The inhibitory effect of Lactobacillus sakei KBL isolated from kimchi on the adipogenesis of 3 T 3L 1 cells

Abnormal adipocyte growth, in terms of increased cell numbers and increased cell differentiation, is considered to be a major pathological feature of obesity. Thus, the inhibition of preadipocyte mitogenesis and differentiation could help prevent and suppress obesity. Some probiotics and cellular components are known to modulate the lipid metabolism in vitro and/or in vivo. The aim of this study was to investigate whether extracts from Lactobacillus sakei KBL cells isolated from kimchi could exert anti-adipogenic action in 3T3-L1 cells (fat cells). Differentiating 3T3-L1 cells were treated with L. sakei KBL cell extracts (L. sakei KBL_CE), and cell viability was assessed by MTT assays. At concentrations below 1 mg/ml, L. sakei KBL_CE did not exert any cytotoxic effect in 3T3-L1 cells. Lipid regulation in the cell culture system was assessed by morphological analysis and oil-red-O staining of fat. Treatment with L. sakei KBL_CE significantly inhibited adipocyte differentiation. L. sakei KBL_CE treatment (1 mg/ml) also reduced lipid accumulation by 25% in fully differentiated 3T3-L1 adipocytes. These findings collectively indicate that L. sakei KBL_CE can reduce fat mass by modulating adipogenesis in maturing preadipocytes.


INTRODUCTION
Probiotics are defined as viable microorganisms that when consumed in adequate amounts, confer health benefits by improving the properties of indigenous microflora (Guillot, 1998).These effects include the amelioration of hypercholesterolemia (Park et al., 2007) and hypertension (Aihara et al., 2005), prevention of cancer (Rafter, 2004), modulation of the immune system (Baken et al., 2006) etc.According to a recent study, live probiotics, dead probiotic cells, and even probiotic cell components can exert significant biological effects outside the gastrointestinal tract (Adam, 2010).Thus, probiotics are believed to be an important part of an overall dietary strategy for maintaining health.However, the identification of various probiotic strains and their mechanisms of action are not fully elucidated, yet.Some probiotics have been found to be effective in regulating *Corresponding author.E-mail: klee02@empal.com.Tel: + 82 32 260 0727.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License adipose tissue in overweight adults and animal models of obesity.In particular, administration of Lactobacillus plantarum to mice led to reductions in adipose tissue weights (Sato et al., 2008;Ma et al., 2008).
Obesity, which occurs when excessive fat accumulates due to an overage of energy intake compared to consumption, is associated with various health consequences, such as cardiovascular disease, diabetes mellitus, and other chronic disorders (Spiegelman and Flier, 2001;Macelin and Chua, 2010).Obesity is widely recognized as a worldwide problem due to its negative health consequences and huge cost to society.Thus, we critically need safe and effective anti-obesity measures.Despite this urgent need and the potential size of the market for anti-obesity drugs, however, the developed drugs have proven unsatisfactory to date, due to their deleterious side effects.Thus, some researchers are turning to edible natural products that have 'historically' been used as dietary supplements for body-weight management and control in many countries (Rayalam et al., 2008).Obesity primarily arises via increased cytoplasmic triglyceride deposition, which leads to adipocyte enlargement, and elevated adipogenesis, which results in the formation of new adipocytes from precursor cells (Gregoire, 2001).Adipose tissue has been shown to secrete a variety of bioactive peptides (that is, adipokines) that can potentially affect glucose and lipid metabolism (Spiegelman and Flier, 2001).As adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and advancement of various diseases, inhibiting the proliferation and differentiation of fat cells is considered to be an important strategy for the potential treatment of obesity (Rayalam et al., 2008;Rosen and Spiegelman, 2006).In many obesity-related studies, the fibroblastic 3T3-L1 preadipocyte line is widely used to investigate the mechanism of preadipocyte proliferation and adipocyte genesis, due to the ability of this cell line to undergo complete differentiation into mature adipocytes.
Kimchi is a fermented cabbage product that is traditional in the Republic of Korea (Chang et al., 2010).Among the lactic acid bacteria (LAB; a subset of probiotic organisms), isolated from kimchi, Lactobacillus sakei is a psychrophilic bacterium that is found as the dominant species in kimchi produced at -1°C (Cho et al., 2006).L. sakei raises interest on anti-obesity LAB.However, there is little information available on the anti-obesity effects of L. sakei LAB.Here, we investigated the effects of L. sakei KBL cell extracts on the cell viability and intracellular lipid accumulation in cultured and differentiating 3T3-L1 (adipocyte) cells.

Isolation of L. sakei KBL and preparation of the cell extract
L. sakei KBL was isolated from kimchi and confirmed by DNA sequencing of 16S rRNA.For experiments, L. sakei KBL was grown anaerobically in deMan-Rogosa-Sharpe (MRS) medium at 37°C for 18 h, and then the cells were collected by centrifugation and washed with phosphate-buffered saline (PBS).The cells were counted and suspended in PBS at 10 10 colony-forming units (CFU)/ml, and then subjected to sonication and centrifugation.Supernatants containing the L. sakei KBL_CE were filter-sterilized (pore size, 0.45 m), lyophilized, and kept at -20°C until use.

The 3T3-L1 cell culture and differentiation assay
The 3T3-L1 preadipocyte cell line was obtained from the American Type Culture Collection (ATCC, USA) and cultured as described elsewhere (Hemati et al., 1997).Cells were cultured in 1% penicillin-streptomycin (PS)/DMEM containing 10% FBS (Gibco-BRL) [Lonza, Walkersville, MD, USA] at 37°C in a 5% CO2 incubator.Differentiation was induced by incubating confluent cells for 6 days in differentiation medium (DM) containing 1% PS/DMEM, 10% FBS, 0.25 mM IBMX, 0.25 µM dexamethasone and 1 µg/ml insulin.The cells were then maintained in post-differentiation medium (DMEM containing 1% PS and 10% FBS), with replacement of the medium every 2 days.To examine the effects of the cell extract on the differentiation of preadipocytes to adipocytes, cells were cultured with MDI in the presence of various concentrations of cell extract (0 to 2.0 mg/ml).

Quantification of cell viability via MTT assay
The MTT assay is a standard colorimetric assay used to measure cellular proliferation (cell growth).Yellow 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced to purple formazan in the mitochondria of living cells.The number of surviving cells is directly proportional to the level of the formazan product created; the amount of colored product is directly proportional to the number of viable cells, and can be read on a multi-well scanning spectrophotometer (Rosen and Spegelman, 2006).The 3T3-L1 preadipocytes were treated with 1 × 10 5 L. sakei KBL_CE/well.After 48 h, the cells were incubated with MTT working solution [Promega] for 3 h.The fat was then removed from the plate, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan dye.The absorbance of the resulting colored solution was quantified (absorbance 570 nm/reference 630 nm) using an ELISA plate reader [Bio-TEK Power-Wave XS, VT, USA]

Oil-red O staining
The cellular lipid content was assessed by Oil Red O staining.After the induction of differentiation, cells were washed twice with PBS, fixed in 3.7% formaldehyde (Sigma-Aldrich) in PBS for 1 h, and stained with Oil Red O [Cayman, USA] for 1 h.The stained fat droplets were dissolved in isopropanol containing 4% Nonidet P-40, and quantified by measuring the absorbance at 520 nm.Pictures were taken using an Olympus microscope.

Statistical analysis
Statistical analyses were performed using Sigma Plot 10.0 [Systat software, USA].Values are expressed as the means ± standard error (SE) from three independent experiments.Statistical significance was determined using the paired Student's t test.

Effects of L. sakei KBL on cell viability in 3T3-L1 preadipocytes
To distinguish any inhibitory effect of L. sakei KBL_CE from a possible cytotoxic effect on 3T3-L1 preadipocytes, we treated cells with various concentrations of L. sakei KBL_CE and performed MTT assays.The extract failed to show any cytotoxic activity against 3T3-L1 cells up to 0.2 mg/ml (Figure 1).

Inhibition of adipocyte differentiation and lipid accumulation
To test whether L. sakei KBL_CE inhibited adipocyte differentiation, we used insulin, dexamethasone, and isobutylmethyl xanthine (differentiation medium, DM) to induce the differentiation of 3T3-L1 preadipocytes, treated the differentiating cells with [0.8 × 10 5 cells/well in a 6-well plate] L. sakei KBL_CE on day 0, and then changed the culture medium every 2 days for a total of 2 days.L. sakei KBL_CE were then switched to add to the 3T3-L1 cells at 2~8 days, and the adipocytes were stained with Oil Red O for visualization of fat droplets.The staining results showed that an 8-day treatment with various concentrations (0 to 1.0 mg/ml) of L. sakei KBL_CE during the differentiation period significantly and dose-dependently inhibited 3T3-L1 adipogenesis (Figure 2, lower panels) in terms of both cell differentiation (Figure 2) and lipid accumulation (Figure 3), compared with control cells.Among the tested concentrations of L. sakei KBL_CE, 1.0 mg/ml was the most effective at reducing the lipid content in differentiated cells (by 25% compared with control cells) (Figure 3).These results suggest that L. sakei KBL_CE inhibited the differentiation of 3T3-L1 preadipocytes by suppressing lipid accumulation.

DISCUSSION
The recent noticeable increases in the worldwide numbers of overweight and obese people are due in part to diet and lifestyle changes (Fuller, 1989).Some natural products have been shown to protect against obesity and have beneficial health effects, and have attracted the attention of researchers because of their relatively good safety profiles (Sato et al., 2008).Among these natural products, probiotic preparations comprising dead cells or their metabolites have been shown to exert biological responses (Ma et al., 2008;Chang et al., 2010).Among the various beneficial health effects of probiotics, their biological impact on obesity has generated considerable interest.For example, the probiotic species, L. plantarum, L. sakei KBL_CE concentration (mg/ml)  Lactobacillus sakei KBL_CE treatment concentration (mg/ml) was shown to have specific biological effects (including anti-obesity effects) in vitro (Cho et al., 2006).In this study, the potential anti-obesity effects of L. sakei KBL_CE were investigated through cell viability assays and Oil red O staining of 3T3-L1 cells treated with various concentrations of the extract either in maintenance culture or during differentiation.For increased adipose tissue to be created, the number or size of adipocytes must increase by either proliferation or differentiation (Ambati et al., 2007).Conversely, the reduction of adipose tissue mass involves the loss of lipids through lipolysis, the inhibition of preadipocyte proliferation, and/or the decreased differentiation of mature adipocytes.Treatment of 3T3-L1 cells with various concentrations (0 to 1.0 mg/ml) of L. sakei KBL_CE did not significantly alter cell viability, indicating that the inhibitory effect of L. sakei KBL_CE on lipid accumulation was due not to reduced cell viability.Such reduction was seen up to a 2.0 mg/ml.Instead, we observed decreased adipogenesis in L. sakei KBL _CE-treated cells compared to control cells (Figure 2), indicating that the extract reduced the lipid accumulation in mature adipocytes.The loss of fat mass can be partly attributed to lipolysis, in which triglycerides are broken down into glycerol and fatty acids in adipocytes.Together, our results indicate that L. sakei KBL_CE may contain components that inhibit lipid accumulation in 3T3-L1 cells.The observed inhibitory effects were more significant in cells treated with a higher concentration of L. sakei KBL_CE versus a lower concentration of L. sakei KBL_CE (Figure 3).Thus, our results confirm the potential anti-obesity effects of L. sakei KBL_CE and suggest that L. sakei KBL may have similar effects.
It is not clear what compounds of L. sakei KBL worked as the main principles of the anti-obesity effects.Therefore, L. sakei KBL can be used as a useful material not only for the production of fermented foods such as kimchi, but also for the investigation of unknown compounds for the anti-obesity effects.Further studies may provide additional insights into the active compounds of L. sakei KBL that have specific effects on obesity.

Conclusion
L. sakei KBL_CE exerted inhibitory effects on adipocyte differentiation and lipid accumulation, suggesting that L. sakei KBL might have further implication for in vivo antiobesity effect and could thus possibly be developed as a therapeutic substance for preventing or treating obesity.

Figure 1 .
Figure 1.Effect of Lactobacillus sakei on the cell viability of post-confluence preadipocytes.The indicated concentrations of L. sakei KBL_CE were added to the differentiation medium at day 0. After 8 days of treatment, viability was determined by MTT assay.No cytotoxic effect was noted up to 1.0 mg/ml.Assays were performed in 3 replicates from 3 independent experiments.Values are means  SEM (p < 0.01).

Figure 2 .
Figure 2. Morphological examination of undifferentiated cells (A), control differentiated cells (B), tumornecrosis factor (TNF)- treated control cells (C), cells treated with various concentrations of L. sakei KBL_CE (D, 0.05 mg/ml; E, 0.4 mg/ml; F, 1.0 mg/ml).Confluent 3T3-L1 cells were treated with various concentrations of L. sakei KBL_CE for 8 days and lipid accumulation was measured by Oil Red O staining.A significant inhibitory effect [using red staining as a proxy for differentiation here and counting the red-stained cells] was noticed.

Figure 3 .
Figure 3. Inhibitory effect of L. sakei KBL_CE on lipid accumulation in 3T3-L1 adipocytes.Confluent 3T3-L1 cells were treated with various concentrations of L. sakei KBL_CE for 8 days, and lipid accumulation was examined by Oil Red O staining.Assays were performed in three replicates from three independent experiments.Values are means  SEM (p<0.01).L. sakei KBL_CE treatment concentration(mg/ml) Lipid concentration(% Control) 0