AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA

ISSN Print: 2151-7517, ISSN Online: 2151-7525 2010, Science Hu, http://www.scihub.org/ABJNA

Animals models for studying diabetes mellitus

Etuk, E.U Department of Pharmacology, College of Health Sciences, Usmanu Danfodiyo University, Sokoto, Nigera Phone: +2348054693770 E-mail: etuk2005@yahoo.co.uk
ABSTRACT Diabetes mellitus is a potentially morbid condition with high prevalence worldwide thus the disease constitutes a major health concern. Presently, it is an incurable metabolic disorder which affects about 2.8% of the global population. The search for compounds with novel properties to deal with the disease condition is still in progress. This makes the use of experimental models for the disease imperative. The current review has attempted to bring together all the reported models, highlighted their short comings and drew the precautions required for each technique. It was noted that, although chemical induction of diabetes mellitus with streptozotocin was the most widely used procedure, alloxan induced diabetes model was the best known drug induced diabetes. Genetic and surgical models were rarely used because they required highly technical skills and the percentage of animals lost during the procedures were higher. From the review it was concluded that, chemical induction with alloxan appears to be the easiest, reliable and the most practicable method of inducing diabetes mellitus in rodents. Keywords: animal model, diabetes mellitus, induction, alloxan, streptozotocin INTRODUCTION The existence of experimental animal model of a disease aids not only the understanding of the pathophysiology of such disease, but also the development of drugs for its treatment. According to the World Health Organization (WHO), there are approximately 160,000 diabetics world wide, the number of diabetics has double in the last few years and is expected to double once again in the year 2025 (Beretta, 2001). Due to its high prevalence and potential deleterious effect on a patient physical and psychological state, diabetes is a major medical concern (Macedo et al., 2002).The disease remains incurable and can only be controlled with drugs. Over the years, several animal models have been developed for studying diabetes mellitus or testing anti-diabetic agents. These models include chemical, surgical (pancreatectomy) and genetic manipulations in several animal species to induce diabetes mellitus. The diabetogenic drugs used include: alloxan monohydrate, streptozotocin with or without nicotinamide , ferric nitrilotriacetate, ditizona and antiinsulin serum. The selection of these models to use for investigating the antidiabetic properties of a new compound may be a very difficult task especially for young researchers. The selections of inappropriate animal models have been identified as one of the common problem associated with ethnobotanical researches (Thatte, 2009). The aim of the present review is to piece together all the various experimental models developed for studying diabetes mellitus, assess the merits and demerits of each model and highlight the precautions needed to avoid erroneous results during the applications of these models. The currently existing animal models include: Normoglycaemic animal model Normal healthy animals can be used for testing potential oral hypoglycaemic agents. This is still a valid screening method which is often used in addition to diabetic animal models (Williamson et al., 1996). This method allows for the effect of the drug to be tested in the animal with an intact pancreatic activity. The comparison may give some information regarding mechanism of action. Hyperglycaemic agent may be detected at the same time. Oral glucose loading animal model This method is often referred to as physiological induction of diabetes mellitus because the blood glucose level of the animal is transiently increased with no damage to the pancreas. In the clinical setting, it is known as Glucose tolerance testing (GTT): a standard procedure often used for the diagnosis of border line diabetic patients. In this procedure, the animals are fasted overnight, then oral glucose load (1- 2.5 g/kg body weight) is given and blood glucose level is monitored over a period of time. Usually rabbits or male rats are used. Etuk and Mohammed (Unpublished) used this procedure to induce hyperglycaemia in wistar rats. The method was found to produce a widely fluctuating level of

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hyperglycaemia when compare to alloxan induction method. Chemical induction of diabetes mellitus: The majority of studies published in the field of ethnopharmacology between 1996 and 2006 employed this model. Streptozotocin (STZ, 69% and alloxan (31%) are by far the most frequently used drugs and this model has been useful for the study of multiple aspects of the disease. Both drugs exert their diabetogenic action when they are administered parenterally (intravenously, intraperitoneally or subcutaneously). The dose of these agents required for inducing diabetes depends on the animal species, route of administration and nutritional status (Federiuk et al., 2004). Streptozotocin model of diabetes mellitus: Streptozotocin or streptozocin or Izostazin or zanosar (STZ) is a synthetic nitrosoureido glucopyranose derivative isolated from fermentations of Streptomyces achromogenes that is classically an anti tumor antibiotic and chemically is related to other nitrosureas used in cancer chemotherapy. Streptozotocin sterile powders are provided and prepared as a chemotherapy agent. Each vial of sterilized Streptozotocin powder contains 1gram of Streptozotocin active ingredient with chemical name 2-Deoxy-2[(methylnitrosoamino)-carbonyl] amino]-Dglucopyranose and 200mg Streptozotocin is available for intravenous use as a dry frozen, pale yellow sterilized product. Streptozotocin prevents DNA synthesis in mammalian and bacterial cells. In the bacterial cells, it renders special reaction with cytocine groups, resulting in degeneration and destruction of DNA. The biochemical mechanism results in mammalian cell death. Streotozotocin prevents cellular reproduction with a much smaller dose than the dose needed for inhibiting the substrate connection to the DNA or inhibiting many of the enzymes involved in DNA synthesis (Holemans and van Assche, 2003). The induction of diabetes mellitus with STZ takes some time, and experiment carried out at suitable time intervals after administration of the agent will give additional information into mechanism of action of the tested compound. During the earlier part of this induction period, hypoglycaemic action of plant extract may be due to stimulation of residual activity of -cells; however after induction of the diabetic state, hypoglycaemic activity will be due to some other mechanism, since in this model of diabetes insulin activity is thought to be negligible. Single dose of STZ in sterile citrate buffer (e.g. pH 4.5 0.1M) may

be used: mice 150 mg/kg; rats 80 mg/kg, administered intraperitoneally. Diabetes develops gradually and may be assessed after a few days, usually four days for mice and seven days for rats. Usually, a serum glucose level of about 180 500 mg/dl indicates the induction of diabetes mellitus. Sometimes diabetic animals are maintained on insulin if the experiments are not to commence immediately to prevent the animals dead (Williamson et al., 1996). Although streptozotocin (STZ) is the most commonly used drug for induction of diabetes in rats (Balamurugan et al., 2003), there are some disadvantages to its use in chronic experiments, especially- spontaneous recovery from high blood glucose levels by the development of functioning insulinoma (Steiner et al., 1970; Yamagami et al., 1985; Iwase et al., 1991) and high incidence of kidney and liver tumours. These problems are due strongly to oncogenic action of STZ (Kazumi et al., 1978). Non insulin dependent diabetes mellitus (NIDDM) was induced by a single intraperitoneal injection of streptozotocin (60mg/kg) and nicotinamide (120mg/kg) to rats (Pellegrino et al., 1998). Paik et al. (2008) reported that, injection of a single high dose of streptozotocin (200 mg/kg body weight) induced rapid and permanent hyperglycaemia in mice. In contrast, the injection of the same total dose divided into multiple subdiabetogenic doses (40 mg/kg per day for five consecutive days) caused the development of delayed but progressive hyperglycemia only in the thymus competent mice. Female mice were less susceptible to streptozotocin at both doses. Alloxan model of diabetes mellitus: Alloxan is the next most commonly used chemical for induction of diabetes mellitus. It is a well- known diabetogenic agent widely used to induce Type 11 diabetes in animals (Viana et al., 2004). Alloxan is a urea derivative which causes selective necrosis of the pancreatic islet -cells. It is used to produce experimental diabetes in animals such as rabbits, rats, mice and dogs. With this agent, it is possible to produce different grades of severity of the disease by varying the dose of alloxan used: these may be classified by measuring fasting blood sugar (FES) levels: e. g. in rabbits moderate diabetes has been defined as an FBS level of 180 250 mg/dl, and severe diabetes as an FBS level of above 250mg/dl (Huralikuppi, 1991). The severe diabetes produced by alloxan results in blood sugar levels equivalent to a total pancreatectomy, hence sulphonylureas such

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as tolbutamide, which act mainly by stimulating insulin release from -cells, show only a small hypoglycaemic effect in this instance. Therefore a test plant extract producing a significant hypoglycaemia (in a severely alloxan- diabetic animal) must be operating through a different mechanism. Moderate diabetic animals are recommended for use in testing drugs for use in Non insulin dependent diabetes mellitus (Williamson et al., 1996). For all animals a single dose of alloxan, 140 180 mg/kg (usually 150 mg/kg) is administered as a 5% w/v in distilled water injected intravenously into the marginal ear vein of rabbit or intraperitoneally in case of mice and rats. A rest period of seven days for rabbits, 12 days for rats and mice is allowed during which the animals have free access to food and water. Alloxan and its reduction product dialuric acid establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. There after, highly reactive hydroxyl radicals are formed by fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of beta cells (Szkudelski, 2001). Thus alloxan induced diabetes mellitus served as a pathological biomodel for testing a substance with supposed antioxidant activities in vivo (Bartosikova et al., 2003). One of the targets of the reactive oxygen species is DNA of pancreatic islets. Its fragmentation takes place in beta cells exposed to alloxan (Takasu, et al., 1991,). The increase in oxygen free radicals in diabetic conditions is mainly because of the effect of the diabetogenic agent alloxan. With this method Macedo et al. (2005) induced diabetes mellitus in experimental rats. The animals were deprived of food for 48 hours, and then weighed and anaesthesized with chloroform in a glass dome. A solution of 2% alloxan (40mg/kg) diluted in 0.9% normal saline was administered to the animals through the iliac vein. The animals were allowed to resume feeding and drinking 30 minutes after the drug administration. In order to asses the effect of alloxan and to chemically establish the diabetic condition, an incision was done in any of the four veins in the tail of the rat with a 15 scapel blade 10 days after induction a blood glucose level determination using a portable glucose analyszer was determined, a serum glucose level of 200 mg/dl was considered hyperglycemic. The most frequently used intravenous dose of alloxan in rats is 65mg/kg, but when it is administered intraperitoneally or

subcutaneously its effective dose must be higher (Federiuk et al., 2004). Alloxan monohydrate 150mg/kg body weight was dissolved in normal saline and injected intraperitoneally after 18 hours fasting to induce hyperglycemia in experimental rats (Yanarday and Colak, 1998). In a separate study, the experimental animals were fasted for 18 hours before alloxan injection and the blood glucose level (BGL) was monitored after alloxanization in blood samples collected by tail tipping method using a Glucometer. Rats with blood glucose level of greater than 150mg/dl were considered diabetic and were selected for study (WHO, 1985). The simplistic argument often made against the use of alloxan to induce type II diabetes mellitus is that, alloxan administration produces beta cells damage and thus leading to type I rather than type II diabetes mellitus. Studies conducted by Etuk and Mohammed (Unpublished) in 2009 showed that, there was no differential response to hypoglycaemic agents by alloxan and glucose loading hyperglycaemic rats. Alloxan administration in experimental animals has been reported to produce pancreatic lesion which is proportional to the dose of the drug administered. And the size of the lesion also correlates with the pancreatic insulin content (McNeill, 1990). This perhaps explains why the drug at a low or medium dose does not produce absolute but insufficient insulin deficiency in experimental animals. Therefore the experimental dose of the drug must be carefully selected in order to avoid excessive pancreatic tissue damage. The most frequently used intravenous dose of alloxan in rats is 65mg/kg, but when it is administered intraperitoneally or subcutaneously its effective dose must be higher (Antia et al., 2005). Ferric nitrilotriacetate induction of diabetes mellitus This is a rarely used procedure. Rats and rabbits parenterally treated with a large daily dose of ferric nitrilotriacetate manifested diabetic symptoms such as hyperglycaemia, glycosuria, ketonemia and ketonuria after approximately 60 days of treatment. The blood insulin response to oral glucose loading was poor (Awai et al., 1979). Induction of diabetes mellitus with ditizona or anti insulin serum has never been reported. Surgical model of diabetes mellitus Another technique used to induce diabetes is complete removal of the pancreas (pancreatectomy). Few researchers have employed this model in the last years to explore effects of natural products with animal species such as rats, pigs, dogs and primates

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(Choi et al., 2004, Rees and Alcolado, 2005: Masiello, 2006). Limitation to this technique include high level of technical expertise and adequate surgical room environment, major surgery and high risk of animal infection, adequate post-operative analgesia and antibiotic administration, supplementation with pancreatic enzymes to prevent malabsorption and loss of pancreatic counter regulatory response to hypoglycemia. More recently, partial pancreatectomy has been employed, but large resection (more than 80% in rats) is required to obtain mild to moderate hyperglycemia. In this case, small additional resection can result in significant hypoinsulinemia (Masiello, 2006). Choi et al., (2004) investigated the action of relative glucose uptake in various tissues of 90% pancreatectomized rats by using either hyperglycemic or euglycemic hyperinsulinemic clamp methodologies. This experimental design permits to evaluate if the compound has some effect upon both resistance to and secretion of insulin. GENETIC MODELS OF DIABETES Spontaneously develop diabetic rats These models permit the evaluation of the effect of a natural product in an animal without the interference of the side effects induced by chemical drugs like alloxan and STZ reported above. Several recent publications summarized the major advances in this field (Masiello, 2006). Example is the spontaneously diabetic Goto-Kakizaki rat which is a genetic lean model of type 11 diabetes originating from selective breeding over many generations of glucose-intolerant nondiabetic wistar rats (Chen and Wang, 2005). Regarding type1 diabetes models, the mouse typically presents hyperglycemia between 12 and 30 weeks of age, whereas in BB rats it occurs around 12 weeks of age. One great advantage of these models is that they can be employed as model of atherosclerosis which represents the long term complication of diabetes mellitus and tested against several natural products (Wu and Huan, 2007). Mutant strains obese diabetic mice are available such as the C57BL/Ksj-db/db. With this model it is possible to test for effects of plant extracts on blood sugar, body weight, insulin production and insulin resistant. Genetically engineered diabetic mice In this case, rodents may be produced to over (transgenic) or under (knockout)-express proteins thought to play a key part in glucose metabolism (Masiello, 2006). Although significant advances in this field have arisen in recent years, especially with the advent of transgenic mice, there have been no studies carried out involving natural products on these models.

Certainly, the high cost restricts their study in sophisticated protocols which explore mechanism of potential therapeutic agents that either stimulates pancreatic -cell death (Meiton, 2006). Insulin dependent diabetes mellitus (IDDM) can be developed by inserting into mice unique viral protein which then express as a self-antigen in the pancreatic islets of langerhans. Autoimmune mediated destruction of beta cells of the islets of langerhans leads to insulin dependent diabetes mellitus (IDDM). Von Herrath and Oldstone (1997) infected mice with lymphocytic choriomeningitis virus (LCMV) to induce IDDM. Similarly, Oldstone et al. (1991) developed a transgenic mouse model that expressed IDDM by inserting a viral gene in the animal egg stage. This procedure is relatively new and rarely used because of the sophisticated techniques and equipment required. Chemical induction appears to be the most popularly used procedure in inducing diabetes mellitus in experimental animals. The best known drug induced diabetic model is the alloxan diabetes. It is capable of inducing both type I and type II diabetes mellitus with proper dosage selection. But the most commonly used drug is streptozotocin for reasons that are not well specified. Experimental animals must be put to use within seven days after induction of diabetes mellitus or maintain with appropriate doses of insulin to prevent animal death. The surgical and genetic methods require highly technical skills, may be associated with a high percentage of animal death and thus are rarely used. Alloxan induced diabetes model appears to be the most reliable and easily reproducible method of inducing diabetes mellitus in experimental animals.
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