Jeju Ground Water Attenuates Adipogenesis in 3T3-L1 Preadipocytes

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231 책임저자：유호진, 󰂕 501-759, 광주시 서석동 375

조선대학교 의과대학 약리학교실, 생물신소재학교실 Tel: 062-230-6337, Fax: 062-230-6586

접수일：2011년 9월 1일, 1차 수정일：2011년 9월 6일, 2차 수정일：2011년 9월 8일, 게재승인일：2011년 9월 15일

Correspondence to：Ho Jin You

Departments of Pharmacology, Bio-meterials, Chosun University School of Medicine, 375, Seosuk-dong, Gwangju 501-759, Korea

Tel: +82-62-230-6337, Fax: +82-62-230-6586 E-mail: [email protected]

Jeju Ground Water Attenuates Adipogenesis in 3T3-L1 Preadipocytes

Seon-Joo Park^1,2, Jin-Won Hyun³ and Ho Jin You^1,2

Departments of ¹Pharmacology and ²Bio-materials, Chosun University School of Medicine, Gwangju 501-759,

3Department of Biochemistry, College of Medicine, Cheju National University, Jeju 690-756, Korea

To investigated the inhibitory effect of Jeju ground water on adipogenesis, 3T3-L1 preadipocyte cells were cultured and differentiated in media made by Jeju ground water and deionized water (DW) containing a mixture of dexamethasone, isobutylmethylxanthine (IBMX) and insulin. Oil Red O staining showed that lipid accumulation attenuated in adipocyte cells treated with Jeju ground water. The mRNA expression levels of peroxisome-activated receptorγ (PPARγ) and CCAAT-enhancer binding proteinα (C/EBPα), which are a key components of the transcriptional control of adipogenesis, were decreased on adipocyte expose to Jeju ground water. Furthermore, the mRNA expression levels of target genes of these factors, such as adipocyte fatty acid (aP2), lipoprotein lipase (LPL) and Leptin, were also decreased by Jeju ground water treatment compared with control water. These results suggest that Jeju ground water containing vanadium components suppressed 3T3-L1 adipocyte differentiation (Cancer Prev Res 16, 231-237, 2011) Key Words: Jeju ground water, Adipogenesis, Adipocyte differentiation, Lipid

INTRODUCTION

Obesity is a prevalent nutrition-related health risk in world- wide and is mostly associated with a lot of pathological dis- orders, type 2 diabetes, hypertension, cancer, and heart disease.^1,2) Obesity results from an excessive accumulation of adipose tissue composed of differentiated adipocyte cells leading to accumulation a larger amount of fat and enlarge adipocyte size.^3,4) Adipogenesis has been extensively studied using in vitro model systems in several adipogenic cell lines. The remarkable characteristic of adipogenesis, is the morphological modification in cells as become rounded in shape. Indeed, adipogenesis is accompanied by increased expression of various transcriptional factors and adipocyte-specific genes such as C/EBPα and PPARγ^5,6) Cross-regulation between both genes is a key component of the transcriptional control of adipogenesis. These fac-

tors direct final phase of adipogenesis by activating expression of adipocyte-specific genes, such as regulate adipocyte differentiation by modulating the expression of their target genes in a coordinated fashion.^7∼10)

Inhibition of adipocyte differentiation is a one of therapeutic trials for prevention and treatment of obesity. Recently, many studies investigates the inhibitory effects of natural product on adipocyte differentiation. To understand the cellular and molec- ular mechanism of function of Jeju ground water on adipocyte differentiation, mouse embryo fibroblast 3T3-L1 cells were differentiated to adipocytes with adipogenic medium made by Jeju ground water containing insulin, dexamethasone, IBMX. The quantitative real-time PCR and western blot demonstrated that Jeju ground water significantly decreased the expression levels of PPARγ and C/EBPα and other transcriptional factor target genes.

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Table 1. Gene-specific primers used for real-time PCR

Gene Forward primer Reverse primer

PPARγ C/EBPα Pref-1 aP2 LPL Leptin TNF-α β-actin

GGTGAAACTCTGGGAGATTC AGGTGCTGGAGTTGACCAGT CTAACCCATGCGAGAACGAT TCACCTGGAAGACAGCTCCT TCCAAGGAAGCCTTTGAGAA GGATCAGGTTTTGTGGTGCT AGGCCTTGTGTTGTGTTTCCA CCACAGCTGAGAGGGAAATC

CAACCATTGGGTCAGCTCTT CAGCCTAGAGATCCAGCGAC GCTTGCACAGACACTCGAAG AATCCCCATTTACGCTGATG CCATCCTCAGTCCCAGAAAA TTGTGGCCCATAAAGTCCTC TGGGGGACAGCTTCCTTCTT AAGGAAGGCTGGAAAAGAGC MATERIALS AND METHODS

1. Reagents

Jeju ground water was provided by the Jeju special self-gov- erning province development corporation (Jeju, Korea). Dexa- methasone, isobutylmethylxanthine, insulin and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO). TRIzol reagent and SuperScript First-Strand Synthesis System for RT-PCR kit were purchased from Invitrogen (Carlsbad, CA).

Brilliant II SYBR Green QPCR Master Mix was obtained from Stratagene (La Jolla, CA). Antibodies were purchased from Cell Signaling (Beverly, MA) and horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Sigma-Aldrich.

2. Cell culture and differentiation

Mouse 3T3-L1 preadipocyte cells were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM (Gibco-BRL, Grand Island, NY, USA) containing 1% penicillin-streptomycin and 10% bovine calf serum (BCS, Gibco-BRL) at 37^oC in a humidified atmosphere of 5%

CO2. For cell viability assay, 3T3-L1 preadipocyte cells were cultured with DW containing 10% BCS. Jeju ground water for 10 passage and then seeded in a 96 well plate at a concentration of 1×10⁵ cells/ml, and incubated for an additional 24, 48 h at 37^oC. 10μl of MTT solution (5 mg/ml) were added to each well of a total reaction volume of 100μl followed by 3 h. Formazan crystals were dissolved in isopropanol containing hydrochloric acid and measured the absorbance at 570 nm. To induce differentiation, 3T3-L1 cells were grown until reaching confluency, and incubated with a mixture of 0.25 M dexamethasone, 0.5 mM IBMX and 1 g/ml insulin in DMEM made by DW or Jeju ground water containing 10% FBS. After

2∼3 days, the induction media were replaced by 10%

FBS/DMEM supplemented with 1μg/ml insulin alone for 2 additional days and then the adipocytes were kept in media containing 10% FBS until experimentation.

3. Oil Red O staining

After the induction of differentiation, the cells were washed twice with PBS, fixed with 10% formalin at room temperature for 1 h, washed with 60% isopropanol, completely dried and then stained with Oil Red O solution (six parts 0.5% Oil Red O in isopropanol and four parts water) for 1 h. The images were photographed using microscope (Nikon). The stained lipid droplets were dissolved in isopropanol for 10 min and quantified by measuring the absorbance at 500 nm.

4. Real-time quantitative RT-PCR

Total RNA was extracted from 3T3-L1 adipocytes using Trizol reagent according to the manufacturer’s instruction. The cDNA was synthesized from 1μg total RNA with SuperScript First-Strand Synthesis System for RT-PCR kit. The quantitative real-time PCR was performed with Brilliant II SYBR Green QPCR Master Mix using the MX3000P real-time PCR system (Stratagene). The specific primer sets for adipogenic marker are listed in Table 1. and relative amounts of mRNA levels were calculated using the comparative cycle threshold (CT) method.¹¹⁾

5. Western blot analysis

3T3-L1 cells were washed twice with PBS and lysed in lysis buffer containing 25 mM Tris-HCl (pH 7.2), 0.1% sodium do- decyl sulfate (SDS), 0.1% Triton X-100, 1% sodium deoxy- cholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM sodium orthovanadate, 1 mM phenylmethyl-

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Fig. 1. Cytotoxicity of Jeju ground water on 3T3-L1 preadipocytes. Cells (1×10⁴ cell/well) were incubated with DW or Jeju ground water for 24 h, 48 h. Cell viability was determined by MTT assay. The result represent the means±SD of data from three experiments.

sulfonylfluoride (PMSF), 10μg/ml aprotinin, and 5μg/ml leu- peptin for 20 min on ice. Equal protein amounts of cell lysate were separated on a 12% SDS-polyacrylamide gel, transferred to a polyvinylidene fluoride (Pall corporation, East Hills, NY).

Antibodies against PPARγ, C/EBPα and adiponectin were used as the primary antibodies, and horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody. The interesting proteins were visualized using an en- hanced chemiluminescence-based detection system (Amersham- Pharmacia Biotech, Piscataway, NJ, USA). For detect the secreted adiponectin in the culture media, differentiated 3T3-L1 adipocytes after 10 days of induction were changed with serum free media. Collected culture media was determined the secretion of adiponectin using western blot analysis.

6. Statistical analysis

Student’s t test and one-way analysis of variance were used to analyze the differences between values obtained in the various experimental and control conditions. p＜0.05 was consid- ered significant.

RESULTS

1. Cytotoxicity of Jeju ground water

The effect of Jeju ground water on 3T3-L1 preadipocytes cell viability and cytotoxicity were investigated by MTT assay.

The cells were cultured with DW or Jeju ground water for

24, 48 h. As shown in Fig. 1. Jeju waters had no significant cytotoxicity against preadipocytes.

2. Inhibitory effects of Jeju ground water on 3T3-L1 adipocyte differentiation

To investigate the effect of Jeju ground water on adipocyte differentiation, 3T3-L1 preadipocyte cells were incubated with culture media made by DW or Jeju ground water each containing dexamethasone, IBMX and insulin for 10 days. The cells were induced differentiation to adipocyte, therefore the morphological changes were observed a rounded phenotype due to the accumulation of lipid droplets in adipocytes. As shown in Fig. 2A, Jeju ground water was shown the reduction of lipid droplets accumulation compared with DW. To confirm these observation, differentiated cells were stained with Oil Red O solution, and then quantified by measuring the absorbance. The staining results revealed that Jeju ground water significantly decreased the cell differentiation (Fig. 2B). Theses results showed the inhibitory effects of Jeju waters on 3T3-L1 adipocyte differentiation.

3. Effects of Jeju ground water on expression of adipogenic transcription factors

PPARγ and C/EBPα is well known as a adipogenic transcriptional factors and show that cross-regulation between both genes is a key component of the transcriptional control of adipogenesis.^12,13) The effect of Jeju ground water on the regulation of PPARγ and C/EBPα expression was analyzed by quantitative real-time PCR. As shown in Fig. 3A, Expression levels of PPARγ and C/EBPα were reduced in Jeju ground water incubated 3T3-L1 adipocytes. We also examined PPARγ and C/EBPα protein expression level by western blotting. In consist of above results, the expression level of PPARγ and C/EBPα protein were also diminished by Jeju ground water (Fig. 3B). These results suggested that Jeju ground water on differentiation of 3T3-L1 preadipocyte to adipocytes through the down-regulation of PPARγ and C/EBPα.

4. Effect of Jeju ground water on expression of adipogenic transcription factor target genes

We further studied whether Jeju ground water regulates the expression of the adipogenic transcription factor target genes adipocyte fatty acid binding protein (aP2), lipoprotein lipase (LPL) and leptin, as well as the preadipocyte factor-1 (Pref-1)

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Fig. 2. Effect of Jeju waters on differentiation of 3T3-L1 adipocytes. (A) Morphological changes were photographed the cell at 10 days after induction of differentiation with DW or Jeju ground waters, respectively. (B) Cells were stained with Oil Red O staining for intracellular lipid accumulation. The results represent the means±SD of data from ten se- parate experiments. *p＜0.05 compared with cells treated with DW.

gene. Pref-1 predominantly expressed in undifferentiated 3T3-L1 cells. Whereas the mRNA level of Pref-1 which predominantly expressed in undifferentiated state was up-regulated, the mRNA levels of aP2, LPL, Leptin were significantly down-regulated by Jeju ground water during adipocyte differentiation (Fig. 4). In addition, adipocytes produce and secrete signaling molecules such as tumor necrosis factor-α (TNFα).

TNFα were also decreased in adipocyte treated with Jeju ground water compared with DW.

5. Effect of Jeju ground water on expression and secretion of adiponectin

Adiponectin is one of multiple adipocytokines secreted by adipose tissue and has been shown to promoters adipocyte differentiation, insulin sensitivity, and lipid accumulation.¹⁴⁾ To determine the expression of adiponectin in adipocyte differentiation induce by Jeju ground water, we assessed protein expression level of adiponectin in cell and culture media by western blot analysis. As shown in Fig. 5, the adiponectin concentration of the media decreased, consistent with the result of the reduction of cellular adiponectin expression in Jeju ground

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Fig. 3. Effect of Jeju ground water on adipogenic transcription factors during differentiation of adipocytes. (A) Expression of PPARγ and C/EBPα mRNA from 3T3-L1 adipocyte cells were measured by quantitative real-time PCR using specific primers. The analysis were performed duplicate experiments (n=9). *p＜0.05, **p＜0.01 compared with cells treated with DW. (B) Western blot analysis were performed with antibodies against PPARγ and C/EBPα. β-actin was used as loading control for same amounts of protein.

water compared with DW.

DISCUSSION

Obesity is most commonly caused by a combination of excessive dietary calories, lack of physical activity, and genetic susceptibility. The most effective treatment for obesity of diet-

ing and physical exercise. Recently, several reports show that natural products have potential as anti-obesity agents that may not have some harmful side-effects.^15∼18)

Vanadium is a well-known antidiabetic metal agent which mimics most of the actions of insulin on mature adipocyte and can also exert anti-tumor effects against chemical carcinogenesis in animals and in various types of malignant cell lines.¹⁹⁾ Several researchers have studied about the effect of vanadium in treatment of adipogenesis as anti-adipogenic agents.^20∼22) Vanadium was found in various natural sources such as vegeta- bles and food.²³⁾

Vanadium is relatively exist at high concentration (26.0±2.0 μg/l) in Jeju ground water obtained from Jeju island in South Korea. It was reported that the Jeju ground water possessed antioxidant effect via ROS scavenging.²⁴⁾ However, the anti-adipogenic mechanisms of Jeju ground water in adipocytes have not yet been investigated. In this study, we demonstrate the effect of Jeju ground water containing vanadium components on 3T3-L1 adipocyte differentiation. Adipocyte differentiation is a multiple process of programmed changes in specific gene expressions. PPARγ and C/EBPα are transiently expressed on early stage for initiating differentiation of preadipocytes. C/EBPα is induced relatively late during adipogenesis and cross-regulation between PPARγ and C/EBPα is important in maintaining the differentiated state.^9,10) Activation of PPARγ induces the expression of genes controlling adipocyte fatty acid metabolism, including aP2, LPL.^25,26) Here we show that Jeju ground water treatment significantly decreased the expression levels of PPARγ and C/EBPα and their adipogenic target genes. Adiponectin is expressed by mature adipocytes and is the most abundant circulating adipokine. Indeed, adiponectin can promote adipocyte differentiation through au- tocrine effects results in increasing lipid content and insulin sensitivity. Jeju ground water was also shown the down-regulation of the expression and secretion of adiponectin on adipocyte differentiation.

CONCLUSION

Taken together, these results indicate that suppression of adipocyte differentiation by Jeju ground water treatment is mediated through down-regulation of the adipogenic transcription factors, PPARγ and C/EBPα and these target genes. It suggests that the ability of Jeju ground water to in-

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Fig. 5. Effect of Jeju ground water on expression of adiponectin. The amounts of adiponectin protein in cell and culture media were assessed by western blot analysis. The graph represents the band intensity of secreted adiponectin.

Fig. 4. Effect of Jeju ground water on Pref-1, aP2, LPL, Leptin and TNFα mRNA expression in 3T3-L1. Cells were cultured in the medium containing DW, Jeju ground water. The data represent the mean±SD of three independent experiments. The analysis were performed duplicate experiments (n=9). *p＜0.05, **p＜0.01 compared with cells treated with DW.

hibit adipocyte differentiation may be contributed as the anti-obesity resource.

ACKNOWLEDGEMENT

This study was supported by the Ministry of Knowledge Economy (MKE), the Korea Institute for Advancement of Technology (KIAT) and Jeju Leading Industry Office through the Leading Industry Development for Economic Region.

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