Intracellular lipid accumulation inhibitory effect of Weissella koreensis OK1-6 isolated from Kimchi on differentiating adipocyte

Authors

  • Y.J. Moon,

    1. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea
    2. Jeonju Makgeolli Research Center, Chonbuk National University, Jeonju, South Korea
    Search for more papers by this author
  • J.R. Soh,

    1. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea
    Search for more papers by this author
  • J.J. Yu,

    1. Department of Food and Biotechnology, Woosuk University, Jeonbuk, South Korea
    Search for more papers by this author
  • H.S. Sohn,

    1. Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea
    2. Jeonju Makgeolli Research Center, Chonbuk National University, Jeonju, South Korea
    Search for more papers by this author
  • Y.S. Cha,

    Corresponding author
    1. Jeonju Makgeolli Research Center, Chonbuk National University, Jeonju, South Korea
    • Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea
    Search for more papers by this author
  • S.H. Oh

    Corresponding author
    1. Department of Food and Biotechnology, Woosuk University, Jeonbuk, South Korea
    • Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea
    Search for more papers by this author

Correspondence

Youn-Soo Cha, Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 561-756, South Korea. E-mail: cha8@jbnu.ac.kr

and Suk-Heung Oh, Department of Food Science and Biotechnology, Woosuk university, 443 Samnye-ro, Samnye-eup, Wanju-gun, Jeollabuk-do, 565-701, South Korea. E-mail:shoh@woosuk.ac.kr

Abstract

Aims

To investigate the intracellular lipid accumulation inhibitory effect of spent culture medium extract and the cytoplasmic fraction of Weissella koreensis OK1-6 cells isolated from kimchi in differentiating 3T3-L1 cells.

Methods and Results

Differentiating 3T3-L1 cells were treated with either cytoplasmic fraction of W. koreensis OK1-6 cells or its spent media for 4 days. Both the spent culture medium extract and cytoplasmic fraction of W. koreensis OK1-6 cells significantly decreased the triglyceride concentration and intracellular lipid accumulation in the treated groups compared with the control group. The mRNA expression levels of C/EBP-α, one of the major transcriptional factors involved in adipocyte differentiation, were significantly less expressed in 3T3-L1 cells treated with the spent medium and cytoplasmic fraction. The expressions of aP2, fatty acid synthase (FAS) and SREBP1 genes were also decreased significantly.

Conclusions

These results suggested that Wkoreensis OK1-6 could play a crucial role in preventing intracellular lipid accumulation by down-regulating the expression of adipocyte-specific genes C/EBPα, aP2, SREBP1 and FAS.

Significance and Impact of the Study

These results may contribute to nutraceutical and food industries in developing probiotic-based therapies for the treatment and prevention of obesity.

Introduction

Kimchi is one of the well-known Korean traditional fermented foods. Kimchi is also known as a functional food rich in lactic acid bacteria (LAB), the major group of bacteria that grows on kimchi. Among LAB found in kimchi includes Lactobacillus plantarum, Lactobacillus sake, Leuconostoc mesenteroides, Leuconostoc lactis, Leuconostoc citreum, Pediococcus pentosaceus, Weissella cibaria, Weissella confusa, Weissella koreensis, etc. (Lee et al. 2002; Cho et al. 2006; Lee and Lee 2010). Probiotic effects of kimchi LAB such as anticarcinogenic, immunomodulatory, antimicrobial, antidiarrhoeal and anti-allergy activities have been reported by several authors (Park 2004; Kim 2005; Kim et al. 2005). These also can be beneficial for the intestinal microflora of humans and animals (Han 1991; Seok et al. 2008). In fact, if a person consumes 300 g of fermented and uncooked kimchi per day, it can increase the colonic LAB above 100-fold comparing with a person who did not consumed kimchi (Park 2004; Oh et al. 2008). In addition, various clinically important amino acids such as γ-aminobutyric acid (GABA) and ornithine are produced as a result of active growth of LAB during fermentation and storage of kimchi (Cho et al. 2007; Seok et al. 2008; Yu et al. 2009).

LAB W. koreensis strains have been isolated from kimchi and characterized (Lee et al. 2002; Yu et al. 2009). W. koreensis is a psychrophilic bacterium and is the dominating species in kimchi produced at −1°C (Cho et al. 2006). Therefore, this dominant species in kimchi produced at −1°C have important roles for kimchi's flavour and taste (Lee 2007). Recently, it has been reported that W. koreensis OK1-6 isolated from kimchi showed ornithine-producing capacity in MRS medium supplemented with 1% arginine (Yu et al. 2009) and showed a growth rate of over 80% in 6% NaCl added MRS medium (Yu and Oh 2010).

l-Ornithine is a non-protein amino acid found in foods such as brackish water bivalve (Uchisawa et al. 2004), wine and cheese (Arena et al. 1999; Liu et al. 2003), and kimchi (Kim 2010). l-ornithine is a medicinal amino acid and used as a food supplement in USA and Europe (Wernerman and Hammarqvist 1987; Muting and Kalk 1992; Kawai et al. 1999, 2000; Robinson et al. 1999). Ornithine affects the growth hormone release, skeletal muscle protein synthesis, immune system stimulation and obesity prevention, etc (Evain-Brion et al. 1982). Specially, ornithine is known as a medicinal agent having an anti-obesity function through growth hormone release and basal metabolism promotion (Evain-Brion et al. 1982; Elam 1988).

Among many bioactive materials found in kimchi, capsaicin derived from red pepper powder has been proposed as an effective component for fat digestion (Choo 2000) and thus as an anti-obesity candidate in kimchi. Recently, with the fact that kimchi contains ornithine-producing W. koreensis strains (Yu et al. 2009), a dominant species produced at −1°C ‘kimchi refrigerator’ (Cho et al. 2006), raises interest on W. koreensis strains as anti-obesity LAB. However, there is little information available on the anti-obesity effects of kimchi LAB and its spent medium. Thus, we investigated the effect of ornithine-producing W. koreensis strains on intracellular lipid accumulation and adipocyte-specific gene expression using the spent medium and cytoplasmic fraction of W. koreensis OK1-6 cells on 3T3-L1 cell line.

Materials and methods

3T3-L1 cell culture

3T3-L1 cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM high glucose) (Lonza, Walksville, MD, USA) supplemented with 10% bovine serum (BS) (Gibco, Grandisland, NY, USA). Two day after postconfluency, adipocyte differentiation was initiated for 2 days using differentiation medium cocktail containing 0∙25 mmol 1−1 dexamethasone, 0∙5 mmol 1−1 3-iso-butyl-1-methylxanthine and 10 μg ml−1 insulin (Sigma-Aldrich, St. Louis, MO, USA) in DMEM supplemented with 10% FBS. Then, cells were cultured for two more days in media containing 5∙0 μg ml−1 of insulin for the completion of differentiation.

Weissella koreensis OK1-6 spent culture medium extract and cytoplasmic fraction

The W. koreensis OK1-6 strain, which was isolated from kimchi and grown in the laboratory condition, was inoculated into MRS broth (pH 6·5) containing (4%, v/v) ± 1% (w/v) of arginine and incubated at 30°C for 72 h and centrifuged for 20 min at 5000 g. The collected supernatants (spent media) and cells were then freeze-dried, dissolved in distilled water to give a concentration of 100 mg ml−1. The samples were sonicated using a sonicator (Fisher Scientific Co., Toronto, ON, Canada) for 2 min followed by 3-min interval (repeated for five times). The suspensions were centrifuged at 1100 g for 15 min at 4°C. The collected supernatants were used as W. koreensis OK1-6 samples for the assays.

Measurement of triglyceride content

Upon differentiation of 3T3-L1 cells with DMI (a cocktail of dexamethasone, 3-isobutyl-1-methylxanthine and insulin), the 3T3-L1 cells were then immediately cultured with 1·0 μg ml−1 of W. koreensis OK1-6 samples for additional 2 days. Triglyceride content was determined using commercially available triglyceride assay kit (Asan pharmaceutical, Seoul, Korea), according to the manufacturer's protocol. The protein concentration was determined using a Bradford reagent (Bio-Rad, Hercules, CA, USA).

Measurement of intracellular lipid accumulation

Intracellular lipid accumulation was measured using Oil-red-O (Sigma, St Louis, MO, USA) staining. After differentiation, the 3T3-L1 cells were then immediately further cultured with 1·0 μg ml−1 of the sample for additional 4 days. After washing cells with PBS, cells were fixed with 10% formaldehyde (Sigma-Aldrich) for overnight and rinsed with 60% isopropanol. Then, cells were stained with 3 mg ml−1 of Oil-red-O for 1 h, and then, the stain solution was removed. After rising with 60% isopropanol, cells were washed with sterile water and observed under a microscope (Olympus, Tokyo, Japan). In addition, the dye was eluted for 1 h with 60% isopropanol. The absorbance (OD 500 nm) was measured by a spectrophotometer (Shimadzu, Kyoto, Japan). The percentage of Oil-red-O-stained material relative to control cells without W. koreensis OK1-6 samples was calculated as A500 nm [LAB sample]/A500 nm [control] × 100.

Measurement of adipocyte-specific gene expression

Total RNA was extracted by trizol reagent; the concentration and the quality of RNA were determined by absorbance at 260–280 nm, respectively. For real-time PCR, 1·0 μg of extracted RNA was reverse transcribed into first-stand cDNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). Amplification was then performed in a cDNA mixture on a 7500 real-time PCR system (Applied Biosystems) using SYBER Green PCR Master Mix (Applied Biosystems, Woolston, Warrington, UK), give a final reaction volume of 20 μl, according to the manufacturer's protocol. PCR was performed with the primers indicated in Table 1.

Table 1. List of probes for genes used in RT-PCR
GeneSense probe sequenceAntisense sequence
  1. PCR was performed using the probes indicated below under optimal amplification conditions (95°C for 10 min; 40 cycles of 95°C for 15 s, 51–58°C for 20 s, 72°C for 35 s) for each gene. The PCR amplification of each cDNA was performed independently using samples in triplicate.

aP25′-CAACCTGTGTGATGCCTTTGTG-3′5′-CTCTTCCTTTGGCTCATGCC-3′
Lipoprotein lipase5′-GCATTTGAGAAAGGGCTCTG-3′5′-CTGACCAGCGGAAGTAGGAG-3′
β-actin5′-TGTTACCAACTGGGACGACA-3′5′-CTCTCAGCTGTGGTGGTGAA-3′
Peroxisome proliferator-activated receptor-γ5′- GCTGTTATGGGTGAAACTCTG-3′5′- ATAAGGTGGAGATGCAGGTTC-3′
SREBP15′- GCGGAGCCATGGATTGCAC-3′5′-CTCTTCCTTGATACCAGGCCC-3′
Fatty acid synthase5′-TCCACCTTTAAGTTGCCCTG-3′5′-TCTGCTCTCGTCATGTCACC-3′
C/EBP-α5′- AGCAACGAGTACCGGGTACG-3′5′-TGTTTGGCTTTATCTCGGCTC-3′

Analysis of ornithine content

Ornithine contents in the spent medium and cytoplasmic fraction of W. koreensis OK1-6 strain were measured by TLC and HPLC analyses as described (Yu et al. 2009). For TLC analysis, silica gel 60 F254 (Merck, Damstadt, Germany), standard ornithine (Sigma-Aldrich), solvent mixture (butanol/acetic acid/dichloromethanol/ water = 5 : 3 : 3 : 3) were used. For HPLC analysis, a 3∙9 × 150 mm AccQ Tag (Nova-Pak C18; Waters Co., Milford, MA, USA) column at 37°C was used, along with mobile phases (AccQ·Tag Eluent A and 60% acetonitrile) with a 1∙0 ml min−1 flow rate and a fluorescence detector (Waters Co.). Ornithine contents were calculated using an ornithine standard (Sigma-Aldrich) based on a standard curve.

Statistical analysis

Data from individual experiments are expressed as then mean ± SD deviation. The data were analysed by one-way anova using the SPSS 12.0 program, and the differences between the means were assessed using Duncan's multiple range test. Statistical significance was considered at P < 0∙05.

Results

Effects of the spent culture medium extract and cytoplasmic fraction of Weissella koreensis OK1-6 on triglyceride and lipid accumulation

The effects of spent culture medium extract and cytoplasmic fraction of W. koreensis OK1-6 on the inhibition of intracellular triglyceride in 3T3-L1 adipocyte are shown in Fig. 1. The data showed that both the cytoplasmic fraction (S3) and spent culture medium extract (S4) inhibited intracellular triglyceride around 20 and 40% of the control, respectively. As shown in Fig. 2, both the spent medium and cytoplasmic fraction of W. koreensis OK1-6 cultured in MRS broth with 1% arginine added also reduced lipid accumulation up to 32 and 28% of the control, respectively, in Oil-red-O-stained 3T3-L1 adipocyte.

Figure 1.

Effect of cytoplasmic fraction and spent medium of Weissella koreensis OK1-6 on triglyceride accumulation in 3T3-L1 adipocyte. Cytoplasmic fraction (S1) and cell-free cultured medium (S2) of W. koreensis OK1-6 cultured without arginine added; cytoplasmic fraction (S3) and cell-free cultured medium (S4) of W. koreensis OK1-6 cultured with 1% arginine. Values with different superscripts are significantly different by anova with Duncan's multiple range tests at P  < 0∙05.

Figure 2.

Effect of Weissella koreensis OK1-6 cytoplasmic fraction and spent medium of W. koreensis OK1-6 on lipid accumulation. (a) Cells were stained with Oil-red-O to label lipids. (b) After Oil-red-O staining, the dye was eluted from the cells with isopropanol and quantified, and the results were plotted. Cytoplasmic fraction (S1) and cell-free cultured medium (S2) of W. koreensis OK1-6 cultured without arginine added; cytoplasmic fraction (S3) and cell-free cultured medium (S4) of W. koreensis OK1-6 cultured with 1% arginine. Values with different superscripts are significantly different by anova with Duncan's multiple range tests at P  < 0∙05.

Effects of the spent culture medium extract and cytoplasmic fraction of Weissella koreensis OK1-6 on adipogenic-specific gene expression

RT-PCR was performed to analyse the expression level of the adipogenic-specific genes, sterol regulatory element-binding protein 1 (SREBP1), adipocyte fatty acid binding protein (aP2), fatty acid synthase (FAS), peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL) and CCAAT/enhancer binding protein-α (C/EBP-α). Differentiating 3T3-L1 adipocyte treated either with the spent medium extract or with the cytoplasmic fraction of W. koreensis OK1-6 showed a significant reduction in the mRNA expression levels of SREBP1, aP2, FAS and C/EBP-α comparing with the untreated cell (Fig. 3).

Figure 3.

Effect of Weissella koreensis OK1-6 cytoplasmic fraction and spent medium of W. koreensis OK1-6 on mRNA expression in 3T3-L1 cell. Pre (preadipocytes), Con (differentiated control adipocytes), cytoplasmic fraction (S1) and cell-free cultured medium (S2) of W. koreensis OK1-6 cultured without arginine added; cytoplasmic fraction (S3) and cell-free cultured medium (S4) of W. koreensis OK1-6 cultured with 1% arginine. LPL, lipoprotein lipase; SREBP1, sterol regulatory element-binding protein-1c; aP2, adipocyte protein 2; FAS, fatty acid synthase; C/EBP-α, CCAAT/enhancer binding protein-α; PPAR-γ, peroxisome proliferator-activated receptor-γ.

Ornithine contents in the spent culture medium extract and cytoplasmic fraction of Weissella koreensis OK1-6

Ornithine contents in the samples prepared were estimated by TLC and HPLC analyses. As shown in Fig. 4, cultured medium extract and cytoplasmic fraction of W. koreensis OK1-6 cells cultured with 1% arginine contained ornithine, and the estimated contents of ornithine were 41∙84 and 0∙23 mmol, respectively. On the other hand, spent medium extract and cytoplasmic fraction of W. koreensis OK1-6 cells cultured without arginine contained relatively far less amounts of ornithine, and the estimated contents were 0∙15 and 0∙01 mmol, respectively.

Figure 4.

TLC analysis of ornithine formed in cytoplasmic fraction and spent medium of Weissella koreensis OK1-6. Cultured medium and cells were lyophilized, and ornithine was extracted as described in Materials and Method. A, spot of standard arginine; O1, O2, O3, O4 and O5, spots of standard ornithine at the concentration of 1∙52, 4∙55, 7∙59, 13∙66 and 15∙18 μg, respectively. C, cytoplasmic fraction; M, cell-free cultured medium. 1% arg, cultured in 1% arginine added MRS broth; MRS, cultured in MRS broth without arginine.

Discussion

Weissella koreensis OK1-6 samples may contain components that inhibit triglyceride and lipid accumulation in 3T3-L1 cells. One of the key components can be ornithine, in fact, the inhibitory effects were observed more significantly in the cultured medium and cytoplasmic fraction with high ornithine contents than with low ornithine contents samples (Figs 1 and 2). In addition, l-ornithine itself showed the triglyceride decreasing effect in a dose-dependent manner (5∙0–500 μmol) (data not shown). Comparing the effects of high ornithine content sample (S4, 41∙84 μmol) (Fig. 1) with that of l-ornithine itself on triglyceride decrease in 3T3-L1 cells suggests that the content of l-ornithine itself (50 μmol) that can cause about 20% decreasing effect is not enough for the all effects of the cell-free cultured medium (S4) treatment as shown in Fig. 1. Therefore, these data also suggested that the W. koreensis OK1-6 samples may contain other factors contributing the lipid lowering effects. Future studies with W. koreensis OK1-6 samples containing enhanced levels of ornithine may provide further insight into the roles of ornithine in the inhibition of lipid accumulation. Also, identification of the other factors exerting the inhibition activity in the LAB samples may provide further insights into approaches for the nutraceutical application of the W. koreensis OK1-6.

The cultured medium extract and cytoplasmic fraction of W. koreensis OK1-6 down-regulated the expression of adipocyte-specific genes. Several studies showed that there were positive correlations between the levels of C/EBP-α gene expression and the lipid accumulation rate (Hu et al. 1995; Jiang et al. 2006; Park et al. 2009). C/EBP-α is known as a potentially central regulator of adipocyte development and regulates the expression of adipogenic-specific genes such as aP2, LPL, FAS and leptin (Hu et al. 1995; Jiang et al. 2006). Thus, it is judged that the decreased expression of C/EBP-α may caused the altered expression of aP2 and FAS and resulted in the reduction in the accumulation of lipid in the differentiated 3T3-L1 cells as shown in Fig. 1 and 2. Previously, it has also been suggested that members of C/EBP family and SREBP1 regulate fatty acid synthesis, while the regulation of lipogenesis at the mRNA level is mainly dependent on FASs that are regulated by C/EBP-α (Zang et al. 2004; Jiang et al. 2006).

Ornithine produced by W. koreensis OK1-6 strain was mainly released into the cultured medium, and the molar ratio of ornithine formed (42∙07 mmol) to arginine added (47∙40 mmol) was about 0∙89. Previously, we reported that the ornithine-producing capacities of W. koreensis strains cultured at a little different conditions (Yu et al. 2009; Yu and Oh 2010). By culturing the W. koreensis OK1-6 strain under the culture conditions used in this study (1% arginine, 1% NaCl in MRS broth, pH 6∙5, 72 h) yield relatively higher amounts of ornithine compared with other conditions used so far (Yu et al. 2009; Yu and Oh 2010). Further studies are required to determine the optimal culture conditions required for a highest ornithine formation and for a conservation of ornithine formed by W. koreensis strains.

LAB fermentation produces a variety of fatty acids and amino acids such as butyrate, conjugated linolecic acid (CLA), GABA and ornithine (Cho et al. 2007; Lee et al. 2007; Park and Oh 2007; Mahkonen et al. 2008; Yu et al. 2009; Yu and Oh 2010). Among the fatty acids and amino acids showing anti-obesity effects in mice and humans are CLA and ornithine (Elam 1988; Park et al. 1997). Previously, it was reported that CLA-producing Lact. plantarum PL62 showed an anti-obesity effect in diet-induced obese mice (Lee et al. 2007). In this study, we showed that ornithine-producing W. koreensis OK1-6 inhibited adipogenesis through down-regulating adipocyte-specific gene expression in differentiated 3T3-L1 adipocyte cells. This is the first study to report the inhibitory effect of W. koreensis strains in intracellular lipid accumulation and triglyceride formation in differentiating 3T3-L1 cell line. The samples as well as ornithine lower the intracellular lipid accumulation in dose-dependent manner (data not shown).

In addition to anti-obesity effect of LAB, anticancer effects, immunopotentiating activities and a neuroprotective effect of several other LAB culture extracts were reported (Jeon et al. 2006; Cho et al. 2007). The activities of several LAB could be attributed to the various fermentative products from LAB. For example, the short-chain fatty acid such as butyrate has a suppressing role in the growth of cancer cells (Mahkonen et al. 2008). In addition, the kimchi LAB-cultured medium extracts containing GABA showed a neuroprotective effect against the neuronal cell death induced by chemicals (Cho et al. 2007). Glycoprotein produced by the LAB isolated from kimchi showed an antimutagenic activity (Chandan 1999). Both the live and heat-killed forms of some species of LAB had strongly activated pan-caspases, resulting in colon cancer cell apopotosis (Gonet-Surowka et al. 2007; Thirabunyanon et al. 2009). From these, it can be concluded that cellular fractions (cell-wall fractions and cytoplasmic fractions), spent media (cell-free supernatants), live whole cells and fermentative products of useful LAB could be beneficial for the improvement of health-related symptoms (Jeon et al. 2006; Cho et al. 2007; Mahkonen et al. 2008; Thirabunyanon et al. 2009). However, the precise mechanisms by which LAB and/or LAB products may modulate many healthful effects are not well studied. Further studies are required to elucidate the molecular pathways of LAB that have specific effects on health-related issues such as obesity will be necessary and continued.

Conclusion

Many studies on different species of LAB reported various health benefiting effects of LAB. To the best of our knowledge, this is the first study to report the anti-obesity effect of W. koreensis OK1-6. Our study demonstrated that both the spent culture media extract and the cytoplasmic fraction of W. koreensis OK1-6, especially with high-ornithine-containing samples, are capable in reducing lipid storage in differentiating adipocytes. Both the spent culture media extract and the cytoplasmic fraction of W. koreensis OK1-6 reduced the expression level of lipogenic gene resulting in the decreased triglyceride level in the treated 3T3-L1 cells comparing with the control group. Our study may be useful for the neutraceutical and food industries in developing probiotic-based anti-obesity therapies.

Acknowledgements

This study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, and by a research grant from Woosuk University in 2012 to S.H. Oh, Republic of Korea and partially supported by the grant for Post-doc progamme, Chonbuk National university (2009).

Ancillary