Effects of antidiabetic drugs in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice

European Journal of Pharmacology 655 (2011) 108–116

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Endocrine Pharmacology

Effects of antidiabetic drugs in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice Atsuo Tahara ⁎, Akiko Matsuyama-Yokono, Masayuki Shibasaki Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Japan

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Article history: Received 9 June 2010 Received in revised form 27 December 2010 Accepted 7 January 2011 Available online 23 January 2011 Keywords: Type 2 diabetes Hyperglycemia Glucose intolerance Insulin resistance Obesity

a b s t r a c t Based on previously established methods, we developed an easily available type 2 diabetic mouse model that exhibits obesity and insulin resistance. We investigated the effects of several antidiabetic drugs on this new model, which was induced by a high-fat diet in combination with streptozotocin and nicotinamide injection. Male ICR mice were fed a high-fat diet (45% of calories as fat) for 3 weeks and then intraperitoneally administered with nicotinamide (1000 mg/kg) and streptozotocin (150 mg/kg). These diabetic mice exhibited hyperglycemia and glucose intolerance as a result of the loss of early-phase insulin secretion. The mice also developed significant insulin resistance, hyperlipidemia and obesity. A single dose of mitiglinide, glibenclamide, sitagliptin, insulin, metformin and voglibose significantly improved glucose tolerance during a liquid meal tolerance test. Repeated administration of sitagliptin and rosiglitazone also improved hyperglycemia and insulin resistance. These results demonstrate that a high-fat diet combined with nicotinamide and streptozotocin injection induces a diabetic mouse model that replicates the metabolic characteristics of human type 2 diabetes. This diabetic model, which exhibits impaired insulin secretion, glucose intolerance, insulin resistance, and obesity, may be suitable to evaluate antidiabetic agents for the treatment of type 2 diabetes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Type 2 diabetes mellitus is a heterogeneous disorder characterized by a progressive decline in insulin action (insulin resistance), followed by the inability of β-cells to compensate for insulin resistance (pancreatic β-cell dysfunction). Insulin resistance is a characteristic metabolic defect that precedes overt β-cell dysfunction and is associated with resistance to insulin-mediated glucose disposal in peripheral tissues, and compensatory hyperinsulinemia. The incidence of type 2 diabetes with insulin resistance is increasing worldwide, in parallel with the obesity epidemic. Therefore, better treatments and novel prevention strategies for type 2 diabetes are urgently needed. To accomplish this goal, appropriate experimental animal models are needed. To date, a number of spontaneous and experimental diabetic animal models have been established, some of which possess pathological features resembling those of type 2 diabetes (Srinivasan and Ramarao, 2007). Although these diabetic animal models do not develop or express the full spectrum of symptoms seen in humans, they are useful in elucidating the pathogenesis and progression of diabetes, as well as in the identification and characterization of antidiabetic drugs. ⁎ Corresponding author at: Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Tel.: +81 29 829 6292; fax: +81 29 852 5391. E-mail address: [email protected] (A. Tahara). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.01.015

We have already reported that streptozotocin-induced diabetic mice exhibit severe hyperglycemia associated with a remarkable depletion (approximately 10% of normal) of pancreatic insulin content (Tahara et al., 2008). Biguanide, insulin and α-glucosidase inhibitors improved glucose tolerance in these mice, whereas sulfonylureas and dipeptidyl peptidase (DPP)-IV inhibitors did not. This model represents type 1 (insulin-dependent) diabetes and appears to be unsuitable for evaluating insulin secretagogues. We used nicotinamide to prevent excessive pancreatic injury induced by streptozotocin and created a novel diabetic model with which it was possible to evaluate insulin secretagogues. The streptozotocin and nicotinamide-induced diabetic mice exhibited moderate hyperglycemia associated with the loss of early-phase insulin secretion, and an approximately 50% decrease in pancreatic insulin content. Antidiabetic drugs, including sulfonylureas and DPP-IV inhibitors, improved glucose tolerance in these mice. Thus, streptozotocin and nicotinamide-induced diabetic mice have many pathological features resembling type 2 diabetes, and can serve as models for the pharmacological evaluation of many diabetic drugs. However, body weight, non-fasting plasma insulin level, and insulin resistance index value (HOMA-R) in these diabetic mice were similar to those of normal mice. Furthermore, these mice did not develop marked obesity or insulin resistance associated with type 2 diabetes. Therefore, nicotinamide and streptozotocin-induced diabetic mice were unsuitable to evaluate insulin-sensitizing drugs, such as thiazolidinediones, or antiobesity drugs.

A. Tahara et al. / European Journal of Pharmacology 655 (2011) 108–116

Several studies have reported that rats fed a high-fat diet develop insulin resistance but not overt hyperglycemia or diabetes (Tanaka et al., 2007; Storlien et al., 1986). It was recently reported that rats fed a highfat diet and treated with low-dose streptozotocin can serve as an alternative animal model for type 2 diabetes because of their impaired insulin secretion, glucose intolerance, insulin resistance and obesity (Srinivasan et al., 2005; Zhang et al., 2008). However, few studies have evaluated the characteristics of glucose metabolism or the effects of the currently available antidiabetic drugs in terms of glucose tolerance and/or insulin resistance. Therefore, the present study aimed to develop a suitable type 2 diabetic mouse model that mimics the metabolic features of type 2 diabetes in humans, yet is cheap, easily available, and useful for experimental studies and preclinical testing of novel drugs for the treatment of type 2 diabetes, including insulinotropic and insulin-sensitizing drugs. Here, we evaluated the characteristics of this model and investigated the effects of several classes of antidiabetic drugs in terms of glucose tolerance and insulin resistance to validate this model for future pharmacological studies of type 2 diabetes. 2. Materials and methods 2.1. Materials Streptozotocin, metformin and glibenclamide were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and insulin (Novolin®R 100) was purchased from Novo Nordisk Pharma Ltd. (Tokyo, Japan). Mitiglinide (Glufast®), voglibose (BASEN®), sitagliptin (Januvia®) and rosiglitazone (Avandia®) were purchased from Kissei Pharmaceutical Co., Ltd. (Nagano, Japan), Takeda Pharmaceutical Company, Ltd. (Osaka, Japan), Merck & Co., Inc. (Whitehouse Station, NJ, USA) and GlaxoSmithKline (Philadelphia, PA, USA), respectively, and purified at Astellas Pharma Inc. (Ibaraki, Japan). Insulin was diluted in physiological saline and injected intraperitoneally. All of the other compounds were dissolved or suspended in 0.5% methylcellulose solution and administered orally. The vehicle-treated group received 0.5% methylcellulose solution. 2.2. Animal models Male 5-week-old ICR mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) and used in this study at 6 weeks of age. Mice were fed either a normal chow diet consisting (as a percentage of total calories [kcal]) of 10% fat, 70% carbohydrate and 20% protein (total caloric energy value = 3.85 kcal/g; D12450B; Research Diets, Inc., New Brunswick, NJ, USA) or a high-fat diet consisting of 45% fat, 35% carbohydrate, and 20% protein (total caloric energy value= 4.73 kcal/g; D12451; Research Diets, Inc.). All mice had free access to food and water, and received their specified diet (normal or high-fat) for the duration of the study. After 3 weeks on either diet, nicotinamide (1000 mg/kg) and streptozotocin (150 mg/kg) were injected intraperitoneally to induce diabetes, as previously described (Tahara et al., 2008). Control mice were intraperitoneally administered with physiological saline. Two weeks later, we assessed the characteristics of the diabetic mice as described below. To investigate the effects of antidiabetic drugs, the diabetic mice were grouped to provide similar mean non-fasting blood glucose levels in each group. The following groups were established: normal chow diet-fed non-diabetic (N) and diabetic mice (N-diabetic), and high-fat diet-fed non-diabetic (HF) and diabetic mice (HF-diabetic). Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, and all experimental procedures were approved by the Animal Ethical Committee of Astellas Pharma Inc.

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collected from the tail vein. Plasma parameters were determined in blood samples collected from the abdominal vena cava under diethyl ether anesthesia. Tissues (pancreas, liver and epididymal adipose tissue) were removed to measure pancreatic insulin content, hepatic lipid content, and fat weight, respectively. 2.4. Oral glucose and intraperitoneal insulin tolerance tests For glucose tolerance testing, overnight-fasted mice were orally administered with 2 g/kg glucose solution, and blood samples were collected from the tail vein at the indicated times to measure blood glucose, plasma insulin and GLP-1 levels. The HOMA-R index value was calculated using the following formula: [fasting blood glucose (mg/dl) x fasting plasma insulin (μU/ml)/405] (Matthews et al., 1985). Because the titer for mouse insulin was not defined, plasma insulin (μU/ml) was calculated using the titer for the international authentic sample of human insulin (26 U/mg). For insulin tolerance testing, an insulin solution (0–1 IU/kg) was intraperitoneally administered to mice fasted for 6 h, and blood samples were collected from the tail vein at the indicated times to measure blood glucose levels. Insulin sensitivity was measured based on the glucose disappearance rate (K-value) within 30 min, as determined by the average slope K in the fitting curve. 2.5. Evaluation of single doses of antidiabetic drugs Normal diet-fed non-diabetic mice were treated with vehicle, and high-fat diet-fed diabetic mice were treated with vehicle or a single dose of mitiglinide (0.3–3 mg/kg), glibenclamide (1–10 mg/kg), sitagliptin (0.3–3 mg/kg), insulin (0.1–0.5 IU/kg), metformin (100–1000 mg/kg), or voglibose (0.1–1 mg/kg). In all groups, except the mitiglinide-treated group, the test drugs were administered and blood samples were taken from the tail vein at −0.5 h and at 0 h before oral administration of a liquid meal (20 ml/kg; containing 206 mg/ml carbohydrate, 53 mg/ml fat, and 53 mg/ml protein; Ensure® H, Abbott, Osaka, Japan). Additional blood samples were collected at 0.5, 1 and 2 h after the liquid meal. In the mitiglinide-treated groups, blood samples (–0.5-h value) were collected from mice after an overnight fast. Mitiglinide was orally administered 25 min later. After 5 min, blood samples (0-h value) were collected and the liquid meal was administered. Additional blood samples were collected at 0.5, 1 and 2 h after liquid meal administration. To measure plasma insulin and GLP-1 levels, the experimental protocol described above was repeated, except blood samples were collected from the abdominal vena cava before drug administration (basal value) or 10 min after liquid meal loading under diethyl ether anesthesia. 2.6. Evaluation of repeated administration of antidiabetic drugs Normal diet-fed non-diabetic and high-fat diet-fed diabetic mice were treated with either vehicle, sitagliptin (1 and 3 mg/kg) or rosiglitazone (1 and 3 mg/kg) once-daily at night for 3 weeks. Glucose tolerance tests were conducted after an overnight fast on Day 19. The following morning, blood samples were collected to measure fasting blood glucose and plasma insulin levels, and an oral glucose tolerance test was performed as described above. Blood samples were collected at 10 min to measure plasma insulin and GLP-1 levels, and at 0.5, 1 and 2 h after glucose loading to measure blood glucose levels. On the morning after the final dose (Day 22), glycolipid metabolic parameters were measured in non-fasting conditions, as described above. 2.7. Biochemical analysis

2.3. Evaluation of the characteristics of the type 2 diabetic mice Glycolipid metabolic parameters were measured under non-fasting conditions. Blood glucose levels were measured from blood samples

Blood glucose levels were measured using Glucose CII-Test reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). HbA1c levels were measured using a DCA2000 system (Bayer Medical, Ltd., Tokyo, Japan).

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Table 1 Glycolipid metabolic parameters in high-fat diet and streptozotocin–nicotinamide-induced diabetic mice.

Body weight (g) Food intake (g/day) Calorie intake (kcal/day) Blood glucose (mg/dl) Plasma insulin (ng/ml) Pancreatic insulin content (ng/mg pancreas) Plasma triglycerides (mg/dl) Plasma NEFA (mEq/l) Plasma total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Liver weight (g) Hepatic triglyceride content (mg/g liver) Hepatic cholesterol content (mg/g liver) Epididymal adipose tissue weight (g)

N

N-diabetic

HF

HF-diabetic

44.7 ± 0.8 4.26 ± 0.17 16.4 ± 0.7 139 ± 7 0.96 ± 0.08 92.3 ± 5.0 107 ± 10 1.11 ± 0.05 123 ± 9 68.4 ± 3.0 54.8 ± 6.7 1.86 ± 0.08 4.48 ± 0.30 0.61 ± 0.03 0.88 ± 0.08

42.7 ± 0.5 4.43 ± 0.27 17.1 ± 1.1 200 ± 11a 1.08 ± 0.10 58.8 ± 4.8a 122 ± 15 1.25 ± 0.04 131 ± 6 69.4 ± 3.7 61.4 ± 4.2 1.92 ± 0.10 5.37 ± 0.68 0.64 ± 0.10 1.01 ± 0.06

50.0 ± 1.1a 4.73 ± 0.23 20.7 ± 1.1a 181 ± 11a 2.09 ± 0.13a,b 80.0 ± 4.0 215 ± 14a,b 1.59 ± 0.08a,b 157 ± 8 79.4 ± 4.0 77.8 ± 8.0 2.31 ± 0.13a,b 9.07 ± 1.05a,b 1.31 ± 0.10a,b 1.56 ± 0.11a,b

48.4 ± 0.7a 4.86 ± 0.15 23.0 ± 0.7a 264 ± 9a,b,c 1.91 ± 0.11a,b 39.8 ± 3.4a,b,c 243 ± 15a,b 1.78 ± 0.10a,b 176 ± 13a,b 86.2 ± 5.2a,b 89.5 ± 9.4a,b 2.36 ± 0.06a,b 11.03 ± 1.72a,b 1.68 ± 0.09a,b,c 1.66 ± 0.09a,b

N, normal diet-fed non-diabetic mice; N-diabetic, normal diet-fed diabetic mice; HF, high-fat diet-fed non-diabetic mice; HF-diabetic, high-fat diet-fed diabetic mice; NEFA, nonesterified fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Data are means ± S.E.M. of five mice per group. The significance of differences between each group was assessed using Tukey's multiple range test. a P b 0.05 vs. N. b P b 0.05 vs. N-diabetic. c P b 0.05 vs. HF mice.

Pancreatic insulin content was measured as follows: a 2-ml aliquot of acid-ethanol solution (75% ethanol, 23.5% purified water, 1.5% concentrated hydrochloric acid) was added to the pancreas samples, and this mixture was homogenized and incubated at 4 °C for 1 h to extract insulin. The supernatant was then centrifuged at 15,000 rpm for 10 min, and the resulting supernatant was used for analysis. To measure hepatic lipid content, approximately 100 mg of liver tissue was transferred to a tube, immersed in 400 μl methanol, and homogenized. After adding chloroform (800 μl) and stirring for 10 min, purified water (200 μl) was added. This mixture was stirred for 10 min and then centrifuged at 2500 rpm for 5 min at room temperature, after which the upper (water) layer was removed using an aspirator. A 50-μl aliquot of the lower (chloroform) layer was transferred to a tube and centrifuged to dryness using an evaporator. The residue was then dissolved in 10 μl ethanol and 40 μl purified water, and the lipid concentrations were measured. Insulin levels in the plasma and pancreas samples were determined using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi, Gunma, Japan). Triglyceride, non-esterified fatty acid (NEFA), and cholesterol levels were measured using triglyceride E-test Wako, NEFA C-test Wako (Wako Pure Chemical Industries, Ltd.), and Determiner L TCII and HDL-C (Kyowa Medex Co., Tokyo, Japan) kits, respectively. Plasma GLP-1, leptin and adiponectin levels were determined using an active GLP-1 ELISA (Linco Research, Inc., St Charles, MO, USA), a mouse/rat leptin ELISA (B-Bridge International, Inc., Mountain View, CA, USA), and a mouse/rat adiponectin ELISA (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), respectively. To prevent degradation of GLP-1 during storage and assay, the DPP-IV inhibitor sitagliptin (1 μM final concentration) was added to the plasma samples.

2.8. Statistical analysis Data are expressed as means±S.E.M. The areas under the concentration–time curves (AUC) were calculated for blood glucose, plasma insulin and GLP-1. Significant differences between pairs of groups were determined using Student's t test, while those between multiple groups were determined using Tukey's or Dunnett's multiple range test. Value of Pb 0.05 was considered significant. All analyses were conducted using SAS 8.2 (SAS Institute Japan, Ltd., Tokyo, Japan) and Prism (GraphPad Software Inc., San Diego, CA, USA) software packages.

3. Results 3.1. Characteristics of high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice As shown in Table 1, mice fed the HF diet for 5 weeks exhibited a significant increase or tendency to increase in food and calorie intake, body and epididymal adipose tissue weight, non-fasting blood glucose, plasma insulin, triglyceride, NEFA and cholesterol levels, and hepatic lipid (triglyceride and cholesterol) content compared with N mice. The N-diabetic mice exhibited a significant increase in non-fasting blood glucose and a decrease in pancreatic insulin content, but no differences in plasma insulin or lipid levels, or hepatic lipid content, compared with N mice. The HF-diabetic mice exhibited significant hyperglycemia, hyperinsulinemia, lipid abnormalities, obesity and decreased pancreatic insulin content. Over 85% of the HF-diabetic mice exhibited significant hyperglycemia, hyperinsulinemia and obesity. Furthermore, preliminary studies revealed that these mice developed a stable diabetic and obese state that lasted for at least 10 weeks after inducing diabetes by intraperitoneal administration of streptozotocin and nicotinamide. Oral glucose tolerance and intraperitoneal insulin tolerance tests were carried out to measure glucose tolerance and insulin sensitivity. Compared with N mice, the HF mice had significantly impaired glucose tolerance as assessed using the oral glucose tolerance test (Fig. 1). Glucose-induced early-phase insulin secretion was not significantly affected by the HF diet, but the fasting plasma insulin level and the plasma insulin AUC determined by the oral glucose tolerance test were significantly increased. Accordingly, HOMA-R values were also significantly increased in HF mice than in N mice (Table 2). Results of the insulin tolerance test showed that insulin at doses ≥0.2 IU/kg significantly reduced blood glucose levels in N mice (Fig. 2). By contrast, in HF mice, insulin only significantly reduced blood glucose levels when administered at the highest dose (i.e., 1 IU/kg). Further results of the insulin tolerance test showed that insulin sensitivity (K value) was significantly lower in HF mice than in N mice. Although the N-diabetic mice showed significantly impaired glucose tolerance as a result of the loss of early-phase insulin secretion, no change was noted in their HOMA-R or K values, and these mice did not exhibit insulin resistance. Meanwhile, HF-diabetic mice also exhibited significantly impaired glucose tolerance as a result

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Fig. 1. Glucose tolerance in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice. (A) Blood glucose, (B) plasma insulin, and (C) GLP-1 levels during the oral glucose tolerance test in normal diet-fed non-diabetic (N) and diabetic (N-diabetic) mice, and in high-fat diet-fed non-diabetic (HF) and diabetic (HF-diabetic) mice. Values are means± S.E.M. of five animals per group. The significance of differences between each group was assessed using Tukey's multiple range test. *P b 0.05 vs. N, #P b 0.05 vs. N-diabetic and $P b 0.05 vs. HF mice.

of the loss of early-phase insulin secretion but had pronounced insulin resistance. Plasma GLP-1 levels did not differ significantly among any of the experimental groups. 3.2. Evaluation of single administration of antidiabetic drugs In the liquid meal tolerance test, mitiglinide, glibenclamide, sitagliptin, insulin, metformin and voglibose dose-dependently and significantly at

each dose improved glucose tolerance in HF-diabetic mice (Fig. 3). In addition, the plasma insulin levels were significantly elevated in the mice treated with mitiglinide, glibenclamide or sitagliptin, which exert insulin-secreting activity, or with insulin (Fig. 4). By contrast, the plasma insulin levels were significantly lower in mice treated with metformin or voglibose. Plasma GLP-1 levels were significantly increased in sitagliptin-treated mice, but not in the other groups, although small non-significant increases were noted with metformin and voglibose.

Table 2 Blood glucose and plasma insulin levels during the oral glucose tolerance test in high-fat diet and streptozotocin–nicotinamide-induced diabetic mice. N Fasting blood glucose (mg/dl) Blood glucose AUC (mg h/dl) Fasting plasma insulin (ng/ml) Plasma insulin AUC (mg h/dl) HOMA-R Fasting plasma GLP-1 (pM) Plasma GLP-1 AUC (pM h)

101 ± 5 339 ± 2 0.61 ± 0.07 63.7 ± 2.2 3.89 ± 0.40 3.53 ± 0.90 292 ± 17

N-diabetic 105 ± 7 463 ± 12a 0.55 ± 0.11 61.0 ± 3.9 3.71 ± 0.78 3.67 ± 0.66 272 ± 17

HF

HF-diabetic a

133 ± 7 432 ± 8a 1.17 ± 0.14a,b 104 ± 1a,b 9.94 ± 1.13a,b 3.61 ± 0.66 312 ± 20

137 ± 9a,b 573 ± 13a,b,c 1.17 ± 0.17a,b 94.4 ± 2.1a,b 10.6 ± 2.1a,b 4.12 ± 0.44 304 ± 18

N, normal diet-fed non-diabetic mice; N-diabetic, normal diet-fed diabetic mice; HF, high-fat diet-fed non-diabetic mice; HF-diabetic, high-fat diet-fed diabetic mice; AUC, area under the curve; HOMA-R, homeostatic model assessment of insulin resistance; GLP-1, glucagon-like peptide-1. Data are means ± S.E.M. of five mice per group. The significance of differences between each group was assessed using Tukey's multiple range test. a P b 0.05 vs. N. b P b 0.05 vs. N-diabetic. c P b 0.05 vs. HF mice.

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Fig. 2. Insulin tolerance in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice. (A) Blood glucose decrease during the insulin tolerance test (ITT) in normal diet-fed non-diabetic (N) and diabetic (N-diabetic) mice, and in high-fat diet-fed non-diabetic (HF) and diabetic (HF-diabetic) mice. (B) Decreases in blood glucose AUC during the ITT. Values are means± S.E.M. of five animals per group. The significance of differences between the vehicle and insulin-treated groups was assessed using Dunnett's multiple range test. *P b 0.05 vs. vehicle-treated mice.

3.3. Evaluation of repeated administration of antidiabetic drugs Repeated administration of sitagliptin or rosiglitazone once daily for 3 weeks significantly reduced non-fasting blood glucose, HbA1c, and plasma insulin levels, increased pancreatic insulin content (Table 4), and improved lipid parameters. Although sitagliptin significantly reduced the plasma leptin levels, the plasma adiponectin levels and body and epididymal adipose tissue weights were unchanged. By contrast, rosiglitazone exhibited a significant increase or tendency to increase in plasma leptin and adiponectin levels as well as body (P b 0.1) and epididymal adipose tissue weight. Administration of these drugs did not significantly affect food intake, liver weight, or hepatic lipid content, although the highest dose of rosiglitazone tended to decrease food intake and increase hepatic lipid content. Results of the oral glucose tolerance test performed on Day 19 showed that sitagliptin and rosiglitazone significantly reduced fasting blood glucose and plasma insulin levels, and improved glucose tolerance (Table 3, Fig. 5). Sitagliptin significantly increased plasma GLP-1 levels and early-phase insulin secretion whereas rosiglitazone significantly increased early-phase insulin secretion but did not affect GLP-1 levels. 4. Discussion Type 2 diabetes is a complex, heterogenous, polygenic disease. The primary defects in insulin secretion and the development of insulin resistance contribute to the etiology of type 2 diabetes. Impaired

postprandial insulin secretion because of functional defects and the loss of surviving pancreatic β-cells leads to hyperglycemia and a subsequent decline in insulin sensitivity (Polonsky et al., 1998; Taylor et al., 1994). Therefore, individuals with type 2 diabetes may experience both reduced insulin secretion and insulin action. Thus, an experimental animal model that mimics the pathogenesis and clinical features of human type 2 diabetes should have both of these traits. Among the animal models currently available, several strains such as KK/Ay, db/db, and ob/ob mice, and Zucker diabetic fatty and OLETF rats exhibit inherited hyperglycemia and insulin resistance, and are widely used in experimental studies (Srinivasan and Ramarao, 2007). However, these inbred diabetic models are relatively expensive and difficult to breed, and maintaining constant pathological conditions for these animals is not easy. In addition, these diabetic animals are unsuitable for studies of insulin secretagogues because of their severe insulin resistance. Thus, given these limitations, many studies have focused on developing a type 2 diabetic rodent model suitable for use in pharmacological research. We recently reported that streptozotocin and nicotinamide-induced diabetic mice exhibited moderate glucose intolerance associated with the loss of early-phase insulin secretion and decreased pancreatic insulin content, and that insulin secretagogues significantly improved glucose tolerance through their insulinotropic action. However, these mice did not exhibit insulin resistance or obesity, and insulin sensitizing agents such as thiazolidinediones failed to improve glucose tolerance (unpublished data). Therefore, these mice were not suitable for the evaluation of insulin-sensitizing drugs. Many studies have reported that

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(mg/kg or IU/kg)

Voglibose

Fig. 3. Effects of (A) mitiglinide, (B) glibenclamide, (C) sitagliptin, (D) insulin, (E) metformin, and (F) voglibose on blood glucose levels during the oral liquid meal tolerance test (LMTT) in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice. (G) Blood glucose AUC during the LMTT. Values are means± S.E.M. of four animals per group. The significance of differences between the normal diet-fed non-diabetic (normal) and high-fat diet-fed diabetic vehicle-treated groups was assessed using Student's t-test. The significance of differences between the vehicle and drug-treated groups was assessed using Dunnett's multiple range test. *P b 0.05 vs. normal mice; #P b 0.05 vs. vehicle-treated mice.

animals fed a HF diet develop insulin resistance. Therefore, we attempted to develop a mouse model by feeding mice a HF diet to induce insulin resistance and then treating them with streptozotocin and nicotinamide. The aim of this study was to develop a relatively inexpensive and more convenient animal model of type 2 diabetes that closely reflects the metabolic characteristics of the disease and would be responsive to antidiabetic drugs. In this study, HF diet feeding for 5 weeks induced insulin resistance, obesity, mild hyperglycemia, hyperlipidemia, and compensatory hyperinsulinemia. This is consistent with findings from previous reports that HF diets induce insulin resistance in rodents, and is also supported by the significantly decreased insulin-mediated glucose clearance noted in isolated adipocytes derived from rats fed a HF diet (Kraegen et al., 1986, 1991; Storlien et al., 1986). Although HF diet-induced insulin resistance has been reported to be caused by various mechanisms, it is considered to be mainly induced through the Randle or glucose–fatty acid cycle (Randle et al., 1963). Briefly, the presence of high levels of triglycerides,

because of excess fat intake, may serve as the main source of fatty acids and thus increase their oxidation. In turn, this preferential use of fatty acids for oxidation blunts insulin-mediated suppression of hepatic glucose output, reduces glucose uptake and utilization in skeletal muscle, and leads to compensatory hyperinsulinemia, a common feature of insulin resistance (Iwanishi and Kobayashi 1993; Rosholt et al., 1994). In contrast, nicotinamide and streptozotocin-administered mice exhibited a marked decline in glucose tolerance because of insulin secretory deficiency, resulting in hyperglycemia with blood glucose levels of ~200 mg/dl, which is an appropriate marker for therapy in clinical settings. However, plasma insulin and lipid levels, and hepatic lipid content were not significantly affected in these animals, neither was insulin resistance nor obesity. Notably, the HF-diabetic mice exhibited significant hyperglycemia, lipid abnormalities, insulin resistance, obesity and decreased pancreatic insulin content. In preliminary studies, HF diet feeding and simultaneous administration of nicotinamide and streptozotocin delayed the onset of insulin resistance and

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A. Tahara et al. / European Journal of Pharmacology 655 (2011) 108–116

A

10 min

Plasma insulin (ng/ml)

Basal

#

4 # #

3

#

*

2

#

*

#

1

Normal Vehicle Mitiglinide (3 mg/kg) Glibenclamide (10 mg/kg) Sitagliptin (3 mg/kg) Insulin (0.5 IU/kg) Metformin (1000 mg/kg) Voglibose (1 mg/kg)

0

B

Basal

10 min

Plasma GLP-1 (pM)

50

#

40 30 20 10 0

Fig. 4. Effects of antidiabetic drugs on plasma insulin and GLP-1 levels during the oral liquid meal tolerance test in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice. (A) Plasma insulin and (B) GLP-1 levels under basal (fasted) conditions and at 10 min after liquid meal loading. Values are means ± S.E.M. of four animals per group. The significance of differences between the normal diet-fed nondiabetic (normal) and high-fat diet-fed diabetic vehicle-treated groups was assessed using Student's t-test. The significance of differences between the vehicle and drugtreated groups was assessed using Dunnett's multiple range test. *P b 0.05 vs. normal mice, #P b 0.05 vs. vehicle-treated mice.

obesity, as compared with the two-stage process used here. Therefore, to create this model, nicotinamide and streptozotocin were administered 3 weeks after starting the HF diet and confirming that the mice exhibited obesity. The success rate was relatively high (N85%), and these mice exhibited a stable diabetic and obese state until at least 10 weeks after inducing diabetes. To characterize these HF-diabetic mice, we examined the effects of single doses of several antidiabetic drugs, including mitiglinide (rapidacting insulin secretagogue), glibenclamide (sulfonylurea), sitagliptin (DPP-IV inhibitor), insulin, metformin (biguanide), and voglibose (α-glucosidase inhibitor). We found that all six drugs significantly improved glucose tolerance in our animal model. In addition, mitiglinide, glibenclamide and sitagliptin, all of which possess insulinsecreting activity, significantly increased plasma insulin levels, and sitagliptin also significantly increased plasma GLP-1 levels. These pharmacologic characteristics of the HF-diabetic mice were similar to those noted in a previous study of normal diet-fed diabetic mice (Tahara et al., 2008). We also examined the effects of repeated doses of sitagliptin and rosiglitazone, a thiazolidinedione with insulin-sensitizing effects. Once-daily administration of sitagliptin or rosiglitazone for 3 weeks reduced non-fasting blood glucose, plasma insulin and lipid parameters, indicating the potent antihyperglycemic and hypolipidemic activities of these drugs. Both drugs also improved insulin resistance. The glucose-lowering effect noted with sitagliptin was associated with

glucose-dependent insulin secretion in response to increased plasma active GLP-1 levels by inhibiting DPP-IV. This antihyperglycemic effect also improved insulin resistance, findings consistent with previously reported results (Tahara et al., 2008; Mu et al., 2009). Rosiglitazone, like other thiazolidinedione-based drugs, reduced blood glucose levels by sensitizing insulin activity in target tissues, mainly by inhibiting lipolysis in adipose tissue and subsequent reduction of glucose production in the liver, and enhancing insulin-mediated glucose disposal in skeletal muscle (Srinivasan et al., 2004). In this animal model, treatment with sitagliptin and rosiglitazone markedly improved the hyperlipidemic state. Other studies have also demonstrated improvements in lipid abnormalities by DPP-IV inhibitors and thiazolidinediones (Srinivasan et al., 2005). Therefore, it seems likely that regulation of lipid metabolism plays an important role in the insulin secreting and sensitizing effects of both drugs. Accordingly, these findings suggest that their mechanism of action might be through stimulating glucosedependent insulin secretion and improving insulin resistance, respectively. As would be expected, the HF-diabetic mice exhibited marked visceral fat accumulation, which preceded the development of insulin resistance. In addition, these mice exhibited significant increases in plasma leptin and decreases in adiponectin levels, which is consistent with previous studies (Ahrén et al., 1997; Sandu et al., 2005). Leptin, the product of the ob gene, is a hormone secreted by adipocytes, and increased body fat content, as in obesity, is closely correlated with the circulating plasma leptin levels (Zhang et al., 1994; Considine et al., 1996). In addition, obesity is accompanied by insulin resistance that induces hyperinsulinemia, which stimulates ob gene expression in adipocytes (Olefsky et al., 1982; Wabitsch et al., 1996). Leptin has been posited as a humoral signal from adipose tissue that acts on the central nervous system to reduce excess food intake and increase energy expenditure in a negative feedback manner (Halaas et al., 1995; Maffei et al., 1995). In the present study, sitagliptin significantly reduced plasma leptin levels, as in other studies (Lamont and Drucker, 2008). This reduction in leptin was associated with improvements in hyperglycemia and insulin resistance. By contrast, rosiglitazone significantly increased plasma leptin levels, despite improving hyperglycemia and insulin resistance. However, this increase in leptin was likely due to the rosiglitazone-induced increase in subcutaneous adiposity (Kim et al., 2008). In addition, we found that rosiglitazone tended to reduce food intake, which may be mediated, in part, by the increased plasma leptin levels. The HF-diabetic mice also exhibited significant reductions in plasma adiponectin levels. Adiponectin, like leptin, is highly expressed in adipose tissue, although the plasma levels of adiponectin are negatively correlated with body mass index and insulin levels, and positively correlated with insulin sensitivity (Majuri et al., 2007). As such, adiponectin may regulate insulin sensitivity by modulating non-oxidative glucose disposal, such as the glycogen synthesis pathway in skeletal muscle. In the present study, rosiglitazone significantly increased plasma adiponectin levels. Thiazolidinediones, such as rosiglitazone, activate peroxisome proliferator-activated receptor-γ,

Table 3 Fasting parameters in high-fat diet and streptozotocin–nicotinamide-induced diabetic mice treated with sitagliptin or rosiglitazone for 3 weeks.

Fasting blood glucose (mg/dl) Fasting plasma insulin (ng/ml) HOMA-R

N

HF-diabetic

Vehicle

Vehicle

109 ± 4 0.74 ± 0.08 5.2 ± 0.6

154 ± 6a 1.18 ± 0.10a 11.6 ± 1.0a

Sitagliptin (mg/kg)

Rosiglitazone (mg/kg)

1

3

1

3

148 ± 7 1.22 ± 0.05 11.6 ± 0.6

128 ± 5b 1.01 ± 0.08b 8.4 ± 0.8b

141 ± 7 1.15 ± 0.07 10.4 ± 0.9

121 ± 5b 0.95 ± 0.06b 7.3 ± 0.4b

N, normal diet-fed non-diabetic mice; HF-diabetic, high-fat diet-fed diabetic mice; HOMA-R, homeostatic model assessment of insulin resistance. Data are means ± S.E.M. of six mice per group. The significance of differences between the N and HF-diabetic groups was assessed using Student's t test. The significance of differences between the HF-diabetic and drug-treated groups was assessed using Dunnett's multiple range test. a P b 0.05 vs. N. b P b 0.05 vs. HF-diabetic mice.

A. Tahara et al. / European Journal of Pharmacology 655 (2011) 108–116

115

Table 4 Non-fasting parameters in high-fat diet and streptozotocin–nicotinamide-induced diabetic mice treated with sitagliptin or rosiglitazone for 3 weeks.

Body weight (g) Food intake (g/day) Calorie intake (kcal/day) Blood glucose (mg/dl) HbA1c (%) Plasma insulin (ng/ml) Pancreatic insulin content (ng/mg pancreas) Plasma triglycerides (mg/dl) Plasma NEFA (mEq/l) Plasma total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Plasma leptin (ng/ml) Plasma adiponectin (μg/ml) Liver weight (g) Hepatic triglyceride content (mg/g liver) Hepatic cholesterol content (mg/g liver) Epididymal adipose tissue weight (g)

N

HF-diabetic

Vehicle

Vehicle

Sitagliptin (mg/kg) 1

3

1

3

43.4 ± 0.9 4.43 ± 0.49 17.1 ± 1.9 157 ± 12 3.17 ± 0.08 1.15 ± 0.13 92.7 ± 8.6 112 ± 15 0.93 ± 0.08 100 ± 7 64.6 ± 5.2 35.6 ± 2.6 3.55 ± 0.51 40.8 ± 1.9 2.19 ± 0.10 6.83 ± 1.11 1.20 ± 0.24 0.94 ± 0.07

49.9 ± 1.0a 5.05 ± 0.24 23.9 ± 1.1a 250 ± 19a 4.07 ± 0.14a 2.53 ± 0.13a 44.9 ± 5.6a 212 ± 25a 1.68 ± 0.12a 159 ± 10a 81.0 ± 5.0a 77.6 ± 8.0a 8.70 ± 0.78a 24.6 ± 2.5a 2.51 ± 0.10a 14.3 ± 1.9a 2.80 ± 0.45a 1.64 ± 0.09a

49.3 ± 1.3 4.83 ± 0.24 22.8 ± 1.1 233 ± 12 4.03 ± 0.15 2.27 ± 0.12 50.5 ± 3.4 187 ± 19 1.40 ± 0.12 152 ± 10 89.2 ± 4.8 62.5 ± 10.7 7.91 ± 0.80 24.0 ± 3.6 2.54 ± 0.07 12.6 ± 1.5 2.91 ± 0.17 1.46 ± 0.08

50.3 ± 1.1 4.76 ± 0.22 22.5 ± 1.0 184 ± 12b 3.40 ± 0.09b 2.03 ± 0.13b 63.1 ± 5.0b 142 ± 17b 1.25 ± 0.09b 129 ± 7 81.8 ± 2.8 46.9 ± 6.4 5.85 ± 0.80b 32.0 ± 2.7 2.39 ± 0.07 10.6 ± 1.2 2.78 ± 0.37 1.44 ± 0.12

49.9 ± 1.0 4.92 ± 0.16 23.3 ± 0.8 254 ± 17 3.87 ± 0.13 2.19 ± 0.12 54.7 ± 6.0 158 ± 13 1.73 ± 0.10 154 ± 9 87.9 ± 6.0 65.6 ± 9.2 8.94 ± 0.65 32.9 ± 3.7 2.49 ± 0.11 15.0 ± 2.1 3.15 ± 0.45 1.62 ± 0.14

52.7 ± 1.7 4.45 ± 0.20 21.1 ± 0.9 191 ± 14b 3.48 ± 0.11b 1.77 ± 0.16b 73.7 ± 4.1b 125 ± 16b 1.24 ± 0.13b 119 ± 10b 84.4 ± 8.2 34.4 ± 7.5b 11.9 ± 0.7b 40.7 ± 1.6b 2.78 ± 0.12 18.1 ± 2.6 3.85 ± 0.27 2.10 ± 0.10b

Rosiglitazone (mg/kg)

N, normal diet-fed non-diabetic mice; HF-diabetic, high-fat diet-fed diabetic mice; NEFA, non-esterified fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Data are means ± S.E.M. of six mice per group. The significance of differences between the N and HF-diabetic groups was assessed using Student's t test. The significance of differences between the HF-diabetic and drug-treated groups was assessed using Dunnett's multiple range test. a P b 0.05 vs. N b P b 0.05 vs. HF-diabetic mice.

Normal

C

Rosiglitazone (3 mg/kg) Blood glucose AUC (mg·h/dl)

300 200 100 0 0.5

1.0

Time (h)

E 30

1

(mg/kg)

10

0

Sitagliptin

Rosiglitazone

or

m

3

1

3

1

cl e hi Ve

N

or

m al

0

#

1

*

2

(mg/kg)

Rosiglitazone

20

ic le

#

al

# 3

Sitagliptin

#

Ve h

#

Plasma GLP-1 (pM)

Plasma insulin (ng/ml)

5 4

300

2.0

Time (h)

N

D

1.5

400

al

0.0

#

m

2.0

# 500

or

1.5

* 600

N

1.0

Sitagliptin

1

0.5

3

0.0

700

3

Blood glucose (mg/dl)

400

3

Rosiglitazone (1 mg/kg)

Sitagliptin (3 mg/kg)

1

Sitagliptin (1 mg/kg)

3

Vehicle

1

Vehicle

le

B

Normal

ic

A

the role of visceral adipose tissue and its products in the development of metabolic syndrome. In conclusion, the present study revealed that a combination of HF diet-feeding plus streptozotocin and nicotinamide administration induces metabolic aberrations, particularly impaired insulin secretion, insulin resistance and obesity, resulting in a phenotype that is very

Ve h

an adipocyte transcription factor, which stimulates adipocyte differentiation into adiponectin-secreting adipocytes (Chou et al., 2007). These adipocytokines may contribute to the improvements in hyperglycemia and insulin resistance observed with sitagliptin and rosiglitazone treatment. Taken together, the findings from the present study suggest that these HF-diabetic mice will be useful in future studies investigating

(mg/kg)

Rosiglitazone

Fig. 5. Effects of repeated administration of sitagliptin and rosiglitazone for 3 weeks on glucose tolerance in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice. (A, B) Time-course of changes in blood glucose levels during the oral glucose tolerance test (OGTT). (C) Blood glucose AUC during the OGTT. (D) Plasma insulin and (E) GLP-1 levels at 10 min after glucose loading. Values are means ± S.E.M. of six animals per group. The significance of differences between the normal diet-fed non-diabetic (normal) and high-fat diet-fed diabetic vehicle-treated groups was assessed using Student's t-test. The significance of differences between the vehicle and drug-treated groups was assessed using Dunnett's multiple range test. *P b 0.05 vs. normal mice; #P b 0.05 vs. vehicle-treated mice.

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