Research Article Alliin, a Garlic ( Allium sativum

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 381815 11 pageshttpdxdoiorg1011552013381815

Research ArticleAlliin a Garlic (Allium sativum) Compound PreventsLPS-Induced Inflammation in 3T3-L1 Adipocytes

Saray Quintero-Fabiaacuten12 Daniel Ortuntildeo-Sahaguacuten1

Manuel Vaacutezquez-Carrera3 and Rociacuteo Ivette Loacutepez-Roa2

1 Laboratorio de Desarrollo y Regeneracion Neural Instituto de Neurobiologıa Departamento de Biologıa Celular y MolecularCUCBA Universidad de Guadalajara Camino Ing R Padilla Sanchez 2100 Las Agujas 44600 Zapopan JAL Mexico

2Departamento de Farmacobiologıa CUCEI Universidad de Guadalajara Boulevard Marcelino Garcıa Barragan No 1421Esquina Calzada Olımpica 44430 Guadalajara JAL Mexico

3 Unidad de Farmacologıa Departamento de Farmacologıa y Quımica Terapeutica Facultad de FarmaciaInstitut de Biomedicina de la UB (IBUB) and CIBER de Diabetes y Enfermedades Metabolicas Asociadas(CIBERDEM)-Instituto de Salud Carlos III 08028 Barcelona Spain

Correspondence should be addressed to Daniel Ortuno-Sahagun danielortunosahagungmailcomand Rocıo Ivette Lopez-Roa rockroa2000yahooes

Received 10 September 2013 Revised 3 November 2013 Accepted 9 November 2013

Academic Editor Giuseppe Valacchi

Copyright copy 2013 Saray Quintero-Fabian et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Garlic (Allium sativum L) has been used to alleviate a variety of health problems due to its high content of organosulfur compoundsand antioxidant activityThemain active component is alliin (S-allyl cysteine sulfoxide) a potent antioxidant with cardioprotectiveand neuroprotective actions In addition it helps to decrease serum levels of glucose insulin triglycerides and uric acid as wellas insulin resistance and reduces cytokine levels However its potential anti-inflammatory effect is unknown We examined theeffects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR Western blot and microarrays analysisof 22000 genes Incubation of cells for 24 h with 100120583molL alliin prevented the increase in the expression of proinflammatorygenes IL-6 MCP-1 and Egr-1 in 3T3-L1 adipocytes exposed to 100 ngmL LPS for 1 h Interestingly the phosphorylation of ERK12which is involved in LPS-induced inflammation in adipocytes was decreased following alliin treatment Furthermore the geneexpression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genesrelated with cancer The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating ananti-inflammatory gene expression profile and by modifying adipocyte metabolic profile

1 Introduction

Obesity has been traditionally linked to metabolic dysfunc-tion led by adipocyte proliferation and hypertrophy [1]However some other systemic alterations are being studiedas related to obesity for example inflammation and immunedysfunction [2] Adipose tissue is now widely considered asan endocrine tissue capable of producing chronic inflamma-tory responses [3ndash5] Obesity has been shown to cause anincrease in plasma concentrations of a number of proinflam-matory markers (eg IL-6 TNF-120572) that are expressed andreleased by adipocytes [6] Diet-induced obesity increases

local and systemic inflammatory adipocytokines in humansand in rodents these factors contribute to adverse health out-comes [7] The homeostatic balance between pro- and anti-inflammatory cytokines and adipokines defines the profileand magnitude of inflammation and its effects on insulinsensitivity and glucose homeostasis [8] Therefore therapiesable to modulate the inflammatory state of adipose tissue arebeing considered for the treatment of obesity [9] Howeverfactors that might mitigate or act against this inflammatoryresponse have remained elusive

Garlic (Allium sativum) is one of the oldest medicinalplants used by different cultures [10] Allium vegetables

2 Mediators of Inflammation

Table 1 Primer sequences used for real-time quantitative PCR

Gene name Ensembl gene Primer sequence (51015840-31015840) bp Tm

Il-6 ENSMUST00000026845 F-TACACATGTTCTCTGGGAAATCGTR-AAGTGCATCATCGTTGTTCATACA 85 75

Aprt ENSMUST00000006764 F-CAGCGGCAAGATCGACTACAR-AGCTAGGGAAGGGCCAAACA 67 60

Mcp-1 ENSMUST00000000193 F-GCTGGAGAGCTACAAGAGGATCAR-CTCTCTCTTGAGCTTGGTGACAAA 79 60

Tnf -120572 ENSMUST00000025263 F-ATGGCCCAGACCCTCACAR-TTGCTACGACGTGGGCTACA 73 60

Egr-1 ENSMUST00000064795 F-GCCGAGCGAACAACCCTATR-CCATCGCCTTCTCATTATTCAGA 77 60

comprise one natural source of organic sulfur-containingcompounds and have been widely investigated regardingtheir therapeutic applications [11 12] mainly due to itscardioprotective effect and to its anticancerogenic properties[10 13] However its anti-inflammatory effects have receivedless attention For example garlic compounds can exert ananti-inflammatory effect by inhibiting the oxidative stress-induced activation of nuclear factor-kappa B (NF-120581B) [14]which is implicated in the expression of proinflammatoryenzymes such as inducible nitric oxide synthase (NOS)and cyclooxygenase-II (COX-II) In particular allicin andajoene two garlic compounds significantly suppressed nitricoxide production by lipopolysaccharide (LPS) stimulationaccompanied by suppression in inducible nitric oxide (iNOS)expression and iNOS activity [15] Although it appearspromising that garlic and its derivatives possess antidiabeticpotential an understanding of the antidiabetic effects of garlicis still in its early stages Furthermore the active compoundsin garlic and doses thereof which can effectively provideantidiabetic effects (ie glycemic control and amelioration ofdiabetic complications) remain to be established

Alliin (S-allyl cysteine sulfoxide) first identified by Stolland Seebeck in 1947 is considered the main specific principleof garlic since that time [16] Alliin can be found in intactgarlic and its reduced form S-allyl-cysteine is the majorcomponent of aged garlic extract (AGE) together with otherderived organosulfur compounds [12 13] It is absorbed inthe intestine via the amino acid transported for cysteine [17]exhibits an hypoglycemic effect and also increases bloodinsulin concentrations [18] and its antioxidant activity hasbeen widely studied [19] Nonetheless to our knowledge itsanti-inflammatory potential has not yet been explored

Mouse preadipocyte 3T3-L1 cells are the most commonlystudied and available adipogenic cell line This cell linehas been quite useful in identifying key molecular mark-ers transcription factors and various interactions that arerequired for preadipocyte differentiation [20] Additionally3T3-L1 adipocytes are able to respond to LPS by meansof a fully intact innate immunity pathway through theproduction and secretion of immunomodulatory (mainlyproinflammatory) molecules such as IL-6 TNF-120572 and TLR-2 [21] and through a TLR-ligand-induced activation whichtriggers a proinflammatory and -diabetic transformation of

adipocytes [22] Interestingly this immune response activa-tion is resembled by human adipose tissue [23] consequentlyonce differentiated 3T3-L1 appears to be a suitable model totest the anti-inflammatory response that affects themetabolicregulation of adipocytes elicited by chemical compoundsand nutraceutics under controlled conditions [24ndash27] Thusto determine the anti-inflammatory effect of alliin in thepresent work we used the LPS-stimulated 3T3-L1 cell lineas an inflammatory model in vitro Our findings indicatethat alliin prevents LPS-induced inflammation by inhibitingERK12 in 3T3-L1 adipocytes

2 Materials and Methods

21 Cell Culture 3T3-L1 cells were obtained from AmericanType Culture Collection (ATCCCL-173) and cultured at 37∘Cin 5 CO

295 humidified air The cells were maintained

in Dulbeccorsquos Eagle modified medium (DMEM) with 25mMHEPES 1 penicillin-streptomycin (100UmL 100 120583gmL)and 10 calf serum The cells were differentiated 2 days afterconfluence (day 0) in DMEM containing 10 Fetal bovineserum (FBS) 025 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine and 5 120583gmL insulin for 48 h the cells werethen incubated in 10 FBSDMEM with insulin for 72 hFinally 10 FBSDMEM was changed every 2 days

22 T3-L1 Cell Treatment On day 8 after differentiation thecells were pretreated during a 24 h periodwith amediumcon-taining 01mM alliin (Sigma Chemical Co) Cells were thenincubatedwith the samemediumalongwith LPS (100 ngmL)for 1 h before being harvested The cultured medium wascollected in tubes and stored at minus80∘C Preliminary exper-iments were performed to determine the concentrations ofalliin during time-course experiments (data not shown)

23 mRNA Expression Quantification by qPCR Total RNAwas isolated from cells by using the Ultraspec reagent(Biotecx Houston TX USA) First-strand cDNA wasreverse-transcribed from 05 120583g of total RNA by employingtheM-MLVReverse Transcriptase protocol (Invitrogen)ThecDNA was amplified with different pairs of specific primersthat were designed utilizing PrimerExpress software (Table 1)

Mediators of Inflammation 3

The real-time quantitative PCR reaction contained02 120583g120583L of reverse-transcribed total RNA 18 120583M for-ward and reverse primers and 10120583L of iQ SYBR GreenSuperMix (Bio-Rad) PCR was performed in 48-wellplates with the MiniOpticon real-time PCR system (Bio-Rad) Quantification was performed by the compara-tive cycle of threshold method with the invariable adeninephosphoribosyltransferase (Aprt) gene used for normal-ization

24 Protein Analysis by Western Blotting To obtain totalprotein from 3T3-L1 adipocytes cells from different treat-ments were homogenized in 50120583L RIPA buffer at 4∘Csupplemented with 10mM sodium orthovanadate 100mMPMSF and 54mgmL aprotinin Insoluble material wasremoved by centrifugation for 30min at 13500 g at 4∘CThe protein concentration of the supernatants was deter-mined by the Bradford method Proteins were denaturedby boiling (5min) then 30 120583g were resolved by SDS-PAGEon 10 separation gels and transferred to immobilon PVDFmembranes (Millipore Bedford MA USA) Nonspecificprotein binding to the PVDF membrane was reduced bypreincubation for 1 h at 22∘C in TBS tween-20 (01) and5 bovine serum albumin (BSA) blocking buffer The PVDFmembranes were incubated overnight at 4∘C with mono-clonal antibodies against total and phosphorylated ERK12(Cell Signaling and Santa Cruz Biotechnology Santa CruzCA USA) which were diluted 1 1000 in blocking bufferAfter incubation themembraneswerewashed for 5min threetimes in TBS tween-20 (01) The blots were subsequentlyincubated with peroxidase-conjugated secondary antibodyfor 1 h at 22∘C in TBS tween-20 (01) and 5 (wv) fat-free milk For evaluation of protein loading the membraneswere incubated and reblotted with anti-120573-actin antibody(Santa Cruz Biotechnology) as appropriate Specific bandswere detected using the EZ-ECL chemiluminescence kit(Amersham) and visualizationcapture was performed byexposure of themembranes toRXfilms Band intensitieswerequantified by Quantity One software (Bio-Rad)

25 Analysis of Cytokine Levels by Enzyme-Linked Immun-osorbent Assays (ELISA) Cytokines and chemokines weremeasured in cell culture supernatants using ELISA tech-niques (Bio-Plex Pro Assays Bio-Rad Laboratories Inc)Thelower detection limit was 38 pgmL for IL-6 21 pgmL forTNF-120572 and 51 pgmL for MCP-1 For data normalizationand to exclude artificial effects of different cell densitiestotal protein concentration was measured for each well andsupernatant cytokine concentrations were divided by proteinconcentration Each sample was measured in triplicate byELISA Values were expressed as mean plusmn standard error ofthemean (SEM) (119899 = 6wells were used for each experimentalgroup)

26 Printing of Arrays Probe Preparation and Hybridizationof Arrays A Mus musculus 22000 65-mer Oligo Libraryfrom Sigma-Genosys sets was used For the hybridizationexperiments the RNA utilized was from cells collected from

cultures For cDNA synthesis 10 120583g of total RNA was usedas the template incorporating dUTP-Cy3 or dUTP-Cy5 andequal quantities of labeled cDNA were hybridized to the22000 oligo mouse arrays as described previously [28 29]

27 Data Acquisition and Analysis of Array Images Acquisi-tion and quantification of the array images were performedin a ScanArray 4000 apparatus using the accompanyingsoftware ScanArray 4000 (Packard BioChips Perkin-ElmerMN USA) All images were captured as described previously[28 29] In all cases the fluorescence signal was from sevento ten times more intensive than the background signal andthe background evaluationwas always evaluated immediatelybeside the labeled spot

28 Data Analysis Microarray data analysis was performedusingGenArise free softwareThe goal of GenArise is to iden-tify genes that are good candidates for differential expressionby calculating an intensity-dependent 119911-score successfullyused and recently reported [28 29] Applying these criteriathe elements with a 119911-score of gt2 standard deviations (SD)are genes likely to be differentially expressed

29 Statistical Analysis The results are expressed as themeanplusmn SEM For statistical analysis GraphPad Prism softwarewas employed and the one-way ANOVA test was applied tocompare the treatment effects Significant differences wereestablished by the Turkey-Kramer test Values of 119875 lt 005were considered statistically significant

3 Results

31 Alliin Pretreatment Significantly Reduces the mRNAExpression and Protein Levels of Proinflammatory MoleculesIL-6 and MCP-1 after LPS Exposure in 3T3-L1 AdipocytesPreviously we determined the alliin concentration that exertsan effect on the expression of the tested genes the concentra-tions probed were 01 03 06 and 10mM (data not shown)From this we selected 01mM as the minimum concentrationable to elicit a clear effect

Cytokine IL-6 is correlated with insulin resistance insubjects with obesity and is inducible throughTLR-4 receptoractivation [30] After the alliin pretreatment mRNA levelsfor IL-6 were significantly reduced (Figure 1(a)) In contrastthe level of TNF-120572 mRNA was apparently not significantlyaffected although a slight tendency toward its decrease inalliin pretreated cells was also noted (Figure 1(b))

Additionally we checked for MCP-1 expression becauseit is produced by a variety of cells including adipocytes inresponse to inflammatory stimuli [31] As expected we founda significant increase in MCP-1 expression in LPS-treatedadipocytes Interestingly we again observed a significantreduction in MCP-1 mRNA levels when LPS-stimulatedadipocytes were pretreated with alliin (Figure 1(c))

Furthermore we verified the expression of Egr-1 whichis described as induced by cytokines and hormones throughactivation of the MAPK pathway and which are related withinsulin resistance [32] Once again the mRNA expression


IL-6

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lowastlowastlowastlowast

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(d)

Figure 1 Messenger RNA (mRNA) expression levels of proinflammatory genes Differentiated adipocytes were incubated with 01mMmLalliin for 24 h and stimulated with 100 ngmL of lipopolysaccharides (LPS) for 1 h Values are expressed as arbitrary units (AU) afternormalization of expression levels against a control gene Results are mean plusmn standard deviations (SD) of three independent experimentslowast119875 le 005 lowastlowast119875 le 001 lowastlowastlowast119875 le 0001

level was significantly reduced by alliin pretreatment evenafter the LPS proinflammatory stimulus (Figure 1(d))

To corroborate these results we evaluated the secretedprotein levels of these cytokines and determined their releaseinto the culture media by ELISA Protein levels detected afterthe LPS stimulus which are significantly reduced by alliinpretreatment are shown in the case of IL-6 (Figure 2(a))and Mcp-1 (Figure 2(d)) Moreover we observed a reductionin TNF-120572 levels (Figure 2(b)) although this was small anddid not reach statistical significance Additionally we testedfor adiponectin levels (Figure 2(c)) because this representsan important union between obesity and insulin resistanceand is considered as an anti-inflammatory protein [33]The control group of adipocytes secretes a large amount ofadiponectin (Figure 2(c)) which is clearly reduced by LPSstimuli In the group pretreated with alliin a slight increasecan be observed in the production of this protein however itcannot overcome the severe reduction elicited by LPS

32 Alliin Exerts Its Anti-Inflammatory Effect at Leastthrough Diminishing the Phosphorylation of ERK12 SinceLPS induces inflammation in adipocytes through ERK12[30] and IL-6 and Egr-1 intracellular signaling mechanismsconverge in this pathway we next examined whether alliinpretreatment affects ERK12 phosphorylation LPS stimulus isable to increase the protein levels of phosphorylated ERK12and alliin pretreatment overwhelms this effect by significantlyreducing this level to nearly reach control levels (Figures 3(a)and 3(b))

33 Gene Expression Profile of Alliin Pretreated 3T3-L1 Cellsafter LPS Stimulus Is Consistent with a Shift in Cell Response toInflammatory Stimulus and Reveals Alliin Action onAdipocytePhysiology Given thatmany othermolecules can be involvedboth in the LPS inflammatory effect and also in the anti-inflammatory effect of alliin in this model we decidedto perform a microarray analysis to identify other genes


IL-6

2 4 6 8 10 12 14 16 18 20 22 240

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(d)

Figure 2 Protein expression levels of proinflammatory and anti-inflammatory proteins secreted by 3T3-L1 adipocytes Cells were incubatedwith 01mM alliin for 24 h and exposed to 100 ngmL of lipopolysaccharides (LPS) for 1 h Cytokine and protein concentration in cell culturesupernatants for 30min 1 3 6 12 and 24 h after LPS exposure were determined by Luminex technology Values are expressed in pgmL ofsupernatant Results are mean plusmn standard deviations (SD) of three independent experiments lowast119875 le 005 lowastlowast119875 le 001 lowastlowastlowast119875 le 0001

Total ERK1Total ERK2

p-ERK1p-ERK2

CT LPS Alliin + LPS Alliin

44kDa42kDa

44kDa42kDa

(a)

000

005

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015

020p-ERK12 versus total ERK12


p-ER

K12

pro

tein

(au

)

lowastlowast

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(b)

Figure 3 Levels of phosphoextracellular signal-regulated kinase (ERK12 p44p42) in mouse 3T3-L1 adipocytes Cells were pretreated for24 h with alliin 01mM and subsequently exposed to 100 ngmL of lipopolysaccharides (LPS) for 1 h afterward (a) Representative Westernblot with phospho-ERK12 and ERK12 antibodies (b) protein levels of phospho-ERK12 and ERK12 in total cell extracts CT control AUarbitrary units Data are expressed as mean plusmn standard deviations (SD) of three independent experiments lowast119875 le 005 lowastlowast119875 le 001


Table 2 Group analysis of genes that increase their expression in 3T3-L1 adipocytes by alliin pretreatment and after LPS stimuli

With known function (12genes 43)

Precise function unknown(16 genesmdash57)

Immune response(10 genesmdash36)

Immunoglobulin related M27752 X53400 AJ400981 X86534X80972 X66457

T-cell receptor related AF041900 AF158133 Z12217 Z86011

Enzymes (8 genesmdash29) Fam213bC1orf93 TdhAcaa1a Pik3cd Phka1 Cbs

Cyp2g1 lactate dehydrogenase A-4pseudogene

Others (8 genesmdash29)B-cell Chemotaxis Cxcl13TLR-4 related Cnpy4

Slirp Tnnt1 Hint2 Nub1 Zfp92 Gm10762Unclassified (2 genesmdash7) AF357337 AK018247

involved in both effects After analysis of the microarrays byGeneArise software we found that of a total of the 22000genes analyzed in themicroarrays a total of 2426 genes (11)modify their expression with a 119911-score between 2 and 62 inat least one of the three comparisons performed which werethe following control versus LPS control versus alliin + LPSand LPS versus alliin + LPS The remainder did not exhibitsignificant variations in expression obtaining a 119911-score ofltplusmn20

Comparison versus control group (nonstimulated adipo-cytes) LPS treatment with or without alliin pretreatmentclearly modifies the expression of 315 genes (upregulating 255and downregulating 60 genes) in common between the twocomparisons (control versus LPS and control versus alliin +LPS) When analyzed by DAVID software [34] at high astrin-gency 237 of 315 (75) correspond to identified genes whichcan be grouped into 64 gene clusters according to functionalcluster analysis This profile is totally consistent with dozensof previous reports regarding the effect of LPS on the geneexpression of numerous genes The clusters that reach anenrichment score of gt15 according to DAVID analysis [34]as well as representative genes whose expression is signifi-cantly modified by LPS in 3T3-L1 differentiated adipocytesindependently of the alliin pretreatment can be consultedin Table 1-SM (See Table 1-SM in Supplementary Mate-rial available online at httpdxdoiorg1011552013381815)Therefore as a first conclusion the 315 genes detected hereas modified by LPS confirms the effect of LPS and alsoenlarges the panorama concerning the effect of LPS ondifferentiated adipocytes in vitro including several new geneswhose mRNA expression are most probably affected by LPSproinflammatory stimuli

On the other hand we found 2108 genes that weremodified by alliin action after proinflammatory LPS stimuliOf these a subgroup of 125 genes was modified in at leasttwo of the comparisons performed (54 were upregulated and71 were downregulated with a 119911-score gt 20) thus we canconsider these genes as thosewhose expression ismodified byalliin pretreatment When analyzed by DAVID software [34]at high astringency 100 of these genes were identified (80)and grouped into 23 functional clusters These functionalclusters can be regrouped into nine functional categories by

obvious similarities in cluster functionData can be consultedin Table 2-SM

Based on previous works [28 29] we further conducteda more detailed analysis of the genes that reach a median119911-score of gt25 (up- or downregulated) between two groupcomparisons We propose that genes fitting these criteria arethose with greatest relevance and whose expression level ismodified by alliin pretreatment in response to proinflamma-tory stimuli All of the genes that were modified by alliinpretreatment in a sole comparison or with a median 119911-scoreof between 20 and 25 were excluded from this analysisWiththis high-stringency criteria we obtain 28 genes upregulatedby alliin and 35 that were downregulated (full results canbe consulted in Tables 3-SM and 4-SM) When individualanalyses were performed among the upregulated genes wefound that the most relevant group included 10 genes (36)and is related with immunoglobulin and the T-cell receptorThe second relevant group includes eight genes than encodefor different enzymes and finally there was a third groupof eight diverse genes and two additional unidentified genes(Table 2) Among the genes downregulated by alliin weidentified amain group that includes 15 genes that are cancer-related (Table 3) in addition to a group of five genes thatencodes for enzymes and a third group of seven genes relatedwith diverse functions It is noteworthy that a fourth groupincludes 10 unknown genes which represent 29 of genesdownregulated by alliin

Thus we present here the gene expression profile elicitedafter LPS stimuli when adipocytes receive an alliin pretreat-mentThese results provide large and clear evidence support-ing alliin action which clearly reverses the proinflammatoryeffect of LPS as well as performing other parallel effectsbecause there is a clear shift in the gene expression profile

4 Discussion

Because obesity is the result of a complex combination ofmultiple genetic and environmental factors the consumptionof certain nutraceuticals such as garlic components insteadof or even in addition to highly energetic fatty and palatablefoods is able to modulate the metabolic functioning ofadipose tissue in a way that results in the attenuation of the


Table 3 Group analysis of genes that decrease their expression in 3T3-L1 adipocytes by alliin pretreatment and after LPS stimuli

Cancer related (15 genesmdash43) Hoxb13 Hmg20b Cxcl16 Uimc1 Elf1 Foxp1 Ptgdr Psmd8 Peg3 Myo15 Lig1Ptbp2 Kif2c Grhpr Fah

Enzymes (5 genesmdash14) Dck Zdhhc4 Pik3c2g Grhpr Fah

Diverse functions (7 genesmdash20)

Adipogenesis AamdcNuclear Rny1 Polr1d Nol12Notch signaling Dtx2Vascular remodeling ReckSt15Ig related S72845

Unknown function (10 genesmdash29) AK010802 AK007003 AK016406 AK021335 AK015506 AK009275 AK007343AK006390 AK006570 AK005580

Bolded gene names correspond to genes that fits into two groups (because are cancer related and also enzymes)

inflammatory state therefore improving the quality of life[35]

41 Inflammatory Induction by LPS in 3T3-L1 Is Coun-teracted by Alliin Treatment Differentiated adipocytes areequipped with the capability to respond directly to innateimmune challenge by LPS from Gram-negative bacteria [2123 36] Some studies have shown the relevance of 3T3-L1adipocytes fully differentiated in vitro and stimulated byLPS as a useful model to test for molecules that exhibitsan anti-inflammatory effect and that are able to modulatethe inflammatory state of adipose tissue [37] LPS causesinduction of IL-6 production upregulation of TLR-2 anda downregulation of adiponectin receptors 1 and 2 [36]Multiple signaling pathways mediate LPS induction of IL-6 [38 39] These pathways include NF-120581B c-JNK ERKinhibitory G protein and PKC mediated processes and Toll-like receptors activate similar but distinct signaling pathwaysdue to their ability to recruit different adapter proteins [22]

LPS induces lipolysis in adipocytes via TLR-4 and ERK12signaling [40] Regarding TLR-4 this receptor recognizesLPS as a ligand [41] and it was demonstrated that stimulationof adipocytes by LPS attenuates insulin signaling by stimulat-ing NF-120581B signaling decreasing AKT phosphorylation andincreasing the expression of inflammatory cytokine genessuch as TNF-120572 and IL-6 in 3T3-L1 adipocytes [42] whichimplicate this receptor in the onset of insulin resistance inobesity and type 2 diabetes On the other hand ERK12 acti-vation is crucial for the induction of inflammatory changesin adipocytes [43] and leads to enhanced NF-120581B activity[44] Activation of PPAR-120573120575 inhibits enhanced cytokineproduction in adipocytes by preventing NF-120581B activation viaERK12 an effect thatmay aid in preventing insulin resistance[30] Additionally stimulation of 3T3-L1 adipocytes by LPSinduces adipocytic insulin resistance via the involvement ofJNK [45] Therefore ERK12 signaling and JNK signalingare relevant in adipose tissue physiology for the regulationof insulin sensitivity and lipolysis [40 45] On the basis ofall of this background and taking into account the resultspresented here it is reasonable to propose one of the possiblemolecular mechanism by which alliin protects against theLPS effect (Figure 4)

Among factors that might mitigate the inflamma-tory response in adipocytes it has been demonstrated

that pretreatment of adipocytes with adiponectin exertsan anti-inflammatory activity by suppressing IL-6 TNF-120572and MCP-1 production from inflamed adipocytes [46 47]This anti-inflammatory action may be mediated throughinhibition of NF-120581B activity as well as through increasedPPAR-120574 expression Recently Gomez-Arbelaez et al showedthat AGE is able to improve adiponectin levels in patientswith metabolic syndrome after 12 weeks [48] Here we showthat a 24 h pretreatment of alliin is able to partially recoveradiponectin levels in LPS-stimulated adipocytesThis furthersupports the anti-inflammatory effect of alliin

In summary we found that pretreatment with alliincounteracted LPS-induced inflammation Our findings showthat alliin downregulates IL-6 andMCP-1 expressions as wellas ERK12 phosphorylation On this basis our data indicatethat speculation can ensue concerning that one of the alliineffects is caused probably through the NF-120581B system as asignaling pathway used to suppress cytokine production inadipocytes (Figure 4) However this proposed mechanismdoes not completely eliminate other possibilities such as forexample the reduction of inflammation through the alreadydescribed antioxidant effect of alliin which can function asa complementary pathway

42 Gene Expression Profile by Microarrays Induced by LPSAffects Several Groups of Genes and This Effect Is Coun-teracted by Alliin Yamashita et al [49] have showed thatwhen stimulated with LPS 3T3-L1 adipocytes coculturedwith murine macrophages these upregulated the expressionof genes associated with inflammation insulin resistanceand angiogenesis The authors analyzed the gene expressionprofile of only 5693 genes Here we analyzed the geneexpression profile of 22000 genes after LPS stimuli with orwithout preincubation with alliin thus our results extendthe gene expression profile of 3T3-L1 adipocytes elicitedby LPS stimulation which is consistent with the regulationof the expression genes related with response to peptidehormone stimulus cytoplasmic membrane-bounded vesicleDNARNA helicase DEADDEAH box type ATP-bindingpositive regulation of phagocytosis regulation of vesicle-mediated transport and ATP-dependent helicase activity asthe main clusters represented in the analysis (Table 1-SM)

We show here the gene expression profile induced byalliin pretreatment in response to LPS inflammatory stimuli


ERK12MAPK

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(b)

Figure 4 A proposal of how alliin could counteract the inflammatory state promoted by lipopolysaccharides (LPS) in 3T3-L1 adipocytes(a) Activation of proinflammatory signaling pathway by lipopolysaccharides (LPS) (b) Alliin could reduce the Toll-like receptor-4 (TLR-4)pathway possibly by diminishing the expression of related genes and proteins such as interleukin-6 (IL-6) monocyte chemostatic protein-1(MCP-1) and early growth receptor-1 (Egr-1) and therefore regulates extracellular signal-regulated kinase (ERK12) activity


(Tables 2 and 3) When we analyzed in detail the genesupregulated by alliin pretreatment with a higher median 119911-score (gt25) in two comparisons we found 10 genes relatedwith immunoglobulin production and with T-cell receptors(Table 2) suggesting that alliin could exert an effect on the3T3-L1 adipocytes inducing lymphopoietic activities This ishighly relevant in the context of the chronic proinflammatorystate in obesity that is induced by adipocytes and that affectsT-cell activation [50] The expression of immunoglobulinreceptors by adipocytes has already been mentioned in part[51 52] however it has received nearly no attention Herewe show that alliin induces a differential expression ofsix immunoglobulin-related genes and four T-cell receptor-related genes in 3T3-L1 adipocytes These immunoglobulinscould act as anti-inflammatory signals or could at leastinfluence adipocyte metabolism and together with T-cellreceptor genes could affect the signaling between adipocytesand T cells

It is also relevant that alliin appears to upregulate Cnpy4(PRAT4B) expression because this molecule is a chaperonethat has been associated with the regulation of TLR-4 mem-brane localization [53] and could act as a negative regulatorof TLR-1 surface trafficking [54] Thus because LPS actionis mediated by TLR-4 it is thus feasible to speculate thatalliin action could modulate this receptor trafficking or itsdistribution on the membrane to counteract the LPS stimuli

On the other hand in relation with genes downregulatedby alliin we found that the gene with the highest down-regulation by alliin was Dck (deoxycytidine kinase) It hasbeen described that 3T3-L1-differentiated adipocytes expressvery low levels when comparedwith proliferating 3T3-L1 cells[55] additionally another two genes related with adipocytedifferentiation process denominated Aamdc [56] and Foxp1[57] were also downregulatedThis reflects and confirms thatthe 3T3-L1 cells were fully differentiated as adipocytes whenpreincubated with alliin and stimulated by LPS

In addition our analysis shows a group of 15 genes that arerelated with cancer (Table 3)The anticarcinogenic propertiesof garlic are well documented Therefore we present herea list of genes whose expression is downregulated by alliinwhich requires more research to unveil their participation inresponse to alliin treatment and their involvement as anti-carcinogenic factors Finally we showed a group of 10 genesthat are downregulated by alliin and whose functions remainunknown suggesting that a relevant portion of alliin effectsis yet to be identified

Taken together our results may partially explain theeffect elicited by alliin however further studies looking atthe upstream and downstream effector molecules will benecessary in order to understand the role of inflammatorysignals in these impairments Analyses of the roles of certainother genes with as yet unknown functions the expressionsof which were significantly changed are currently in progressin our laboratory Additionally it will be of interest to test theeffect of alliin also in immune cells such as macrophages orin an in vivo study where macrophage infiltration is presentin the adipose tissue Thus other yet unknown mechanismsappear to account for the loss of LPS responsibility inadipocytes isolated from inflamed adipose tissue

5 Concluding Remarks

In conclusion the results presented here demonstrate thepossible mechanism by which alliin a garlic compoundcontrols the inflammatory state of adipocytes by decreasingIL-6 and MCP-1 expressions (both at mRNA and proteinlevels) as well as diminishing ERK12 phosphorylation inLPS-stimulated 3T3-L1 adipocytes and generating an anti-inflammatory gene expression profile in adipocytes andmodifying their metabolic profile

Additionally we confirmed that alliin modifies themRNA expression of genes involved in phospholipid andorganophosphate metabolic processes in positive regulationof the related immune process (expressing immunoglobulin(Ig) and in T-cell receptor-related genes) some enzymesfor metabolic and energy process and genes involved incancer All of these processes can be somehow involved inadipocyte protection against a proinflammatory stimulusthus they constitute interesting groups of genes to be furtherexplored as involved in adipocyte physiology derived fromthe alliin action A deeper understanding of the mechanismsthat regulate anti-inflammatory signaling in adipocytesby alliin action may contribute to unraveling possibletreatments for obesity-induced inflammation and insulinresistance

Conflict of Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Acknowledgments

This work was partially supported by CONACyT-MexicoGrants 295457 to SQ-F PROMEP1035082919 to RIL-R and funds from the Spanish Ministerio de Economiay Competitividad (SAF2012-30708) CIBER de Diabetes yEnfermedades Metabolicas Asociadas (CIBERDEM) is anInstituto de Salud Carlos III Project The authors thankDr Ana Marıa Puebla-Perez Dr Miguel Cruz and Biolo-gist Claudia Elizabeth Morales Martinez for their valuableassistance and fruitful discussions They also appreciatethe technical assistance of Lorena Chavez Simon GuzmanJose Luis Santillan and Jorge Ramirez in the microarraydeterminations

References

[1] 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

[2] F P de Heredia S Gomez-Martınez and A Marcos ldquoObesityinflammation and the immune systemrdquo Proceedings of theNutrition Society vol 71 no 2 pp 332ndash338 2012

[3] E E Kershaw and J S Flier ldquoAdipose tissue as an endocrineorganrdquo Journal of Clinical Endocrinology and Metabolism vol89 no 6 pp 2548ndash2556 2004


[4] P Trayhurn and I S Wood ldquoSignalling role of adipose tissueadipokines and inflammation in obesityrdquo Biochemical SocietyTransactions vol 33 no 5 pp 1078ndash1081 2005

[5] A W Ferrante Jr ldquoObesity-induced inflammation a metabolicdialogue in the language of inflammationrdquo Journal of InternalMedicine vol 262 no 4 pp 408ndash414 2007

[6] P D Cani J AmarM A Iglesias et al ldquoMetabolic endotoxemiainitiates obesity and insulin resistancerdquo Diabetes vol 56 no 7pp 1761ndash1772 2007

[7] C N Lumeng J L Bodzin and A R Saltiel ldquoObesity induces aphenotypic switch in adipose tissue macrophage polarizationrdquoJournal of Clinical Investigation vol 117 no 1 pp 175ndash184 2007

[8] O Osborn and J M Olefsky ldquoThe cellular and signaling net-works linking the immune system and metabolism in diseaserdquoNature Medicine vol 18 no 3 pp 363ndash374 2012

[9] M S Kim M S Lee and D Y Kown ldquoInflammation-mediatedobesity and insulin resistance as targets for nutraceuticalsrdquoAnnals of the New York Academy of Sciences vol 1229 no 1 pp140ndash146 2011

[10] M Iciek I Kwiecien and L Włodek ldquoBiological properties ofgarlic and garlic-derived organosulfur compoundsrdquo Environ-mental and Molecular Mutagenesis vol 50 no 3 pp 247ndash2652009

[11] P Rose M Whiteman P K Moore and Z Z Yi ldquoBioactiveS-alk(en)yl cysteine sulfoxide metabolites in the genus Alliumthe chemistry of potential therapeutic agentsrdquo Natural ProductReports vol 22 no 3 pp 351ndash368 2005

[12] X Gu and Y Z Zhu ldquoTherapeutic applications of organosulfurcompounds as novel hydrogen sulfide donors andor media-torsrdquo Expert Review of Clinical Pharmacology vol 4 no 1 pp123ndash133 2011

[13] T Ariga and T Seki ldquoAntithrombotic and anticancer effects ofgarlic-derived sulfur compounds a reviewrdquo BioFactors vol 26no 2 pp 93ndash103 2006

[14] N Ide and B H S Lau ldquoGarlic compounds minimize intracel-lular oxidative stress and inhibit nuclear factor-120581B activationrdquoJournal of Nutrition vol 131 no 3 pp 1020Sndash1026S 2001

[15] V M Dirsch A K Kiemer H Wagner and A M VollmarldquoEffect of allicin and ajoene two compounds of garlic oninducible nitric oxide synthaserdquo Atherosclerosis vol 139 no 2pp 333ndash339 1998

[16] A Stoll and E Seebeck ldquoUber Alliin die genuine Muttersub-stanz des Knoblaucholsrdquo Experientia vol 3 no 3 pp 114ndash1151947

[17] H Sakurai N Tsuchimoto T Fukumoto et al ldquoDecompositionand absorption of S-allyl-L-cysteine sulfoxide in the intestineand culture cellsrdquo in Proceedings of the Annual Meeting ofthe Japanese Society Society for Bioscience Biotechnology andAgrochemistry 2000 abstract 139

[18] K T Augusti and C G Sheela ldquoAntiperoxide effect of S-allylcysteine sulfoxide an insulin secretagogue in diabetic ratsrdquoExperientia vol 52 no 2 pp 115ndash119 1996

[19] A L Colın-Gonzalez R A Santana C A Silva-Islas M EChanez-Cardenas A Santamarıa and P D Maldonado ldquoTheantioxidantmechanisms underlying the aged garlic extract- andS-allylcysteine-induced protectionrdquo Oxidative Medicine andCellular Longevity Article ID 907162 16 pages 2012

[20] S P PoulosMVDodson andG J Hausman ldquoCell linemodelsfor differentiation preadipocytes and adipocytesrdquoExperimentalBiology and Medicine vol 235 no 10 pp 1185ndash1193 2010

[21] Y Lin H Lee A H Berg M P Lisanti L Shapiro and PE Scherer ldquoThe lipopolysaccharide-activated Toll-like receptor(TLR)-4 induces synthesis of the closely related receptor TLR-2in adipocytesrdquo Journal of Biological Chemistry vol 275 no 32pp 24255ndash24263 2000

[22] A Kopp C Buechler M Bala et al ldquoToll-like receptorligands cause proinflammatory and prodiabetic activation ofadipocytes via phosphorylation of extracellular signal-regulatedkinase and c-Jun N-terminal kinase but not interferon regula-tory factor-3rdquo Endocrinology vol 151 pp 1097ndash1108 2010

[23] S J Creely P G McTernan C M Kusminski et al ldquoLipo-polysaccharide activates an innate immune system response inhuman adipose tissue in obesity and type 2 diabetesrdquo AmericanJournal of PhysiologymdashEndocrinology and Metabolism vol 292no 3 pp E740ndashE747 2007

[24] R Garcia-Macedo F Sanchez-Munoz J C Almanza-Perez G Duran-Reyes F Alarcon-Aguilar and M CruzldquoGlycine increases mRNA adiponectin and diminishes pro-inflammatory adipokines expression in 3T3-L1 cellsrdquo EuropeanJournal of Pharmacology vol 587 no 1ndash3 pp 317ndash321 2008

[25] S K Chakrabarti B K Cole Y Wen S R Keller and J LNadler ldquo1215-Lipoxygenase products induce inflammation andimpair insulin signaling in 3t3-l1 adipocytesrdquoObesity vol 17 no9 pp 1657ndash1663 2009

[26] Y An K Liu Y Zhou and B Liu ldquoSalicylate inhibitsmacrophage-secreted factors induced adipocyte inflammationand changes of adipokines in 3T3-L1 adipocytesrdquo Inflammationvol 32 no 5 pp 296ndash303 2009

[27] C Freise U Erben U Neuman et al ldquoAn active extract ofLindera obtusiloba inhibits adipogenesis via sustained Wntsignaling and exerts anti-inflammatory effects in the 3T3-L1preadipocytesrdquo Journal of Nutritional Biochemistry vol 21 no12 pp 1170ndash1177 2010

[28] A E Rojas-MayorquınNMTorres-Ruız DOrtuno-SahagunandG Gudino-Cabrera ldquoMicroarray analysis of striatal embry-onic stem cells induced to differentiate by ensheathing cellconditioned mediardquo Developmental Dynamics vol 237 no 4pp 979ndash994 2008

[29] D Ortuno-Sahagun M C Rivera-Cervantes G Gudino-Cabrera et al ldquoMicroarray analysis of rat hippocampus exposedto excitotoxicity reversal Na+Ca2+ exchanger NCX3 is over-expressed in glial cellsrdquoHippocampus vol 22 no 2 pp 128ndash1402012

[30] R Rodrıguez-Calvo L Serrano T Coll et al ldquoActivationof peroxisome proliferator-activated receptor 120573120575 inhibitslipopolysaccharide-induced cytokine production in adipocytesby lowering nuclear factor-120581B activity via extracellular signal-related kinase 12rdquo Diabetes vol 57 no 8 pp 2149ndash2157 2008

[31] H Xu G T Barnes Q Yang et al ldquoChronic inflammation in fatplays a crucial role in the development of obesity-related insulinresistancerdquo Journal of Clinical Investigation vol 112 no 12 pp1821ndash1830 2003

[32] N Shen X Yu F Y Pan X Gao B Xue and C J Li ldquoAn earlyresponse transcription factor Egr-1 enhances insulin resistancein type 2 diabetes with chronic hyperinsulinismrdquo Journal ofBiological Chemistry vol 286 no 16 pp 14508ndash14515 2011

[33] K Robinson J Prins and B Venkatesh ldquoClinical reviewadiponectin biology and its role in inflammation and criticalillnessrdquo Critical Care vol 15 pp 1ndash9 2011


[34] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[35] D Ortuno Sahagun A L Marquez-Aguirre S Quintero-Fabian R I Lopez-Roa and A E Rojas-Mayorquın ldquoModu-lation of PPAR-120574 by nutraceutics as complementary treatmentfor obesity-related disorders and inflammatory diseasesrdquo PPARResearch vol 2012 Article ID 318613 17 pages 2012

[36] K M Ajuwon W Banz and T A Winters ldquoStimulation withPeptidoglycan induces interleukin 6 and TLR2 expression anda concomitant downregulation of expression of adiponectinreceptors 1 and 2 in 3T3-L1 adipocytesrdquo Journal of Inflammationvol 6 article 8 2009

[37] E Zoico V Di Francesco D Olioso et al ldquoIn vitro aging of 3T3-L1 mouse adipocytes leads to altered metabolism and responseto inflammationrdquo Biogerontology vol 11 no 1 pp 111ndash122 2010

[38] R A Frost G J Nystrom and C H Lang ldquoLipopolysac-charide and proinflammatory cytokines stimulate interleukin-6expression in C2C12 myoblasts role of the Jun NH2-terminalkinaserdquo American Journal of PhysiologymdashRegulatory Integrativeand Comparative Physiology vol 285 no 5 pp R1153ndashR11642003

[39] K M Ajuwon S K Jacobi J L Kuske and M E SpurlockldquoInterleukin-6 and interleukin-15 are selectively regulated bylipopolysaccharide and interferon-120574 in primary pig adipocytesrdquoAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 286 no 3 pp R547ndashR553 2004

[40] L Zu J He H Jiang C Xu S Pu and G Xu ldquoBacterialendotoxin stimulates adipose lipolysis via toll-like receptor 4and extracellular signal-regulated kinase pathwayrdquo Journal ofBiological Chemistry vol 284 no 9 pp 5915ndash5926 2009

[41] A Poltorak X He I Smirnova et al ldquoDefective LPS signalingin C3HHeJ and C57BL10ScCr mice mutations in Tlr4 generdquoScience vol 282 no 5396 pp 2085ndash2088 1998

[42] M J Song K H Kim J M Yoon and J B Kim ldquoActivationof Toll-like receptor 4 is associated with insulin resistance inadipocytesrdquo Biochemical and Biophysical Research Communica-tions vol 346 no 3 pp 739ndash745 2006

[43] T Suganami K Tanimoto-Koyama J Nishida et al ldquoRoleof the Toll-like receptor 4NF-120581B pathway in saturated fattyacid-induced inflammatory changes in the interaction betweenadipocytes and macrophagesrdquo Arteriosclerosis Thrombosis andVascular Biology vol 27 no 1 pp 84ndash91 2007

[44] S Chung K LaPoint K Martinez A Kennedy M BSandberg and M K McIntosh ldquoPreadipocytes mediatelipopolysaccharide-induced inflammation and insulin resis-tance in primary cultures of newly differentiated humanadipocytesrdquo Endocrinology vol 147 no 11 pp 5340ndash5351 2006

[45] J E Davis N K Gabler J Walker-Daniels and M E Spur-lock ldquoThe c-Jun N-terminal kinase mediates the induction ofoxidative stress and insulin resistance by palmitate and toll-likereceptor 2 and 4 ligands in 3T3-L1 adipocytesrdquo Hormone andMetabolic Research vol 41 no 7 pp 523ndash530 2009

[46] K M Ajuwon and M E Spurlock ldquoAdiponectin inhibitsLPS-induced NF-120581B activation and IL-6 production andincreases PPAR1205742 expression in adipocytesrdquo American Journalof PhysiologymdashRegulatory Integrative and Comparative Physiol-ogy vol 288 no 5 pp R1220ndashR1225 2005

[47] E Zoico U Garbin D Olioso et al ldquoThe effects of adiponectinon interleukin-6 and MCP-1 secretion in lipopolysaccharide-treated 3T3-L1 adipocytes role of the NF-120581B pathwayrdquo Interna-tional Journal of Molecular Medicine vol 24 no 6 pp 847ndash8512009

[48] D Gomez-Arbelaez V Lahera P Oubina et al ldquoGarlicextract improves adiponectin levels in subjects with metabolicsyndrome a double-blind placebo-controlled randomizedcrossover studyrdquo Mediators of Inflammation vol 2013 ArticleID 285795 6 pages 2013

[49] A Yamashita Y Soga Y Iwamoto et al ldquoDNA microarrayanalyses of genes expressed differentially in 3T3-L1 adipocytesco-cultured withmurinemacrophage cell line RAW2647 in thepresence of the toll-like receptor 4 ligand bacterial endotoxinrdquoInternational Journal of Obesity vol 32 no 11 pp 1725ndash17292008

[50] M Papatriantafyllou ldquoImmunometabolism CD4(+) T cell acti-vation by adipocytes in obesityrdquo Nature Reviews Immunologyvol 13 no 4 article 223 2013

[51] J Palming B G Gabrielsson E Jennische et al ldquoPlasmacells and Fc receptors in human adipose tissue-lipogenic andanti-inflammatory effects of immunoglobulins on adipocytesrdquoBiochemical and Biophysical Research Communications vol 343no 1 pp 43ndash48 2006

[52] H K Lee B H Lee S A Park and C W Kim ldquoThe proteomicanalysis of an adipocyte differentiated from human mesenchy-mal stem cells using two-dimensional gel electrophoresisrdquoProteomics vol 6 no 4 pp 1223ndash1229 2006

[53] K Konno Y Wakabayashi S Akashi-Takamura et al ldquoAmolecule that is associated with Toll-like receptor 4 and reg-ulates its cell surface expressionrdquo Biochemical and BiophysicalResearch Communications vol 339 no 4 pp 1076ndash1082 2006

[54] B E Hart and R I Tapping ldquoCell surface trafficking of TLR1is differentially regulated by the chaperones PRAT4A andPRAT4Brdquo Journal of Biological Chemistry vol 287 pp 16550ndash16562 2012

[55] S N Rylova F Albertioni G Flygh and S Eriksson ldquoActivityprofiles of deoxynucleoside kinases and 51015840-nucleotidases in cul-tured adipocytes and myoblastic cells insights into mitochon-drial toxicity of nucleoside analogsrdquo Biochemical Pharmacologyvol 69 no 6 pp 951ndash960 2005

[56] X Ma W Ding J Wang et al ldquoLOC66273 isoform 2 anovel protein highly expressed in white adipose tissue inducesadipogenesis in 3T3-L1 cellsrdquo Journal of Nutrition vol 142 no3 pp 448ndash455 2012

[57] I Gerin G T BommerM E Lidell A Cederberg S EnerbackandOAMcDougald ldquoOn the role of FOX transcription factorsin adipocyte differentiation and insulin-stimulated glucoseuptakerdquo Journal of Biological Chemistry vol 284 no 16 pp10755ndash10763 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Primer sequences used for real-time quantitative PCR

Gene name Ensembl gene Primer sequence (51015840-31015840) bp Tm

Il-6 ENSMUST00000026845 F-TACACATGTTCTCTGGGAAATCGTR-AAGTGCATCATCGTTGTTCATACA 85 75

Aprt ENSMUST00000006764 F-CAGCGGCAAGATCGACTACAR-AGCTAGGGAAGGGCCAAACA 67 60

Mcp-1 ENSMUST00000000193 F-GCTGGAGAGCTACAAGAGGATCAR-CTCTCTCTTGAGCTTGGTGACAAA 79 60

Tnf -120572 ENSMUST00000025263 F-ATGGCCCAGACCCTCACAR-TTGCTACGACGTGGGCTACA 73 60

Egr-1 ENSMUST00000064795 F-GCCGAGCGAACAACCCTATR-CCATCGCCTTCTCATTATTCAGA 77 60

comprise one natural source of organic sulfur-containingcompounds and have been widely investigated regardingtheir therapeutic applications [11 12] mainly due to itscardioprotective effect and to its anticancerogenic properties[10 13] However its anti-inflammatory effects have receivedless attention For example garlic compounds can exert ananti-inflammatory effect by inhibiting the oxidative stress-induced activation of nuclear factor-kappa B (NF-120581B) [14]which is implicated in the expression of proinflammatoryenzymes such as inducible nitric oxide synthase (NOS)and cyclooxygenase-II (COX-II) In particular allicin andajoene two garlic compounds significantly suppressed nitricoxide production by lipopolysaccharide (LPS) stimulationaccompanied by suppression in inducible nitric oxide (iNOS)expression and iNOS activity [15] Although it appearspromising that garlic and its derivatives possess antidiabeticpotential an understanding of the antidiabetic effects of garlicis still in its early stages Furthermore the active compoundsin garlic and doses thereof which can effectively provideantidiabetic effects (ie glycemic control and amelioration ofdiabetic complications) remain to be established

Alliin (S-allyl cysteine sulfoxide) first identified by Stolland Seebeck in 1947 is considered the main specific principleof garlic since that time [16] Alliin can be found in intactgarlic and its reduced form S-allyl-cysteine is the majorcomponent of aged garlic extract (AGE) together with otherderived organosulfur compounds [12 13] It is absorbed inthe intestine via the amino acid transported for cysteine [17]exhibits an hypoglycemic effect and also increases bloodinsulin concentrations [18] and its antioxidant activity hasbeen widely studied [19] Nonetheless to our knowledge itsanti-inflammatory potential has not yet been explored

Mouse preadipocyte 3T3-L1 cells are the most commonlystudied and available adipogenic cell line This cell linehas been quite useful in identifying key molecular mark-ers transcription factors and various interactions that arerequired for preadipocyte differentiation [20] Additionally3T3-L1 adipocytes are able to respond to LPS by meansof a fully intact innate immunity pathway through theproduction and secretion of immunomodulatory (mainlyproinflammatory) molecules such as IL-6 TNF-120572 and TLR-2 [21] and through a TLR-ligand-induced activation whichtriggers a proinflammatory and -diabetic transformation of

adipocytes [22] Interestingly this immune response activa-tion is resembled by human adipose tissue [23] consequentlyonce differentiated 3T3-L1 appears to be a suitable model totest the anti-inflammatory response that affects themetabolicregulation of adipocytes elicited by chemical compoundsand nutraceutics under controlled conditions [24ndash27] Thusto determine the anti-inflammatory effect of alliin in thepresent work we used the LPS-stimulated 3T3-L1 cell lineas an inflammatory model in vitro Our findings indicatethat alliin prevents LPS-induced inflammation by inhibitingERK12 in 3T3-L1 adipocytes

2 Materials and Methods

21 Cell Culture 3T3-L1 cells were obtained from AmericanType Culture Collection (ATCCCL-173) and cultured at 37∘Cin 5 CO

295 humidified air The cells were maintained

in Dulbeccorsquos Eagle modified medium (DMEM) with 25mMHEPES 1 penicillin-streptomycin (100UmL 100 120583gmL)and 10 calf serum The cells were differentiated 2 days afterconfluence (day 0) in DMEM containing 10 Fetal bovineserum (FBS) 025 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine and 5 120583gmL insulin for 48 h the cells werethen incubated in 10 FBSDMEM with insulin for 72 hFinally 10 FBSDMEM was changed every 2 days

22 T3-L1 Cell Treatment On day 8 after differentiation thecells were pretreated during a 24 h periodwith amediumcon-taining 01mM alliin (Sigma Chemical Co) Cells were thenincubatedwith the samemediumalongwith LPS (100 ngmL)for 1 h before being harvested The cultured medium wascollected in tubes and stored at minus80∘C Preliminary exper-iments were performed to determine the concentrations ofalliin during time-course experiments (data not shown)

23 mRNA Expression Quantification by qPCR Total RNAwas isolated from cells by using the Ultraspec reagent(Biotecx Houston TX USA) First-strand cDNA wasreverse-transcribed from 05 120583g of total RNA by employingtheM-MLVReverse Transcriptase protocol (Invitrogen)ThecDNA was amplified with different pairs of specific primersthat were designed utilizing PrimerExpress software (Table 1)










3 Results






IL-6

00

05

10

15

20

Control LPS Alliin

lowastlowastlowast


IL-6

RNA

(au

)

Alliin + LPS

(a)

TNF-120572

00

05

10

15

20

25

Control LPS Alliin

TNF-120572

RNA

(au

)

Alliin + LPS

(b)

MCP-1

00

05

10

15

20

25

30

35

Control LPS Alliin

lowastlowast

lowastlowast

lowast

Mcp

-1RN

A (a

u)

Alliin + LPS

(c)

EGR-1

00

25

50

75

Control LPS Alliin


lowast

Egr-1

RNA

(au

)

Alliin + LPS

(d)







IL-6

2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

IL-6

(pg

mL)

Secretion time (h)

(a)

2 4 6 8 10 12 14 16 18 20 22 240

5

10

15

Secretion time (h)

TNF-120572

(pg

mL)

TNF-120572

(b)

Adiponectin

2 4 6 8 10 12 14 16 18 20 22 240

5000

10000

15000

20000

Adip

onec

tin (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(c)

MCP-1

0 2 4 6 8 10 120

5000

10000

15000

20000

25000

MCP

-1 (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(d)



p-ERK1p-ERK2


44kDa42kDa

44kDa42kDa

(a)

000

005

010

015



p-ER

K12

pro

tein

(au

)

lowastlowast

lowastlowast

(b)



















4 Discussion



















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results






IL-6

00

05

10

15

20

Control LPS Alliin

lowastlowastlowast


IL-6

RNA

(au

)

Alliin + LPS

(a)

TNF-120572

00

05

10

15

20

25

Control LPS Alliin

TNF-120572

RNA

(au

)

Alliin + LPS

(b)

MCP-1

00

05

10

15

20

25

30

35

Control LPS Alliin

lowastlowast

lowastlowast

lowast

Mcp

-1RN

A (a

u)

Alliin + LPS

(c)

EGR-1

00

25

50

75

Control LPS Alliin


lowast

Egr-1

RNA

(au

)

Alliin + LPS

(d)







IL-6

2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

IL-6

(pg

mL)

Secretion time (h)

(a)

2 4 6 8 10 12 14 16 18 20 22 240

5

10

15

Secretion time (h)

TNF-120572

(pg

mL)

TNF-120572

(b)

Adiponectin

2 4 6 8 10 12 14 16 18 20 22 240

5000

10000

15000

20000

Adip

onec

tin (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(c)

MCP-1

0 2 4 6 8 10 120

5000

10000

15000

20000

25000

MCP

-1 (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(d)



p-ERK1p-ERK2


44kDa42kDa

44kDa42kDa

(a)

000

005

010

015



p-ER

K12

pro

tein

(au

)

lowastlowast

lowastlowast

(b)



















4 Discussion



















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-6

00

05

10

15

20

Control LPS Alliin

lowastlowastlowast


IL-6

RNA

(au

)

Alliin + LPS

(a)

TNF-120572

00

05

10

15

20

25

Control LPS Alliin

TNF-120572

RNA

(au

)

Alliin + LPS

(b)

MCP-1

00

05

10

15

20

25

30

35

Control LPS Alliin

lowastlowast

lowastlowast

lowast

Mcp

-1RN

A (a

u)

Alliin + LPS

(c)

EGR-1

00

25

50

75

Control LPS Alliin


lowast

Egr-1

RNA

(au

)

Alliin + LPS

(d)







IL-6

2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

IL-6

(pg

mL)

Secretion time (h)

(a)

2 4 6 8 10 12 14 16 18 20 22 240

5

10

15

Secretion time (h)

TNF-120572

(pg

mL)

TNF-120572

(b)

Adiponectin

2 4 6 8 10 12 14 16 18 20 22 240

5000

10000

15000

20000

Adip

onec

tin (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(c)

MCP-1

0 2 4 6 8 10 120

5000

10000

15000

20000

25000

MCP

-1 (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(d)



p-ERK1p-ERK2


44kDa42kDa

44kDa42kDa

(a)

000

005

010

015



p-ER

K12

pro

tein

(au

)

lowastlowast

lowastlowast

(b)



















4 Discussion



















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-6

2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

IL-6

(pg

mL)

Secretion time (h)

(a)

2 4 6 8 10 12 14 16 18 20 22 240

5

10

15

Secretion time (h)

TNF-120572

(pg

mL)

TNF-120572

(b)

Adiponectin

2 4 6 8 10 12 14 16 18 20 22 240

5000

10000

15000

20000

Adip

onec

tin (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(c)

MCP-1

0 2 4 6 8 10 120

5000

10000

15000

20000

25000

MCP

-1 (p

gm

L)

Secretion time (h)

CTLPS

Alliin + LPSAlliin

(d)



p-ERK1p-ERK2


44kDa42kDa

44kDa42kDa

(a)

000

005

010

015



p-ER

K12

pro

tein

(au

)

lowastlowast

lowastlowast

(b)



















4 Discussion



















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































4 Discussion



















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ERK12MAPK

JAK

RAF

PP

Egr-1

TLR-4

CD-14


Nucleus

IL-6

gp130

Cytoplasm

LPS1h

+

+

++

TNF-120572IL-6MCP-1NF120581B

(a)

Nucleus

Cytoplasm

ERK12MAPK

TLR-4

CD-14

LPS1h

IL-6

gp130

NF120581B Egr-1

Alliin24h

TNF-120572IL-6MCP-1

O

NH2


ndash



Response to stress

(b)













Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Alliin, a Garlic ( Allium sativum