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Chemico-Biological Interactions 188 (2010) 635–642

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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rsolic acid ameliorates thymic atrophy and hyperglycemia intreptozotocin–nicotinamide-induced diabetic mice

in Leea, Sung-Tae Yeeb, Jong-Jin Kimb, Myung-Sook Choic, Eun-Young Kwonc,won-Il Seoa, Mi-Kyung Leea,∗

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

r t i c l e i n f o

rticle history:eceived 29 June 2010eceived in revised form5 September 2010ccepted 17 September 2010vailable online 24 September 2010

eywords:mmune response

etforminon-obese type 2 diabeteshymusrsolic 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 regulationand immune responses in streptozotocin–nicotinamide (STZ/NA)-induced diabetic mice. Diabetic micewere supplemented with two different doses of UA (0.01 and 0.05%, w/w) or metformin (0.5%, w/w) for4 weeks. Compared with the untreated diabetic group, UA and metformin significantly improved bloodglucose, glycosylated hemoglobin, glucose tolerance, insulin tolerance and plasma leptin levels as wellas aminotransferase activity. The plasma and pancreatic insulin concentrations were significantly higherin both UA groups than in the untreated diabetic group. Supplementation with metformin increasedthe pancreatic insulin level without a change in the plasma insulin level. The relative thymus weightswere lower in the untreated diabetic group compared to the non-diabetic group; however, the UA ormetformin group had significantly improved thymus weights. Mice receiving UA or metformin supple-mentation had increased CD4+CD8+ subpopulations in the thymus compared to the untreated diabeticmice. 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 untreateddiabetic group, but lipopolysaccharide-stimulated B-lymphocyte proliferation and the CD19+ subpopula-tion were not significantly different among the groups. In the STZ/NA-induced diabetic mice, metforminincreased the splenic T-lymphocyte CD4+ and CD8+ cell numbers without any change in T-lymphocyteproliferation. Both doses of UA lowered splenic IL-6 levels, whereas metformin increased IFN-�, IL-6 andTNF-� levels compared to the untreated diabetic mice. These results suggest that low-dose UA may be

gent

used as a hypoglycemic a

. Introduction

The incidence of type 2 diabetes mellitus is increasing annu-lly worldwide and is characterized by insulin resistance andmpaired insulin secretion. In Western countries, many type 2iabetic patients are obese, whereas in Asian countries includingorea, a large proportion of type 2 diabetic patients are non-obese

1,2]. The non-obese type 2 diabetes phenotype is characterized by

educed insulin secretion and less insulin resistance compared withhe obese type 2 diabetes phenotype. Importantly, non-obese typediabetic patients have a similar increased risk of cardiovascular

isease compared to obese type 2 diabetic patients [3].

∗ Corresponding author at: Department of Food and Nutrition, Sunchon Nationalniversity, 413 Jungangro, Suncheon, Jeonnam 540-742, Republic of Korea.el.: +82 61 750 3656; fax: +82 61 752 3657.

E-mail address: leemk@sunchon.ac.kr (M.-K. Lee).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.09.019

and immune modulator in non-obese type 2 diabetic mice.© 2010 Elsevier Ireland Ltd. All rights reserved.

Diabetes mellitus is a complex metabolic disorder resulting inhyperglycemia [4]. Hyperglycemia may be attributed to defectsin pancreatic �-cells, insulin secretion, hepatic glucose output,glucose uptake of peripheral tissues [5] and immune function[6]. Insulin resistance, hyperglycemia, inflammatory disordersand immune dysfunction cause high morbidity and mortality inpatients with severe trauma, burn injuries or sepsis [7]. It has beenreported that diabetic patients exhibit lower resistance to com-mon infections than their healthy counterparts [8]. High bloodglucose is toxic to multiple cell populations, including immune andimmune-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 medicinalherbs. UA has been reported to have several pharmacologicaleffects, including anti-hepatitis, anti-inflammatory, anti-ulcer andanti-hyperlipidemic effects [11]. Recently, UA has been found tobe an anti-diabetic agent [12,13]. Our previous study showed that

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A (0.05%, w/w) exhibited anti-diabetic and immunomodulatoryctivities in STZ-induced diabetic mice that were fed a high-fatiet, which was mediated by inhibition of hepatic glucose produc-ion, the preservation of pancreatic �-cells and enhancement of themmune system [14,15].

Metformin, a biguanide glucose-lowering agent, is commonlysed for the treatment of type 2 diabetes due to decreasing hep-tic glucose production through gluconeogenesis suppression andctivating peripheral glucose utilization in muscle, intestine andiver [16]; however, the effect of metformin on the immune sys-em in organisms with diabetes is not yet understood. Furthermore,ng et al. [17] suggested that metformin was at least as efficacious

n the non-obese as it was in the obese and supported metforminse in non-obese diabetic patients. Therefore, we conducted theresent study to investigate whether low-dose UA can modulatehe immune system and hyperglycemia in non-obese type 2 dia-etic mice and compared the results to the actions of metformin.

. Materials and methods

.1. Animals and experimental design

Sixty-five male ICR mice that were 8-weeks-old were purchasedrom Biogenomics, Inc. (Seoul, Korea). The mice were individuallyoused in polycarbonate cages at 22 ± 2 ◦C on a 12-h light-darkycle. The mice were all treated in strict accordance with Sunchonational University’s guidelines for the care and use of labora-

ory animals. The mice were acclimatized for seven days; then,hey were randomly divided into non-diabetic (n = 10) and dia-etic (n = 55) groups. Non-obese type 2 diabetes was induced bysingle injection of STZ (50 mg/kg body weight in 0.1 M citrate

uffer, pH 4.2; Sigma, St. Louis, MO, USA) into the peritoneum onwo consecutive days. Nicotinamide (NA, 120 mg/kg body weight;igma, St. Louis, MO, USA) was dissolved in saline and injectedntraperitoneally 15 min before the administration of STZ on therst day [18]. The non-diabetic mice were injected with cit-ate buffer or saline alone. After seven days, only STZ/NA-treatedice that exhibited a fasting blood glucose level ≥11 mmol/Lere used in the study. The diabetic mice were randomly subdi-

ided into four groups of nine mice each; the untreated diabeticDM) group, the diabetic-low-dose ursolic acid (DM-lowUA) group,he diabetic-high-dose ursolic acid (DM-highUA) group and theiabetic-metformin (positive control, DM-Metformin) group. Theice were fed an AIN-76 semi-synthetic diet [19,20] with UA (0.01

nd 0.05 g/100 g diet; TCI Co., Ltd., Japan) or metformin (0.5 g/100 giet; Sigma, St. Louis, MO, USA) for 4 weeks. We established theose of metformin based on our previous data (not published),hich was performed based on previous human [21] and animal

22] studies. The mice had access to food and water ad libitum. Athe end of the experimental period, food was withheld for 12 h; the

ice were then anesthetized with ether, and blood samples wereaken from the inferior vena cava to determine the levels of plasmaiomarkers.

.2. Blood glucose and glycosylated hemoglobin levels

The fasting blood glucose concentration was monitored usingglucometer (GlucoDr SuperSensor, Allmedicus, Korea) to test

enous blood drawn from the tail vein every week after a 6-h fast.on-fasting blood glucose concentrations were also determined

sing a glucometer (GlucoDr SuperSensor) at the end of the exper-

ment.The glycosylated hemoglobin (HbA1c) concentration was mea-

ured after hemolysis of the anticoagulated whole blood specimen.bA1c was determined immuno-turbidimetrically.

ractions 188 (2010) 635–642

2.3. Intraperitoneal glucose and insulin tolerance tests

Intraperitoneal glucose and insulin tolerance tests (IPGTT andIPITT) were performed at the third and fourth week, respectively.Following a 6-h fast, the mice were injected intraperitoneally withglucose (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 and90 min after the insulin injection.

2.4. Plasma and pancreatic insulin levels

Blood was collected in a heparin-coated tube and centrifugedat 600 × g for 15 min at 4 ◦C. The plasma insulin levels were thendetermined using a quantitative sandwich enzyme immunoassaykit (ELISA kit, Crystal Chem Inc., IL, USA).

The pancreatic insulin concentration was determined asdescribed by Shima et al. [23] with a slight modification. The pan-creas was homogenized with 10 volumes of a cold acid-ethanolsolution and kept for 72 h at 4 ◦C. Thereafter, the homogenate wascentrifuged at 600 × g for 30 min at 4 ◦C, and the supernatant wastested for insulin concentration. The insulin concentration wasdetermined using a quantitative sandwich enzyme immunoassaykit (ELISA kit, Shibaygi Co., Ltd., Japan).

2.5. Plasma leptin level and aminotransferase activity

The plasma leptin levels were determined using a quantita-tive sandwich enzyme immunoassay kit (R&D Systems, USA). Theaspartate transaminase (AST) and alanine transaminase (ALT) activ-ities 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 wereprepared from individual mice by mincing the thymus and spleen,respectively. The lymphocytes were prepared by density gradientcentrifugation in a Ficoll-Hypaque solution (Sigma Co., St. Louis,MO, USA). All cells were maintained in RPMI 1640 media supple-mented with 10% fetal bovine serum (FBS), 20 mM HEPES buffer,2 m l-glutamine, 1 mM pyruvate, 100 U/mL penicillin, 50 �g/mLstreptomycin, 50 �M 2-mercaptoethanol, and 1% nonessentialamino acids. All supplements, as well as RPMI 1640, were pur-chased from Gibco BRL (Grand Island, USA).

2.7. Lymphocyte population assays

In the lymphocyte population assays, 5 × 106 cells were blockedwith anti-CD16/CD32(Fc�Ш/

∏receptor) mAb for 30 min at 4 ◦C

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-CD19mAb for 30 min at 4 ◦C. The stained cells were washed and detectedusing flow cytometry (COULTER, Epics XL, USA).

2.8. Lymphocyte proliferation assays

The cell proliferation assay was conducted using the CellTiter96® AQueous One Solution Cell Proliferation Assay (Promega, Madi-son, WI, USA), which is a colorimetric method for determining the

number of viable, proliferating cells. The spleen lymphocytes pre-viously prepared were seeded at a density of 5 × 105 cells per wellin 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 Inte

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ig. 1. Effect of ursolic acid supplementation on changes in body weight of STZ/NA-nduced diabetic mice. The values are expressed as the means ± S.E. NSNot significant,he values not sharing a common letter differ significantly at p < 0.05.

fter 48 h of incubation, 15 �L of the CellTiter 96® AQueous Oneolution Reagent was added directly to the culture wells. The cellsere incubated for 3–8 h, and the absorbance was then recorded at

90 nm using a 96-well plate reader (Titertek Multiscan Plus, Fin-and). The quantity of formazan product measured by the 490 nmbsorbance was directly proportional to the number of living cellsn culture. The stimulation index (SI) was calculated as follows:

I = O.D. of stimulated cellsO.D. of negative control cells

.9. Cytokine production assays

Splenocytes (5 × 106 cells/mL) were treated with 1 �g/mL Con Ar 10 �g/mL LPS for 24 h, and cell supernatants were collected. IL-, IL-6, IFN-� and TNF-� levels were measured by ELISA kits (R&Dystems, USA) according to the manufacturer’s instructions.

.10. Statistical analysis

All data are presented as the means ± standard error (S.E.), andhe data were evaluated by one-way ANOVAs using SPSS (SPSSnc., Chicago). The differences between the means were assessedsing Duncan’s multiple-range test. Differences were consideredtatistically significant when p < 0.05.

ig. 2. Effect of ursolic acid supplementation on fasting blood glucose levels (A) and non-re expressed as the means ± S.E. The values not sharing a common letter differ significan

ractions 188 (2010) 635–642 637

3. Results

3.1. Change in body weight

The untreated diabetic mice had a significant loss in body weightafter the second week. At the end of the experimental period, theweight of the untreated diabetic mice was significantly (15%) lowerthan that of the non-diabetic mice. However, metformin supple-mentation prevented the weight loss, and the mice with both dosesof UA supplementation had a tendency to increase body weightcompared to the untreated diabetic mice (Fig. 1).

Daily food intake was significantly higher in the untreated dia-betic mice than in the non-diabetic mice. Supplementation withUA did not affect food intake, but metformin effectively low-ered food intake in non-obese type 2 diabetic mice (data notshown).

3.2. Blood glucose, glycosylated hemoglobin, IPGTT and IPITT

Fig. 2 shows the blood glucose levels in the non-diabetic andexperimental mice from each group. The fasting blood glucose con-centration in the untreated diabetic mice was significantly higherthan in the non-diabetic mice during the 4 weeks (Fig. 2A). The fast-ing and non-fasting blood glucose concentrations in the untreateddiabetic mice were increased by 2.3- and 3.3-fold, respectively,compared to the non-diabetic mice (Fig. 2A and B). However, bothdoses of UA and metformin supplementation significantly loweredblood glucose concentrations compared to the untreated diabeticgroup (Fig. 2A). After 4 weeks, the fasting blood glucose levels ofthe low-dose UA-, high-dose UA- and metformin-supplementedgroups were significantly lower (by 29%, 22% and 30%, respectively)than the level in the untreated diabetic group. Mice with UA or met-formin supplementation also had significantly lowered non-fastingblood glucose (Fig. 2B) and glycosylated hemoglobin concentra-tions (Table 1) compared to the untreated diabetic mice. In thisstudy, low-dose UA supplementation efficiently lowered both fast-ing and non-fasting blood glucose concentrations. Compared withUA, metformin was more efficient in lowering the non-fasting bloodglucose concentration.

In addition, UA and metformin supplementation significantlyimproved the glucose tolerance and insulin tolerance in the non-obese type 2 diabetic animal model. The efficacy of both dosesof UA on IPGTT and IPITT was similar to those of metformin(Table 2).

fasting blood glucose levels (B, 4th week) in STZ/NA-induced diabetic mice. Valuestly at p < 0.05.

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

Table 1Effects of ursolic acid supplementation on glycosylated hemoglobin concentration and plasma biomarkers in STZ/NA-induced diabetic mice*.

NonDM DM DM-lowUA

DM-highUA

DM-Metformin

HbA1c (%) 6.22 ± 0.10a 11.10 ± 0.74c 9.67 ± 0.37b 9.54 ± 0.40b 7.46 ± 0.43b

PlasmaLeptin (ng/mL) 1.72 ± 0.16b 1.19 ± 0.09a 1.71 ± 0.16b 1.89 ± 0.09b 1.67 ± 0.18b

AST (U/L) 34.90 ± 0.95a 47.25 ± 4.77c 37.88 ± 1.81a,b 35.14 ± 1.01a 43.00 ± 1.90b,c

ATL (U/L) 13.40 ± 0.54a 20.00 ± 1.24b 15.28 ± 1.62a 16.00 ± 0.88a 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 2Effects of ursolic acid supplementation on intraperitoneal glucose and insulin tolerance tests in STZ/NA-induced diabetic mice*.

NonDM DM DM- lowUA DM- highUA DM-Metformin

IPGTT (mmol/L)0 min 7.22 ± 0.24a 16.95 ± 1.71c 11.72 ± 1.38b 12.35 ± 1.99b 11.89 ± 0.83b

30 min 10.76 ± 0.53a 23.48 ± 2.09c 18.70 ± 1.67b 20.63 ± 1.32b,c 19.20 ± 1.21b

60 min 9.59 ± 0.46a 24.26 ± 2.48c 17.30 ± 1.67b 18.76 ± 1.77b 17.41 ± 1.00b

120 min 7.05 ± 0.14a 19.75 ± 1.56c 14.65 ± 1.86b 15.02 ± 1.73b 13.43 ± 1.56b

IPITT (mmol/L)0 min 6.86 ± 0.32a 16.09 ± 1.44c 12.18 ± 1.20b 12.41 ± 1.18b 9.88 ± 1.19a,b

30 min 3.75 ± 0.26a 8.09 ± 0.64c 5.98 ± 0.53b 6.13 ± 0.54b 5.24 ± 0.62a,b

60 min 2.45 ± 0.22a 5.49 ± 0.52c 3.87 ± 0.39b 3.98 ± 0.39b 3.91 ± 0.64b

3.47 ± 0.17b 3.06 ± 0.25b 3.48 ± 0.50b

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.3. Plasma leptin concentration and aminotransferase activity

The plasma leptin concentration in the untreated diabeticice was significantly lower than that in the non-diabetic group.owever, UA and metformin significantly elevated the leptin con-entration compared to the untreated diabetic group (Table 1). Bothoses of UA effectively suppressed the increase in the plasma ASTnd ALT activities in the non-obese type 2 diabetic mice. Metforminlso significantly lowered the plasma ALT activity compared to thentreated diabetic group (Table 1).

.4. Insulin concentrations in plasma and pancreas

The plasma and pancreatic insulin concentrations in thentreated diabetic mice were 21% and 32%, respectively, of those inon-diabetic mice (Fig. 3). Both doses of UA significantly elevatedhe plasma insulin concentrations compared to the untreated dia-etic group; however, metformin did not alter the plasma insulinoncentration (Fig. 3A). The pancreatic insulin concentration wasignificantly higher in the UA- and metformin-supplementedroups than in the untreated diabetic group (Fig. 3B).

.5. Relative thymus weight and cellular subpopulations

The thymic index was calculated as organ weight (mg)/bodyeight (g) × 100, and it is presented in Fig. 4A. The relative thy-us weight in the untreated diabetic mice was significantly lower

ompared to the non-diabetic mice. However, both doses of UA andetformin supplementation suppressed the thymic atrophy in the

on-obese type 2 diabetic mice.When thymocyte subpopulations were evaluated by flow

ytometry analyses, the numbers of the double-positive immaturecells (CD4+CD8+) in the diabetic mice were significantly lower

han that in the non-diabetic mice; in contrast, double-negativeCD4−CD8−) and single-positive (CD4+ or CD8+) subpopulationsere significantly increased in the untreated diabetic group.owever, supplementation with both doses of UA or metformin sig-ificantly 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 asthe 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

F ymocyte T-lymphocyte subpopulations (B) in STZ/NA-induced diabetic mice. Values aree p < 0.05.

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

.6. Splenic lymphocyte proliferation and cellular subpopulations

Con A-induced splenic T-lymphoctye proliferation was signif-cantly inhibited in the untreated diabetic mice when comparedo the non-diabetic mice; there were no significant differencesetween these groups in LPS-induced B-lymphocyte prolifera-ion (Fig. 5). Both doses of UA significantly improved splenic-lymphocyte proliferation, but UA supplementation had no effectn B-lymphocyte proliferation. Metformin did not affect either T or-lymphocyte proliferation in the non-obese type 2 diabetic miceFig. 5).

The splenic lymphocyte populations of CD4+ (helper T) and CD8+

cytotoxic T) cells were significantly decreased in the untreated dia-etic mice compared to the non-diabetic mice (Table 3). However,A and metformin increased the numbers of CD4+ and CD8+ lym-hocyte subpopulations compared to the untreated diabetic group.he counts of CD19+ cells were not different among the groupsTable 3).

.7. Splenic cytokine production

IL-2 and IFN-� production by splenocytes was lower in the

ntreated diabetic mice than in the non-diabetic mice, whereas IL-production was higher in the untreated diabetic group (Table 4).A supplementation lowered the splenic IL-6 level, whereas met-

ormin significantly increased the IFN-�, IL-6 and TNF-� levelsompared to the untreated diabetic group (Table 4).

able 3ffects of ursolic acid supplementation on splenic lymphocyte subpopulations in STZ/NA-

NonDM DM

T-cells (%)CD4+ 16.72 ± 1.14b 12.44 ± 0.83a

CD8+ 5.90 ± 0.20b,c 3.72 ± 0.22a

B-cells (%)CD19+ 37.51 ± 1.11NS 34.45 ± 2.23

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

A (1 �g/mL) or LPS (10 �g/mL) for 48 h. The data are presented as the stimulationindex (O.D. of stimulated cells/O.D. of negative control cells). Values are expressedas the means ± S.E. The values not sharing a common letter differ significantly atp < 0.05.

4. Discussion

Increasing evidence has suggested that the development of type2 diabetes and its complications are closely associated with the

immune system. In Asians, a number of diabetic patients havenon-obese type 2 diabetes, and 50% of adult non-obese type 2diabetics are latent autoimmune diabetic patients [24]. DepressedT-lymphocyte function has been known to be a result of hyper-glycemia [25] or a toxic side effect of the diabetic agent [26,27].

induced diabetic mice*.

DM- lowUA DM- highUA DM-Metformin

16.84 ± 1.16b 16.88 ± 1.52b 19.68 ± 0.88b

5.04 ± 0.55b 5.10 ± 0.52b 6.32 ± 0.14c

36.91 ± 2.49 40.30 ± 3.72 39.25 ± 2.86

0.05.

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

Table 4Effects of ursolic acid supplementation on splenic cytokine productions in STZ/NA-induced diabetic mice*.

Splenic cytokines (ng/mL) NonDM DM DM-lowUA

DM-highUA

DM-Metformin

IL-2 1.16 ± 0.09b 0.62 ± 0.04a 0.64 ± 0.08a 0.59 ± 0.04a 0.69 ± 0.05a

IFN-� 4.34 ± 0.53b 1.38 ± 0.13a 1.10 ± 0.42a 1.23 ± 0.23a 3.84 ± 0.80b

IL-6 0.77 ± 0.10a 1.27 ± 0.11b 0.58 ± 0.10a 0.64 ± 0.06a 1.76 ± 0.09c

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any traditional medicines or natural compounds from medicinallants have been used to modulate immune function. We pre-iously showed that dietary supplementation with 0.05% (w/w)A can cause immunomodulation in STZ-induced diabetic mice

ed a high-fat diet [14]. The aim of this study was to investigatehether low-dose UA (0.01%, w/w) supplementation improvesyperglycemia and immune function in STZ/NA-induced diabeticice, a non-obese type 2 diabetes animal model [18].This study demonstrated that both doses of UA resulted in the

ecovery of the splenic T-lymphocyte proliferative response andubpopulations in the non-obese type 2 diabetic mice. In STZ/NA-nduced diabetic mice, Con A-induced T-lymphocyte proliferationnd the numbers of single-positive (CD4+ and CD8+) subpopula-ions were significantly lower than those of non-diabetic mice.D4+ T-lymphocytes protect the body from infection by bacteria,iruses and other pathogens [28]. Once activated, T-helper cellsctivate other immune cells such as macrophages, B-lymphocytesnd cytotoxic T-lymphocytes to defend against these infections29]. In this study, regardless of dose, mice receiving UA supple-

entation significantly increased splenic T-cell proliferation andD4+ and CD8+ subpopulations compared to the untreated dia-etic mice. Thus, the increase in T-lymphocytes proliferation inhe mice supplemented with UA was consistent with our pre-ious results [14]. This study indicates the potential stimulatingffects of UA on T-lymphocytes, which may lead to an increasedmmune response. Supplementation with metformin did not affecthe proliferative response of splenic T-lymphocytes, although it didignificantly increase the T-lymphocyte subpopulations (CD4+ andD8+) in STZ/NA-induced diabetic mice. In an in vitro study, met-ormin inhibited Con A-stimulated T-lymphocyte proliferation in

ice [30]. As such, UA may especially affect T-cells in the immuneystem through proliferation and activation of CD4+ and CD8+ cellsn the non-obese type 2 diabetic mice, whereas metformin did notnduce proliferation of T-lymphocytes.

Many pro-inflammatory cytokines cause diabetes-associatedbnormalities, such as insulin resistance, impaired insulin secre-ion, and increased capillary permeability; inflammatory cytokineslso accelerate atherosclerosis [31] and may also cause associatedomplications, such as dyslipidemia, cardiovascular disease andenal failure [32]. Thus, there is a close relationship between themmunological state and the development of type 2 diabetes [33].horvaldson et al. [34] reported that elevated levels of glucosend free fatty acids as well as changes in cytokine production areommon features of both type 1 and type 2 diabetes. The presenttudy observed that IL-2 and IFN-� levels in splenic T-lymphocytesere significantly lower in the diabetic mice compared to theon-diabetic mice, whereas IL-6 levels in B-lymphocytes wereignificantly higher. Both doses of UA normalized IL-6 levels com-ared to the untreated diabetic group. However, supplementationith metformin significantly increased IFN-�, IL-6 and TNF-� lev-

ls compared to the untreated diabetic and UA-supplementedroups. Thus, UA exhibits anti-inflammatory properties in non-bese type 2 diabetic mice, but metformin increased the levelsf pro-inflammatory cytokines, namely IFN-�, IL-6 and TNF-. Both in vitro and in vivo studies have demonstrated that

0.57 ± 0.01a 0.47 ± 0.03a 1.14 ± 0.07b

0.05.

UA suppressed the expression of LPS-induced pro-inflammatorymediators in RAW264.7 mouse macrophages [35] and 12-O-tetradecanoylphorbol-13-acetate-induced skin tumor promotion[36].

The majority of cells located in peripheral blood, spleen andlymphoids are dependent on the thymus for proper differentia-tion. Interestingly, we found that both doses of UA and metforminrestored depressed relative thymus weight by hyperglycemia. Thethymus is the primary lymphoid organ that is crucial for thedevelopment of immature T-cells into cells that execute immunefunctions in the periphery against several pathological conditions[37]. Many researchers have shown that thymic atrophy may becaused by diabetes [37,38]. Dagistanli et al. [39] reported thatthymic atrophy is caused by elevated intracellular calcium lev-els leading to apoptosis in STZ-induced diabetes. In this study,we also found that the relative thymus weight was dramaticallylowered by 61% in STZ/NA-induced diabetic mice compared to thenon-diabetic group; however, 0.01% UA, 0.05% UA and metforminsupplementation significantly improved thymic atrophy by 1.9-,1.5- and 1.7-fold, respectively, compared to the untreated diabeticgroup, which indicated that UA and metformin may enhance theimmune response. Furthermore, hyperglycemia is toxic to multi-ple immune cell populations, including lymphocytes [9]. Therefore,we determined whether UA or metformin might affect the thymiccellular differentiation in non-obese type 2 diabetic mice. The rel-ative thymic CD4+CD8+ subpopulation was significantly lower inthe untreated diabetic group than in the non-diabetic group. Thisresult was in agreement with previous works [38,40]. The loss ofthymic double-positive T cells has been shown to occur in other dis-eases, mainly infectious diseases [41,42]. T-cell precursors come tothe thymus from bone marrow via the bloodstream and undergodevelopment to mature T-cells. The thymocytes early in devel-opment lack detectable CD4 and CD8, which they are referred toas double-negative (CD4−CD8−) cells. CD4 and CD8 coreceptorsare then expressed; this double positive (CD4+CD8+) stage is aperiod of rapid proliferation. Double positive thymocytes developinto mature single-positive CD4+ thymocytes or single-positiveCD8+ thymocytes. These single-positive cells undergo additionalnegative selection and migrate from the cortex to the medulla,where they pass from the thymus into the peripheral immune sys-tem [43]. These results indicated that increases in the numbers ofCD4+ and CD8+ of splenic T-lymphocytes in the UA or metformin-supplemented group might be associated with the increased in thethymic CD4+CD8+ subpopulation.

Recently, it has been shown that leptin is controlled by glu-cose, insulin, glucocorticoids and cytokines and that leptin playsan important role in thymic organization and maintenance [44,45].Leptin-knockout mice or mice without the functional leptin recep-tor 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 leptinlevels in diabetics were associated with thymic atrophy [46,47]. Inthis study, we found that UA and metformin supplementation ele-vated plasma leptin levels along with the insulin concentration innon-obese type 2 diabetic mice. Plasma leptin levels were positively

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orrelated with plasma insulin levels (r = 0.382, p < 0.01) (data nothown).

Elmas et al. [40] reported that the thymus is ultrastructurallyffected by diabetes and concomitant fasting and that insulinreatment was able to reverse these pathological changes. Someesearchers have suggested that almost all of the benefits fromntensive insulin therapy may be attributed to its effect in lower-ng blood glucose concentration [7]. Other studies have shown thatnsulin is not only the main anabolic and anti-catabolic hormoneut also a direct regulator of inflammation and inmmunocom-etence [48]. In this study, both doses of UA and metforminignificantly lowered the fasting and non-fasting blood glucose lev-ls. Thus, low-dose UA exhibited a blood glucose-lowering effect inhe non-obese type 2 diabetic mice. In this study, despite plasmansulin concentrations being slightly lower in the metformin grouphan the UA group, insulin sensitivity was similar. Metformin was

ore efficient than UA at lowering non-fasting blood glucose lev-ls but not fasting glucose, glucose tolerance and insulin tolerance.oshida et al. [49] also reported that metformin decreased plasmalucose more effectively in the fed state than in the fasted stateithout insulin secretion in GK rats, a non-obese type 2 diabetic

nimal model. Glucose utilization and disposal is a major determi-ant of the plasma glucose levels in the fed state [50]. In addition,etformin but not UA lowered daily food intake in non-obese typediabetic mice. The pancreatic insulin concentration was higher

n the low-dose UA-, high-dose UA- and metformin-supplementedroups; however, the plasma insulin level was only higher in the UAroup than in the untreated diabetic mice. These results support theypothesis that metformin decreases plasma glucose by activatinglucose utilization independent of insulin secretion [49].

UA is well known for its hepatoprotective effect on acutehemically-induced liver injury as well as liver fibrosis and cirrhosis51]. Insulin decreases the serum concentrations of liver enzymesAST and ALT), indicating that insulin alleviates liver injury andmproves the production of liver constructive proteins, such aslbumin, that lead to the preservation of liver function [52]. SerumST or ALT is generally used primary screening parameters for liverisease. Therefore, we assessed the activities of AST and ALT in thistudy. Mice with UA and metformin supplementation exhibited sig-ificantly lower plasma AST and/or ALT activities compared to thentreated diabetic group. The beneficial effects of UA on the liverould be due to its antioxidant and anti-inflammatory effects [53].

. Conclusion

Our findings suggested that UA and metformin may enhancemmune function by suppressing thymic atrophy and acting uponmmune cells, and low-dose UA may be an efficient anti-diabeticgent that increases plasma and pancreatic insulin concentrationsn the non-obese type 2 diabetic patients.

onflict of interest

There are no conflicts of interest.

cknowledgements

This work was supported by National Research Foundation oforea Grant funded by the Korean Government (KRF-2007-331-00058).

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