Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 460927 7 pageshttpdxdoiorg1011552013460927
Research ArticleLactobacillus plantarum LG42 Isolated from Gajami Sik-HaeInhibits Adipogenesis in 3T3-L1 Adipocyte
Jeong-Eun Park12 Suk-Heung Oh3 and Youn-Soo Cha12
1 Department of Food Science and Human Nutrition Chonbuk National University Jeonju 561-756 Republic of Korea2 Jeonju Makgeolli Research Center Chonbuk National University Jeonju 561-756 Republic of Korea3 Department of Food and Biotechnology Woosuk University Jeonbuk 565-701 Republic of Korea
Correspondence should be addressed to Youn-Soo Cha cha8jbnuackr
Received 3 October 2012 Accepted 17 December 2012
Academic Editor Fabio Ferreira Perazzo
Copyright copy 2013 Jeong-Eun Park et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
We investigated whether lactic acid bacteria isolated from gajami sik-hae (GLAB) are capable of reducing the intracellular lipidaccumulation by downregulating the expression of adipogenesis-related genes in differentiated 3T3-L1 cells The GLAB Lacto-bacillus plantarum LG42 significantly decreased the intracellular triglyceride storage and the glycerol-3-phosphate dehydrogenase(GPDH) activity in a dose-dependent manner mRNA expression of transcription factors like peroxisome proliferator-activatedreceptor (PPAR) 120574 and CCAATenhancer-binding protein (CEBP) 120572 involved in adipogenesis was markedly decreased by theGLAB treatment Moreover the GLAB also decreased the expression level of adipogenic markers like adipocyte fatty acid bindingprotein (aP2) leptin GPDH and fatty acid translocase (CD36) significantly These results suggest that the GLAB inhibits lipidaccumulation in the differentiated adipocyte through downregulating the expression of adipogenic transcription factors and otherspecific genes involved in lipid metabolism
1 Introduction
Gajami sik-hae is a fermented fish product popular in thenortheastern coastal area of Korea Sik-hae is a traditionalKorean fermented seafood and has long been used for season-ing and is the generic name for a class of Korean lactic acidfermented fish products [1] It is prepared through blending ofvarious kinds of seafood including cooked rice red pepperradish garlic ginger malt meal and salt that becomes palat-able through the subsequent preservation and fermentation[2] The unique taste of sik-hae is due to the presence of theaforementioned ingredients and also due to the fermentingaction of various microorganisms during the fermentationperiod [3] Lactic acid bacteria (LAB) are the most pre-dominant microorganisms involved in sik-hae fermentationSome LABs were isolated from fish fermented food in Korea[4] LAB is a viable bacteria that beneficially influence thehealth of the host Recently LAB has been revaluated fortheir nutritional physiological and pharmacological aspectswhich have raised attention towards the functional effect ofKorean traditional foods including kimchi and joet-gal [5]
Various nutritional and therapeutic effects ascribed to LABin the human body are the metabolic stimulation of vitaminsynthesis and enzyme production antimutation anticancergastric secretomotor and immune function [6]
Nowadays the consumer pays a lot of attention in lookingfor the relation between food and its health benefit As a con-sequence the market for foods with health-promoting prop-erties the so-called functional foods has shown a remarkablegrowth over the past few years [7] The development offood which utilizes the functionality and effectiveness ofprobiotics has been officially recognized as an important fieldof study Consequently numerous studies for using probioticorganisms as a functional food have been investigated Earlierstudies on probiotics have been brought into effect on thestabilization of gastrointestinal microflora the reduction ofsaprogenic products the prevention of degenerative diseaseactivation of the immune system anticancer activities anti-obesity lowering of cholesterol and the suppression andprevention of constipation [8ndash12]120574-Aminobutyric acid (GABA) is a four-carbon nonpro-
tein amino acid conserved from bacteria to plants and
2 BioMed Research International
vertebrates It was discovered in plants more than half acentury ago but the interest in GABA shifted to animalswhen it was revealed that GABA occurs at high levelsin the brain playing a major role in neurotransmission[13] The pathway for GABA synthesis is composed of thecytosolic enzyme glutamate decarboxylase (GAD) and themitochondrial enzymes GABA transaminase (GABA-T) andsuccinic semialdehyde dehydrogenase (SSADH) [14] Theconsumption ofGABA-enriched foods such asmilk soybeanand gabaron tea has been reported to suppress the elevationof systolic blood pressure in spontaneously hypertensiverats (SHRs) [15ndash17] In this study isolated LAB with GABAproducing ability from traditional Korean fermented foodssuch as kimchi [18] and sik-hae (SH Oh) The isolatedLABs were Lactobacillus brevis OPK-3 Lactobacillus sakeiOPK2-59 and Lactobacillus plantarum LG42 Therefore itwas our interest to find whether the isolated LAB can bea novel nutraceuticals This study was carried out with theobjective of testing the antiobesity property of Lactobacillusplantarum LG42 (GLAB) with GABA producing abilityOur finding reveals the inhibitory effect of the GLAB onadipocyte differentiation through modulating the expressionof adipogenic transcription factors and other adipogenesisspecific genes suggesting the antiobesity property of GLAB
2 Materials and Methods
21 Cell Culture 3T3-L1 cells (American Type Culture Col-lection (ATCC)) were cultured in DMEM containing highglucose supplemented with 10 fetal bovine serum (FBS)and penicillinstreptomycin in 6 well culture plates Twodays after confluence cells were cultured in the adipocytedifferentiation cocktail media containing 5mM 3-isobutyl-1-methylxanthine (IBMX) 1mM dexamethasone (SigmaUSA) and insulin (10mgmL) in DMEM supplemented with10 fetal bovine serum (FBS) for 2 days The differentiationwas complete after 6 days
22 GLAB Sample and Treatment L plantarum LG42 alactic acid bacteria havingGABAproducing capacity isolatedfrom gajami sik-hae (GLAB) was supplied by Woosuk Uni-versity genetic engineering laboratory Korea For TLC (thinlayer chromatography) analysis silica gel 60 F254 (MerckGermany) standard GABA (Sigma USA) solvent mixture(butanol acetic acid dichloromethanol water in 5 3 3 3ratio) were usedThe culturedmedium cell-free supernatantand cytoplasmic fraction samples (03 120583L each) were spotted3 times The GLAB was incubated at 37∘C for 16sim18 hr inMRS agar plates (Difco Detroit USA) All purified strainswere kept at minus70∘C until use After culturing the GLAB allstrains were harvested in a refrigerated centrifuge (1100timesgfor 3min at 4∘C) and washed three times with distilled waterfor the removal of MRS brothThe washed GLAB was freeze-dried and resuspended in distilled water at a concentrationof 10mgmL and homogenized for 50 sec followed by 1minrest (repeated 3 times) using a sonicator (Fisher ScientificCo Toronto ON USA) The suspension was centrifugedat 1100 timesg for 15min at 4∘C The 3T3-L1 cells were treated
with five different concentrations of the supernatant that is0 (control) 10 20 30 and 40120583gmL
23 Oil RedOStaining of 3T3-L1Adipocyte Intracellular lipidaccumulationwasmeasured using oil redO (Sigma St LouisMO) 3T3-L1 cells were fixed with 37 formaldehydePBSand stained with oil red O Quantification of lipidaccumulation was achieved by oil red O from stained cellswith isopropyl alcohol andmeasured spectrophotometricallyat 510 nm The oil red O stained material was expressed on aper cell basis using the cell number determined from similarplates The percentage of oil red O stained material relativeto control wells containing cell culture medium withoutcompounds was calculated as 119860
510 nm (GLAB)119860510 nm
(control) times 100
24 Triglyceride Content Triglyceride content was deter-mined using a commercial triglyceride assay kit (Zen-bioResearch Triangle Park NC) according to themanufacturerrsquosprotocolThe protein concentration was determined by usinga Bradford reagent (Sigma St Louis MO)
25 Glycerol-3-Phosphate Dehydrogenase Activity GPDHwas measured by following the disappearance of NADH dur-ing enzyme-catalysed dihydroxyacetone phosphate reduc-tion using the GPDH activity assay kit (TAKARA BIOINC Japan) GPDH activity was spectrophotometricallydetermined at 340 nm One unit was defined as the amountof enzyme required for the consumption of 1 120583mol of NADHfor one minute at 30∘C The enzyme activity was calculatedwith the following formula GPDH activity (unitsmL) =(ΔOD
340times 119860 (mL) times dilution ratio of the test sample)(622
times119861 (mL) times 119862 (cm))
ΔOD340
decrease in the absorbance at 340 nm perminute119860 (mL) total reaction volume119861 (mL) the volume of enzyme solution (dilutedsample) added119862 (cm) optical path length of the cell usedlowast622 millimolar absorption coefficient of NADHmolecules
26 Quantitative Real-Time PCR Analysis Total RNA wasextracted from 3T3-L1 cells at various times after adipogenicinduction using Trizol reagent (Invitrogen Life TechnologiesCarlsbad CA USA) and the concentration measuredspectrophotometrically The extracted RNA was reversetranscribed into complementary DNA using a high capacitycDNA reverse transcription kit (Applied Biosystems FosterCity CA USA) Then the RNA expression level was quanti-fied by a quantitative real-time PCR using SYBR Green PCRMaster Mix (Applied Biosystems Woolston WarringtonUK) and the 7500 real-timePCR system (AppliedBiosystemsFoster City CA USA) according to the manufacturersquosprotocol The Sequences of primers used for quantitativereal-time PCR are as follows fatty acid binding protein (aP2)
BioMed Research International 3
F 51015840-AGTGAAAACTTCGATGATTACATGAA-31015840 and R51015840-GCCTGCCACTTTCCTTGTG-31015840 fatty acid translocase(CD36) F 51015840-TTGTACCTATACTGTGGCTAAATGAGA-31015840 and R 51015840-CTTGTGTTTTGAACATTTCTGCTT-31015840CCAATenhancer-binding protein 120572 (CEBP120572) F 51015840-AGCAACGAGTACCGGGTACG-31015840 and R 51015840-TGTTTGG-CTTTATCTCGGCTC-31015840 peroxisome proliferator-activatedreceptor 120574 (PPAR120574) F 51015840-CAAGAATACCAAAGTGCG-ATCAA-31015840 and R 51015840-GAGCTGGGTCTTTTCAGAAT-AATAAG-31015840 (leptin) F 51015840-CCGCCAAGCAGAGGGT-CAC-31015840 and R 51015840-GCATTCAGGGCTAACATCCAACT-31015840glycerol-3-phosphate dehydrogenase(GPDH) F 51015840-CTCT-TCTTGCCGCTTCAGTTT-31015840 and R 51015840-CATGTAGGCCA-TGAGGTCCACCAC-31015840 120573-actin F 51015840-ATGGATGACGA-TATCGCT-31015840 and R 51015840-ATGAGGTAGTCTGTCAGGT-31015840Relative quantification of gene expression with real-timePCR data was calculated relative to 120573-actin
27 Analysis of GABA Production GABA formation in cul-tured medium and cytoplasmic fraction of L plantarumLG42 cells were analyzed by TLC as described (25) In orderto produce GABA in the culture medium 1 MSG wasadded to MRS broth and then cultured for 48 h at 30∘Cand verified the existence or nonexistence of GABA in thebacterial cytoplasmic fraction by using TLC
28 Statistical Analysis All values are expressed as mean plusmnSD The data were analyzed by one-way ANOVA using SPSSversion 160 The differences among groups were assessedusingDuncanrsquosmultiple range test Statistical significancewasconsidered at 119875 value lt 005
3 Results
31 Oil Red O Staining and Intracellular Triglyceride Theeffects of GLAB on oil red O stained 3T3-L1 adipocyteare shown in Figure 1 Cells treated with 10 120583g and 20120583gconcentrations did not show any significant effects comparedwith untreated cells Differentiated cells treated with 30120583gof GLAB accumulated about a 30 decrease in intracellularlipid compared to control Also the treatment with 40120583gGLAB resulted in further reduction to approximately 58 ofthe lipid accumulation of the control
The effect of GLAB on the inhibition of intracellulartriglyceride in 3T3-L1 adipocyte is shown in Figure 2 Theresults demonstrated that GLAB caused an inhibition onintracellular triglyceride accumulation Especially 40 120583g ofGLAB treatment which was most effective compared with10 120583g treated and untreated cells (Figure 2)
32 Measurement of GPDH Activity The effects of GLAB onGPDH activity in 3T3-L1 adipocyte are shown in Figure 3GPDH activity which indicates the late phase of adipocytedifferentiation was also determined The effect of GLAB onadipogenesis was clearly dose dependent The high dose 30and 40120583g of GLAB significantly decreased GPDH activity(Figure 3)
33 mRNA Expression of Lipid Metabolism-Related Gene inDifferentiated 3T3-L1 Adipocyte PPAR120574 and CEBP120572mRNAlevels in differentiated 3T3-L1 adipocyte were significantlydecreased in cells treated with GLAB compared withuntreated cell especially 40 120583g treated cell was most effective(Figure 4) In addition the expression of PPAR120574 target geneaP2 was significantly decreased in GLAB treated cells andCD 36 was significantly decreased in 20 30 and 40 120583gtreated cells compared with untreated cell during adipocytedifferentiation (Figure 4) Cells treatedwithGLAB resulted ina significant decrease in the mRNA levels of leptin comparedto cell untreated with GLAB The GPDH mRNA level wassignificantly decreased in 30 and 40 120583gmL treated cellscompared with 10120583gmL treated and untreated cells
34 GABA Production by L plantarum LG42 GABA pro-duction by L plantarum LG42 strain was studied in MRSbroth containing 1 MSG Based on the TLC analysis datathe cultured medium and cell-free culture supernatant ofL plantarum LG42 cells contained GABA but not thecytoplasmic fraction (Figure 5) This data indicated that Lplantarum LG42 cells have GABA producing ability and theGABA was excreted mainly in the cell culture medium
4 Discussion
Obesity threatens to become the 21st centuryrsquos leadingmetabolic disease in the world [19] Complications associatedwith obesity are responsible for the most obesity related mor-bidity andmortality Obesity increases circulating cholesteroland triglyceride levels and is closely associatedwith hyperten-sion cardiovascular disease type 2 diabetes mellitus cancerrespiratory complications and osteoarthritis [20] Obesity isa condition in which adipocytes not only accumulate a largeamount of fat but also become enlarged At a cellular levelit is characterized by an increase in the number and sizeof adipocytes differentiated from fibroblastic preadipocytesin the adipose tissue [21] The severity of obesity is corre-lated with the degree of differentiation of preadipocytes toadipocytes and the enlargement of adipocytes in adipose tis-sues [22] PPAR120574 and CEBP120572 are transcriptional activatorswhich play a major role in coordinating the adipocyte geneexpression during adipocyte differentiation [23] Thereforewe investigated the effects of lactic acid bacteria isolatedgajami sik-hae (GLAB) on the differentiation of 3T3-L1cells to clarify its antiobesity mechanism GLAB significantlysuppressed lipid accumulation in 3T3-L1 cells in a nontoxicconcentration GPDH activity was decreased in 3T3-L1 cellstreated with GLAB compared to control cells incubated inthe differentiation medium (Figure 3) (119875 lt 005) GPDHis a key enzyme important for triacylglycerol synthesis [24]the present result indicates that GLAB suppresses adipocytedifferentiation resulted in reducing GAPDH level
It is noteworthy that PPAR120574 and CEBP120572 levels weredownregulated in 3T3-L1 cells treated with GLAB (Figure 4)PPAR120574 and CEBP120572 play vital roles in the early stage ofadipose differentiation [25] PPAR120574 and CEBP120572 regulatethe expression of adipogenic genes such as CD36 leptin
4 BioMed Research International
0 10 20 30 40(120583gmL)
(a)
aab ab
bc
b
0
20
40
60
80
100
120
0 10 20 30 40
Rela
tive o
il re
d O
stai
n(P
erce
ntag
e of c
ontr
ol)
H2
(120583gmL)
(b)
Figure 1The effect of lactic acid bacteria isolated gajami sik-hae on oil redO stained in 3T3-L1 adipocyte (a) Photograph of oil redO staining(b) Quantification of oil red O staining Values with different superscripts are significantly different by ANOVAwith Duncanrsquos multiple rangetests at 119875 lt 005
TGp
rote
in (m
g)
(120583gmL)0 10 20 30 40
aa
ab ab
b
0
05
1
15
2
25
3
Figure 2The effect of lactic acid bacteria isolated gajami sik-hae ontriglyceride accumulation in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
GPDH and aP2 triggering the accumulation of fat in thecells [26 27] In this study the expression of PPAR120574 andCEBP120572was inhibited byGLAB together resulting in reducedadipogenesis indirectly confirmed by measuring oil red stainand GPDH activity Therefore it appears that GLAB inhibitsadipogenesis by reducing or suppressing the expression ofPPAR120574 and CEBP120572 levels It is also reasonable to articulatethat GLAB acts directly on PPAR120574 and CEBP120572 Kim et al(2008) showed that treating 3T3-L1 cells with milk fermentedwith lactic acid bacteria isolated from kimchi decreased thelevels of PPAR120574 and CEBP-120572 expression [28] Thus it couldbe possible that lactic acid bacteria isolated from gajami sik-hae affects the downstreamgenes of PPAR120574 andCEBP120572 thusinhibiting adipogenesis in 3T3-L1 cells
a
ab ab
b
c
0
5
10
15
20
25
0 10 20 30 40
Pro
tein
(U
mg)
Figure 3 The effect of lactic acid bacteria isolated gajami sik-hae on GPDH activity in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
aP2 gene is central to the pathway that links obesity toinsulin resistance possibly by linking fatty acid metabolismto the expression of (tumor necrosis factor-120572) TNF-120572 [29 30]CD36mRNAexpression is activated during 3T3-L1 adipocytedifferentiation and CD36 protein levels are positively cor-related with PPAR120574 and CEBP120572 [31] CD36 is a long chainfatty acid transporter present on the plasma membrane aswell as in intracellular pools of the skeletal muscle [32]High level CD36 might result in lipid accumulation whichis supported by studies using CD36 null mice [33] Leptin ahormone and the product of the ob gene is primarily secretedby adipose tissue It is involved in the regulation of energyexpenditure and food intake [34] The expression of leptin isregulated by several substances like insulin glucocorticoidsand TNF-120572 Here in this study we found that aP2 CD36 and
BioMed Research International 5
ab b
c
d
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(a)
a
b b
cd
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(b)
a
b b
cc
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
aP2
(c)
aa
abb
c
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
GPDH
(d)
a
b bc
d
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
Leptin
0
1
(e)
a ab b
c c
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
CD36
0
1
(f)
Figure 4 The effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of PPAR120574 CEBP120572 aP2 GPDH leptin and CD36in 3T3-L1 cells Values with different superscripts are significantly different by ANOVA with Duncanrsquos multiple range tests at 119875 lt 005Peroxisome proliferator-activated receptor 120574 (PPAR120574) CCAATenhancer-binding protein 120572 (CEBP120572) fatty acid binding protein (aP2)glycerol-3-phosphate dehydrogenase(GPDH) fatty acid translocase (CD36)
leptinmRNAexpressionswere inhibited byGLAB (Figure 4)aP2 CD36 and leptin are the target genes of CEBP120572 andPPAR120574 and are regulated by these transcription factorsThus our results suggest that the inhibition of adipogenicgene expression induced by GLAB may be mediated by theinhibition of CEBP120572 and PPAR120574 expression
Gajami sik-hae is a fermented food containing manykinds of microorganisms with Lactobacillus and Strepto-coccaceae as the dominant species [35] Lactobacillus andLeuconostoc species among lactobacillus produces variousmaterials by proliferation fermentation and metabolismFunctional organic acids like lactic acid and citric acidsare the major products of fermentation by these bacteriaFunctional materials such as acetylcholine dextran bac-teriocin and 120574-aminobutyric acid are also produced bythese microbes depending on the origin of the fermenting
materials The fermenting materials containing carotenoidsascorbic acids phenolic compounds and amino acids playa major role in this process [18 36] Among the variousbeneficial health effects of the probiotics [37 38] theirbiological impact on obesity has generated a considerableinterest Regarding the anti-obesity property of probioticssome reports demonstrated that dairy products fermentedwith lactic acid bacteria exert anti-obesity effects [28 39 40]Previously it has been shown that cytoplasmic fractions ofuseful LAB have some beneficial effects for the improvementof health related symptoms [41 42] Among various com-pounds from LAB contributing to anti-obesity effects andimproving lipid profiles are conjugated linoleic acid (CLA)ornithine GABA hydroxy methyl glutaric acid orotic acidand so forth [18 43ndash46] Although GABA can exert an anti-obesity effect and the GLAB has GABA producing capacity
6 BioMed Research International
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
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[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
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the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
vertebrates It was discovered in plants more than half acentury ago but the interest in GABA shifted to animalswhen it was revealed that GABA occurs at high levelsin the brain playing a major role in neurotransmission[13] The pathway for GABA synthesis is composed of thecytosolic enzyme glutamate decarboxylase (GAD) and themitochondrial enzymes GABA transaminase (GABA-T) andsuccinic semialdehyde dehydrogenase (SSADH) [14] Theconsumption ofGABA-enriched foods such asmilk soybeanand gabaron tea has been reported to suppress the elevationof systolic blood pressure in spontaneously hypertensiverats (SHRs) [15ndash17] In this study isolated LAB with GABAproducing ability from traditional Korean fermented foodssuch as kimchi [18] and sik-hae (SH Oh) The isolatedLABs were Lactobacillus brevis OPK-3 Lactobacillus sakeiOPK2-59 and Lactobacillus plantarum LG42 Therefore itwas our interest to find whether the isolated LAB can bea novel nutraceuticals This study was carried out with theobjective of testing the antiobesity property of Lactobacillusplantarum LG42 (GLAB) with GABA producing abilityOur finding reveals the inhibitory effect of the GLAB onadipocyte differentiation through modulating the expressionof adipogenic transcription factors and other adipogenesisspecific genes suggesting the antiobesity property of GLAB
2 Materials and Methods
21 Cell Culture 3T3-L1 cells (American Type Culture Col-lection (ATCC)) were cultured in DMEM containing highglucose supplemented with 10 fetal bovine serum (FBS)and penicillinstreptomycin in 6 well culture plates Twodays after confluence cells were cultured in the adipocytedifferentiation cocktail media containing 5mM 3-isobutyl-1-methylxanthine (IBMX) 1mM dexamethasone (SigmaUSA) and insulin (10mgmL) in DMEM supplemented with10 fetal bovine serum (FBS) for 2 days The differentiationwas complete after 6 days
22 GLAB Sample and Treatment L plantarum LG42 alactic acid bacteria havingGABAproducing capacity isolatedfrom gajami sik-hae (GLAB) was supplied by Woosuk Uni-versity genetic engineering laboratory Korea For TLC (thinlayer chromatography) analysis silica gel 60 F254 (MerckGermany) standard GABA (Sigma USA) solvent mixture(butanol acetic acid dichloromethanol water in 5 3 3 3ratio) were usedThe culturedmedium cell-free supernatantand cytoplasmic fraction samples (03 120583L each) were spotted3 times The GLAB was incubated at 37∘C for 16sim18 hr inMRS agar plates (Difco Detroit USA) All purified strainswere kept at minus70∘C until use After culturing the GLAB allstrains were harvested in a refrigerated centrifuge (1100timesgfor 3min at 4∘C) and washed three times with distilled waterfor the removal of MRS brothThe washed GLAB was freeze-dried and resuspended in distilled water at a concentrationof 10mgmL and homogenized for 50 sec followed by 1minrest (repeated 3 times) using a sonicator (Fisher ScientificCo Toronto ON USA) The suspension was centrifugedat 1100 timesg for 15min at 4∘C The 3T3-L1 cells were treated
with five different concentrations of the supernatant that is0 (control) 10 20 30 and 40120583gmL
23 Oil RedOStaining of 3T3-L1Adipocyte Intracellular lipidaccumulationwasmeasured using oil redO (Sigma St LouisMO) 3T3-L1 cells were fixed with 37 formaldehydePBSand stained with oil red O Quantification of lipidaccumulation was achieved by oil red O from stained cellswith isopropyl alcohol andmeasured spectrophotometricallyat 510 nm The oil red O stained material was expressed on aper cell basis using the cell number determined from similarplates The percentage of oil red O stained material relativeto control wells containing cell culture medium withoutcompounds was calculated as 119860
510 nm (GLAB)119860510 nm
(control) times 100
24 Triglyceride Content Triglyceride content was deter-mined using a commercial triglyceride assay kit (Zen-bioResearch Triangle Park NC) according to themanufacturerrsquosprotocolThe protein concentration was determined by usinga Bradford reagent (Sigma St Louis MO)
25 Glycerol-3-Phosphate Dehydrogenase Activity GPDHwas measured by following the disappearance of NADH dur-ing enzyme-catalysed dihydroxyacetone phosphate reduc-tion using the GPDH activity assay kit (TAKARA BIOINC Japan) GPDH activity was spectrophotometricallydetermined at 340 nm One unit was defined as the amountof enzyme required for the consumption of 1 120583mol of NADHfor one minute at 30∘C The enzyme activity was calculatedwith the following formula GPDH activity (unitsmL) =(ΔOD
340times 119860 (mL) times dilution ratio of the test sample)(622
times119861 (mL) times 119862 (cm))
ΔOD340
decrease in the absorbance at 340 nm perminute119860 (mL) total reaction volume119861 (mL) the volume of enzyme solution (dilutedsample) added119862 (cm) optical path length of the cell usedlowast622 millimolar absorption coefficient of NADHmolecules
26 Quantitative Real-Time PCR Analysis Total RNA wasextracted from 3T3-L1 cells at various times after adipogenicinduction using Trizol reagent (Invitrogen Life TechnologiesCarlsbad CA USA) and the concentration measuredspectrophotometrically The extracted RNA was reversetranscribed into complementary DNA using a high capacitycDNA reverse transcription kit (Applied Biosystems FosterCity CA USA) Then the RNA expression level was quanti-fied by a quantitative real-time PCR using SYBR Green PCRMaster Mix (Applied Biosystems Woolston WarringtonUK) and the 7500 real-timePCR system (AppliedBiosystemsFoster City CA USA) according to the manufacturersquosprotocol The Sequences of primers used for quantitativereal-time PCR are as follows fatty acid binding protein (aP2)
BioMed Research International 3
F 51015840-AGTGAAAACTTCGATGATTACATGAA-31015840 and R51015840-GCCTGCCACTTTCCTTGTG-31015840 fatty acid translocase(CD36) F 51015840-TTGTACCTATACTGTGGCTAAATGAGA-31015840 and R 51015840-CTTGTGTTTTGAACATTTCTGCTT-31015840CCAATenhancer-binding protein 120572 (CEBP120572) F 51015840-AGCAACGAGTACCGGGTACG-31015840 and R 51015840-TGTTTGG-CTTTATCTCGGCTC-31015840 peroxisome proliferator-activatedreceptor 120574 (PPAR120574) F 51015840-CAAGAATACCAAAGTGCG-ATCAA-31015840 and R 51015840-GAGCTGGGTCTTTTCAGAAT-AATAAG-31015840 (leptin) F 51015840-CCGCCAAGCAGAGGGT-CAC-31015840 and R 51015840-GCATTCAGGGCTAACATCCAACT-31015840glycerol-3-phosphate dehydrogenase(GPDH) F 51015840-CTCT-TCTTGCCGCTTCAGTTT-31015840 and R 51015840-CATGTAGGCCA-TGAGGTCCACCAC-31015840 120573-actin F 51015840-ATGGATGACGA-TATCGCT-31015840 and R 51015840-ATGAGGTAGTCTGTCAGGT-31015840Relative quantification of gene expression with real-timePCR data was calculated relative to 120573-actin
27 Analysis of GABA Production GABA formation in cul-tured medium and cytoplasmic fraction of L plantarumLG42 cells were analyzed by TLC as described (25) In orderto produce GABA in the culture medium 1 MSG wasadded to MRS broth and then cultured for 48 h at 30∘Cand verified the existence or nonexistence of GABA in thebacterial cytoplasmic fraction by using TLC
28 Statistical Analysis All values are expressed as mean plusmnSD The data were analyzed by one-way ANOVA using SPSSversion 160 The differences among groups were assessedusingDuncanrsquosmultiple range test Statistical significancewasconsidered at 119875 value lt 005
3 Results
31 Oil Red O Staining and Intracellular Triglyceride Theeffects of GLAB on oil red O stained 3T3-L1 adipocyteare shown in Figure 1 Cells treated with 10 120583g and 20120583gconcentrations did not show any significant effects comparedwith untreated cells Differentiated cells treated with 30120583gof GLAB accumulated about a 30 decrease in intracellularlipid compared to control Also the treatment with 40120583gGLAB resulted in further reduction to approximately 58 ofthe lipid accumulation of the control
The effect of GLAB on the inhibition of intracellulartriglyceride in 3T3-L1 adipocyte is shown in Figure 2 Theresults demonstrated that GLAB caused an inhibition onintracellular triglyceride accumulation Especially 40 120583g ofGLAB treatment which was most effective compared with10 120583g treated and untreated cells (Figure 2)
32 Measurement of GPDH Activity The effects of GLAB onGPDH activity in 3T3-L1 adipocyte are shown in Figure 3GPDH activity which indicates the late phase of adipocytedifferentiation was also determined The effect of GLAB onadipogenesis was clearly dose dependent The high dose 30and 40120583g of GLAB significantly decreased GPDH activity(Figure 3)
33 mRNA Expression of Lipid Metabolism-Related Gene inDifferentiated 3T3-L1 Adipocyte PPAR120574 and CEBP120572mRNAlevels in differentiated 3T3-L1 adipocyte were significantlydecreased in cells treated with GLAB compared withuntreated cell especially 40 120583g treated cell was most effective(Figure 4) In addition the expression of PPAR120574 target geneaP2 was significantly decreased in GLAB treated cells andCD 36 was significantly decreased in 20 30 and 40 120583gtreated cells compared with untreated cell during adipocytedifferentiation (Figure 4) Cells treatedwithGLAB resulted ina significant decrease in the mRNA levels of leptin comparedto cell untreated with GLAB The GPDH mRNA level wassignificantly decreased in 30 and 40 120583gmL treated cellscompared with 10120583gmL treated and untreated cells
34 GABA Production by L plantarum LG42 GABA pro-duction by L plantarum LG42 strain was studied in MRSbroth containing 1 MSG Based on the TLC analysis datathe cultured medium and cell-free culture supernatant ofL plantarum LG42 cells contained GABA but not thecytoplasmic fraction (Figure 5) This data indicated that Lplantarum LG42 cells have GABA producing ability and theGABA was excreted mainly in the cell culture medium
4 Discussion
Obesity threatens to become the 21st centuryrsquos leadingmetabolic disease in the world [19] Complications associatedwith obesity are responsible for the most obesity related mor-bidity andmortality Obesity increases circulating cholesteroland triglyceride levels and is closely associatedwith hyperten-sion cardiovascular disease type 2 diabetes mellitus cancerrespiratory complications and osteoarthritis [20] Obesity isa condition in which adipocytes not only accumulate a largeamount of fat but also become enlarged At a cellular levelit is characterized by an increase in the number and sizeof adipocytes differentiated from fibroblastic preadipocytesin the adipose tissue [21] The severity of obesity is corre-lated with the degree of differentiation of preadipocytes toadipocytes and the enlargement of adipocytes in adipose tis-sues [22] PPAR120574 and CEBP120572 are transcriptional activatorswhich play a major role in coordinating the adipocyte geneexpression during adipocyte differentiation [23] Thereforewe investigated the effects of lactic acid bacteria isolatedgajami sik-hae (GLAB) on the differentiation of 3T3-L1cells to clarify its antiobesity mechanism GLAB significantlysuppressed lipid accumulation in 3T3-L1 cells in a nontoxicconcentration GPDH activity was decreased in 3T3-L1 cellstreated with GLAB compared to control cells incubated inthe differentiation medium (Figure 3) (119875 lt 005) GPDHis a key enzyme important for triacylglycerol synthesis [24]the present result indicates that GLAB suppresses adipocytedifferentiation resulted in reducing GAPDH level
It is noteworthy that PPAR120574 and CEBP120572 levels weredownregulated in 3T3-L1 cells treated with GLAB (Figure 4)PPAR120574 and CEBP120572 play vital roles in the early stage ofadipose differentiation [25] PPAR120574 and CEBP120572 regulatethe expression of adipogenic genes such as CD36 leptin
4 BioMed Research International
0 10 20 30 40(120583gmL)
(a)
aab ab
bc
b
0
20
40
60
80
100
120
0 10 20 30 40
Rela
tive o
il re
d O
stai
n(P
erce
ntag
e of c
ontr
ol)
H2
(120583gmL)
(b)
Figure 1The effect of lactic acid bacteria isolated gajami sik-hae on oil redO stained in 3T3-L1 adipocyte (a) Photograph of oil redO staining(b) Quantification of oil red O staining Values with different superscripts are significantly different by ANOVAwith Duncanrsquos multiple rangetests at 119875 lt 005
TGp
rote
in (m
g)
(120583gmL)0 10 20 30 40
aa
ab ab
b
0
05
1
15
2
25
3
Figure 2The effect of lactic acid bacteria isolated gajami sik-hae ontriglyceride accumulation in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
GPDH and aP2 triggering the accumulation of fat in thecells [26 27] In this study the expression of PPAR120574 andCEBP120572was inhibited byGLAB together resulting in reducedadipogenesis indirectly confirmed by measuring oil red stainand GPDH activity Therefore it appears that GLAB inhibitsadipogenesis by reducing or suppressing the expression ofPPAR120574 and CEBP120572 levels It is also reasonable to articulatethat GLAB acts directly on PPAR120574 and CEBP120572 Kim et al(2008) showed that treating 3T3-L1 cells with milk fermentedwith lactic acid bacteria isolated from kimchi decreased thelevels of PPAR120574 and CEBP-120572 expression [28] Thus it couldbe possible that lactic acid bacteria isolated from gajami sik-hae affects the downstreamgenes of PPAR120574 andCEBP120572 thusinhibiting adipogenesis in 3T3-L1 cells
a
ab ab
b
c
0
5
10
15
20
25
0 10 20 30 40
Pro
tein
(U
mg)
Figure 3 The effect of lactic acid bacteria isolated gajami sik-hae on GPDH activity in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
aP2 gene is central to the pathway that links obesity toinsulin resistance possibly by linking fatty acid metabolismto the expression of (tumor necrosis factor-120572) TNF-120572 [29 30]CD36mRNAexpression is activated during 3T3-L1 adipocytedifferentiation and CD36 protein levels are positively cor-related with PPAR120574 and CEBP120572 [31] CD36 is a long chainfatty acid transporter present on the plasma membrane aswell as in intracellular pools of the skeletal muscle [32]High level CD36 might result in lipid accumulation whichis supported by studies using CD36 null mice [33] Leptin ahormone and the product of the ob gene is primarily secretedby adipose tissue It is involved in the regulation of energyexpenditure and food intake [34] The expression of leptin isregulated by several substances like insulin glucocorticoidsand TNF-120572 Here in this study we found that aP2 CD36 and
BioMed Research International 5
ab b
c
d
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(a)
a
b b
cd
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(b)
a
b b
cc
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
aP2
(c)
aa
abb
c
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
GPDH
(d)
a
b bc
d
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
Leptin
0
1
(e)
a ab b
c c
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
CD36
0
1
(f)
Figure 4 The effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of PPAR120574 CEBP120572 aP2 GPDH leptin and CD36in 3T3-L1 cells Values with different superscripts are significantly different by ANOVA with Duncanrsquos multiple range tests at 119875 lt 005Peroxisome proliferator-activated receptor 120574 (PPAR120574) CCAATenhancer-binding protein 120572 (CEBP120572) fatty acid binding protein (aP2)glycerol-3-phosphate dehydrogenase(GPDH) fatty acid translocase (CD36)
leptinmRNAexpressionswere inhibited byGLAB (Figure 4)aP2 CD36 and leptin are the target genes of CEBP120572 andPPAR120574 and are regulated by these transcription factorsThus our results suggest that the inhibition of adipogenicgene expression induced by GLAB may be mediated by theinhibition of CEBP120572 and PPAR120574 expression
Gajami sik-hae is a fermented food containing manykinds of microorganisms with Lactobacillus and Strepto-coccaceae as the dominant species [35] Lactobacillus andLeuconostoc species among lactobacillus produces variousmaterials by proliferation fermentation and metabolismFunctional organic acids like lactic acid and citric acidsare the major products of fermentation by these bacteriaFunctional materials such as acetylcholine dextran bac-teriocin and 120574-aminobutyric acid are also produced bythese microbes depending on the origin of the fermenting
materials The fermenting materials containing carotenoidsascorbic acids phenolic compounds and amino acids playa major role in this process [18 36] Among the variousbeneficial health effects of the probiotics [37 38] theirbiological impact on obesity has generated a considerableinterest Regarding the anti-obesity property of probioticssome reports demonstrated that dairy products fermentedwith lactic acid bacteria exert anti-obesity effects [28 39 40]Previously it has been shown that cytoplasmic fractions ofuseful LAB have some beneficial effects for the improvementof health related symptoms [41 42] Among various com-pounds from LAB contributing to anti-obesity effects andimproving lipid profiles are conjugated linoleic acid (CLA)ornithine GABA hydroxy methyl glutaric acid orotic acidand so forth [18 43ndash46] Although GABA can exert an anti-obesity effect and the GLAB has GABA producing capacity
6 BioMed Research International
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
[1] H S Jung S H Lee and K L Woo ldquoEffect of salting levelson the changes of taste constituents of domestic fermentedflounder sikhae of Hamkyeng-Dordquo Journal of Food Science andBiotechnology vol 24 no 1 pp 59ndash64 1992
[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
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the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
F 51015840-AGTGAAAACTTCGATGATTACATGAA-31015840 and R51015840-GCCTGCCACTTTCCTTGTG-31015840 fatty acid translocase(CD36) F 51015840-TTGTACCTATACTGTGGCTAAATGAGA-31015840 and R 51015840-CTTGTGTTTTGAACATTTCTGCTT-31015840CCAATenhancer-binding protein 120572 (CEBP120572) F 51015840-AGCAACGAGTACCGGGTACG-31015840 and R 51015840-TGTTTGG-CTTTATCTCGGCTC-31015840 peroxisome proliferator-activatedreceptor 120574 (PPAR120574) F 51015840-CAAGAATACCAAAGTGCG-ATCAA-31015840 and R 51015840-GAGCTGGGTCTTTTCAGAAT-AATAAG-31015840 (leptin) F 51015840-CCGCCAAGCAGAGGGT-CAC-31015840 and R 51015840-GCATTCAGGGCTAACATCCAACT-31015840glycerol-3-phosphate dehydrogenase(GPDH) F 51015840-CTCT-TCTTGCCGCTTCAGTTT-31015840 and R 51015840-CATGTAGGCCA-TGAGGTCCACCAC-31015840 120573-actin F 51015840-ATGGATGACGA-TATCGCT-31015840 and R 51015840-ATGAGGTAGTCTGTCAGGT-31015840Relative quantification of gene expression with real-timePCR data was calculated relative to 120573-actin
27 Analysis of GABA Production GABA formation in cul-tured medium and cytoplasmic fraction of L plantarumLG42 cells were analyzed by TLC as described (25) In orderto produce GABA in the culture medium 1 MSG wasadded to MRS broth and then cultured for 48 h at 30∘Cand verified the existence or nonexistence of GABA in thebacterial cytoplasmic fraction by using TLC
28 Statistical Analysis All values are expressed as mean plusmnSD The data were analyzed by one-way ANOVA using SPSSversion 160 The differences among groups were assessedusingDuncanrsquosmultiple range test Statistical significancewasconsidered at 119875 value lt 005
3 Results
31 Oil Red O Staining and Intracellular Triglyceride Theeffects of GLAB on oil red O stained 3T3-L1 adipocyteare shown in Figure 1 Cells treated with 10 120583g and 20120583gconcentrations did not show any significant effects comparedwith untreated cells Differentiated cells treated with 30120583gof GLAB accumulated about a 30 decrease in intracellularlipid compared to control Also the treatment with 40120583gGLAB resulted in further reduction to approximately 58 ofthe lipid accumulation of the control
The effect of GLAB on the inhibition of intracellulartriglyceride in 3T3-L1 adipocyte is shown in Figure 2 Theresults demonstrated that GLAB caused an inhibition onintracellular triglyceride accumulation Especially 40 120583g ofGLAB treatment which was most effective compared with10 120583g treated and untreated cells (Figure 2)
32 Measurement of GPDH Activity The effects of GLAB onGPDH activity in 3T3-L1 adipocyte are shown in Figure 3GPDH activity which indicates the late phase of adipocytedifferentiation was also determined The effect of GLAB onadipogenesis was clearly dose dependent The high dose 30and 40120583g of GLAB significantly decreased GPDH activity(Figure 3)
33 mRNA Expression of Lipid Metabolism-Related Gene inDifferentiated 3T3-L1 Adipocyte PPAR120574 and CEBP120572mRNAlevels in differentiated 3T3-L1 adipocyte were significantlydecreased in cells treated with GLAB compared withuntreated cell especially 40 120583g treated cell was most effective(Figure 4) In addition the expression of PPAR120574 target geneaP2 was significantly decreased in GLAB treated cells andCD 36 was significantly decreased in 20 30 and 40 120583gtreated cells compared with untreated cell during adipocytedifferentiation (Figure 4) Cells treatedwithGLAB resulted ina significant decrease in the mRNA levels of leptin comparedto cell untreated with GLAB The GPDH mRNA level wassignificantly decreased in 30 and 40 120583gmL treated cellscompared with 10120583gmL treated and untreated cells
34 GABA Production by L plantarum LG42 GABA pro-duction by L plantarum LG42 strain was studied in MRSbroth containing 1 MSG Based on the TLC analysis datathe cultured medium and cell-free culture supernatant ofL plantarum LG42 cells contained GABA but not thecytoplasmic fraction (Figure 5) This data indicated that Lplantarum LG42 cells have GABA producing ability and theGABA was excreted mainly in the cell culture medium
4 Discussion
Obesity threatens to become the 21st centuryrsquos leadingmetabolic disease in the world [19] Complications associatedwith obesity are responsible for the most obesity related mor-bidity andmortality Obesity increases circulating cholesteroland triglyceride levels and is closely associatedwith hyperten-sion cardiovascular disease type 2 diabetes mellitus cancerrespiratory complications and osteoarthritis [20] Obesity isa condition in which adipocytes not only accumulate a largeamount of fat but also become enlarged At a cellular levelit is characterized by an increase in the number and sizeof adipocytes differentiated from fibroblastic preadipocytesin the adipose tissue [21] The severity of obesity is corre-lated with the degree of differentiation of preadipocytes toadipocytes and the enlargement of adipocytes in adipose tis-sues [22] PPAR120574 and CEBP120572 are transcriptional activatorswhich play a major role in coordinating the adipocyte geneexpression during adipocyte differentiation [23] Thereforewe investigated the effects of lactic acid bacteria isolatedgajami sik-hae (GLAB) on the differentiation of 3T3-L1cells to clarify its antiobesity mechanism GLAB significantlysuppressed lipid accumulation in 3T3-L1 cells in a nontoxicconcentration GPDH activity was decreased in 3T3-L1 cellstreated with GLAB compared to control cells incubated inthe differentiation medium (Figure 3) (119875 lt 005) GPDHis a key enzyme important for triacylglycerol synthesis [24]the present result indicates that GLAB suppresses adipocytedifferentiation resulted in reducing GAPDH level
It is noteworthy that PPAR120574 and CEBP120572 levels weredownregulated in 3T3-L1 cells treated with GLAB (Figure 4)PPAR120574 and CEBP120572 play vital roles in the early stage ofadipose differentiation [25] PPAR120574 and CEBP120572 regulatethe expression of adipogenic genes such as CD36 leptin
4 BioMed Research International
0 10 20 30 40(120583gmL)
(a)
aab ab
bc
b
0
20
40
60
80
100
120
0 10 20 30 40
Rela
tive o
il re
d O
stai
n(P
erce
ntag
e of c
ontr
ol)
H2
(120583gmL)
(b)
Figure 1The effect of lactic acid bacteria isolated gajami sik-hae on oil redO stained in 3T3-L1 adipocyte (a) Photograph of oil redO staining(b) Quantification of oil red O staining Values with different superscripts are significantly different by ANOVAwith Duncanrsquos multiple rangetests at 119875 lt 005
TGp
rote
in (m
g)
(120583gmL)0 10 20 30 40
aa
ab ab
b
0
05
1
15
2
25
3
Figure 2The effect of lactic acid bacteria isolated gajami sik-hae ontriglyceride accumulation in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
GPDH and aP2 triggering the accumulation of fat in thecells [26 27] In this study the expression of PPAR120574 andCEBP120572was inhibited byGLAB together resulting in reducedadipogenesis indirectly confirmed by measuring oil red stainand GPDH activity Therefore it appears that GLAB inhibitsadipogenesis by reducing or suppressing the expression ofPPAR120574 and CEBP120572 levels It is also reasonable to articulatethat GLAB acts directly on PPAR120574 and CEBP120572 Kim et al(2008) showed that treating 3T3-L1 cells with milk fermentedwith lactic acid bacteria isolated from kimchi decreased thelevels of PPAR120574 and CEBP-120572 expression [28] Thus it couldbe possible that lactic acid bacteria isolated from gajami sik-hae affects the downstreamgenes of PPAR120574 andCEBP120572 thusinhibiting adipogenesis in 3T3-L1 cells
a
ab ab
b
c
0
5
10
15
20
25
0 10 20 30 40
Pro
tein
(U
mg)
Figure 3 The effect of lactic acid bacteria isolated gajami sik-hae on GPDH activity in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
aP2 gene is central to the pathway that links obesity toinsulin resistance possibly by linking fatty acid metabolismto the expression of (tumor necrosis factor-120572) TNF-120572 [29 30]CD36mRNAexpression is activated during 3T3-L1 adipocytedifferentiation and CD36 protein levels are positively cor-related with PPAR120574 and CEBP120572 [31] CD36 is a long chainfatty acid transporter present on the plasma membrane aswell as in intracellular pools of the skeletal muscle [32]High level CD36 might result in lipid accumulation whichis supported by studies using CD36 null mice [33] Leptin ahormone and the product of the ob gene is primarily secretedby adipose tissue It is involved in the regulation of energyexpenditure and food intake [34] The expression of leptin isregulated by several substances like insulin glucocorticoidsand TNF-120572 Here in this study we found that aP2 CD36 and
BioMed Research International 5
ab b
c
d
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(a)
a
b b
cd
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(b)
a
b b
cc
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
aP2
(c)
aa
abb
c
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
GPDH
(d)
a
b bc
d
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
Leptin
0
1
(e)
a ab b
c c
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
CD36
0
1
(f)
Figure 4 The effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of PPAR120574 CEBP120572 aP2 GPDH leptin and CD36in 3T3-L1 cells Values with different superscripts are significantly different by ANOVA with Duncanrsquos multiple range tests at 119875 lt 005Peroxisome proliferator-activated receptor 120574 (PPAR120574) CCAATenhancer-binding protein 120572 (CEBP120572) fatty acid binding protein (aP2)glycerol-3-phosphate dehydrogenase(GPDH) fatty acid translocase (CD36)
leptinmRNAexpressionswere inhibited byGLAB (Figure 4)aP2 CD36 and leptin are the target genes of CEBP120572 andPPAR120574 and are regulated by these transcription factorsThus our results suggest that the inhibition of adipogenicgene expression induced by GLAB may be mediated by theinhibition of CEBP120572 and PPAR120574 expression
Gajami sik-hae is a fermented food containing manykinds of microorganisms with Lactobacillus and Strepto-coccaceae as the dominant species [35] Lactobacillus andLeuconostoc species among lactobacillus produces variousmaterials by proliferation fermentation and metabolismFunctional organic acids like lactic acid and citric acidsare the major products of fermentation by these bacteriaFunctional materials such as acetylcholine dextran bac-teriocin and 120574-aminobutyric acid are also produced bythese microbes depending on the origin of the fermenting
materials The fermenting materials containing carotenoidsascorbic acids phenolic compounds and amino acids playa major role in this process [18 36] Among the variousbeneficial health effects of the probiotics [37 38] theirbiological impact on obesity has generated a considerableinterest Regarding the anti-obesity property of probioticssome reports demonstrated that dairy products fermentedwith lactic acid bacteria exert anti-obesity effects [28 39 40]Previously it has been shown that cytoplasmic fractions ofuseful LAB have some beneficial effects for the improvementof health related symptoms [41 42] Among various com-pounds from LAB contributing to anti-obesity effects andimproving lipid profiles are conjugated linoleic acid (CLA)ornithine GABA hydroxy methyl glutaric acid orotic acidand so forth [18 43ndash46] Although GABA can exert an anti-obesity effect and the GLAB has GABA producing capacity
6 BioMed Research International
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
[1] H S Jung S H Lee and K L Woo ldquoEffect of salting levelson the changes of taste constituents of domestic fermentedflounder sikhae of Hamkyeng-Dordquo Journal of Food Science andBiotechnology vol 24 no 1 pp 59ndash64 1992
[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
BioMed Research International 7
the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
Figure 1The effect of lactic acid bacteria isolated gajami sik-hae on oil redO stained in 3T3-L1 adipocyte (a) Photograph of oil redO staining(b) Quantification of oil red O staining Values with different superscripts are significantly different by ANOVAwith Duncanrsquos multiple rangetests at 119875 lt 005
TGp
rote
in (m
g)
(120583gmL)0 10 20 30 40
aa
ab ab
b
0
05
1
15
2
25
3
Figure 2The effect of lactic acid bacteria isolated gajami sik-hae ontriglyceride accumulation in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
GPDH and aP2 triggering the accumulation of fat in thecells [26 27] In this study the expression of PPAR120574 andCEBP120572was inhibited byGLAB together resulting in reducedadipogenesis indirectly confirmed by measuring oil red stainand GPDH activity Therefore it appears that GLAB inhibitsadipogenesis by reducing or suppressing the expression ofPPAR120574 and CEBP120572 levels It is also reasonable to articulatethat GLAB acts directly on PPAR120574 and CEBP120572 Kim et al(2008) showed that treating 3T3-L1 cells with milk fermentedwith lactic acid bacteria isolated from kimchi decreased thelevels of PPAR120574 and CEBP-120572 expression [28] Thus it couldbe possible that lactic acid bacteria isolated from gajami sik-hae affects the downstreamgenes of PPAR120574 andCEBP120572 thusinhibiting adipogenesis in 3T3-L1 cells
a
ab ab
b
c
0
5
10
15
20
25
0 10 20 30 40
Pro
tein
(U
mg)
Figure 3 The effect of lactic acid bacteria isolated gajami sik-hae on GPDH activity in 3T3-L1 adipocyte Values with differentsuperscripts are significantly different by ANOVA with Duncanrsquosmultiple range tests at 119875 lt 005
aP2 gene is central to the pathway that links obesity toinsulin resistance possibly by linking fatty acid metabolismto the expression of (tumor necrosis factor-120572) TNF-120572 [29 30]CD36mRNAexpression is activated during 3T3-L1 adipocytedifferentiation and CD36 protein levels are positively cor-related with PPAR120574 and CEBP120572 [31] CD36 is a long chainfatty acid transporter present on the plasma membrane aswell as in intracellular pools of the skeletal muscle [32]High level CD36 might result in lipid accumulation whichis supported by studies using CD36 null mice [33] Leptin ahormone and the product of the ob gene is primarily secretedby adipose tissue It is involved in the regulation of energyexpenditure and food intake [34] The expression of leptin isregulated by several substances like insulin glucocorticoidsand TNF-120572 Here in this study we found that aP2 CD36 and
BioMed Research International 5
ab b
c
d
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(a)
a
b b
cd
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
(b)
a
b b
cc
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
aP2
(c)
aa
abb
c
0
02
04
06
08
1
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
GPDH
(d)
a
b bc
d
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
Leptin
0
1
(e)
a ab b
c c
02
04
06
08
12
0 10 20 30 40
Rel
ativ
e m
RN
A le
vel
CD36
0
1
(f)
Figure 4 The effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of PPAR120574 CEBP120572 aP2 GPDH leptin and CD36in 3T3-L1 cells Values with different superscripts are significantly different by ANOVA with Duncanrsquos multiple range tests at 119875 lt 005Peroxisome proliferator-activated receptor 120574 (PPAR120574) CCAATenhancer-binding protein 120572 (CEBP120572) fatty acid binding protein (aP2)glycerol-3-phosphate dehydrogenase(GPDH) fatty acid translocase (CD36)
leptinmRNAexpressionswere inhibited byGLAB (Figure 4)aP2 CD36 and leptin are the target genes of CEBP120572 andPPAR120574 and are regulated by these transcription factorsThus our results suggest that the inhibition of adipogenicgene expression induced by GLAB may be mediated by theinhibition of CEBP120572 and PPAR120574 expression
Gajami sik-hae is a fermented food containing manykinds of microorganisms with Lactobacillus and Strepto-coccaceae as the dominant species [35] Lactobacillus andLeuconostoc species among lactobacillus produces variousmaterials by proliferation fermentation and metabolismFunctional organic acids like lactic acid and citric acidsare the major products of fermentation by these bacteriaFunctional materials such as acetylcholine dextran bac-teriocin and 120574-aminobutyric acid are also produced bythese microbes depending on the origin of the fermenting
materials The fermenting materials containing carotenoidsascorbic acids phenolic compounds and amino acids playa major role in this process [18 36] Among the variousbeneficial health effects of the probiotics [37 38] theirbiological impact on obesity has generated a considerableinterest Regarding the anti-obesity property of probioticssome reports demonstrated that dairy products fermentedwith lactic acid bacteria exert anti-obesity effects [28 39 40]Previously it has been shown that cytoplasmic fractions ofuseful LAB have some beneficial effects for the improvementof health related symptoms [41 42] Among various com-pounds from LAB contributing to anti-obesity effects andimproving lipid profiles are conjugated linoleic acid (CLA)ornithine GABA hydroxy methyl glutaric acid orotic acidand so forth [18 43ndash46] Although GABA can exert an anti-obesity effect and the GLAB has GABA producing capacity
6 BioMed Research International
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
[1] H S Jung S H Lee and K L Woo ldquoEffect of salting levelson the changes of taste constituents of domestic fermentedflounder sikhae of Hamkyeng-Dordquo Journal of Food Science andBiotechnology vol 24 no 1 pp 59ndash64 1992
[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
BioMed Research International 7
the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
Figure 4 The effect of lactic acid bacteria isolated gajami sik-hae on mRNA levels of PPAR120574 CEBP120572 aP2 GPDH leptin and CD36in 3T3-L1 cells Values with different superscripts are significantly different by ANOVA with Duncanrsquos multiple range tests at 119875 lt 005Peroxisome proliferator-activated receptor 120574 (PPAR120574) CCAATenhancer-binding protein 120572 (CEBP120572) fatty acid binding protein (aP2)glycerol-3-phosphate dehydrogenase(GPDH) fatty acid translocase (CD36)
leptinmRNAexpressionswere inhibited byGLAB (Figure 4)aP2 CD36 and leptin are the target genes of CEBP120572 andPPAR120574 and are regulated by these transcription factorsThus our results suggest that the inhibition of adipogenicgene expression induced by GLAB may be mediated by theinhibition of CEBP120572 and PPAR120574 expression
Gajami sik-hae is a fermented food containing manykinds of microorganisms with Lactobacillus and Strepto-coccaceae as the dominant species [35] Lactobacillus andLeuconostoc species among lactobacillus produces variousmaterials by proliferation fermentation and metabolismFunctional organic acids like lactic acid and citric acidsare the major products of fermentation by these bacteriaFunctional materials such as acetylcholine dextran bac-teriocin and 120574-aminobutyric acid are also produced bythese microbes depending on the origin of the fermenting
materials The fermenting materials containing carotenoidsascorbic acids phenolic compounds and amino acids playa major role in this process [18 36] Among the variousbeneficial health effects of the probiotics [37 38] theirbiological impact on obesity has generated a considerableinterest Regarding the anti-obesity property of probioticssome reports demonstrated that dairy products fermentedwith lactic acid bacteria exert anti-obesity effects [28 39 40]Previously it has been shown that cytoplasmic fractions ofuseful LAB have some beneficial effects for the improvementof health related symptoms [41 42] Among various com-pounds from LAB contributing to anti-obesity effects andimproving lipid profiles are conjugated linoleic acid (CLA)ornithine GABA hydroxy methyl glutaric acid orotic acidand so forth [18 43ndash46] Although GABA can exert an anti-obesity effect and the GLAB has GABA producing capacity
6 BioMed Research International
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
[1] H S Jung S H Lee and K L Woo ldquoEffect of salting levelson the changes of taste constituents of domestic fermentedflounder sikhae of Hamkyeng-Dordquo Journal of Food Science andBiotechnology vol 24 no 1 pp 59ndash64 1992
[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
BioMed Research International 7
the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
Figure 5 TLC analysis of GABA in the culture medium andcytoplasmic fraction of L plantarum LG42 cells A spot of standardMSG B spot of standard GABA CndashE spots of cell culture medium(C) cell-free supernatant (D) and cytoplasmic fraction of cells (E)cultured in MRS broth with 1MSG F spot of cell culture mediumcultured without added MSG
and since the cytoplasmic fraction used in this study containsa negligible amount of GABA (Figure 5) it is not clearwhat compounds of GLAB worked as the main principlesof the anti-obesity effects Therefore GLAB having GABAproducing ability can be used as a useful material not onlyfor the production of fermented foods such as sik-hae withan enhanced level of GABA but also for the investigationof unknown compounds except GABA for the anti-obesityeffects Further studies are required to identify the activecompounds of GLAB that have specific effects on obesity
In conclusion the inhibitory effects of GLAB on 3T3-L1 adipocyte as indicated by a decrease in intracellulartriglyceride content andGPDH activity have been elucidatedIt appears to bemediated throughdownregulating the expres-sion of adipogenic transcription factor PPAR120574 and CEBP120572and adipocyte-specific gene such as aP2 leptin GPDH andCD36These results indicate that GLABmay play a role in thecontrol of adipogenesis and might have further implicationfor in vivo antiobesity effect
Conflict of Interests
The authors declare that they have no conflict of interests
Acknowledgments
This paper was supported by the Globalization of KoreanFoods RampD program (Grant no 911049-1) funded by theMinistry of Food Agriculture Forestry and Fisheries Repub-lic of KoreaThe authors thankNUCElectronics Co Ltd fortheir assistance
References
[1] H S Jung S H Lee and K L Woo ldquoEffect of salting levelson the changes of taste constituents of domestic fermentedflounder sikhae of Hamkyeng-Dordquo Journal of Food Science andBiotechnology vol 24 no 1 pp 59ndash64 1992
[2] J H Yoon S S Kang K C Lee et al ldquoBacillus jeotgalisp nov isolated from jeotgal Korean traditional fermentedseafoodrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 3 pp 1087ndash1092 2001
[3] C H Lee ldquoLactic acid fermented foods and their benefits inAsiardquo Food Science and Biotechnology vol 5 no 3 pp 187ndash1971996
[4] H J Lee Y J Joo C S Park et al ldquoPurification and character-ization of a bacteriocin produced by Lactococcus lactis subsplactis H-559 isolated from kimchirdquo Journal of Bioscience andBioengineering vol 88 no 2 pp 153ndash159 1999
[5] S J Kim ldquoPotential probiotic properties of lactic acid bacteriaisolated from kimchirdquo Food Science and Biotechnology vol 14no 4 pp 547ndash550 2005
[6] A S Naidu W R Bidlack and R A Clemens ldquoProbioticspectra of lactic acid bacteria (LAB)rdquo Critical Reviews in FoodScience and Nutrition vol 39 no 1 pp 13ndash126 1999
[7] F Leroy and L De Vuyst ldquoLactic acid bacteria as functionalstarter cultures for the food fermentation industryrdquo Trends inFood Science amp Technology vol 15 no 2 pp 67ndash78 2004
[8] B Goldin and S L Gorbach ldquoAlterations in fecal microfloraenzymes related to diet age lactobacillus supplements anddimethylhydrazinerdquo Cancer vol 40 no 5 pp 2421ndash2426 1977
[9] S E Gilliland and D K Walker ldquoFactors to consider whenselecting a culture of Lactobacillus acidophilus as a dietaryadjunct to produce a hypocholesterolemic effect in humansrdquoJournal of Dairy Science vol 73 no 4 pp 905ndash911 1990
[10] R Fuller ldquoProbiotics in human medicinerdquo Gut vol 32 no 4pp 439ndash442 1991
[11] H Poo H M Pyo T Y Lee et al ldquoOral administration ofhuman papillomavirus type 16 E7 displayed on Lactobacilluscasei induces E7-specific antitumor effects in C57BL6 micerdquoInternational Journal of Cancer vol 119 no 7 pp 1702ndash17092006
[12] H Y Lee J H Park S H Seok et al ldquoHuman originatedbacteria Lactobacillus rhamnosus PL60 produce conjugatedlinoleic acid and show anti-obesity effects in diet-induced obesemicerdquo Biochimica et Biophysica Acta vol 1761 no 7 pp 736ndash744 2006
[13] P Krogsgaard-Larsen ldquoGABA receptorsrdquo in Receptor Phama-cology and Function M Williams R A Glennon and P M WM Timmermans Eds pp 349ndash383 Marcel Dekker New YorkNY USA 1989
[14] B V Manyam L Katz T A Hare K Kaniefski and R DTremblay ldquoIsoniazid-induced elevation of CSF GABA levelsand effects on chorea in Huntingtonrsquos diseaserdquo Annals ofNeurology vol 10 no 1 pp 35ndash37 1981
[15] C F Liu Y T Tung C L Wu B H Lee W H Hsu and T MPan ldquoAntihypertensive effects of Lactobacillus-fermented milkorally administered to spontaneously hypertensive ratsrdquo Journalof Agricultural and FoodChemistry vol 59 no 9 pp 4537ndash45432011
[16] M Omori T Yano J Okamoto T Tsushida T Murai andM Higuchi ldquoEffect of anaerobic treated tea (Gabaron Tea) onblood pressure of spontaneously hypertensive ratsrdquo Journal of
BioMed Research International 7
the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
the Agricultural Chemical Society of Japan vol 61 no 11 pp1449ndash1451 1987
[17] H Aoki Y Furuya Y Endo and K Fujimoto ldquoEffect of 120574-aminobutyric acid-enriched tempeh-like fermented soybean(GABA-tempeh) on the blood pressure of spontaneously hyper-tensive ratsrdquo Bioscience Biotechnology and Biochemistry vol 67no 8 pp 1806ndash1808 2003
[18] M O Bae H J Kim Y S Cha M K Lee and S H OhldquoEffects of Kimchi lactic acid bacteria Lactobacillus sp OPK2-59 with high GABA producing capacity on liver functionimprovementrdquo Journal of the Korean Society of Food Science andNutrition vol 38 no 11 pp 1499ndash1505 2009
[19] Formiguera andACanton ldquoObesity epidemiology and clinicalaspectsrdquo Best Practice amp Research Clinical Gastroenterology vol18 no 6 pp 1125ndash1146 2004
[20] P G Kopelman ldquoObesity as a medical problemrdquo Nature vol404 no 6778 pp 635ndash643 2000
[21] T Furuyashiki H Nagayasu Y Aoki et al ldquoTea catechinsuppresses adipocyte differentiation accompanied by down-regulation of PPAR1205742 and CEBP120572 in 3T3-L1 cellsrdquo BioscienceBiotechnology and Biochemistry vol 68 no 11 pp 2353ndash23592004
[22] Y W Wang and P J Jones ldquoConjugated linoleic acid andobesity control efficacy andmechanismsrdquo International Journalof Obesity and Related Metabolic Disorders vol 28 no 8 pp941ndash955 2004
[23] X Guo and K Liao ldquoAnalysis of gene expression profile during3T3-L1 preadipocyte differentiationrdquo Gene vol 251 no 1 pp45ndash53 2000
[24] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979
[25] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000
[26] J Berger and D E Moller ldquoThe mechanisms of action ofPPARsrdquo Annual Review of Medicine vol 53 pp 409ndash435 2002
[27] T Kawada N Takahashi and T Fushiki ldquoBiochemical andphysiological characteristics of fat cellrdquo Journal of NutritionalScience and Vitaminology vol 47 no 1 pp 1ndash12 2001
[28] N H Kim P D Moon S J Kim et al ldquoLipid profile loweringeffect of Soypro fermented with lactic acid bacteria isolatedfrom Kimchi in high-fat diet-induced obese ratsrdquo BioFactorsvol 33 no 1 pp 49ndash60 2008
[29] P Tontonoz E Hu R A Graves A I Budavari and B MSpieglman ldquomPPAR gamma 2 tissue-specific regulator of anadipocyte enhancerrdquo Genes and Development vol 8 no 10 pp1224ndash1234 1994
[30] G S Hotamisligil R S Johnson R J Distel R Ellis V EPapaioannou and B M Spiegelman ldquoUncoupling of obesityfrom insulin resistance through a targeted mutation in aP2 theadipocyte fatty acid binding proteinrdquo Science vol 274 no 5291pp 1377ndash1379 1996
[31] L Qiao C Zou P Shao J Schaack P F Johnson and J ShaoldquoTranscriptional regulation of fatty acid translocaseCD36expression by CCAATenhancer-binding protein 120572rdquo The Jour-nal of Biological Chemistry vol 283 no 14 pp 8788ndash8795 2008
[32] H Muller K Deckers and J Eckel ldquoThe fatty acid translocase(FAT)CD36 and the glucose transporter GLUT4 are localizedin different cellular compartments in rat cardiac musclerdquo
Biochemical and Biophysical Research Communications vol 293no 2 pp 665ndash669 2002
[33] B I Frohnert T Y Hui and D A Bernlohr ldquoIdentificationof a functional peroxisome proliferator-responsive element inthe murine fatty acid transport protein generdquo The Journal ofBiological Chemistry vol 274 no 7 pp 3970ndash3977 1999
[34] D S Weigle T R Bukowski D C Foster et al ldquoRecombinantob protein reduces feeding and body weight in the obobmouserdquo The Journal of Clinical Investigation vol 96 no 4 pp2065ndash2070 1995
[35] H J Lee and W J Kim ldquoIsolation and characterization of anti-listerial and amylase sensitive enterocin producingEnterococcusfaeciumDB1 from gajami-sikhae a fermented flat fish in KoreardquoFood Science and Biotechnology vol 19 no 2 pp 373ndash381 2010
[36] K Y Park and H S Cheigh ldquoAntimutagenic and anticancereffects of lactic acid bacteria isolated from Kimchirdquo BioindustryNews vol 13 no 3 pp 11ndash17 2000
[37] J Schrezenmeir and M de Vrese ldquoProbiotics prebiotics andsynbioticsmdashapproaching a definitionrdquoThe American Journal ofClinical Nutrition vol 73 no 2 supplement pp 361Sndash364S2001
[38] F P J Martin Y Wang N Sprenger et al ldquoProbiotic modu-lation of symbiotic gut microbial-host metabolic interactionsin a humanized microbiome mouse modelrdquoMolecular SystemsBiology vol 4 article 157 2008
[39] K Manabu and H Fang ldquoAnti-obesity effects of probiotics infermented milkrdquo Journal of Intestinal Microbiology vol 24 no4 pp 265ndash271 2010
[40] J Z Xiao S KondoN Takahashi et al ldquoEffects ofmilk productsfermented by Bifidobacterium longum on blood lipids in ratsand healthy adult male volunteersrdquo Journal of Dairy Science vol86 no 7 pp 2452ndash2461 2003
[41] MThirabunyanon P Boonprasom and P Niamsup ldquoProbioticpotential of lactic acid bacteria isolated from fermented dairymilks on antiproliferation of colon cancer cellsrdquo BiotechnologyLetters vol 31 no 4 pp 571ndash576 2009
[42] H J Hur K W Lee H Y Kim D K Chung and H J Lee ldquoInvitro immunopotentiating activities of cellular fractions of lacticacid bacteria isolated from Kimchi and bifidobacteriardquo Journalof Microbiology and Biotechnology vol 16 no 5 pp 661ndash6662006
[43] Y J Moon J R Soh J J Yu H S Sohn Y S Cha and S HOh ldquoIntracellular lipid accumulation inhibitory effect of Weis-sella koreensis OK1-6 isolated from Kimchi on differentiatingadipocyterdquo Journal of Applied Microbiology vol 113 no 3 pp652ndash658 2012
[44] J R Soh N S Kim C H Oh S H Oh and Y S ChaldquoCarnithine andor GABA supplementation increases immunefunction and changes lipid profiles and some lipid solublevitamins in mice chronically administered alcoholrdquo Journal ofFood Science and Nutrition vol 15 no 3 pp 196ndash205 2010
[45] Y G An ldquoProbiotic lactic acid bacteriardquo Journal of KoreanSociety of Food Science and Nutrition vol 24 no 4 pp 817ndash8322011
[46] Y Xu G OWilliam C C Lee G MMartin and T QingchunldquoRole of GABA release from leptin receptor-expressing neuronsin body weight regulationrdquo Endocrinology vol 153 no 5 pp2223ndash2233 2012
