Ursolic acid ameliorates thymic atrophy and hyperglycemia in streptozotocin–nicotinamide-induced diabetic mice

Chemico-Biological Interactions 188 (2010) 635–642

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Ursolic acid ameliorates thymic atrophy and hyperglycemia in streptozotocin–nicotinamide-induced diabetic mice Jin Lee a , Sung-Tae Yee b , Jong-Jin Kim b , Myung-Sook Choi c , Eun-Young Kwon c , Kwon-Il Seo a , Mi-Kyung Lee a,∗ a b c

Department of Food and Nutrition, Sunchon National University, Suncheon 540-742, Republic of Korea Department of Biology, Sunchon National University, Suncheon 540-742, Republic of Korea Department of Food Science and Nutrition, Kyungpook National University, Daegu 702-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 June 2010 Received in revised form 15 September 2010 Accepted 17 September 2010 Available online 24 September 2010 Keywords: Immune response Metformin Non-obese type 2 diabetes Thymus Ursolic acid

a b s t r a c t The purpose of this study was to assess the effects of low-dose ursolic acid (UA) on glycemic regulation and immune responses in streptozotocin–nicotinamide (STZ/NA)-induced diabetic mice. Diabetic mice were supplemented with two different doses of UA (0.01 and 0.05%, w/w) or metformin (0.5%, w/w) for 4 weeks. Compared with the untreated diabetic group, UA and metformin significantly improved blood glucose, glycosylated hemoglobin, glucose tolerance, insulin tolerance and plasma leptin levels as well as aminotransferase activity. The plasma and pancreatic insulin concentrations were significantly higher in both UA groups than in the untreated diabetic group. Supplementation with metformin increased the pancreatic insulin level without a change in the plasma insulin level. The relative thymus weights were lower in the untreated diabetic group compared to the non-diabetic group; however, the UA or metformin group had significantly improved thymus weights. Mice receiving UA or metformin supplementation had increased CD4+ CD8+ subpopulations in the thymus compared to the untreated diabetic mice. Concanavalin A-stimulated splenic T-lymphocyte proliferation and single-positive (CD4+ and CD8+ ) subpopulations were significantly higher in the UA-supplemented diabetic groups than in the untreated diabetic group, but lipopolysaccharide-stimulated B-lymphocyte proliferation and the CD19+ subpopulation were not significantly different among the groups. In the STZ/NA-induced diabetic mice, metformin increased the splenic T-lymphocyte CD4+ and CD8+ cell numbers without any change in T-lymphocyte proliferation. Both doses of UA lowered splenic IL-6 levels, whereas metformin increased IFN-␥, IL-6 and TNF-␣ levels compared to the untreated diabetic mice. These results suggest that low-dose UA may be used as a hypoglycemic agent and immune modulator in non-obese type 2 diabetic mice. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The incidence of type 2 diabetes mellitus is increasing annually worldwide and is characterized by insulin resistance and impaired insulin secretion. In Western countries, many type 2 diabetic patients are obese, whereas in Asian countries including Korea, a large proportion of type 2 diabetic patients are non-obese [1,2]. The non-obese type 2 diabetes phenotype is characterized by reduced insulin secretion and less insulin resistance compared with the obese type 2 diabetes phenotype. Importantly, non-obese type 2 diabetic patients have a similar increased risk of cardiovascular disease compared to obese type 2 diabetic patients [3].

∗ Corresponding author at: Department of Food and Nutrition, Sunchon National University, 413 Jungangro, Suncheon, Jeonnam 540-742, Republic of Korea. Tel.: +82 61 750 3656; fax: +82 61 752 3657. E-mail address: [email protected] (M.-K. Lee). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.09.019

Diabetes mellitus is a complex metabolic disorder resulting in hyperglycemia [4]. Hyperglycemia may be attributed to defects in pancreatic ␤-cells, insulin secretion, hepatic glucose output, glucose uptake of peripheral tissues [5] and immune function [6]. Insulin resistance, hyperglycemia, inflammatory disorders and immune dysfunction cause high morbidity and mortality in patients with severe trauma, burn injuries or sepsis [7]. It has been reported that diabetic patients exhibit lower resistance to common infections than their healthy counterparts [8]. High blood glucose is toxic to multiple cell populations, including immune and immune-related cells such as lymphocytes [9] and endothelial cells [10]. Ursolic acid (UA) is a pentacyclic triterpenoid compound that naturally occurs in berries, leaves, flowers, fruits and medicinal herbs. UA has been reported to have several pharmacological effects, including anti-hepatitis, anti-inflammatory, anti-ulcer and anti-hyperlipidemic effects [11]. Recently, UA has been found to be an anti-diabetic agent [12,13]. Our previous study showed that

636

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

UA (0.05%, w/w) exhibited anti-diabetic and immunomodulatory activities in STZ-induced diabetic mice that were fed a high-fat diet, which was mediated by inhibition of hepatic glucose production, the preservation of pancreatic ␤-cells and enhancement of the immune system [14,15]. Metformin, a biguanide glucose-lowering agent, is commonly used for the treatment of type 2 diabetes due to decreasing hepatic glucose production through gluconeogenesis suppression and activating peripheral glucose utilization in muscle, intestine and liver [16]; however, the effect of metformin on the immune system in organisms with diabetes is not yet understood. Furthermore, Ong et al. [17] suggested that metformin was at least as efficacious in the non-obese as it was in the obese and supported metformin use in non-obese diabetic patients. Therefore, we conducted the present study to investigate whether low-dose UA can modulate the immune system and hyperglycemia in non-obese type 2 diabetic mice and compared the results to the actions of metformin. 2. Materials and methods 2.1. Animals and experimental design Sixty-five male ICR mice that were 8-weeks-old were purchased from Biogenomics, Inc. (Seoul, Korea). The mice were individually housed in polycarbonate cages at 22 ± 2 ◦ C on a 12-h light-dark cycle. The mice were all treated in strict accordance with Sunchon National University’s guidelines for the care and use of laboratory animals. The mice were acclimatized for seven days; then, they were randomly divided into non-diabetic (n = 10) and diabetic (n = 55) groups. Non-obese type 2 diabetes was induced by a single injection of STZ (50 mg/kg body weight in 0.1 M citrate buffer, pH 4.2; Sigma, St. Louis, MO, USA) into the peritoneum on two consecutive days. Nicotinamide (NA, 120 mg/kg body weight; Sigma, St. Louis, MO, USA) was dissolved in saline and injected intraperitoneally 15 min before the administration of STZ on the first day [18]. The non-diabetic mice were injected with citrate buffer or saline alone. After seven days, only STZ/NA-treated mice that exhibited a fasting blood glucose level ≥11 mmol/L were used in the study. The diabetic mice were randomly subdivided into four groups of nine mice each; the untreated diabetic (DM) group, the diabetic-low-dose ursolic acid (DM-lowUA) group, the diabetic-high-dose ursolic acid (DM-highUA) group and the diabetic-metformin (positive control, DM-Metformin) group. The mice were fed an AIN-76 semi-synthetic diet [19,20] with UA (0.01 and 0.05 g/100 g diet; TCI Co., Ltd., Japan) or metformin (0.5 g/100 g diet; Sigma, St. Louis, MO, USA) for 4 weeks. We established the dose of metformin based on our previous data (not published), which was performed based on previous human [21] and animal [22] studies. The mice had access to food and water ad libitum. At the end of the experimental period, food was withheld for 12 h; the mice were then anesthetized with ether, and blood samples were taken from the inferior vena cava to determine the levels of plasma biomarkers.

2.3. Intraperitoneal glucose and insulin tolerance tests Intraperitoneal glucose and insulin tolerance tests (IPGTT and IPITT) were performed at the third and fourth week, respectively. Following a 6-h fast, the mice were injected intraperitoneally with glucose (1 g/kg body weight) or insulin (1 unit/kg body weight). Blood glucose levels were determined from tail vein blood at 0, 30, 60, and 120 min after the glucose injection and at 0, 30, 60 and 90 min after the insulin injection. 2.4. Plasma and pancreatic insulin levels Blood was collected in a heparin-coated tube and centrifuged at 600 × g for 15 min at 4 ◦ C. The plasma insulin levels were then determined using a quantitative sandwich enzyme immunoassay kit (ELISA kit, Crystal Chem Inc., IL, USA). The pancreatic insulin concentration was determined as described by Shima et al. [23] with a slight modification. The pancreas was homogenized with 10 volumes of a cold acid-ethanol solution and kept for 72 h at 4 ◦ C. Thereafter, the homogenate was centrifuged at 600 × g for 30 min at 4 ◦ C, and the supernatant was tested for insulin concentration. The insulin concentration was determined using a quantitative sandwich enzyme immunoassay kit (ELISA kit, Shibaygi Co., Ltd., Japan). 2.5. Plasma leptin level and aminotransferase activity The plasma leptin levels were determined using a quantitative sandwich enzyme immunoassay kit (R&D Systems, USA). The aspartate transaminase (AST) and alanine transaminase (ALT) activities were measured using an automatic biochemistry analyzer (FUJIFILM DRI-Chem 3500i, Japan). 2.6. Isolation of lymphocytes from the thymus and spleen Single cells suspensions of thymocytes and splenocytes were prepared from individual mice by mincing the thymus and spleen, respectively. The lymphocytes were prepared by density gradient centrifugation in a Ficoll-Hypaque solution (Sigma Co., St. Louis, MO, USA). All cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 20 mM HEPES buffer, 2 m l-glutamine, 1 mM pyruvate, 100 U/mL penicillin, 50 ␮g/mL streptomycin, 50 ␮M 2-mercaptoethanol, and 1% nonessential amino acids. All supplements, as well as RPMI 1640, were purchased from Gibco BRL (Grand Island, USA). 2.7. Lymphocyte population assays 6 In the lymphocyte population  assays, 5 × 10 cells were blocked receptor) mAb for 30 min at 4 ◦ C with anti-CD16/CD32(Fc␥Ш/ and then washed with a PBS solution containing 1% FBS and 0.1% NaN3 . The cells were stained with PE-conjugated anti-CD8 mAb, FITC-conjugated anti-CD4 mAb and FITC-conjugated anti-CD19 mAb for 30 min at 4 ◦ C. The stained cells were washed and detected using flow cytometry (COULTER, Epics XL, USA).

2.2. Blood glucose and glycosylated hemoglobin levels 2.8. Lymphocyte proliferation assays The fasting blood glucose concentration was monitored using a glucometer (GlucoDr SuperSensor, Allmedicus, Korea) to test venous blood drawn from the tail vein every week after a 6-h fast. Non-fasting blood glucose concentrations were also determined using a glucometer (GlucoDr SuperSensor) at the end of the experiment. The glycosylated hemoglobin (HbA1c ) concentration was measured after hemolysis of the anticoagulated whole blood specimen. HbA1c was determined immuno-turbidimetrically.

The cell proliferation assay was conducted using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), which is a colorimetric method for determining the number of viable, proliferating cells. The spleen lymphocytes previously prepared were seeded at a density of 5 × 105 cells per well in 96-well plates with RPMI-1640 medium containing 10% FBS. The cells were incubated with or without concanavalin A (Con A, 1 ␮g/mL) and lipopolysaccharide (LPS, 10 ␮g/mL) at 37 ◦ C in 5% CO2 .

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

637

3. Results 3.1. Change in body weight

Fig. 1. Effect of ursolic acid supplementation on changes in body weight of STZ/NAinduced diabetic mice. The values are expressed as the means ± S.E. NS Not significant, the values not sharing a common letter differ significantly at p < 0.05.

96®

After 48 h of incubation, 15 ␮L of the CellTiter AQueous One Solution Reagent was added directly to the culture wells. The cells were incubated for 3–8 h, and the absorbance was then recorded at 490 nm using a 96-well plate reader (Titertek Multiscan Plus, Finland). The quantity of formazan product measured by the 490 nm absorbance was directly proportional to the number of living cells in culture. The stimulation index (SI) was calculated as follows: SI =

O.D. of stimulated cells O.D. of negative control cells

2.9. Cytokine production assays Splenocytes (5 × 106 cells/mL) were treated with 1 ␮g/mL Con A or 10 ␮g/mL LPS for 24 h, and cell supernatants were collected. IL2, IL-6, IFN-␥ and TNF-␣ levels were measured by ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. 2.10. Statistical analysis All data are presented as the means ± standard error (S.E.), and the data were evaluated by one-way ANOVAs using SPSS (SPSS Inc., Chicago). The differences between the means were assessed using Duncan’s multiple-range test. Differences were considered statistically significant when p < 0.05.

The untreated diabetic mice had a significant loss in body weight after the second week. At the end of the experimental period, the weight of the untreated diabetic mice was significantly (15%) lower than that of the non-diabetic mice. However, metformin supplementation prevented the weight loss, and the mice with both doses of UA supplementation had a tendency to increase body weight compared to the untreated diabetic mice (Fig. 1). Daily food intake was significantly higher in the untreated diabetic mice than in the non-diabetic mice. Supplementation with UA did not affect food intake, but metformin effectively lowered food intake in non-obese type 2 diabetic mice (data not shown). 3.2. Blood glucose, glycosylated hemoglobin, IPGTT and IPITT Fig. 2 shows the blood glucose levels in the non-diabetic and experimental mice from each group. The fasting blood glucose concentration in the untreated diabetic mice was significantly higher than in the non-diabetic mice during the 4 weeks (Fig. 2A). The fasting and non-fasting blood glucose concentrations in the untreated diabetic mice were increased by 2.3- and 3.3-fold, respectively, compared to the non-diabetic mice (Fig. 2A and B). However, both doses of UA and metformin supplementation significantly lowered blood glucose concentrations compared to the untreated diabetic group (Fig. 2A). After 4 weeks, the fasting blood glucose levels of the low-dose UA-, high-dose UA- and metformin-supplemented groups were significantly lower (by 29%, 22% and 30%, respectively) than the level in the untreated diabetic group. Mice with UA or metformin supplementation also had significantly lowered non-fasting blood glucose (Fig. 2B) and glycosylated hemoglobin concentrations (Table 1) compared to the untreated diabetic mice. In this study, low-dose UA supplementation efficiently lowered both fasting and non-fasting blood glucose concentrations. Compared with UA, metformin was more efficient in lowering the non-fasting blood glucose concentration. In addition, UA and metformin supplementation significantly improved the glucose tolerance and insulin tolerance in the nonobese type 2 diabetic animal model. The efficacy of both doses of UA on IPGTT and IPITT was similar to those of metformin (Table 2).

Fig. 2. Effect of ursolic acid supplementation on fasting blood glucose levels (A) and non-fasting blood glucose levels (B, 4th week) in STZ/NA-induced diabetic mice. Values are expressed as the means ± S.E. The values not sharing a common letter differ significantly at p < 0.05.

638

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

Table 1 Effects of ursolic acid supplementation on glycosylated hemoglobin concentration and plasma biomarkers in STZ/NA-induced diabetic mice* . NonDM HbA1c (%) Plasma Leptin (ng/mL) AST (U/L) ATL (U/L)

DM

DMlowUA

6.22 ± 0.10a

11.10 ± 0.74c

9.67 ± 0.37b

1.72 ± 0.16b 34.90 ± 0.95a 13.40 ± 0.54a

1.19 ± 0.09a 47.25 ± 4.77c 20.00 ± 1.24b

1.71 ± 0.16b 37.88 ± 1.81a,b 15.28 ± 1.62a

DMhighUA 9.54 ± 0.40b 1.89 ± 0.09b 35.14 ± 1.01a 16.00 ± 0.88a

DMMetformin 7.46 ± 0.43b 1.67 ± 0.18b 43.00 ± 1.90b,c 16.57 ± 1.49a

Means in the same row not sharing a common superscript letter differ significantly at p < 0.05. * Means ± S.E. Table 2 Effects of ursolic acid supplementation on intraperitoneal glucose and insulin tolerance tests in STZ/NA-induced diabetic mice* . NonDM

DM

DM- lowUA

DM- highUA

IPGTT (mmol/L) 0 min 30 min 60 min 120 min

7.22 10.76 9.59 7.05

± ± ± ±

0.24a 0.53a 0.46a 0.14a

16.95 23.48 24.26 19.75

± ± ± ±

1.71c 2.09c 2.48c 1.56c

11.72 18.70 17.30 14.65

± ± ± ±

1.38b 1.67b 1.67b 1.86b

12.35 20.63 18.76 15.02

± ± ± ±

1.99b 1.32b,c 1.77b 1.73b

IPITT (mmol/L) 0 min 30 min 60 min 90 min

6.86 3.75 2.45 2.84

± ± ± ±

0.32a 0.26a 0.22a 0.20a

16.09 8.09 5.49 4.77

± ± ± ±

1.44c 0.64c 0.52c 0.26c

12.18 5.98 3.87 3.47

± ± ± ±

1.20b 0.53b 0.39b 0.17b

12.41 6.13 3.98 3.06

± ± ± ±

1.18b 0.54b 0.39b 0.25b

DM-Metformin 11.89 19.20 17.41 13.43

± ± ± ±

0.83b 1.21b 1.00b 1.56b

9.88 5.24 3.91 3.48

± ± ± ±

1.19a,b 0.62a,b 0.64b 0.50b

Means in the same row not sharing a common superscript letter differ significantly at p < 0.05. * Means ± S.E.

3.3. Plasma leptin concentration and aminotransferase activity The plasma leptin concentration in the untreated diabetic mice was significantly lower than that in the non-diabetic group. However, UA and metformin significantly elevated the leptin concentration compared to the untreated diabetic group (Table 1). Both doses of UA effectively suppressed the increase in the plasma AST and ALT activities in the non-obese type 2 diabetic mice. Metformin also significantly lowered the plasma ALT activity compared to the untreated diabetic group (Table 1). 3.4. Insulin concentrations in plasma and pancreas The plasma and pancreatic insulin concentrations in the untreated diabetic mice were 21% and 32%, respectively, of those in non-diabetic mice (Fig. 3). Both doses of UA significantly elevated the plasma insulin concentrations compared to the untreated diabetic group; however, metformin did not alter the plasma insulin concentration (Fig. 3A). The pancreatic insulin concentration was significantly higher in the UA- and metformin-supplemented groups than in the untreated diabetic group (Fig. 3B). 3.5. Relative thymus weight and cellular subpopulations The thymic index was calculated as organ weight (mg)/body weight (g) × 100, and it is presented in Fig. 4A. The relative thymus weight in the untreated diabetic mice was significantly lower compared to the non-diabetic mice. However, both doses of UA and metformin supplementation suppressed the thymic atrophy in the non-obese type 2 diabetic mice. When thymocyte subpopulations were evaluated by flow cytometry analyses, the numbers of the double-positive immature T cells (CD4+ CD8+ ) in the diabetic mice were significantly lower than that in the non-diabetic mice; in contrast, double-negative (CD4− CD8− ) and single-positive (CD4+ or CD8+ ) subpopulations were significantly increased in the untreated diabetic group. However, supplementation with both doses of UA or metformin significantly reversed such changes so that the thymocyte populations

Fig. 3. Effect of ursolic acid supplementation on plasma (A) and pancreatic (B) insulin concentrations in STZ/NA-induced diabetic mice. Values are expressed as the means ± S.E. The values sharing a common letter differ significantly at p < 0.05.

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

639

Fig. 4. Effect of ursolic acid supplementation on relative thymus weight (A) and thymocyte T-lymphocyte subpopulations (B) in STZ/NA-induced diabetic mice. Values are expressed as means ± S.E. The values sharing a common letter differ significantly at p < 0.05.

in UA-supplemented mice were similar to those in the non-diabetic group (Fig. 4B).

3.6. Splenic lymphocyte proliferation and cellular subpopulations Con A-induced splenic T-lymphoctye proliferation was significantly inhibited in the untreated diabetic mice when compared to the non-diabetic mice; there were no significant differences between these groups in LPS-induced B-lymphocyte proliferation (Fig. 5). Both doses of UA significantly improved splenic T-lymphocyte proliferation, but UA supplementation had no effect on B-lymphocyte proliferation. Metformin did not affect either T or B-lymphocyte proliferation in the non-obese type 2 diabetic mice (Fig. 5). The splenic lymphocyte populations of CD4+ (helper T) and CD8+ (cytotoxic T) cells were significantly decreased in the untreated diabetic mice compared to the non-diabetic mice (Table 3). However, UA and metformin increased the numbers of CD4+ and CD8+ lymphocyte subpopulations compared to the untreated diabetic group. The counts of CD19+ cells were not different among the groups (Table 3).

Fig. 5. Effect of ursolic acid supplementation on the proliferation of spleen cells in STZ/NA-induced diabetic mice. The spleen cells (5 × 105 ) were stimulated with Con A (1 ␮g/mL) or LPS (10 ␮g/mL) for 48 h. The data are presented as the stimulation index (O.D. of stimulated cells/O.D. of negative control cells). Values are expressed as the means ± S.E. The values not sharing a common letter differ significantly at p < 0.05.

4. Discussion 3.7. Splenic cytokine production IL-2 and IFN-␥ production by splenocytes was lower in the untreated diabetic mice than in the non-diabetic mice, whereas IL6 production was higher in the untreated diabetic group (Table 4). UA supplementation lowered the splenic IL-6 level, whereas metformin significantly increased the IFN-␥, IL-6 and TNF-␣ levels compared to the untreated diabetic group (Table 4).

Increasing evidence has suggested that the development of type 2 diabetes and its complications are closely associated with the immune system. In Asians, a number of diabetic patients have non-obese type 2 diabetes, and 50% of adult non-obese type 2 diabetics are latent autoimmune diabetic patients [24]. Depressed T-lymphocyte function has been known to be a result of hyperglycemia [25] or a toxic side effect of the diabetic agent [26,27].

Table 3 Effects of ursolic acid supplementation on splenic lymphocyte subpopulations in STZ/NA-induced diabetic mice* . NonDM

DM

DM- lowUA

DM- highUA

DM-Metformin

T-cells (%) CD4+ CD8+

16.72 ± 1.14b 5.90 ± 0.20b,c

12.44 ± 0.83a 3.72 ± 0.22a

16.84 ± 1.16b 5.04 ± 0.55b

16.88 ± 1.52b 5.10 ± 0.52b

19.68 ± 0.88b 6.32 ± 0.14c

B-cells (%) CD19+

37.51 ± 1.11NS

34.45 ± 2.23

36.91 ± 2.49

40.30 ± 3.72

39.25 ± 2.86

Means in the same row not sharing a common superscript letter differ significantly at p < 0.05. NS, Not significant. * Means ± S.E.

640

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

Table 4 Effects of ursolic acid supplementation on splenic cytokine productions in STZ/NA-induced diabetic mice* . Splenic cytokines (ng/mL)

NonDM

IL-2 IFN-␥ IL-6 TNF-␣

1.16 4.34 0.77 0.51

± ± ± ±

0.09b 0.53b 0.10a 0.02a

DM 0.62 1.38 1.27 0.55

DMhighUA

DMlowUA ± ± ± ±

0.04a 0.13a 0.11b 0.04a

0.64 1.10 0.58 0.57

± ± ± ±

0.08a 0.42a 0.10a 0.01a

0.59 1.23 0.64 0.47

± ± ± ±

DMMetformin 0.04a 0.23a 0.06a 0.03a

0.69 3.84 1.76 1.14

± ± ± ±

0.05a 0.80b 0.09c 0.07b

Means in the same row not sharing a common superscript letter differ significantly at p < 0.05. * Means ± S.E.

Many traditional medicines or natural compounds from medicinal plants have been used to modulate immune function. We previously showed that dietary supplementation with 0.05% (w/w) UA can cause immunomodulation in STZ-induced diabetic mice fed a high-fat diet [14]. The aim of this study was to investigate whether low-dose UA (0.01%, w/w) supplementation improves hyperglycemia and immune function in STZ/NA-induced diabetic mice, a non-obese type 2 diabetes animal model [18]. This study demonstrated that both doses of UA resulted in the recovery of the splenic T-lymphocyte proliferative response and subpopulations in the non-obese type 2 diabetic mice. In STZ/NAinduced diabetic mice, Con A-induced T-lymphocyte proliferation and the numbers of single-positive (CD4+ and CD8+ ) subpopulations were significantly lower than those of non-diabetic mice. CD4+ T-lymphocytes protect the body from infection by bacteria, viruses and other pathogens [28]. Once activated, T-helper cells activate other immune cells such as macrophages, B-lymphocytes and cytotoxic T-lymphocytes to defend against these infections [29]. In this study, regardless of dose, mice receiving UA supplementation significantly increased splenic T-cell proliferation and CD4+ and CD8+ subpopulations compared to the untreated diabetic mice. Thus, the increase in T-lymphocytes proliferation in the mice supplemented with UA was consistent with our previous results [14]. This study indicates the potential stimulating effects of UA on T-lymphocytes, which may lead to an increased immune response. Supplementation with metformin did not affect the proliferative response of splenic T-lymphocytes, although it did significantly increase the T-lymphocyte subpopulations (CD4+ and CD8+ ) in STZ/NA-induced diabetic mice. In an in vitro study, metformin inhibited Con A-stimulated T-lymphocyte proliferation in mice [30]. As such, UA may especially affect T-cells in the immune system through proliferation and activation of CD4+ and CD8+ cells in the non-obese type 2 diabetic mice, whereas metformin did not induce proliferation of T-lymphocytes. Many pro-inflammatory cytokines cause diabetes-associated abnormalities, such as insulin resistance, impaired insulin secretion, and increased capillary permeability; inflammatory cytokines also accelerate atherosclerosis [31] and may also cause associated complications, such as dyslipidemia, cardiovascular disease and renal failure [32]. Thus, there is a close relationship between the immunological state and the development of type 2 diabetes [33]. Thorvaldson et al. [34] reported that elevated levels of glucose and free fatty acids as well as changes in cytokine production are common features of both type 1 and type 2 diabetes. The present study observed that IL-2 and IFN-␥ levels in splenic T-lymphocytes were significantly lower in the diabetic mice compared to the non-diabetic mice, whereas IL-6 levels in B-lymphocytes were significantly higher. Both doses of UA normalized IL-6 levels compared to the untreated diabetic group. However, supplementation with metformin significantly increased IFN-␥, IL-6 and TNF-␣ levels compared to the untreated diabetic and UA-supplemented groups. Thus, UA exhibits anti-inflammatory properties in nonobese type 2 diabetic mice, but metformin increased the levels of pro-inflammatory cytokines, namely IFN-␥, IL-6 and TNF␣. Both in vitro and in vivo studies have demonstrated that

UA suppressed the expression of LPS-induced pro-inflammatory mediators in RAW264.7 mouse macrophages [35] and 12-Otetradecanoylphorbol-13-acetate-induced skin tumor promotion [36]. The majority of cells located in peripheral blood, spleen and lymphoids are dependent on the thymus for proper differentiation. Interestingly, we found that both doses of UA and metformin restored depressed relative thymus weight by hyperglycemia. The thymus is the primary lymphoid organ that is crucial for the development of immature T-cells into cells that execute immune functions in the periphery against several pathological conditions [37]. Many researchers have shown that thymic atrophy may be caused by diabetes [37,38]. Da˘gistanli et al. [39] reported that thymic atrophy is caused by elevated intracellular calcium levels leading to apoptosis in STZ-induced diabetes. In this study, we also found that the relative thymus weight was dramatically lowered by 61% in STZ/NA-induced diabetic mice compared to the non-diabetic group; however, 0.01% UA, 0.05% UA and metformin supplementation significantly improved thymic atrophy by 1.9-, 1.5- and 1.7-fold, respectively, compared to the untreated diabetic group, which indicated that UA and metformin may enhance the immune response. Furthermore, hyperglycemia is toxic to multiple immune cell populations, including lymphocytes [9]. Therefore, we determined whether UA or metformin might affect the thymic cellular differentiation in non-obese type 2 diabetic mice. The relative thymic CD4+ CD8+ subpopulation was significantly lower in the untreated diabetic group than in the non-diabetic group. This result was in agreement with previous works [38,40]. The loss of thymic double-positive T cells has been shown to occur in other diseases, mainly infectious diseases [41,42]. T-cell precursors come to the thymus from bone marrow via the bloodstream and undergo development to mature T-cells. The thymocytes early in development lack detectable CD4 and CD8, which they are referred to as double-negative (CD4− CD8− ) cells. CD4 and CD8 coreceptors are then expressed; this double positive (CD4+ CD8+ ) stage is a period of rapid proliferation. Double positive thymocytes develop into mature single-positive CD4+ thymocytes or single-positive CD8+ thymocytes. These single-positive cells undergo additional negative selection and migrate from the cortex to the medulla, where they pass from the thymus into the peripheral immune system [43]. These results indicated that increases in the numbers of CD4+ and CD8+ of splenic T-lymphocytes in the UA or metforminsupplemented group might be associated with the increased in the thymic CD4+ CD8+ subpopulation. Recently, it has been shown that leptin is controlled by glucose, insulin, glucocorticoids and cytokines and that leptin plays an important role in thymic organization and maintenance [44,45]. Leptin-knockout mice or mice without the functional leptin receptor exhibit a striking decrease in thymus size and loss of the cortex [45]. Insulin has an in vivo stimulatory effect on human and rodent leptin [44,46]. Some studies have reported that decreased leptin levels in diabetics were associated with thymic atrophy [46,47]. In this study, we found that UA and metformin supplementation elevated plasma leptin levels along with the insulin concentration in non-obese type 2 diabetic mice. Plasma leptin levels were positively

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

correlated with plasma insulin levels (r = 0.382, p < 0.01) (data not shown). Elmas et al. [40] reported that the thymus is ultrastructurally affected by diabetes and concomitant fasting and that insulin treatment was able to reverse these pathological changes. Some researchers have suggested that almost all of the benefits from intensive insulin therapy may be attributed to its effect in lowering blood glucose concentration [7]. Other studies have shown that insulin is not only the main anabolic and anti-catabolic hormone but also a direct regulator of inflammation and inmmunocompetence [48]. In this study, both doses of UA and metformin significantly lowered the fasting and non-fasting blood glucose levels. Thus, low-dose UA exhibited a blood glucose-lowering effect in the non-obese type 2 diabetic mice. In this study, despite plasma insulin concentrations being slightly lower in the metformin group than the UA group, insulin sensitivity was similar. Metformin was more efficient than UA at lowering non-fasting blood glucose levels but not fasting glucose, glucose tolerance and insulin tolerance. Yoshida et al. [49] also reported that metformin decreased plasma glucose more effectively in the fed state than in the fasted state without insulin secretion in GK rats, a non-obese type 2 diabetic animal model. Glucose utilization and disposal is a major determinant of the plasma glucose levels in the fed state [50]. In addition, metformin but not UA lowered daily food intake in non-obese type 2 diabetic mice. The pancreatic insulin concentration was higher in the low-dose UA-, high-dose UA- and metformin-supplemented groups; however, the plasma insulin level was only higher in the UA group than in the untreated diabetic mice. These results support the hypothesis that metformin decreases plasma glucose by activating glucose utilization independent of insulin secretion [49]. UA is well known for its hepatoprotective effect on acute chemically-induced liver injury as well as liver fibrosis and cirrhosis [51]. Insulin decreases the serum concentrations of liver enzymes (AST and ALT), indicating that insulin alleviates liver injury and improves the production of liver constructive proteins, such as albumin, that lead to the preservation of liver function [52]. Serum AST or ALT is generally used primary screening parameters for liver disease. Therefore, we assessed the activities of AST and ALT in this study. Mice with UA and metformin supplementation exhibited significantly lower plasma AST and/or ALT activities compared to the untreated diabetic group. The beneficial effects of UA on the liver could be due to its antioxidant and anti-inflammatory effects [53]. 5. Conclusion Our findings suggested that UA and metformin may enhance immune function by suppressing thymic atrophy and acting upon immune cells, and low-dose UA may be an efficient anti-diabetic agent that increases plasma and pancreatic insulin concentrations in the non-obese type 2 diabetic patients. Conflict of interest There are no conflicts of interest. Acknowledgements This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2007-331F00058). References [1] T. Nagaya, H. Yoshida, H. Takahashi, M. Kawai, Increases in body mass index, even within non-obese levels, raise the risk for Type 2 diabetes mellitus: a follow-up study in a Japanese population, Diabetes Med. 22 (2005) 1107–1111.

641

[2] D.J. Kim, K.E. Song, J.W. Park, H.K. Cho, K.W. Lee, K.B. Huh, Clinical characteristics of Korean type 2 diabetic patients in 2005, Diabetes Res. Clin. Pract. 77S (2007) S252–S257. [3] A. Vaag, S.S. Lund, Non-obese patients with type 2 diabetes and prediabetic subjects: distinct phenotypes requiring special diabetes treatment and (or) prevention? Appl. Physiol. Nutr. Metab. 32 (5) (2007) 912–920. [4] P.J. Klover, R.A. Mooney, Hepatocytes: critical for glucose homeostasis, Int. J. Biochem. 36 (2004) 753–758. [5] R.A. DeFronzo, D. Simonson, E. Ferrannini, Hepatic and peripheral insulin resistance: a common feature of type 2 (non-insulin-dependent) and type 1 (insulin-dependent) diabetes mellitus, Diabetologia 23 (1982) 313–319. [6] D.C. Gore, S.E. Wolf, D.N. Herndon, R.R. Wolfe, Metformin blunts stress-induced hyperglycemia after thermal injury, J. Trauma 54 (2003) 555–561. [7] H.P. Deng, J.K. Chai, The effects and mechanisms of insulin on systemic inflammatory response and immune cells severe trauma, burn injury, and sepsis, Int. Immunopharmacol. 9 (2005) 1251–1259. [8] A Marble, H.J. White, A.T. Fernald, The nature of the lowered resistance to infection in diabetes mellitus, J. Clin. Invest. 17 (1938) 423–430. [9] P.C. Calder, G. Dimitriadis, P. Newsholme, Glucose metabolism in lymphoid and inflammatory cells and tissues, Curr. Opin. Clin. Nutr. Metab. Care 10 (4) (2007) 531–540. [10] R.J. Esper, J.O. Vilarino, R.A. Machado, A. Paragano, Endothelial dysfunction in normal and abnormal glucose metabolism, Adv. Cardiol. 45 (2008) 17–43. [11] S.F. Ahmad, B. Khan, S. Bani, K.A. Suri, N.K. Satti, G.N. Qazi, Amelioration of adjuvant-induced arthritis by ursolic acid through altered Th1/Th2 cytokine production, Pharmacol. Res. 53 (2006) 233–240. [12] W. Zhang, D. Hong, Y. Zhou, Y. Zhang, Q. Shen, J.Y. Li, L.H. Hu, J. Li, Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing insulin receptor phosphorylation and stimulating glucose uptake, Biochim. Biophys. Acta 1760 (2006) 1505–1512. [13] S.H. Jung, Y.J. Ha, E.K. Shim, S.Y. Choi, J.L. Jin, H.S.Y. Choi, J.R. Lee, Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activatior, Biochem. J. 403 (2007) 243–250. [14] S.M. Jang, S.T. Yee, J. Choi, M.S. Choi, G.M. Do, S.M. Jeon, J. Yeo, M.J. Kim, K.I. Seo, M.K. Lee, Ursolic acid enhances the cellular immune system and pancreatic beta-cell function in streptozotocin-induced diabetic mice fed a high-fat diet, Int. Immunopharmacol. 9 (2009) 113–119. [15] S.M. Jang, M.J. Kim, M.S. Choi, E.Y. Kwon, M.K. Lee, Inhibitory effects of ursolic acid on hepatic polyol pathway and glucose production in streptozotocininduced diabetic mice, Metab. Clin. Exp. 59 (2010) 512–519. [16] R.S. Hundal, M. Krssak, S. Dufour, D. Laurent, V. Lebon, V. Chandramouli, S.E. Inzucchi, W.C. Schumann, K.F. Petersen, B.R. Landau, G.I. Shulman, Mechanism by which metformin reduces glucose production in type 2 diabetes, Diabetes 49 (2000) 2063–2069. [17] C.R. Ong, S.M. Twigg, L.M. Molyneaux, D.K. Yue, M.I. Constantino, Long-term efficacy of metformin therapy in nonobese individuals with type 2 diabetes, Diabetes Care 29 (2006) 2361–2364. [18] T. Nakamura, T. Terajima, T. Ogata, K. Ueno, N. Hashimoto, K. Ono, S. Yano, Establishment and pathophysiological characterization of Type 2 diabetic mouse model produced by streptozotocin and nicotinamide, Biol. Pharm. Bull. 29 (6) (2006) 1167–1174. [19] American Institute of Nutrition, Report of Ad Hoc committee on standards for nutritional studies, J. Nutr. 110 (1980) 1717–1726. [20] American Institute of Nutrition, Report of the American Institute of Nutrition. Ad Hoc committee on standards for nutritional studies, J. Nutr. 107 (1977) 1340–1348. [21] C.J. Glueck, D. Aregawi, M. Winiarska, M. Agloria, G. Luo, L. Sieve, P. Wang, Metformin-diet ameliorates coronary heart disease risk factors and facilitates resumption of regular menses in adolescents with polycystic ovary syndrome, J. Pediatr. Endocrinol. Metab. 19 (6) (2006) 831–842. [22] M. Suwa, T. Egashira, H. Nakano, H. Sasaki, S. Kumagai, Metformin increases the PGC-1␣ protein and oxidative enzyme activities possibly via AMPK phosphorylation in skeletal muscle in vivo, J. Appl. Physiol. 101 (2006) 1685– 1692. [23] K. Shima, M. Zhu, Y. Noma, A. Mizuno, T. Murakami, T. Sano, M. Kuwajima, Exercise training in Otsuka Long-Evans Tokushima Fatty rat, a model of spontaneous non-insulin-dependent diabetes mellitus: effects on the B-cell mass, insulin content and fibrosis in the pancreas, Diabetes Res. Clin. Pract. 35 (1997) 11–19. [24] T. Tuomi, A. Carlsson, H. Li, B. Isomaa, A. Miettinen, A. Nilsson, M. Nissen, B.O. Ehrnstrom, B. Forsen, B. Snickars, K. Lahti, C. Forsblom, C. Saloranta, M.R. Taskinen, L.C. Groop, Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies, Diabetes 48 (1999) 150–157. [25] T. Tabata, Y. Okuna, S. Fujni, S. Kimura, Y. Kinoshita, Maturational impairment of thymic lymphocytes in streptozotocin-induced diabetes in rats, Cell Immunol. 89 (1984) 250–258. [26] W.K. Nichols, L.L. Vann, J.B. Spellman, Streptozotocin effects on T lymphocytes and bone marrow cells, Clin. Exp. Immunol. 46 (1981) 627–632. [27] S.R. Wellhausen, Defination of streptozotocin toxicity for primary lymphoidal tissues, Diabetes 35 (1986) 1404–1411. [28] P. Scott, S.H. Kamfmann, The role of T-cell subsets and cytokines in the regulation of infection, Immunol. Today 12 (10) (1991) 346–348. [29] P.R. Hanlon, M.G. Robbins, C. Scholl, D.M. Barnes, Aqueous extracts from dietary supplements influence the production of inflammatory cytokines in immortalized and primary T lymphocytes, BMC Complement. Altern. Med. 9 (2009) 51–61.

642

J. Lee et al. / Chemico-Biological Interactions 188 (2010) 635–642

[30] M.E. Solano, V. Sander, M.R. Wald, A.B. Motta, Dehydroepiandrosterone and metformin regulate proliferation of murine T lymphocytes, Clin. Exp. Immunol. 153 (2008) 289–296. [31] J.M. Fernandez-Real, J.D. Pickup, Innate immunity, insulin resistance and type 2 diabetes, Trends Endocrinol. Metabol. 19 (2007) 10–16. [32] M. Dworacka, H. Winiarska, M. Borowska, M. Abramczyk, T. BobkiewiczKozlowska, G. Dworacki, Pro-atherogenic alterations in T-lymphocyte subpopulations related to acute hyperglycaemia in type 2 diabetic patients, Circ. J. 71 (2007) 962–967. [33] T. Takahiro, M. Yoshinobu, K. Takahiro, S. Kumi, I. Jiro, Correlations of sleep disturbance with the immune system in type 2 diabetes mellitus, Diabetes Res. Clin. Pract. 85 (2009) 286–292. [34] L. Thorvaldson, S. Stalhammar, S. Sandler, Effects of a diabetes-like environment in vitro on cytokine production by mouse splenocytes, Cytokine 43 (2008) 93–97. [35] N. Suh, T. Honda, H.J. Finlay, A. Barchowsky, C. Williams, N.E. Benoit, Q.W. Xie, C. Nathan, G.W. Gribble, M.B. Sporn, Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages, Cancer Res. 58 (1998) 717–723. [36] H. Tokuda, H. Ohigashi, K. Koshimizu, Y. Ito, Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion by 12-O-tetradecanoylphorbol-13acetate, Cancer Lett. 33 (1986) 279–285. [37] P.R. Nagib, J. Gameiro, L. Guilherme Stivanin-Silva, M. Sueli Parreira de Arruda, D. Maria Serra Villa-Verde, W. Savino, L. Verinaud, Thymic microenvironmental alterations in experimentally induced diabetes, Immunobiology, in press, doi:10.1016/j.imbio.2010.02.001. [38] E.O. Barreto, I. Riederer, A.C. Arantes, V.F. Carvalho, F.A. Farias-Filho, R.S.B. Cordeiro, M.A. Martins, W. Savino, P.M.R. Silva, Thymus involution in alloxan diabetes: analysis of mast cells, Mem. Inst. Oswaldo Cruz 100 (Suppl. 1) (2005) 127–130. [39] F.K. Da˘gistanli, B.S. Duman, M. Öztürk, Protective effects of a calcium channel blocker on apoptosis in thymus of neonatal STZ-diabetic rats, Acta Histochem. 107 (2005) 207–214. [40] C. Elmas, D. Erdogan, G. Take, C. Ozogul, A. Nacar, M. Koksal, Ultrastructure of the thymus in diabetes mellitus and starvation, Adv. Ther. 25 (2008) 67–76. [41] W. Savino, The thymus is a common target organ in infectious diseases, PLoS Pathog. 2 (2006) 472–483.

[42] C.F. Andrade, J. Gameiro, P.R. Nagib, B.O. Carbalho, R.L. Talaisys, F.T. Costa, L. Verinaud, thymic alterations in Plasmodium berghei-infected mice, Cell Immunol. 253 (2008) 1–4. [43] A Singer, R. Bosselut, CD4/CD8 coreceptors in thymocyte development, selection, and lineage commitment: analysis of the CD4/CD8 lineage decision, Adv. Immunol. 83 (2004) 91–131. [44] A.L. Gruver, G.D. Sempowski, Cytokines, leptin, and stress-induced thymic atrophy, J. Leukoc. Biol. 84 (2008) 915–923. [45] L.A. Velloso, W. Savino, E. Mansour, Leptin action in the thymus, Ann. N.Y. Acad. Sci. 1156 (2009) 29–34. [46] W. Savino, M. Dardenne, L.A. Velloso, S.D. Silva-Barvosa, The thymus is a common target in malnutrition and infection, Br. J. Nutr. 98 (2007) S11–S16. [47] E. Bernotiene, G. Palmer, C. Gabay, The role of leptin in innate and adaptive immune responses, Arthritis Res. Ther. 8 (2006) 217–226. [48] B. Ellger, Y. Debaveye, I. Vanhorebeek, L. Langouche, A. Giulietti, E. Van Etten, P. Herijgers, C. Mathieu, G. Van den Berghe, Survival benefits of intensive insulin therapy in critical illness: impact of maintaining normoglycemia versus glycemia-independent actions of insulin, Diabetes 55 (4) (2006) 1096– 1105. [49] T. Yoshida, A. Okuno, J. Tanaka, K. Takahashi, R. Nakashima, S. Kanda, J. Ogawa, Y. Hagisawa, T. Fujiwara, Metformin primarily decreases plasma glucose not by gluconeogenesis suppression but by activating glucose utilization in a non-obese type 2 diabetes Goto-Kakizaki rats, Eur. J. Pharmacol. 623 (2009) 141–147. [50] T. Issad, L. Penicaud, P. Ferre, J. Kande, M.A. Baudon, J. Girard, Effects of fasting on tissue glucose utilization in conscious resting rats, Biochem. J. 246 (1987) 241–244. [51] J. Liu, Pharmacology of oleanolic acid and ursolic acid, J. Ethnopharmacol. 49 (1995) 57–68. [52] M.G. Jeschke, H. Rensing, D. Klein, T. Schubert, A.E. Mautes, U. Bolder, R.S. Croner, Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats, J. Hepatol. 42 (6) (2005) 870–879. [53] Y. Ikeda, A. Murakami, H. Ohigashi, Ursolic acid: an anti-and pro-inflammatory triterpenoid, Mol. Nutr. Food Res. 52 (2008) 26–42.