WO2014028737A1

WO2014028737A1 - Diabetic animal model for diabetes research

Info

Publication number: WO2014028737A1
Application number: PCT/US2013/055143
Authority: WO
Inventors: James A. Eldridge; William C. CAMPAIGNE
Original assignee: Campaigne Enterprise Llc
Priority date: 2012-08-16
Filing date: 2013-08-15
Publication date: 2014-02-20

Abstract

A method for producing an animal model for insulin resistance, obesity and/or type 2 diabetes may include administering a dose of streptozotocin or functional equivalents or derivatives thereof to a pregnant animal and/or feeding a diet comprising a high fructose- and/or sucrose containing diet.

Description

DIABETIC ANIMAL MODEL FOR DIABETES RESEARCH

BACKGROUND Technical Field

[0001] Embodiments disclosed herein relate generally to diabetic animal models. In particular, embodiments disclosed herein relate to diabetic animal models for use in diabetes research.

Background Art

[0002] Type 2 diabetes is a complex syndrome affecting a growing number of people worldwide. This polygenic disease characterized by insulin resistance, results in many pathophysiologic conditions such as hyperglycemia, steatohepatitis, and nephropathy. The key driving forces for the increased prevalence of type 2 diabetes are modern Westernized diets, dietary habits, and sedentary lifestyles associated with the dramatic rises in obesity. The primary feature of type 2 diabetes is insulin resistance and defect in insulin secretion. Insulin is a key hormone synthesized and secreted by pancreatic beta-cells that stimulates glucose uptake in various organs (particularly muscle, liver, and adipose tissue). Insulin also regulates hepatic glucose production via controlling the expression of the gene encoding glucose-6-phosphatase and inhibits lipolysis in adipose tissue. Impaired insulin action (i.e. insulin resistance) occurs when target tissues are unable to respond to normal concentrations of insulin.

[0003] Once this dysregulation begins and in absence of treatment, beta-cells secrete increased amount of insulin (hyperinsulinemia) to maintain euglycemia (normal circulating glucose levels). However, in the absence of treatment, beta-cells fail to produce enough insulin, leading to increase in circulating glucose (hyperglycaemia). As long as enough beta-cells are viable and secreting the appropriate rate of insulin to maintain euglycemia, type 2 diabetes does not arise. It is widely accepted that the accumulation of free fatty acids in insulin-sensitive non adipose tissues (i.e., liver and muscles), can impair insulin-mediated-glucose uptake in these tissues. Moreover, increased lipid production by the liver enhances fatty acid oxidation, decreases insulin-dependent inhibition of hepatic glucose production and, therefore, increases gluconeogenesis, further worsening the hyperglycaemia. [0004] Generally, therapeutic strategies for type 2 diabetes involve insulin and antidiabetic agents falling within one of several classes to stimulate pancreatic insulin secretion, reduce hepatic glucose production, delay digestion and absorption of intestinal carbohydrate, or improve insulin action.

[0005] With a disease such as type 2 diabetes, the development of animal models is required to understand the basic processes and physiological consequences of the disease as well as potential therapies. A number of models have been developed to study the effects of type 2 diabetes including genetically derived, chemically induced, surgically induced, and transgenic or knockout models. These models are invaluable for studying type 2 diabetes at the cellular and molecular level. Many models require costly use of insulin and/or streptozotocin (or a similar drug). For a model to be particularly useful, it should develop the complications of diabetes with an etiology similar to that observed in humans.

SUMMARY

[0006] This summary is provided to introduce a selection of concepts that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0007] In one aspect, embodiments disclosed herein relate to a method for producing an animal model for insulin resistance, obesity and/or type 2 diabetes that include administering a dose of streptozotocin or functional equivalents or derivatives thereof to a pregnant animal.

[0008] In another aspect, embodiments disclosed herein relate to a method for producing an animal model for insulin resistance, obesity and/or type 2 diabetes that includes feeding a diet to a pig or rat comprising 40-80 percent by weight carbohydrates, 3-35 percent by weight fats, 10-25 percent by weight proteins, wherein at least 10% of the diet is fructose and/or sucrose and at least 5% of the diet is fiber.

[0009] In yet another aspect, embodiments disclosed herein relate to a method of screening for a therapeutic agent useful for treating or preventing a diabetic complication, comprising providing, by the methods disclosed herein, a test animal and a substantially identical control animal; administering a candidate agent to the test animal; maintaining the test animal and the control animal under conditions appropriate for development of at least one diabetic complication in the control animal; assessing said at least one diabetic complication in the test animal and the control animal; and comparing the severity and/or onset of the diabetic complication in the test animal with that of the control animal, wherein reduced severity and/or delay in the onset of the diabetic complication in the test animal indicates that the candidate agent is the therapeutic agent useful for treating or preventing the diabetic complication.

[0010] Other aspects and advantages of the claimed subject matter will be apparent from the following description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 shows hepatic fat deposition results for the examples disclosed herein.

[0012] FIG. 2 shows glomerular hypertrophy results for the examples disclosed herein.

[0013] FIG. 3 shows pancreatic tissues for the examples disclosed herein.

[0014] FIG. 4 shows results of streptozotocin administration for the examples disclosed herein.

DETAILED DESCRIPTION

[0015] In one aspect, embodiments disclosed herein relate to methods of producing a diabetic animal which may be used in a method of identifying compounds that can reverse diabetes and are suitable for interventive therapy in diabetes and its complications. In another aspect, the present disclosure provides a method for identifying and/or testing compounds for the treatment of type 2 diabetes and/or its complications.

[0016] Diabetic animal models may be produced by the administration of a high fructose corn syrup-based Western diet to the subject animals alone or in combination with prenatal introduction of the antibiotic streptozotocin or functional equivalents or derivatives thereof during gestation of the subject animals. The methods of the present disclosure also relate to the introduction of the antibiotic streptozotocin or functional equivalents or derivatives thereof to a pregnant animal for the production of hypoglycemic and hyperglycemic neonates.

[0017] While prior diet-induced diabetic animal models have been known, the diets do not accurately reflect a "Westernized" diet, in particular, a diet having a significant level of high fructose corn syrup. Thus, in accordance with embodiments of the present disclosure, a high fructose-containing diet may be administered to an animal to trigger onset of type 2 diabetes, as well as one or more type 2 diabetes complications.

[0018] In one or more embodiments, the animal to which the high fructose- or sucrose-based diet is administered is a pig or a rat. In one or more embodiments, the rat may be a Neotoma micropus. In one or more embodiments, the pig may be any species falling with the Sus genus, including, but not limited to, Sus scrofa domesticus.

[0019] In a particular embodiment, the high-fructose- or sucrose-based diet may be formed from at least 10 percent fructose and/or sucrose, or at least 12 percent, 15 percent, or 18 percent fructose in other embodiments. The total amount of fructose present in the diet may be based on the addition of fructose through high fructose corn syrup and/or through fructose added alone. As defined herein, high fructose corn syrup (HFCS) generally defined as having a combination of sugars (including fructose) and a balance of water. The most widely used varieties of high-fructose corn syrup are: HFCS 55 (mostly used in soft drinks), approximately 55% fructose and 42% glucose; and HFCS 42 (used in beverages, processed foods, cereals and baked goods), approximately 42% fructose and 53% glucose. Further, because high fructose corn syrup is provided in liquid form (due to the water), the addition of high fructose corn syrup may be limited (depending on the animal being fed the food) largely be based on the ability to keep the food in a substantially solid state, if desired. For example, the amount of high fructose corn syrup may be less than 15 weight percent. However, depending on the desired fructose content, additional fructose may be added to the diet formulation directly, not through high fructose corn syrup. Further, in some embodiments, both fructose and sucrose may be present, and together may constitute at least 10 weight percent of the diet or at least 12, 15, or 18 weight percent of the diet in other embodiments. Further, when both fructose and sucrose are both used in combination, the relative ratios between the two (fructose to sucrose) may broadly encompass the entire range from 0: 1 to 1 :0, but in particular embodiments, may range from 1 :2 to 2: 1 or 1.5:1 to 1 : 1.5.

[0020] The carbohydrates of the present disclosure may include fructose (or other sugars such as glucose and sucrose), as well as fiber and non-fiber carbohydrate components. Fiber may be added through inulin and/or powdered cellulose, for example, as well as any other dietary fiber such as non-starch polysaccharides, and many other plant components. In accordance with embodiments of the present disclosure, within the carbohydrate content, fiber may be incorporated in an amount that is in excess of 4 percent by weight, or at least 5, 6, 7, or percent by weight in various other embodiments. Non-fiber carbohydrates may be sourced, for example, starchy polysaccharides, such as from corn starch, dextrin, cereal grains, etc. Further, it may also be desirable to include vitamins and minerals in amounts representing average dietary levels.

[0021] The fats of the present disclosure may be derived a variety of sources, such as, for example, milk fat, lard, shortening, and/or plant-based oils (vegetable, safflower, peanut, palm, etc.). Proteins, for example, may be derived from casein and/or any balanced amino acid product. Further, one skilled in the art would appreciate that other food sources falling within these categories may also be used.

[0022] In formulating the diet, the different food sources may be combined to reflect an average Western diet. Further, one of ordinary skill in the art would appreciate that the ranges may vary depending on the food sources selected. For example, in one embodiment, fats may be present in an amount ranging from about 20 to 35 percent by weight, and proteins may be present in an amount ranging from 10 to 25 percent by weight, vitamins and other minerals in an amount up to 6 percent, where the balance is carbohydrates (for example, ranging from 40 to 65 percent by weight). In another embodiment, a lower-fat, higher-carbohydrate diet may be used. For example, in such an embodiment, fats may be present in an amount ranging from about 3 to 20 percent by weight, and proteins may be present in an amount ranging from 10 to 25 percent by weight, vitamins and other minerals in an amount up to 6 percent, where the balance is carbohydrates (for example, ranging from 50 to 80 percent by weight). In an even more particular embodiment, the high fructose based diet may include the nutritional components listed in Table 1 below.

Table 1

Selenium 0.17 ppm In an even more particular embodiment, the high fructose based diet may include the nutritional components listed in Table 2 below.

Table 2

Chromium 1.1 ppm

Molybdenum 0.17 ppm

Selenium 0.17 ppm In yet another particular embodiment, a high fructose/sucrose based diet may include the nutritional components listed in Table 3 below.

Table 3

Selenium 0.17 ppm In yet another particular embodiment, a high fructose/sucrose, low-fat based diet may include the nutritional components listed in Table 4 below.

Table 4

[0027] As mentioned above, some embodiments of the present disclosure may also involve the administration of streptozotocin or a functional equivalent or derivative thereof during to a pregnant animal to induce a toxic effect on the pancreatic beta cells of the gestating animals. In one embodiment, the streptozotocin may be administered in an amount ranging from 5 to 9 grams to the sow. However, this amount may vary depending on the species to which the drug is administered, depending, for example, on the size of the animal. For example, such dosage may range from 0.05 grams/kg of animal to 0.09 grams/kg animal.

[0028] The administration of streptozotocin or a functional equivalent or derivative thereof may occur during the last 20% of the gestation period. Additionally, it may also be desirable to administer the streptozotocin or a functional equivalent or derivative thereof with at least 10% or in more particular embodiments, at least 15% of the gestation cycle remaining. Thus, for example, a swine, having a gestation period of 112-114 days, may be administered streptozotocin at around day 95. Further, it is noted that for other species, the administration of the streptozotocin (or functional equivalent or derivative thereof) may occur during the development of the pancreas, and thus may be varied accordingly. It is also noted that additional administration of streptozotocin or a functional equivalent or derivative thereof may be given to the neonates, in a similar g/kg dosage as discussed above to decrease the number of beta cells within the pancreas.

[0029] A "functional equivalent or derivative" of streptozotocin is defined as a streptozotocin compound that has been altered such that the pancreatic beta-cells' destroying properties of the compound are essentially the same in kind, but not necessarily in amount. Streptozotocin is a substance that has little side effects. Another exemplary beta-cell-toxic substance is alloxan. Alloxan is a substance with well-known characteristics and is easy to obtain.

[0030] In one or more embodiments, the animals may be challenged by additional environmental factors that contribute to the induction of type II diabetes mellitus and/or Syndrome X. Examples of environmental factors that the animals of the present disclosure may be exposed to are a specific diet and/or low physical activity. Exposure to low physical activity is, for example, established by restraining pigs in their motion, for instance, by accommodating them in small cages. Thus, in one or more embodiments, the animal may be also challenged by low physical activity. Further, even if the animal is not subjected to the high fructose diet of the present disclosure, the animal may be subjected to a high fat, low protein diet.

[0031] As mentioned above, in addition to producing a diabetic animal model, the present disclosure also involves methods of identifying and/or testing candidate compounds for the treatment of type 2 diabetes and/or its complications. Said method comprises the steps of administering a compound of interest to an animal produced by a method of the present disclosure and determining whether diabetes and/or at least one type 2 diabetes complication are reversed by said compound. In an exemplary embodiment of the method for identifying and/or testing compounds for the treatment of type 2 diabetes, a candidate compound is administered to an animal which has been produced according to any of the embodiments disclosed herein, and an indicator value (e.g. a blood glucose level, or an insulin level in blood or in other tissues) having a correlation with insulin resistance, obesity and/or type 2 diabetes is then measured in the animal. Thereafter, the obtained indicator value is compared with that of a control animal. Based on the comparative results, it is confirmed whether or not the candidate compound is able to alleviate or eliminate the symptoms of type 2 diabetes. Specifically, the blood glucose level of an animal of the present disclosure, to which a candidate compound has been administered, is measured. When the measured blood glucose level is lower than that of a control animal, which has not been in contact with the candidate compound, the candidate compound may be selected as a therapeutic agent for the treatment of type 2 diabetes.

[0032] Moreover, after an animal of the present disclosure has been administered a candidate compound, the insulin level of an animal of the present disclosure may be measured as an indicator value having correlation with insulin resistance, obesity and/or type 2 diabetes. When the measured insulin level is lower than that of a control animal, which has not been in contact with the candidate compound, the candidate compound may be selected as a therapeutic agent for the treatment of type 2 diabetes. [0033] Further, a candidate compound may be tested for its ability to treat type 2 diabetes as described above and for its ability to treat complications associated with type 2 diabetes by determining whether the compound can revert and/or alleviate type 2 diabetes complications developed by the animals of the present disclosure such as, for example, cardiovascular disease, retinopathy, neuropathy, nephropathy and nonalcoholic fatty liver disease.

[0034] Examples of candidate compounds may include a peptide, a protein, a non- peptide compound, a synthetic compound, a fermented product, a cell extract, a cell culture supernatant, a plant extract, a tissue extract and blood plasma of mammal (e.g. a mouse, a rat, a swine, a bovine, a sheep, a monkey, a human, etc.). Such compounds may be either novel compounds or known compounds. These candidate compounds may form salts. Examples of such salts of candidate compounds include salts with physiologically acceptable acids (e.g. organic acids and inorganic acids, etc.) or with bases (e.g. metal salts, etc.). Examples of such salts include salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or salts with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

[0035] In a further embodiment, embodiments of the present disclosure also include evaluating effects of a treatment on the manifestation of type II diabetes mellitus or Syndrome X, wherein treatment comprises pancreatic beta-cell transplant. Symptoms of diabetes mellitus type II or Syndrome X in the aforementioned animal model can be evaluated before and after transplant of pancreatic beta-cells. Thus, in one or more embodiments, the present disclosure, therefore, provides the use of an animal for evaluating effects of a pancreatic beta-cell transplant.

[0036] EXAMPLES

[0037] Example 1

[0038] Ten adult male Neotoma micropus were live-trapped between from two sites.

Both sites are located in Ector County, Texas. One site located on the University of Texas Permian Basin (UTPB) campus (31 °53'6.94"N, 102°19'3.93"W). The other located on the Hurt Ranch (31°56'30.66"N, 102°20'13.63"W) approximately 3km northeast of the UTPB campus. Only males were tested in this initial phase of model development to eliminate the potentially confounding effects of pregnancy and lactation on the factors of interest. After capture, animals were taken to the UTPB campus and the initial mass of each animal was determined. Animals were housed in wired bottom cages (67.056cm X 16.74cm X 26.67cm) in the UTPB animal facility in a temperature control environment of 27°C ± 5°C with a 12:12 light/dark cycle. On 31 August, 2007, 9 wild caught N. micropus (6 males, 3 females), were taken from the Hurt Ranch to serve as field controls. Females used as field controls were neither lactating nor pregnant. The experiment was concluded at the end of 12 weeks by euthanizing all animals in the experimental group as well as the field control animals that had been captured within 24 hours of the conclusion of the experiment.

[0039] At the end of a 24 hour acclimation period, an initial blood glucose reading

(mg/dL) was obtained from all animals. Glucose was measured with a glucometer (Home Diagnostics, Ft. Lauderdale, FL). A final blood glucose measurement was obtained for experimental animals at the end of the 12 week experimental period following euthanasia. Blood glucose was measured using blood withdrawn from the left ventricle.

[0040] Experimental animals were fed a high fat and high carbohydrate experimental diet similar to that shown in Table 2 above, containing 19.75% casein, 15% HFCS-55, 12% fructose, 12% sucrose, 10% dextrin, 6% Crisco, 6% lard, 6% milk fat, 5%AIN Mineral mix/Fiber, 2.5% Inulin, 2.5% powdered cellulose, 1.27% soybean oil, 1.15% AIN95 Vitamin mix/fiber, 0.54% corn oil, 0.3% L-cystine, 0.2% chloine bitartrate, 0.15%) chlolesterol, and minor amounts of red dye and t-butylhydroquinone. All animals were provided with 20g/day of their assigned diet which represents ad lib and allowed us to measure the amount diet (g/day) consumed by all animals. Animals had free access to water.

[0041] Immediately after euthanasia, Full body scans were made of each animal to measure fat deposition. Scans were obtained using a Hologic QDR series Discovery- A model dual energy x-ray analysis (DEXA) (Hologic Incorporated, Bedford, MA.).

[0042] After euthanasia, organs from all animals were harvested for histological analysis. Organs harvested included a transverse section from the left lobe of the liver, and a cross section of the left kidney. Additionally, the entire pancreas was harvested from 3 experimental and 3 field control animals. Tissue samples were preserved in 10% neutral buffered formalin and paraffin embedded for light microscopic analyses. Tissue samples were sectioned at ΙΟμιη and stained using standard Mayer's hematoxylin and eosin techniques (Spector and Goldman, 2006) and analyzed by two physicians trained in pathology at Texas Tech University Health Science Center (CWS) and Medical Center Hospital (ME), Odessa, Texas.

[0043] Hepatic lipid droplets from five random fields of view from each liver section were quantified by hand and a mean was calculated for each subject. Droplets were quantified on a droplet/mm basis. Glomerular hypertrophy was assessed by measuring the relative Bowman's space using the Micron 2.0 software. To assess the space, 5 glomeruli from each kidney were chosen and 5 randomly selected measurement angles on each glomeruli were chosen. The space was measured at each angle and a mean was calculated for each of the subjects.

[0044] The results of show that when N. micropus is brought into the laboratory and placed on a diet high in fat and carbohydrates, they show a significant increase in body fat. Specifically, the results indicate that percent body fat of field control and experimental animals is significantly different (F— 9.85, P < 0.05; Table 3 below). Percent body fat of the 9 field control animals was 1.67% ± 0.24 (X± SE), whereas percent of body fat at the conclusion of the study for the 10 experimental animals was 5.65 ± 1.18 ( ± SE).

[0045] Excess dietary fat is stored as adipose tissue. Likewise, excess dietary carbohydrates, those not converted to glycogen or used up for immediate energy demands, are also stored as hepatic fat. N. micropus placed on a westernized diet showed a decrease in mass (shown in Table 3); however, as is evident from the increase in percent body fat, the lean mass was replaced with fat mass.

[0046] Pre-diet and post-diet blood glucose levels of the experimental group was significantly different. The pre-diet glucose level was 89 ± 18.82 mg/dL (x ± SE), while the post-diet glucose level was 132.42 ± 6.73 mg/dL (X ± SE). The difference between the pre diet and post diet glucose levels is significant (t = 8.15, P < 0.05).

[0047] Hepatic fat deposition was measured in the field control and experimental groups, as shown in Figure 1. The average number of hepatic lipid droplets found in the field control group was 13.11 ± 4.16 droplets/mm² (X ± SE), and average number of lipid droplets found in the experimental population was 717.63 ± 287.99 droplets/mm (X ± SE). The difference in the number of lipid droplets/mm is significant (F = 5.01, P < 0.05).

[0048] The results also show that glomerular hypertrophy developed in the experimental group (Figure 2). Bowman's space for the field control group was 7.39 ± 1.61 μ ( X ± SE), while mean Bowman's space for the experimental group was 3.69 ± 1.83μ (X ± SE). The difference in Bowman's space between the two groups is significant F = 21.71, P < .05).

[0049] Finally, a qualitative histological analysis of the pancreata show a marked decline in the presence of pancreatic islets in the experimental group that consumed the Western diet compared to the field control group. Intact islets were observed in H&E stained pancreatic tissue of field control subjects of the field control group (Figure 3a). Only one visible islet (indicated by the arrow) could be located in the pancreata of the 3 animals surveyed in the experimental group (Figure 3b). H&E stained pancreas (Figure 3 c) exhibits transition of normal (bottom left) to diabetic (upper right) pancreatic tissue (400x) in the experimental group.

[0050] Example 2

[0051] Three separate studies were run to determine the dosage response of streptozotocin (STZ) in the pregnant swine model. Study one determined the effects of 3 grams of STZ given in the 90^th day of gestation on blood glucose levels of new born piglets. Study two determined the effects of 3 grams of STZ given in the 95^th day of gestation on blood glucose levels of new born piglets. Finally, study three determined the effects of 5 grams of STZ given in the 95^th day of gestation on blood glucose levels of new born piglets. Blood glucose levels were examined for piglets in the 1^st, 2^nd, 5^th, 7^th, and 14^th day after birth. The data (shown in FIG. 4) shows that a 5 gram bolus of STZ given at the 95^th day of gestation elicits a positive response to blood glucose levels similar to those seen in type 2 diabetics. Further, hyperglycemic piglets born from sows given the 5 gram dose of STZ and fed a 25% high fructose corn syrup diet exhibited hyperglycemia through 24 weeks of growth.

[0052] One or more embodiments may provide at least one of the following advantages. Administration of streptozotocin (or functional equivalents or derivatives thereof) to a pregnant animal and/or a high fructose-based diet represents a means for developing a larger animal model for the study of the onset and progression of obesity, hyperglycemia, fat proliferation of the liver (fatty liver disease), glomerular swelling, fatty infiltration and obliteration of the pancreatic beta cells, and diabetic retinopathy. All of these symptoms are associated with the occurrence of diabetes in humans. These models would be a means to generate a more cost effective large animal model to create diabetic conditions and could replace the currently available model that requires dosages of 15g to 20g of streptozotocin to generate diabetes in full grown animals. Furthermore it will allow researchers to study the onset and progression of diabetes as well as allowing for the study of the regeneration of beta cells so as to develop new treatments for diabetes. Specifically, the neonatal diabetic swine model may be used for diabetic studies, drug discovery studies, metabolic studies associated with high fat and high sugar diets, liver disease studies, kidney disease studies, and retinopathy studies.

[0053] Genetically altered strains of rats currently used in such studies (such as THE

POUND MOUSE, OP-CD, OR-CD, Dahl/SS, SS-13BN, SHR, Stroke Prone, SHROB, SHROB Lean, GK, ZDF, ZSF1, Zucker) all result from the genetic manipulation through inbreeding, outbreeding, or hybridization to create the potential for a diabetic model. Each of these are good scientific models studying specific genetic weaknesses or abnormalities related to a specific diabetic condition; however they lack the genetic diversity to be generalized to the entirety of the disease. Prior to the discovery of the proposed models, only mature and miniature bred swine have been used as a possible large animal non-genetically manipulated candidate for the study of diabetes. The uniqueness of neonatal diabetic model is that this animal model allows for the study of diabetic complications from birth to death with a much lower dosage of streptozotocin than previously used in the adult counterparts. The representative characteristics of diabetes shown in neonatal diabetic swine model include obesity, hyperglycemia, nephropathy, retinopathy, fatty infiltration of the liver, and fatty infiltration and obliteration of the pancreatic beta cells. Furthermore, like humans, neonatal diabetic swine model's presentation of increased fat mass occurs when caloric intake exceeds caloric output.

[0054] Furthermore, the use of the Westernized, high-fructose-based diet may allow for the spontaneous development of obesity, hyperflycemia, fat proliferation of the liver (fatty liver disease), glomerular swelling, fatty infiltration and obliteration of the pancreatic beta cells, and diabetic retinopathy, all of which are associated with the occurrence of type 2 diabetes in humans. Thus, the model would be one of the few animal models that exhibit spontaneous representation of obesity and type 2 diabetes without genetic manipulation or hydrbidization. Further, because the diet is similar to that consumed by North American humans, it may be more representative of current human behaviors and disease progression than genetically manipulated models.

[0055] Although only a few example embodiments have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the example embodiments without materially departing from this invention. Accordingly, all such modifications are intended to be included within the scope of this disclosure as defined in the following claims. In the claims, means-plus- function clauses are intended to cover the structures described herein as performing the recited function and not only structural equivalents, but also equivalent structures. Thus, although a nail and a screw may not be structural equivalents in that a nail employs a cylindrical surface to secure wooden parts together, whereas a screw employs a helical surface, in the environment of fastening wooden parts, a nail and a screw may be equivalent structures. It is the express intention of the applicant not to invoke 35 U.S.C. § 112, paragraph 6 for any limitations of any of the claims herein, except for those in which the claim expressly uses the words 'means for' together with an associated function.

Claims

CLAIMS What is claimed:

1. A method for producing an animal model for insulin resistance, obesity and/or type 2 diabetes:

administering a dose of streptozotocin or functional equivalents or derivatives thereof to a pregnant animal.

2. The method of claim 1, wherein the animal is a swine.

3. The method of claim 1, wherein the dose ranges from 5 to 9 grams.

4. The method of claim 1, wherein the administration occurs at a time ranging from 80 to 85% of the gestation period for the pregnant animal.

5. The method of claim 1, wherein upon delivery of neonates from the pregnant animal, the neonates are administered an addition dose of streptozotocin or functional equivalents or derivatives thereof.

6. The method of claim 1, wherein upon delivery of neonates from the pregnant animal, the neonates are fed a diet containing at least 10% fructose.

7. The method of claim 6, further comprising: administering a candidate compounds for the treatment of type 2 diabetes and/or its complications to the animal.

8. The method of claim 7, further comprising: measuring an indicator value in the animal and comparing the indicator value to a control animal.

9. A method for producing an animal model for insulin resistance, obesity and/or type 2 diabetes:

feeding a diet to a pig or rat comprising 40-80 percent by weight carbohydrates, 3-35 percent by weight fats, 10-25 percent by weight proteins, wherein at least 10% of the diet is fructose and/or sucrose and at least 5% of the diet is fiber.

10. The method of claim 9, further comprising: restraining the motion of the pig or the rat.

1 1. The method of claim 9, wherein the rat is a Neotoma micropus.

12. The method of claim 9, wherein the animal is a neonate.

13. The method of claim 9, wherein the fructose is sourced in the diet through high fructose corn syrup.

14. The method of claim 13, wherein fructose is further sourced in the diet through an additional quantity of fructose.

15. The method of claim 6, further comprising: administering a candidate compounds for the treatment of type 2 diabetes and/or its complications to the animal.

16. The method of claim 7, further comprising: measuring an indicator value in the animal and comparing the indicator value to a control animal.

17. The method of claim 9, wherein the carbohydrates are less than 65 percent by weight of the diet.

18. The method of claim 9, wherein the fats are at least 20 percent by weight of the diet.

19. A method of screening for a therapeutic agent useful for treating or preventing a diabetic complication, comprising:

providing, by the method of claim 1 or claim 9, a test animal and a substantially identical control animal;

administering a candidate agent to the test animal;

maintaining the test animal and the control animal under conditions appropriate for development of at least one diabetic complication in the control animal;

assessing said at least one diabetic complication in the test animal and the control animal; and,

comparing the severity and/or onset of the diabetic complication in the test animal with that of the control animal,

wherein reduced severity and/or delay in the onset of the diabetic complication in the test animal indicates that the candidate agent is the therapeutic agent useful for treating or preventing the diabetic complication.