**2. Zucker diabetic fatty rats**

An animal model for biomedical investigation is one in which normative biology, behavior, and pathological process can be studied and in which the phenomenon in one or more respects resembles the same phenomenon in humans [35]. Research in diabetes on humans is not possible or only partially possible. Hence, animal model of DM is very useful and advantageous [36]. Animal disease models are essential tools for studying the pathophysiology of DM enabling therapeutic interventions to be developed [37]. It is true that the present therapeutic approaches to treat DM and obesity, which are saving many lives every day, were invented, validated, and optimized on animal models [38]. When studying T2DM the use of an animal model with a homogenous genetic background is advised [39]. Most of the available models are based on rodents [36]. Rodents are most commonly utilized due to their small size, short generation interval, and easy availability [39] and because of economic consideration [36]. Being mammals, the physiology of rats is similar to humans than nonmammalian species [40]. Nevertheless, nonrodent models of diabetes are needed as a valuable supplement to rodents for both practical and physiological reasons with respect to humans [36]. Many animal models for DM research are obese, reflecting the human condition where obesity is closely related to T2DM development [41]. Animals exhibiting a syndrome of insulin resistance

and T2DM reflecting the human disease involve many species with genetic, nutritional, or experimental causation [36]. There are a lot of rodent models available for the research in T2DM, but some of them may not always be satisfactory to mirror human T2DM due to the large heterogeneity in the latter. There are no fully unified classification criteria for this type of animal model. But, the spontaneous type 2 diabetic rodent models are considered the most outstanding and most useful [42].

According to Srinivasan and Ramarao [36], spontaneous diabetic animal models have special advantages and also disadvantages. The advantages are:


Among the disadvantages are mainly:


One of the rodent models that reflect human form of T2DM is Zucker diabetic fatty (ZDF) rats. ZDF rats as spontaneous diabetic animal model exhibit both the prediabetic and the end stage observed in human T2DM patients [43]. Spontaneously diabetic animals of T2DM may be acquired from the individuals with one or several genetic mutations transmitted from generation to generation or selected from nondiabetic outbred animals by repeated breeding through several generations. The result is that these animals inherited DM either as single or multigene defects. The metabolic particularities result from single gene defect (monogenic) which is due to dominant gene or recessive gene, or it can be of polygenic origin [36].

ZDF rats come from a colony of outbred Zucker rats in the laboratory of Dr. Walter Shaw at Eli Lilly Research Laboratories in Indianapolis (USA) during the years 1974–1975. In early 1981, some animals with diabetic lineage were designated and redefined. An inbred line of ZDF rats was established in 1985. Development to a genetic model was established in 1991 [44]. The Zucker fatty (ZF) rats carry a spontaneous mutation in the leptin receptor gene (fa) [45]. ZF rats resulted from the simple autosomal recessive (fa) gene on chromosome 5 [36]. This mutation causes hyperphagia, early onset of obesity, and insulin resistance [14] along with increased growth of subcutaneous fat depot [46]. At the age of 4 weeks, ZF rats gain weight more rapidly due to increased growth of subcutaneous fat depot, and

**79**

*Zucker Diabetic Fatty Rats for Research in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.88161*

linemia [42].

they have a noticeably higher body weight at about the age of 9 weeks [47]. The hyperphagia and obesity in ZF rats are attributed to hypothalamic defect in leptin receptor signaling that is related to mild hyperglycemia, mild glucose intolerance, insulin resistance, hyperlipidemia, and moderate hypertension [46]. ZF rats have

Thereafter, a mutation in ZF strain led to a substrain with an evident diabetic phenotype—the Zucker diabetic fatty (ZDF) rats [42]. ZDF rats are less obese than ZF rats having a decrease beta cell mass which resulted in inability to compensate for severe insulin resistance [48]. The ZDF rats were derived by selective inbreeding of hyperglycemic ZF rats [49] within the first months of life due to leptin receptor defect and a genetically reduced insulin promoter activity [17]. ZF rats maintain normoglycemia despite their obese phenotype, hyperlipidemia, and hyperinsu-

The ZDF male rats became an experimental model for type 2 diabetes mellitus (T2DM). They have a predictable progression from prediabetic to diabetic state [50]. The ZDF rats carry a genetic defect in β-cell transcription. It is inherited independently of the leptin receptor mutation and insulin resistance [17]. In prediabetic stage of ZDF rats, there is no change in insulin mRNA levels. But, significant reduction (30–70%) of other islet mRNA levels, such as glucokinase, mitochondrial

Ca2+-ATPase, and transcription factor islet-1 may be detected [51]. It is known that FFA-induced suppression of insulin output in prediabetic stage of ZDF rats is conveyed by nitric oxide (NO) [52]. ZDF rats start to develop T2DM as early as 10 weeks of age, reaching 100% incidence at around 20 weeks of age [53]. It is possible to shorten prediabetic state and reach the symptoms of T2DM after high-energy diet. But, the animals receiving this diet are in the risky group because the diabetic state with its complications arrives quickly and rats can perish. In our experiment with ZDF rats, high-energy diet caused ketoacidosis that meant two cases of animal death

Blood glucose concentrations in ZDF rats usually increase from 7 to 10 weeks of age and impaired glucose tolerance at 5–7 weeks of age. At the age of 12 weeks, glucose intolerance becomes more severe than at 5–7 weeks of age [55]. Chronic and increasing hyperglycemia in ZDF rats is related to the loss of insulin and pancreatic duodenal homeobox (PDX-1) mRNAs. The lack of glucose stimulated insulin secretion. The possible prevention of hyperglycemia could block the deficit in insulin

Male ZDF rats that are homozygous recessive have nonfunctional leptin receptors (fa/fa) and develop hyperlipidemia, obesity, and hyperglycemia. Rats that are homozygous dominant (+/+) or heterozygous (fa/+) are lean with normoglycemia. They are healthy, display no symptoms of diabetes, and are usually used as agematch control rats in the experiments. In young fa/fa rats, insulin resistance appears which extends to a deployed insulin secretory defect that initiates hyperglycemia and inadequate β-cell compensation [17, 49]. The insulin resistance is a result of a

In ZDF rats, there are sex differences for phenotypes of diet-induced insulin resistance and glucose intolerance. The most affected are male individuals [57]. On normal diet, male rats from ZDF strain develop severe hypoinsulinemia and hyperglycemia by 4 months of age. Female individuals maintain normal level of blood glucose and insulin despite advanced obesity [38]. Female ZDF rats with fa/fa genotype become also obese and insulin resistant, but do not progress to hyperglycemia, except when fed a high-fat diet [58, 59]. The female

channels,

glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+

in 7th week after initializing feeding with this caloric diet [54].

mutant leptin receptor that causes obesity [17].

amount and PDX-1 gene expression and improve insulin secretion [56].

impaired glucose tolerance rather than apparent diabetes [42].

*Animal Models in Medicine and Biology*

multifactorial disease easy.

Among the disadvantages are mainly:

d.Require sophisticated maintenance.

and T2DM reflecting the human disease involve many species with genetic, nutritional, or experimental causation [36]. There are a lot of rodent models available for the research in T2DM, but some of them may not always be satisfactory to mirror human T2DM due to the large heterogeneity in the latter. There are no fully unified classification criteria for this type of animal model. But, the spontaneous type 2 diabetic rodent models are considered the most outstanding and most useful [42]. According to Srinivasan and Ramarao [36], spontaneous diabetic animal models

a.The development of T2DM is of spontaneous origin involving genetic factors.

c.Most of inbred animal model in which the genetic background is homogeneous and environmental factors can be controlled allow genetic dissection of this

a.Highly inbred, homogenous, and mostly monogenic inheritance and development of diabetes are highly genetically determined unlike heterogeneity in humans.

c.Mortality due to ketosis problem is high in the case of animals with brittle pancreas

One of the rodent models that reflect human form of T2DM is Zucker diabetic fatty (ZDF) rats. ZDF rats as spontaneous diabetic animal model exhibit both the prediabetic and the end stage observed in human T2DM patients [43]. Spontaneously diabetic animals of T2DM may be acquired from the individuals with one or several genetic mutations transmitted from generation to generation or selected from nondiabetic outbred animals by repeated breeding through several generations. The result is that these animals inherited DM either as single or multigene defects. The metabolic particularities result from single gene defect (monogenic) which is due to dominant gene or recessive gene, or it can be of polygenic

ZDF rats come from a colony of outbred Zucker rats in the laboratory of Dr. Walter Shaw at Eli Lilly Research Laboratories in Indianapolis (USA) during the years 1974–1975. In early 1981, some animals with diabetic lineage were designated and redefined. An inbred line of ZDF rats was established in 1985. Development to a genetic model was established in 1991 [44]. The Zucker fatty (ZF) rats carry a spontaneous mutation in the leptin receptor gene (fa) [45]. ZF rats resulted from the simple autosomal recessive (fa) gene on chromosome 5 [36]. This mutation causes hyperphagia, early onset of obesity, and insulin resistance [14] along with increased growth of subcutaneous fat depot [46]. At the age of 4 weeks, ZF rats gain weight more rapidly due to increased growth of subcutaneous fat depot, and

have special advantages and also disadvantages. The advantages are:

b.Animals develop characteristic features resembling human T2DM.

d.Variability of results is minimal and it required smaller sample size.

b.Limited availability and expensive for the diabetes study.

and requires insulin treatment in later stage for survival.

**78**

origin [36].

they have a noticeably higher body weight at about the age of 9 weeks [47]. The hyperphagia and obesity in ZF rats are attributed to hypothalamic defect in leptin receptor signaling that is related to mild hyperglycemia, mild glucose intolerance, insulin resistance, hyperlipidemia, and moderate hypertension [46]. ZF rats have impaired glucose tolerance rather than apparent diabetes [42].

Thereafter, a mutation in ZF strain led to a substrain with an evident diabetic phenotype—the Zucker diabetic fatty (ZDF) rats [42]. ZDF rats are less obese than ZF rats having a decrease beta cell mass which resulted in inability to compensate for severe insulin resistance [48]. The ZDF rats were derived by selective inbreeding of hyperglycemic ZF rats [49] within the first months of life due to leptin receptor defect and a genetically reduced insulin promoter activity [17]. ZF rats maintain normoglycemia despite their obese phenotype, hyperlipidemia, and hyperinsulinemia [42].

The ZDF male rats became an experimental model for type 2 diabetes mellitus (T2DM). They have a predictable progression from prediabetic to diabetic state [50]. The ZDF rats carry a genetic defect in β-cell transcription. It is inherited independently of the leptin receptor mutation and insulin resistance [17]. In prediabetic stage of ZDF rats, there is no change in insulin mRNA levels. But, significant reduction (30–70%) of other islet mRNA levels, such as glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca2+-ATPase, and transcription factor islet-1 may be detected [51]. It is known that FFA-induced suppression of insulin output in prediabetic stage of ZDF rats is conveyed by nitric oxide (NO) [52]. ZDF rats start to develop T2DM as early as 10 weeks of age, reaching 100% incidence at around 20 weeks of age [53]. It is possible to shorten prediabetic state and reach the symptoms of T2DM after high-energy diet. But, the animals receiving this diet are in the risky group because the diabetic state with its complications arrives quickly and rats can perish. In our experiment with ZDF rats, high-energy diet caused ketoacidosis that meant two cases of animal death in 7th week after initializing feeding with this caloric diet [54].

Blood glucose concentrations in ZDF rats usually increase from 7 to 10 weeks of age and impaired glucose tolerance at 5–7 weeks of age. At the age of 12 weeks, glucose intolerance becomes more severe than at 5–7 weeks of age [55]. Chronic and increasing hyperglycemia in ZDF rats is related to the loss of insulin and pancreatic duodenal homeobox (PDX-1) mRNAs. The lack of glucose stimulated insulin secretion. The possible prevention of hyperglycemia could block the deficit in insulin amount and PDX-1 gene expression and improve insulin secretion [56].

Male ZDF rats that are homozygous recessive have nonfunctional leptin receptors (fa/fa) and develop hyperlipidemia, obesity, and hyperglycemia. Rats that are homozygous dominant (+/+) or heterozygous (fa/+) are lean with normoglycemia. They are healthy, display no symptoms of diabetes, and are usually used as agematch control rats in the experiments. In young fa/fa rats, insulin resistance appears which extends to a deployed insulin secretory defect that initiates hyperglycemia and inadequate β-cell compensation [17, 49]. The insulin resistance is a result of a mutant leptin receptor that causes obesity [17].

In ZDF rats, there are sex differences for phenotypes of diet-induced insulin resistance and glucose intolerance. The most affected are male individuals [57]. On normal diet, male rats from ZDF strain develop severe hypoinsulinemia and hyperglycemia by 4 months of age. Female individuals maintain normal level of blood glucose and insulin despite advanced obesity [38]. Female ZDF rats with fa/fa genotype become also obese and insulin resistant, but do not progress to hyperglycemia, except when fed a high-fat diet [58, 59]. The female

ZDF rats develop T2DM just on a diabetogenic diet [60]. Thus, male ZDF rats are widely used as animal models for human T2DM and diabetic nephropathy and neuropathy [58, 61]. Hyperglycemia in diabetic ZDF rats was recorded at 2.5 month of age, and then blood glucose increased and reached an average value of 29.5 ± 0.9 mM at the age of 5 months. At the age of 5 months, fatty ZDF rats developed significant symptoms of thermal hypoalgesia indicated by prolonged response latencies in a tail-flick test. With progressing diabetes, the markers of thermal hypoalgesia increased at the age of 7 months and persisted till the 10th month [10]. The ZDF rats undergo a rapid transition between 10 and 15 weeks of age. At 10 weeks of age, they are insulin resistant, hyperlipidemic, and hyperinsulinemic. But, the high plasma insulin levels are insufficient to control glucose level, and the animals are hyperglycemic. Between 10 and 15 weeks of age, a loss in insulin secretory function occurs which leads to a marked decline in plasma insulin levels along with hyperglycemia [43]. The ability to secrete insulin to compensate peripheral insulin resistance is limited. β cells of ZDF rats are brittle and easily succumb to over-secretion pressure. The primary defect lies not in the ability of β cells to proliferate but rather in an enhanced rate of apoptosis. It shows impaired insulin secretory β-cell response to glucose, while it remains untouched to non-glucose secretogogues like arginine, a phenomenon similar to human T2DM. Downregulation of β-cell GLUT 2 transporters together with impaired insulin synthesis is probably responsible for hyperglycemia in ZDF animals. Decreased glucose transport activity and lowered GLUT 4 levels are present in the skeletal muscle and adipose tissue [34, 36, 48, 62]. Generally, it was reported that in the progression of ZDF rats, the decline of β-cell glucose transporter 2 (GLUT 2) membrane receptors and the incidental loss of muscle glucose transporter 4 (GLUT 4) are responsible for the impaired insulin secretion and subsequent hyperglycemia. The activity of GLUT 4 receptors decreased in adipose tissue and skeletal muscle. This results in reduced β-cell transport ability together with the peripheral insulin resistance [50].

Siwy et al. [63] characterized the strain of ZDF rats as appropriate model for human disease based on urinary peptidomic profiles. In the study the diabetic rats were heavier than lean individuals. Consistent with a diabetic phenotype, ZDF rats were hyperglycemic and dyslipidemic already at early (2 months of age) and more severely at late (8 month of age). Renal function was markedly impaired at 8 months. Proteinuria was present at 2 months and progressively increased at 8 months. At 8 months, ZDF rats' renal histology showed pathological changes, including glomerular sclerosis with thickening of the Bowman capsule and retraction of the tuft, tubular atrophy and dilatation, and hyaline casts. Lean rats did not develop any histopathological changes. Chen and Wang [44] introduced the following pharmacologically related characteristics of ZDF rats: 25–55% reduction of GLUT 4 in the adipose tissue, heart, and skeletal muscle; loss of pancreatic duodenal homeobox gene expression; and free fatty acids and nitric oxide induced suppression of insulin output (**Table 1**).

Obesity of ZDF rats (fa/fa) is caused by hyperphagia [59], and food restriction can counteract or delay development of T2DM [55]. Hyperphagia leads to hyperinsulinemia, which upregulates transcription factors that stimulate lipogenesis. This results in ectopic deposition of triacylglycerol in non-adipocytes, thereby providing fatty acid substrate for pathological non-oxidative metabolism, such as ceramide synthesis [64].

The severity of DM in the adult hyperglycemic ZDF rats is reflected in body weight and food consumption [42]. High-energy diet in animal's models inclined to DM leads to obesity, hyperinsulinemia, and altered glucose metabolism

**81**

muscle wasting.

patients [66].

*Zucker Diabetic Fatty Rats for Research in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.88161*

T2DM

human type

• Hyperphagia • Polyuria • Polydipsia • Hyperglycemia • Hyperlipidemia • Hyperinsulinemia • Insulin resistance

tissue

Obesity

Genetic feature Leptin receptors

• Associated with obesity

Type of diabetes

Using in research

**Table 1.**

Progression of diabetes

**Main feature Characteristic Description**

Development spontaneously involving

Acceleration of symptoms by high-fat diet Mortality due to ketosis after high-fat diet

Homozygous recessive (fa/fa) rats diabetic homozygous dominant (+/+) and heterozygous (fa/+) remain lean and

Minimal variability of results, the possibility to use small size groups

Predictable progression from prediabetic

genetic factors

Brittle pancreas

normoglycemic

to diabetic state

• Characteristic features resembling

• Reduction of GLUT 4 in adipose

• Genetic defect in β cell transcription

• Defect in leptin signaling

• Insulin receptor deletion

Mechanism of T2DM

Insulin secretory defects Inadequate β cell compensation

*Major genetic, physiological, and pathophysiological characteristics of ZDF rats.*

because of insufficient compensation by the pancreatic islets [31]. In our experiment [54] high-energy diet immediately induced hyperglycemia in ZDF rats, animals developed obesity, and we observed disturbance in some hematology parameters as neutrophils, mean platelet volume (MPV), and platelet count (PLT), which is a possible marker of angiopathy. In the groups of diabetic rats, we observed the significant weight decrease when compared to the control animals. It is probably linked to accelerated switch from prediabetic to diabetic state. It suggests the inability to utilize the calories consumed [42] and the degradation of structural proteins and muscle wasting in diabetic individuals [65]. Hempe et al. [59] found that body weight of ZDF obese rats was higher than the lean rats at the beginning of their study. Later, at around 16 weeks of age, body weight of obese animals started to decline. Lean rats reached the same body weight as obese rats at 25 weeks of age. The decrease of weight in obese diabetic rats on high energy is linked to accelerated switch from prediabetic to diabetic accomplished with associated complications of T2DM. Oyedemi et al. [65] explained this reduction in body weight as degradation of structural proteins and

Hempe et al. [59] evaluated if nephropathy and neuropathy in ZDF rats are linked to the hyperglycemic state of the rats and are real diabetic late complications or are related to other characteristics of the fa/fa genotype. Good glycemic control may be effective in delaying the neuropathic symptoms in diabetic


*Zucker Diabetic Fatty Rats for Research in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.88161*

#### **Table 1.**

*Animal Models in Medicine and Biology*

with the peripheral insulin resistance [50].

suppression of insulin output (**Table 1**).

ZDF rats develop T2DM just on a diabetogenic diet [60]. Thus, male ZDF rats are widely used as animal models for human T2DM and diabetic nephropathy and neuropathy [58, 61]. Hyperglycemia in diabetic ZDF rats was recorded at 2.5 month of age, and then blood glucose increased and reached an average value of 29.5 ± 0.9 mM at the age of 5 months. At the age of 5 months, fatty ZDF rats developed significant symptoms of thermal hypoalgesia indicated by prolonged response latencies in a tail-flick test. With progressing diabetes, the markers of thermal hypoalgesia increased at the age of 7 months and persisted till the 10th month [10]. The ZDF rats undergo a rapid transition between 10 and 15 weeks of age. At 10 weeks of age, they are insulin resistant, hyperlipidemic, and hyperinsulinemic. But, the high plasma insulin levels are insufficient to control glucose level, and the animals are hyperglycemic. Between 10 and 15 weeks of age, a loss in insulin secretory function occurs which leads to a marked decline in plasma insulin levels along with hyperglycemia [43]. The ability to secrete insulin to compensate peripheral insulin resistance is limited. β cells of ZDF rats are brittle and easily succumb to over-secretion pressure. The primary defect lies not in the ability of β cells to proliferate but rather in an enhanced rate of apoptosis. It shows impaired insulin secretory β-cell response to glucose, while it remains untouched to non-glucose secretogogues like arginine, a phenomenon similar to human T2DM. Downregulation of β-cell GLUT 2 transporters together with impaired insulin synthesis is probably responsible for hyperglycemia in ZDF animals. Decreased glucose transport activity and lowered GLUT 4 levels are present in the skeletal muscle and adipose tissue [34, 36, 48, 62]. Generally, it was reported that in the progression of ZDF rats, the decline of β-cell glucose transporter 2 (GLUT 2) membrane receptors and the incidental loss of muscle glucose transporter 4 (GLUT 4) are responsible for the impaired insulin secretion and subsequent hyperglycemia. The activity of GLUT 4 receptors decreased in adipose tissue and skeletal muscle. This results in reduced β-cell transport ability together

Siwy et al. [63] characterized the strain of ZDF rats as appropriate model for human disease based on urinary peptidomic profiles. In the study the diabetic rats were heavier than lean individuals. Consistent with a diabetic phenotype, ZDF rats were hyperglycemic and dyslipidemic already at early (2 months of age) and more severely at late (8 month of age). Renal function was markedly impaired at 8 months. Proteinuria was present at 2 months and progressively increased at 8 months. At 8 months, ZDF rats' renal histology showed pathological changes, including glomerular sclerosis with thickening of the Bowman capsule and retraction of the tuft, tubular atrophy and dilatation, and hyaline casts. Lean rats did not develop any histopathological changes. Chen and Wang [44] introduced the following pharmacologically related characteristics of ZDF rats: 25–55% reduction of GLUT 4 in the adipose tissue, heart, and skeletal muscle; loss of pancreatic duodenal homeobox gene expression; and free fatty acids and nitric oxide induced

Obesity of ZDF rats (fa/fa) is caused by hyperphagia [59], and food restriction can counteract or delay development of T2DM [55]. Hyperphagia leads to hyperinsulinemia, which upregulates transcription factors that stimulate lipogenesis. This results in ectopic deposition of triacylglycerol in non-adipocytes, thereby providing fatty acid substrate for pathological non-oxidative metabolism, such as ceramide

The severity of DM in the adult hyperglycemic ZDF rats is reflected in body weight and food consumption [42]. High-energy diet in animal's models inclined to DM leads to obesity, hyperinsulinemia, and altered glucose metabolism

**80**

synthesis [64].

*Major genetic, physiological, and pathophysiological characteristics of ZDF rats.*

because of insufficient compensation by the pancreatic islets [31]. In our experiment [54] high-energy diet immediately induced hyperglycemia in ZDF rats, animals developed obesity, and we observed disturbance in some hematology parameters as neutrophils, mean platelet volume (MPV), and platelet count (PLT), which is a possible marker of angiopathy. In the groups of diabetic rats, we observed the significant weight decrease when compared to the control animals. It is probably linked to accelerated switch from prediabetic to diabetic state. It suggests the inability to utilize the calories consumed [42] and the degradation of structural proteins and muscle wasting in diabetic individuals [65]. Hempe et al. [59] found that body weight of ZDF obese rats was higher than the lean rats at the beginning of their study. Later, at around 16 weeks of age, body weight of obese animals started to decline. Lean rats reached the same body weight as obese rats at 25 weeks of age. The decrease of weight in obese diabetic rats on high energy is linked to accelerated switch from prediabetic to diabetic accomplished with associated complications of T2DM. Oyedemi et al. [65] explained this reduction in body weight as degradation of structural proteins and muscle wasting.

Hempe et al. [59] evaluated if nephropathy and neuropathy in ZDF rats are linked to the hyperglycemic state of the rats and are real diabetic late complications or are related to other characteristics of the fa/fa genotype. Good glycemic control may be effective in delaying the neuropathic symptoms in diabetic patients [66].
