical induced models, nonrodent models for type II diabetes, diabetic animal models like canine (dog), porcine (pig) models, feline (cat), obese rhesus monkey, virally induced diabetic type I animal models, transgenic/ knock-out diabetic type I animals, and the cell line models.

| OBJEC TIVE
In this this review article, we discuss diabetes complications, diabetes around the world, and diabetes models, including in vivo models and in vitro models for DM.

| ANIMAL MODEL S FOR DIAB E TIC RE S E ARCH
To accomplish diabetic research, scientists have relied on animal models.Pioneering animal studies on DM in dogs were conducted by Nobel laureates Ivan Pavlov, Fedrick Banting, and Charles Best early last century. 15Recently small animals like rodents (mice and rats) are more often exploited for diabetic research, 16,17 with the ability to delete or overexpress a specific gene by knockout and transgenic technologies making them popular models. 18Large animals like porcine (pig) models, 19 canine (dog) models, 20 and nonhuman primate models, 21 as well as Zebrafish models 22 are outlined in Table 1.

| In vivo models for type 1 diabetes
Type 1 diabetes is a condition involving beta cells in the pancreas, 1 and therefore diabetic models are created using chemical induction, 26,27 genetically derived or spontaneously diabetic animals, 28 or genetically or virally induced animals, 29,30 in which the functions of pancreatic beta cells in the experimental animals are ultimately destroyed or modified, eventually leading to hyperglycemia, weight loss, hyperphagia etc. 31,32

| Chemically induced diabetes type 1 model
Chemical agent-induced diabetes in lab animals is the most prevalent option.Among the agents used are streptozotocin (STZ) and alloxan (ALX), both of which achieve a rapid outcome, resulting in an experimental model useful for elucidating the causes of human DM. 17,33,34The toxic effects are only specific to pancreatic beta cells, other organs are spared, mortality is low and doses of these diabetogens are specified and have been optimized by many researchers. 26,35Due to the rapid rate of beta cell regeneration, therapy is less durable and reversible. 368][39] Other diabetogens used in experimental models are dithizone, 40 cyclosporine, tacrolimus, 41 dehydroascorbic acid, dehydroisoascorbic acid, 42 sodium diethyl dithiocarbonate, 43 potassium xanthate, uric acid, and lithium. 44

| Genetically derived or spontaneous diabetic type 1 animals
The most commonly used animals for genetically derived type 1 DM are NOD mouse, 52 BB rat, 53 LETL rat, 54 KDP rat, 54 and LEW-IDDM rat. 55her animal models less frequently used are New Zealand rabbit, 56 Keeshond dog, 57 Chinese hamster, 58 and different monkeys such as Macaca nemestrina, Fascicularis, and Nigra papio hamadryas. 59Genetic mutations that are naturally occurring frequently exhibit an isomorphic phenotypic resemblance between the diabetic animal and the diabetic person, and animals with these mutations are utilized in DM research. 60The contrast between the more frequently used animals and humans are detailed in Table 3. 29,44,54 These animal models are generally monogenic and demonstrate distinct mechanisms of action, whereas the human ADME system is much more complicated. 61,62In addition, these animal models are naturally rare and post-diabetes care aimed at maintaining the animals' health is difficult. 63,64

| Transgenic/knock-out diabetic type 1 animals
Powerful techniques for determining the role of particular genes in glucose metabolism and the etiology of diabetes include knock-out and transgenic mice. 29Pronuclear microinjection produces transgenic animals that often overexpress the transgene, while gene targeting produces animals with an endogenous target gene deleted or altered (knockout/knockin). 65This method can elucidate which TA B L E 1 Animal models of type 1 and type 2 DM.   30 Consequently, beta-cell destruction has been initiated using viruses in several animal models.Direct infection of the beta cell or the start of an autoimmune reaction against the beta cell can both result in destruction. 74The various viruses used to induce DM are Coxsackie B virus, 75 encephalomyocarditis virus, 76 Kilham rat virus, 77 lymphocytic choriomeningitis virus (LCMV) under insulin promoter, 78 rubella, 79 and the mumps virus. 80e virus-induced approach can be challenging because the result depends on the virus replicability as well as the time of the infection. 29deed, research has revealed that, depending on the circumstances, viruses can both cause and prevent autoimmunity. 81While viruses have been connected to some type 1 diabetes cases in humans, the extent of the role they play in the disease's development is unknown. 30,821.5| Surgically induced models (SIM) or pancreatectomy type 1 diabetic animal models Non-rodent animals like pigs, 83,84 dogs, 23,24 and primates 24,25 have hyperglycemia after having a pancreatectomy.This model is a trusted way to cause hyperglycemia when a highly skilled and qualified surgeon is involved. However, th animal undergoes a fairly invasive procedure that raises the risk of hypoglycemia and causes pancreatic exocrine insufficiency.

| In vivo models for type 2 diabetes
Insulin resistance and the beta cell's failure to produce insulin to compensate are hallmarks of type 2 diabetes. 2Consequently, types of animal models for type 2 diabetes include models of beta cell loss and/or insulin resistance. 44,50,51Obesity is prevalent in animal models of type 2 diabetes, mimicking the human scenario where obesity is directly associated with the development of type 2 diabetes. 292.1 | Genetically derived or spontaneous diabetic type 2 animals (obese model) The most commonly used animals for type 2 DM are ob/ob (obese) mouse, 85 db/db mouse, 86 KK (Kuo Kondo) mouse, 87 KK/ Ay (Kuo Kondo/Ay) mouse, 88 NZO (New Zealand Obese) mouse, 89 NONc/New Zealand obese 10 mouse, 90 TSOD (Tsumara Suzuki Obese diabetes) mouse, 91 M16 mouse, 92 Zucker fatty rat, 93,94 ZDF (Zucker diabetic fatty) rat, 95 and WDF (Wistar diabetic fatty) rat. 96In the above models, development of diabetes is spontaneous and shares many characteristics with typical human type 2 DM.The majority of inbred animal models are homogeneous and under environmental control, which makes genetic analysis simple.Minimum outcome variability necessitates a small sample size. 18The characteristics of some of these animals are described in Table 4.

| Genetically derived or spontaneous diabetic type 2 animals (non-obese model)
Lean animal models of type 2 diabetes must also be explored because not all people with DM type 2 are obese.These include models with inadequate beta cells, which eventually results in overt type 2 diabetes in humans (Table 5). 99,100These models, which include Goto Kakizaki (GK) rats, 101 Cohen diabetic rat (CDR), 102 spontaneously diabetic torii (SDT) rat, 102 Alloxan susceptible/Lt mouse, 103 human islet amyloid polypeptide (hIAPP) mice are rare.In these animal models, diabetes is not induced by chemicals or by genetic changes. 12Due to insufficient islet compensation, high fat intake can result in obesity, insulin resistance, and impaired glucose homeostasis. 110,111Examples of animals in this category are C57/BL 6J mouse, desert gerbil or sand rat, 12,112 spiny mouse, and Nile grass rat.The characteristics of these animal models are given in Table 6.

| Non-rodent models for type 2 diabetic animal models
Non-rodent animal models includes cats and obese rhesus monkeys.[122] The rhesus monkey (Macaca mulatta), a non-rodent model of T2DM, offers the most comparable representation of metabolic problems in diabetes.If kept on an ad libitum laboratory diet, especially fructose, it develops obesity, hyperinsulinemia, and insulin resistance.Over several years, it proceeds to necrosis of beta cells, a sharp drop in insulin levels, and hyperglycemia. 59,123,124e Zebrafish model is an attractive model system for the study of metabolic abnormalities.Zebrafish have preserved energy balance and cholesterol metabolism.They are the perfect model for studying lipid metabolism and also, when given an abundance of laboratory nutrients, zebrafish are shown to have hepatic steatosis and higher plasma triglyceride levels.[127][128]

| ANIMAL MODEL S FOR DIAB E TIC COMPLIC ATIONS
Diabetes mellitus is a chronic, sapping metabolic condition that can cause an enormous long-lasting increase in blood sugar levels.The resulting hyperglycemia plays a key role in the development of diabetic complications, such as damage to organs, both structural and functional, resulting in damage to the kidneys (diabetic nephropathy), eyes (diabetic retinopathy), and nerves (diabetic neuropathy). 129,130 is also linked to chronic macrovascular problems such as peripheral vascular disease, coronary heart disease, and stroke (diabetic cardiomyopathy).It has also been discovered that the primary mechanism behind the pathogenesis of such diabetic complications is the generation of oxygen free radical species (ROS). 11,131The animal models that are used to analyze these complications are listed in Table 7, along with their characteristics.

Diabetes development Cause of DM Physiological changes
Goto Kakizaki (GK) 101 rats 2-8 weeks Inadequate pancreatic growth factors and compromised insulin sensitivity in the liver, skeletal muscle and adipose tissues Hyperglycemia, retinopathy, nephropathy, decreased immune markers 105 Cohen diabetic rat (CDR) 102 2 months Diet changes, reduced insulin secretion Retinopathy, nephropathy, reduced fertility, testicular degeneration, 106 hyperglycemia can be retrieved by adjusting diet 39 Spontaneously Diabetic Torii (SDT) rat 107

| IN VITRO TECHNI QUE S FOR A SS E SS ING D IAB E TE S MILLITUS
In vitro (cell or tissue culture) 145 diabetic models are frequently employed by pharmaceutical companies in the search for new treatments.In vitro models can be employed initially for the screening of test materials or to characterize the cellular or molecular actions of lead chemical substances in advanced phases of development.
In vitro models of diabetes are also used in some basic pharmacological research to identify new treatment targets and gain a better understanding of the cellular and molecular mechanisms underlying the illness. 146The primary tissues implicated in the TA B L E 7 Experimental models for diabetic complications.In vitro models include primary cell cultures generated from normal, diabetic, transgenic animals, or cell lines derived from normal, or transgenic animals. 130,131,147The advantages and disadvantages of in vitro models for human diabetes research are specified in Table 8.9][150] Diabetic complications like diabetic nephropathy, and neuropathy can be assessed by optical fluorescence imaging of the structure of the kidney and nerves.Western blot, ELISA, and PCR can be used to analyze gene expression of inflammatory and oxidative markers.Flow cytometry can be used to investigate the degree of retinal endothelial cell death, 150 and concurrently it can enumerate overall beta islet cell health and beta cell glucose sensitivity. 151Insulin secretion can determined by ELISA. 152The luciferase base assay 153,154 and the glucose uptake assay 155 can be done using radiolabeling methods. 156,157Reporter gene assays can identify PPARg and GLUT-4. 158

| CON CLUS IONS
Studying the pathophysiology and clinical aspects of diabetes mellitus in humans is important because 537 million people are suffering from the disease and 240 million people remain undiagnosed.In addition, the health expenditure incurred in 2021 was 966 billion USD and is expected to increase to 1 trillion USD by 2045.To overcome this endemic condition many new drugs have been introduced into the market after testing in both in vivo and in vitro models.These models of diabetes mellitus are very helpful research tools for testing any new synthetic or herbal drug.In this review, we have summarized the development of various models, including those induced by alloxan and streptozotocin, various models using small animal such as rodents, models involving deletion or overexpression of a specific gene using knockout and transgenic animals (immunogenic), and virally induced diabetic models.We also summarize obese and non-obese models, and diet or nutrition-induced models, as well as non-rodent models which are unique for assessing type 2 DM.The Zebrafish model is considered the most appropriate and advanced model for the screening of diabetes and its complications, that is microvascular complications and retinopathy, but it cannot be used for assessing diabetic nephropathy because of primitive renal cells.Diabetes and diabetic neuropathy can be tested best using rodent models due to their similarity structurally, molecularly, and functionally to humans.These models are also the cheapest and mostly easily available, and are easy to handle and maintain compared to other models.The use of large animals like pigs, monkeys, cats, and dogs is considered for pharmacological screening of diabetes induced by chemicals like alloxan, streptozotocin and even by pancreatectomy, but cost, handling and maintenance are some of the issues to be considered.In vitro models like murine and human beta and pancreatic islet cell lines, human stem cells and organoid culture are also discussed, with their advantages and disadvantages.
Although no known animal species closely resembles human diabetes, each model serves as a vital tool for research into genetic, endocrine, metabolic, and morphologic changes, and underlying etiopathogenic changes.The study's design will determine the animal model to use.
More suitable animal models could be used if the subsets of type 1 and type 2 diabetes are better understood.The wide range of disease manifestations in either type 1 or type 2 diabetes makes it unlikely that one animal model (or one treatment) will fit.Therefore, it is important TA B L E 8 Advantages and disadvantages of in vitro models for human diabetes research.

In vitro model Advantages Disadvantages
Murine beta-cell lines 159,160 Simple to culture.There are numerous cell types readily available.A good opportunity to research cell physiology and test medications The mouse cell line can be challenging to select because of differences from humans.Vascularization and cell-to-cell contact are absent Human beta-cell lines 161 Simple to culture.Established human betacell lines permit progress in human diabetes research and clinical applicability Stable human cell lines are hard to make, and there aren't many of them.Genetic flaws are present in the majority of human cell lines.Grow slowly or respond poorly to glucose.Vascularization and cell-to-cell contact are absent Murine pancreatic islets 162,163 Can be isolated more quickly and inexpensively than human islets.Simple to genetically modify Human islets have different islet morphology, vascularization, and blood flow to murine pancreatic islets Human pancreatic islets 164,165 Maintain the islet structure.Used to study the biology of the human pancreas Few donors supply.Don't allow long functional studies.Heterogeneity in their characteristics: size, genetics Human stem cells 166,167 A renewable source of beta-cells.Can be genetically modified.Allow longer studies than pancreatic islets To obtain them, a long and expensive process is needed Organoid cultures 168,169 Resemble the diseased organ architecture better than traditional 2D cultures

ACK N OWLED G M ENTS
The authors are thankful to the School of Pharmacy and Health Sciences, USIU-A and Geethanjali College of Pharmacy for providing all the necessary tools and sources for writing this review.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

E TH I C S S TATEM ENT
Not applicable.
Characteristics of difference between humans and genetically derived or spontaneous diabetic animals.
Type 1 diabetes development has been attributed to viral infections. 104 Characteristics of genetically derived or spontaneous diabetic animals (obese model).