**2. Obese animal model**

#### **2.1. Genetic mouse model**

#### *2.1.1. ob/ob mouse*

*Lepob* mutation on chromosome 6 was discovered at the Jackson laboratory in a multiple recessive stock in 1949 [20], and the *Lepob* mutation was subsequently transferred to B6 inbred strain background. *Lepob* mutation on the B6 background (ob/ob) mice shows obesity, hyperinsulinemia, and relatively mild hyperglycemia.

Body weights in ob/ob mice significantly increased as compared with those in lean mice at 7 weeks of age (mean values; ob/ob mice, 44.0 g vs. lean mice, 23.1 g). The body weights periodically increased, reaching a maximum level of approximately 55 g at 11 weeks of age. With overt obesity, blood insulin levels in ob/ob mice also significantly increased as compared with those in lean mice. The blood insulin levels in ob/ob mice showed a remarkable increase at 7 weeks of age (mean values; ob/ob mice, 23.7 ng/ml vs. lean mice, 4.2 ng/ml). Blood glucose levels in ob/ob mice increased as compared with those in lean mice from 7 to 11 weeks of age, but the levels decreased with aging and normalized after 12 weeks of age. Since the pancreatic islets in ob/ob mice have a proliferative activity with an increase of blood insulin levels, the hyperglycemia is improved with aging. Moreover, ob/ob mice show the overt fat accumulation with hyperphagia. In ob/ob mice, the *de novo* lipogenesis and the hepatic fatty acid synthesis are significantly elevated [21].

The ob/ob mice fed a standard diet show fatty liver, but do not represent NASH-like lesion. NASH-like lesion is induced in ob/ob mice by methionine-choline deficient (MCD) and highfat (HF) diets [22, 23].

#### *2.1.2. db/db mouse*

obesity [6]. The basic therapies for obesity are appropriate dietary restriction for the purpose of decreasing energy intake and effective exercise for the purpose of promoting energy expenditure. The life style modifications, such as diet therapy and exercise, mainly occupy the treatments for obesity; however, medical therapy is performed on patients who do not show

Medical therapy is a fundamental step in reducing the accumulation of excess fat. To reduce excess fat accumulation and excess body weight, antiobesity drugs that reduce lipid absorption in the intestine or appetite have been developed. In past years, centrally acting drugs, such as phentermine, mazindol, and fenfluramine, had been approved as antiobesity drugs, but the drugs have since been withdrawn in the USA and Europe [7, 8]. Mazindol is now available only in Japan [9]. In the 1990s, another type of antiobesity drug, orlistat, which inhibits lipid absorption in the intestine, was approved in the USA and Europe and is now also available [10]. Thereafter, sibutramine and rimonabant were developed; however, both drugs were withdrawn because of adverse effects [11]. Development of drug combinations, such as qsymia and contrave, has been recently promoted [12], and serotonin (5HT2c)-R agonist lorcaserin was accepted by the FDA in 2012 [13]. In addition, a variety of drugs with various mechanisms, such as protein tyrosine phosphatase (PTP) 1B inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, diacylglycerol acyltransferase (DGAT) 1 inhibitors, and monoacylglycerol acyltransferase (MGAT) inhibitors, have been investigated in clinical and basic stages

Animal models have played important roles in the development of these antiobesity drugs. Obese animal models are essential to elucidate the etiology for the drug development. In the first half of this chapter, we introduce the characteristics of obese animal models. Obese animal models are divided into two types: genetic and nongenetic models. An overview of the pathophysiological features, such as body weight, blood chemical parameters, and histopathology of microangiopathy, is presented for both types. Moreover, an obese model is expected to be used as a NASH model. An overview of the development of NASH-like hepatic lesions in each model is also presented. In the second half of this chapter, results of pharmacological studies using the obese animal models for new antiobesity drugs are shown. The pharmaco-

logical effects were investigated using both genetic and nongenetic animal models.

*Lepob* mutation on chromosome 6 was discovered at the Jackson laboratory in a multiple recessive stock in 1949 [20], and the *Lepob* mutation was subsequently transferred to B6 inbred strain background. *Lepob* mutation on the B6 background (ob/ob) mice shows obesity, hyper-

weight loss effect by the life style modifications.

50 Adiposity - Omics and Molecular Understanding

[14–19].

**2. Obese animal model**

**2.1. Genetic mouse model**

insulinemia, and relatively mild hyperglycemia.

*2.1.1. ob/ob mouse*

In 1966, a recessive *Leprdb* mutation (db/db) was found on chromosome 4 in C57BL KS/J inbred strain [24]. The db/db mouse was produced by backcrossing among the C57BL KS/J inbred strains.

db/db mice show a development of obesity after weaning, but the metabolic abnormalities, including hypeglycemia, are more severe as compared with those in ob/ob mice. Body weights in db/db mice significantly increased as compared with those in lean mice at 7 weeks of age (mean values; db/db mice, 42.4 g vs. lean mice, 28.4 g). The body weights periodically increased, reaching a maximum level of approximately 50 g at 11 weeks of age. The degree of weight gain in db/db mice was mild as compared with that in ob/ob mice. With obesity, the blood insulin levels in db/db mice increased as compared with those in lean mice at 7 weeks of age (mean values; db/db mice, 10.8 ng/ml vs. lean mice, 3.2 ng/ml). However, the insulin levels decreased gradually with aging, and the level in db/db mice at 11 weeks of age was comparable with that in lean mice. Blood glucose level at 7 weeks of age in db/db mice was about 700 mg/dl, and the hyperglycemia is sustained over the life span. The fluctuation in blood insulin and glucose levels is associated with the pancreatic β cell mass in db/db mice.

In examination of renal lesions in db/db mice, the creatinine clearance decreases after 20 weeks of age, and the substantial glomerular changes, such as albuminuria, mesangial area enlargement, and basement membrane thickening, are observed [25]. There are some reports of neuropathy and retinopathy in db/db mice [26, 27]. Impaired motor nerve conduction velocities (MNCV) are observed during the early phase of the diabetic syndrome. In morphological studies, db/db mice show loss or shrinkage of myelinated fibers in sural nerve and ventral root, and axonal atrophy after 25 weeks of age [28]. In the retina, pathological changes, such as loss of pericytes, acellular capillaries, and blood-retinal barrier breakdown, are observed.

The db/db mice fed a standard diet show fatty liver, but do not represent NASH-like lesion. Like ob/ob mice, NASH-like lesion is induced in ob/ob mice by methionine-choline deficient (MCD) and high-fat (HF) diets [29, 30].

#### *2.1.3. KKAy mouse*

KK mouse, which is a spontaneously diabetic model, was established by Kondo et al. [31]. Furthermore, Nakamura et al. established KKAy mouse, which is an obese diabetic model, by introducing the yellow obese gene (Ay) into the KK mice [32, 33].

In KKAy mice, metabolic abnormalities, such as obesity, hyperinsulinemia, and hyperglycemia, are observed from 6 weeks of age, but the abnormalities are improved with aging [34].

Glomerular lesions, such as glomerulosclerosis, glomerular basement membrane (GBM) thickening, and nodular-like changes, are observed after 16 weeks of age [35]. Moreover, in retina of KKAy mice, the apoptosis cell number for retinal neural cells in the ganglion cell layer increased with aging [36].

It is reported that NASH-like lesions are observed in KKAy mice fed a MCD diet [37].

#### *2.1.4. Tsumura Suzuki obese diabetics (TSOD) mouse*

In 1992, two inbred strains: Tsumura Suzuki obese diabetics and Tsumura Suzuki nonobese (TSNO) mice were established by selective breeding of obese mice in ddy strain [38, 39].

In the male TSOD mice, metabolic abnormalities, such as hyperinsulinemia, hyperglycemia, and dyslipidemia, are developed with the increase of body weight. In the examination of pancreatic islets, the hypertrophy is observed with the increase in number of β cells and the degranulation of β cells [38].

In histopathological analyses in kidney, glomerular lesions, such as GBM thickening and mesangial area enlargement, are observed after 18 weeks of age [40]. The sensory neuropathy is observed after 12 months of age, and the motor neuropathy is also shown after 14 months of age. In histological analyses in sciatic nerves, a decrease in the density of nerve fibers is observed after 18 months of age. Moreover, the degenerative changes of myelinated fibers and the separation of myelin sheaths are observed with intralamellar edema and remyelination. Retinal lesions in TSOD mice are not reported.

#### **2.2. Genetic rat model**
