Research Article Forskolin Inhibits Lipopolysaccharide-Induced...

Research ArticleForskolin Inhibits Lipopolysaccharide-InducedModulation of MCP-1 and GPR120 in 3T3-L1 Adipocytesthrough an Inhibition of NF120581B

Jeanne Durendale Chiadak12 Tatjana Arsenijevic1 Kevin Verstrepen1

Franccediloise Gregoire1 Nargis Bolaky1 Valeacuterie Delforge1 Veacuteronique Flamand3

Jason Perret1 and Christine Delporte1

1Laboratory of Pathophysiological and Nutritional Biochemistry Universite Libre de Bruxelles Brussels Belgium2Department of Biochemistry Faculty of Sciences University of Dschang Dschang Cameroon3Institute for Medical Immunology Faculty of Medicine Universite Libre de Bruxelles Gosselies Belgium

Correspondence should be addressed to Christine Delporte cdelportulbacbe

Received 13 June 2016 Accepted 13 October 2016

Academic Editor Julio Galvez

Copyright copy 2016 Jeanne Durendale Chiadak et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In an obese state Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotacticprotein-1 (MCP-1) in adipose tissue In contrast G-protein coupled receptor 120 (GPR120) mediates antiobesity effects The aimof this study was to determine the signaling pathway by which Forskolin (FK) a cyclic adenosine monophosphate- (cAMP-)promoting agent causing positive changes in body composition in overweight and obese adult men affects MCP-1 and GPR120expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes such as in an obese state 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4and inhibitor of kappa B (I120581B120572) In DC LPS increased MCP-1 TLR-4 and nuclear factor-120581B1 (NF120581B1) mRNA levels whereas itdecreased GPR120mRNA levels In DC FK inhibited the LPS-induced increase inMCP-1 TLR-4 andNF120581B1mRNA levels and theLPS-induced decrease in GPR120mRNA BAY11-7082 and CLI-095 abolished these LPS-induced effects In conclusion FK inhibitsLPS-induced increase inMCP-1mRNA levels and decrease in GPR120mRNA levels in adipocytes andmay be a potential treatmentfor inflammation in obesity Furthermore TLR-4-induced activation of NF120581B may be involved in the LPS-induced regulation ofthese genes

1 Introduction

In addition to storing excess energy adipose tissue isnow widely recognized as an important endocrine organIndeed it secretes numerous cytokines called ldquoadipokinesrdquowhich have various functions includingmacrophage recruit-ment regulation of feeding behavior energy homeosta-sis and insulin sensitivity [1] Obesity is characterized byadipose tissue inflammation and macrophage infiltration[2ndash4] Adipose tissue expression of monocyte chemotacticprotein-1 (MCP-1) and circulatingMCP-1 levels are increasedupon obesity in rodent suggesting that MCP-1-mediatedmacrophage infiltration of adipose tissue may contribute to

the metabolic disequilibrium associated with obesity andinsulin resistance [5 6] Evidence providing an inflammatorylink between obesity and type 2 diabetes is accumulating Innumerous animal and clinical studies obesity is associatedwith a state of low-grade chronic inflammation in liverand adipose tissue which includes activation of the innateimmune system and the appearance of proinflammatoryimmune cells [7 8] Nutritional strategies designed to alle-viate adipokines dysregulation include modulating dietaryfatty acids with the majority of evidence suggesting the anti-inflammatory effects of the marine derived long-chain n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid(205n-3 EPA) and docosahexaenoic acid (226n-3 DHA)

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 1431789 11 pageshttpdxdoiorg10115520161431789

2 Mediators of Inflammation

which can blunt lipopolysaccharide- (LPS-) stimulated ele-vations in adipokines [9ndash13] Furthermore activation of G-protein coupled receptor 120 (GPR120) by DHA antagonizesthe proinflammatory effects of tumor necrosis factors-alpha(TNF-120572) and lipopolysaccharide in a macrophage cell line[14]The inhibitory effects of DHA on LPS-Toll-like receptor-4- (TLR-4-) induced activation of nuclear factor-kappa B(NFkB) activity [15 16] might be mediated by a GPR120 and120573-Arrestin 2-dependent mechanism [14] 120573-Arrestin proteinsplay an important role in regulating the responsiveness ofG-protein coupled receptors (GPCRs) by contributing tomechanisms involved in both GPCR desensitization andresensitization [17]

Previous studies suggest that saturated fatty acids pro-mote inflammation by activating TLR-4 on adipocytes andmacrophages [18] Indeed mice lacking TLR-4 are pro-tected against high-fat diet-induced obesity inflammationand insulin resistance because they are resistant to thesuppression of insulin signaling during lipid infusion andexhibit reduced insulin-mediated changes in systemic glu-cose metabolism [19] Forskolin (FK) is a labdane diterpeneisolated from the roots of the Coleus forskohlii plant aperennial herb with fleshy fibrous roots belonging to themintfamily of plants In the early-to-mid-1980s Forskolin wasprimarily used as an agent to help a number of cardiovasculardisease conditions mainly through a vasodilator effect [20]This effect resulted from increased adenylate cyclase activitywithin the body Forskolin causes positive changes in bodycomposition in overweight and obese adult men [21] Oneof the potential explanations for the decrease in fat massand body fat percentage may be adenylate cyclase activa-tion and thus cyclic adenosine monophosphate (cAMP)accumulation within adipose tissue which stimulates freefatty acid release and lipolysis Indeed Forskolin has beenwidely used as a potent activator of adenylate cyclase incellular preparations to study cAMP-dependent transductionpathways [22ndash25] As circulating MCP-1 levels are increasedin rodent obesity and the role of Forskolin in fat massreduction is clearly established the aim of this study wasto determine on one hand the expression of MCP-1 TLR-4 GPR120 120573-Arrestin 2 and NF120581B1 expression in DC andon the other hand the signaling pathway by which Forskolinaffects MCP-1 and GPR120 expression in the LPS-inducedinflammatory response in 3T3-L1 cells differentiated intoadipocytes

2 Materials and Methods

21 Reagents Dulbeccorsquos modified Eaglersquos medium (DMEM45 gL glucose) streptomycinpenicillin fetal bovine serumhorse serum and calf serum were provided by Invitrogen(Carlsbad CA USA) Bovine insulin 3-isobutyl-1-methyl-xanthine (IBMX) dexamethasone lipopolysaccharides andForskolin were purchased from Sigma (St Louis MO USA)TLR-4 signaling inhibitor (CLI-095) and kappa B (I120581B120572)inhibitor (BAY11-7082) were purchased from InvivoGen (SanDiego CA USA) Anti-120573-actin and anti-I120581B120572 came fromMillipore (Temecula CA USA) and anti-phospho-I120581B120572

(Ser3236) (5A5)was purchased fromCell Signaling Technol-ogy Inc (Danvers MA USA)

22 Cell Culture and Treatments 3T3-L1 murine preadipo-cyte cells were grown in DMEM supplemented with 10 calfserum 200UmL penicillin and 200UmL streptomycin in8 CO

2humidified atmosphere at 37∘C until confluence

Two days after confluence to induce adipocyte differentia-tion cells were incubated for 60 h in DMEM supplementedwith 10 fetal bovine serum and containing 500 120583M IBMX025 120583Mdexamethasone and 10 120583gmL insulinThe cells werethen maintained in the culture medium supplemented withinsulin only and this media was changed every 2 days (day 5and day 7) until complete differentiation (monitored by lipiddroplet accumulation under the microscope and confirmedby Oil Red Coloration) had occurred (day 9) On day 9 thedifferentiated 3T3-L1 cells (DC) were treated for 4 h withwater followed by 4 h with water and ethanol (CTL) or 4 hwith water and 4 h with 10 120583M Forskolin (FK) or 4 h with1 120583gmL LPS followed by 4 h with LPS and ethanol (LPS) or4 h with LPS followed by 4 h LPS and FK (LPS + FK) Bothcells and culture media from cultured undifferentiated cells(UDC at day 0) and from DC (at day 9 following treatment)were harvested To determine how the expressions of thegenes of interest are modulated by NF120581B activation the DCwere treated with I120581B120572 inhibitor BAY11-7082 at 10120583M TheTLR-4 signaling inhibitor CLI-095 at 3 120583M was also usedFor inhibitor studies DC were pretreated with the inhibitorfor 1 hour before exposure to LPS stimulation and thencoincubated with LPS and the inhibitor for 4 h prior to RNAextraction and 24 h prior to protein extraction

23 RNA Isolation Isolation of RNA as well as assessmentof RNA concentration and purity and RNA integrity wereperformed as previously described [26 27]

24 RT-qPCR Design of qPCR primers cDNA synthesisand qPCR reactions were performed as previously described[26]The primer pairs which were used are shown in Table 1

25 Analysis of Gene Expression Stability Gene expressionstability analysis and matching statistics were performedusing Biogazelle qBASE Plus software [28] Data were nor-malized using the references genes tyrosine 3-monoox-ygenasetryptophan 5-monooxygenase activation proteinzeta polypeptide (mmYwhaz) non-POU-domain contain-ing octamer binding protein (mmNONO) and 120573-actin(mmACTB) which were previously validated for this cellularand experimental system [26]

26 MCP-1 ELISA Assay MCP-1 protein levels of the culturemedia were determined by ELISA using a Duoset ELISA kitfrom RampD Diagnostics (Minneapolis MN USA)

27 Protein Extraction and Western Blot Analysis DC werewashed with calcium- and magnesium-free PBS and lysedin 1mL of lysis buffer containing 50mM TrisHCl (pH75) 150mM NaCl 05 Nonidet P40 50mM NaF 1mM

Mediators of Inflammation 3

Table 1 Real-time PCR primer sequences

Gene Forward primer (51015840 rArr 31015840) Reverse primer (51015840 rArr 31015840)mmNONO TGCTCCTGTGCCACCTGGTACTC CCGGAGCTGGACGGTTGAATGCmmACTB CCTGTGCTGCTCACCGAGGC GACCCCGTCTCCGGAGTCCATCmmYwhaz AAAAACAGCTTTCGATGAAGCC GCCGGTTAATTTTCCCCTCCmmMCP-1 TTCACCAGCAAGATGATCCCA TCCTTCTTGGGGTCAGCACAmmTLR-4 AGGACTCTGATCATGGCACTG GGAATGTCATCAGGGACTTTGCmmGPR120 GGTGCCGGGACTGGTCATTGT AGAGCGTGCGGAAGAGTCGGTmm120573-Arrestin 2 ATGGGAGAAAAACCCGGGAC CACAGGGTCCACTTTGTCCAmmNF120581B1 CTGCAGCTCTTACCCTGGAG GTAATTGCGTGGCAGAGTGG

sodium orthovanadate dithiothreitol and a cocktail of pro-tease inhibitors (cOmplete EDTA-free Roche) Whole celllysates were prepared and submitted to SDS-polyacrylamidegel electrophoresis (SDS-PAGE) in the presence of 5 120573-mercaptoethanol using 12 polyacrylamide gels Harvest ofDC and preparation of whole cell lysates were performedas previously described [27] Proteins were transferred topolyvinylidene difluoride membranes and immunolabeledusing primary antibodies against I120581B120572 phospho-I120581B120572 and120573-actin The bound primary antibodies were detected usingsecondary anti-mouse or anti-rabbit antibodies (GE Health-care Little Chalfont Buckinghamshire UK) and ECL chemi-luminescence detection kit (PerkinElmer Waltham MAUSA)The protein bands were scanned and digitized and thedensity of each band was determined using the Quantity Onesoftware (Bio-Rad Laboratories Hercules CA USA)

28 Statistical Analysis Data are presented as mean plusmn SEMof 3 experiments Group means were compared by paired 119905-test and 119905-test for unique sample Differences were consideredstatistically significant at 119901 lt 005 All statistical analyseswere performed using SPSS 22 (IBM Corp version 22000)

3 Results

31 Expression of MCP-1 TLR-4 GPR120 and 120573-Arrestin2 mRNA Levels in UDC and DC MCP-1 TLR-4 GPR120and 120573-Arrestin 2 mRNA levels were measured by RT-qPCRin UDC and DC Upon adipocyte differentiation TLR-4GPR120 and 120573-Arrestin 2 mRNA levels were significantlyupregulated 5-fold (119901 lt 005) 130-fold (119901 lt 0005) and16-fold (119901 lt 005) respectively (Figure 1) In contrast MCP-1 mRNA levels were significantly downregulated 008-fold(119901 lt 0005) upon adipocyte differentiation (Figure 1)

32 Effect of FK and LPS Modulation of MCP-1 mRNA andProtein Levels in DC In DC LPS significantly upregulatedboth mRNA (40-fold Figure 2(a)) and protein (128-foldFigure 2(b)) levels of MCP-1 (119901 lt 005) as compared to CTLIn DC FK did not significantly modify both MCP-1 mRNAand protein levels as compared to CTL (Figure 2) Upontreatment of DC with both LPS and FK the MCP-1 mRNAlevel was significantly decreased by 957 as compared toLPS-treated DC (119901 lt 005 Figure 2(a)) However MCP-1

protein level was not significantly modified under LPS andFK treatment as compared to LPS treatment alone at the timepoints assessed (Figure 2(b))

33 Effect of FK and LPS on TLR-4 mRNA Levels in DCLPS significantly increased TLR-4 mRNA level (16-fold)as compared to CTL (119901 lt 005) while FK significantlydecreased it (05-fold 119901 lt 005) (Figure 3) TLR-4 mRNAlevel was significantly decreased by 735 in response to LPSand FK treatment as compared to LPS treatment (119901 lt 005)(Figure 3)

34 Effect of FK and LPS on GPR120 mRNA Levels in DCLPS significantly decreasedGPR120mRNA level (06-fold) ascompared toCTL (119901 lt 005) while FK significantly increasedit (22-fold 119901 lt 005) (Figure 4) LPS and FK significantlyincreased 28-fold the GPR120 mRNA level as compared toLPS treatment (119901 lt 001) (Figure 4)

35 Effect of FK and LPS on 120573-Arrestin 2 mRNA in DC LPShad no effect on 120573-Arrestin 2 mRNA level whereas FK treat-ment significantly decreased it (06-fold119901 lt 005) (Figure 5)In addition 120573-Arrestin 2 mRNA level was not significantlymodified under LPS and FK treatment as compared to that ofLPS treatment alone (Figure 5)

36 Effects of LPS and FK on NF120581B1 mRNA and I120581B120572 ProteinLevels in DC LPS have been shown to stimulate secretionof inflammatory adipokines through activation of Toll-likereceptor-4 (TLR-4) and the downstream transcription factornuclear factor NF120581B in adipocytes [29ndash31] In additiona small increase in cytosolic concentrations of I120581B120572 nega-tively affects NF120581B nuclear translocation [32] We thereforeexamined the effect of LPS and FK on NF120581B1 mRNA levelsand I120581B120572 protein levels in DC and the result shows thatthe NF120581B1 mRNA level was significantly upregulated in thepresence of LPS as compared to CTL (119901 lt 005) FKsignificantly decreased by 875 the NF120581B1 mRNA level ascompared to that of LPS-treated DC (119901 lt 005) (Figure 6(a))To determine if Forskolin affected I120581B120572 proteolysis andthereby NF120581B1 activation total and phosphorylated I120581B120572protein levels were analyzed by semiquantitative Westernblotting (Figures 6(b) and 6(c)) In DC treated with andwithout LPS in the presence or absence of Forskolin no


0

02

04

06

08

1

12

UDC DC

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowastlowastlowast

(a)

0

1

2

3

4

5

6

7

UDC DC

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(b)

0

20

40

60

80

100

120

140

160

UDC DC

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

2

UDC DC

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

(d)

Figure 1 Expression ofmRNAofMCP-1 (a) TLR-4 (b) GPR120 (c) and120573-Arrestin 2 (d) in undifferentiated versus differentiated 3T3-L1 cellsUDC and DC were obtained as described under Materials and Methods The results are expressed as relative mRNA levels (fold stimulationover UDC set to 1) and are the means plusmn SEM of 3 independent experiments Data were analyzed by 119905-test for unique sample lowast119901 lt 005 andlowastlowastlowast119901 lt 0005 versus UDC

significant differences were observed in terms of I120581B120572 (totaland phosphorylated) protein level (Figures 6(b) and 6(c))

37 Effects of a TLR-4 Inhibitor on MCP-1 GPR120 TLR-4and NF120581B1 mRNA Levels upon LPS Treatment To determinewhether the regulation of MCP-1 GPR-120 and TLR-4mRNA levels induced by LPS was mediated by TLR-4 a LPSreceptor DC were preincubated for 1 hour in the absence orpresence of a TLR-4 inhibitor (3120583M of CLI095) prior to a4 h treatment with LPS (1 120583gmL) The MCP-1 mRNA levelwas significantly increased by LPS (80-fold 119901 lt 005) ascompared to CTL while CLI095 significantly decreased it by80 (119901 lt 005) (Figure 7) LPS decreased the GPR120mRNA(06-fold 119901 lt 005) as compared to CTL while CLI095abolished it (119901 lt 005) (Figure 7) In addition both TLR-4 and NF120581B1 mRNA levels were significantly upregulated

upon LPS treatment (3-fold and 2-fold resp 119901 lt 005) ascompared to CTL but they were abolished in the presence ofCLI095 (Figure 7)

38 Effects of an I120581B120572 Inhibitor on MCP-1 GPR120 TLR-4andNF120581B1mRNALevels upon LPSTreatment To specificallyassess the involvement of an NF120581B1-dependent pathway inthe regulation of MCP-1 and GPR120 mRNA levels inducedby LPS DC were pretreated with an I120581B120572 inhibitor BAY11-7082 prior to LPS treatment The LPS-induced increase inMCP-1 mRNA level was significantly decreased by 54 byBAY11-7082 (119901 lt 005) (Figure 7) The LPS-induced decreasein GPR120 mRNA level was abolished by BAY11-7082 (119901 lt005) (Figure 7) LPS-induced increase in both TLR-4 andNF120581B1 mRNA levels was abolished in the presence of BAY11-7082 (Figure 7)


0

5

10

15

20

25

30

35

40

45

50

CTL FK LPS LPS + FK

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(a)

0

10000

20000

30000

40000

50000

60000

70000

CTL FK LPS LPS + FK

Secr

eted

MCP

-1 p

rote

in (p

gm

L)

lowast

(b)

Figure 2 mRNA expression of MCP-1 (a) and MCP-1 protein secretion (b) in differentiated 3T3-L1 cells treated with LPS in the presence orabsence of FK Differentiated 3T3-L1 cells were treated as described inMaterials andMethods under the following conditions CTL 10 120583MFK1 120583gmL LPS and LPS + FK (a)The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and (b) secretedMCP-1 proteinlevels are the means plusmn SEM of 3 independent experiments Data were analyzed using 119905-test for unique sample and paired 119905-test lowast119901 lt 005versus CTL 119901 lt 005 versus LPS

4 Discussion

Obesity is strongly associated with increased risk of cardio-vascular disorders Recent studies have shown that increasedlevels of proinflammatory cytokines including MCP-1 areinvolved in obesity and insulin resistance [4 33] and thatexcess intake and endogenous release (lipolysis) of saturatedfatty acids might enhance expression of TLR-4 target genesincludingMCP-1 Indeed TLR-4-deficient knockoutmice fedwith a diet rich in saturated fatty acid had lower macrophageinfiltration and MCP-1 expression in their visceral adiposetissue as compared to wild-type mice [34] LPS have beenshown to stimulate secretion of inflammatory adipokinesthrough activation of Toll-like receptor-4 (TLR-4) and thedownstream transcription factor nuclear factor NF120581B inadipocytes [29ndash31] In addition Forskolin caused positivechanges in body composition in overweight and obese adultmen [21] In the present study we analyzed for the first timethe effect of Forskolin on LPS-inducedmodulation ofMCP-1TLR-4 GPR120 120573-Arrestin 2 and NF120581B1 gene expression

We confirmed that MCP-1 mRNA and proteins levelswere increased following LPS treatment in 3T3-L1 cells dif-ferentiated into adipocytes and that this effect was mediatedby TLR-4 as a TLR-4 inhibitor abolished the effect of LPSFurthermore we showed for the first time that Forskolincan inhibit the LPS-induced increase of MCP-1 mRNAlevel Although MCP-1 protein levels were also increasedin response to LPS Forskolin did not significantly affectthis LPS-induced increase in MCP-1 protein levels Directrelationship between the mRNA and protein levels is notalways observed as the levels of both mRNA and proteins

levels result from a ratio between synthesis and degradationIn addition half-lives of proteins within cells vary widelyfrom minutes to several days [35] As both MCP-1 mRNAand secreted MCP-1 protein levels were not determined atsimilar times following cell treatment (4 hours for mRNAand 24 hours for proteins) distinct incubation times and theobvious distinct effects of FK on mRNA and protein half-lives (ie affecting synthesis andor degradation) are likelyto account for the observed data Further work will includekinetic studies to determine the half-lives of mRNA andprotein

Our data suggest that Forskolinmight therefore representan interesting approach to decrease inflammation related toobesity in adipose tissue The signaling pathway involvedin the inhibition of LPS-induced MCP-1 mRNA levels byForskolin was also investigated Toll-like receptors expressedon virtually all human cells and binding a wide spectrumof exogenous and endogenous ligands such as bacterial LPSare involved in metabolic disorders [36 37] Our resultconfirmed that LPS alone increased TLR-4 mRNA levels indifferentiated adipocytes and this increase was abolished byCLI095 a TLR-4 inhibitor These data suggest a positivefeedback loop between LPS and TLR-4 Furthermore ourdata are in agreement with those showing that TLR-4 knock-out mice are protected against high-fat diet-induced obesityinflammation and insulin resistance and exhibited reducedinsulin-mediated changes in systemic glucose metabolism[19] Our data also show for the first time that Forskolin caninhibit LPS-induced increase in TLR-4 mRNA levels Thesedata clearly suggest that Forskolin could play a protectiverole in the inflammatory environment of adipose tissue


0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

Figure 3 mRNA expression of TLR-4 in differentiated 3T3-L1 cellstreated with LPS in the presence or absence of FK Differentiated3T3-L1 cells were treated as described in Materials and Methodsunder the following conditions CTL 10 120583M FK 1 120583gmL LPS andLPS+FKThe results are expressed asmRNA levels (fold stimulationover CTL set to 1) and are the means plusmn SEM of 3 independentexperiments Data were analyzed using 119905-test for unique sample andpaired 119905-test lowast119901 lt 005 versus CTL 119901 lt 005 versus LPS

In addition our study originally reported that Forskolinincreased GPR120mRNA level both in the absence and in thepresence of LPS that is a proinflammatory cue These dataare particularly interesting as in amacrophage cell line DHA(a GPR120 agonist) antagonizes the proinflammatory effectsof TNF-120572 and LPS and requires the presence of GPR120 tocounteract the proinflammatory actions of LPS on cytokinesrsquogene expression and protein secretion [14] In the presenceof LPS DHA significantly decreased MCP-1 mRNA levelbut increased both 120573-Arrestin 2 and GPR120 mRNA levels[38]The inhibitory effects of DHA on LPS-(TLR-4)-inducedactivation of NFkB activity might be mediated by a GPR120-120573-Arrestin 2-dependent mechanism [14] We hypothesizedthat the effect of Forskolin on LPS-induced increase ofMCP-1mRNA level could involve a GPR120-120573-Arrestin 2-dependentanti-inflammatorymechanism requiringNF120581B activation viaI120581B120572 degradation upon phosphorylation Thereby we exam-ined the effect of an I120581B120572 inhibitor BAY11-7082 on MCP-1 GPR120 TLR-4 and NF120581B1 mRNA levels We observedthat BAY11-7082 abolished the effect of LPS on MCP-1GPR120 TLR-4 and NF120581B1 mRNA levels this confirmedthe involvement of an NF120581B pathway in the regulation ofexpression of these genes In addition our results showedthat Forskolin alone or in the presence of LPS inhibitsNF120581B1 mRNA levels in DC Furthermore no change inboth total and phosphorylated I120581B120572 protein levels couldbe detected by semiquantitative Western blot analysis uponadipocyte treatment with LPS and Forskolin alone or incombination Recent reports have hypothesized that smallincreases in cytosolic concentrations of I120581B120572 negatively affect

0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast

Figure 4mRNAexpression ofGPR120 in differentiated 3T3-L1 cellstreated with LPS in the presence or absence of FK Differentiated3T3-L1 cells were treated as described in Materials and Methodsunder the following conditions CTL 10 120583M FK 1 120583gmL LPS andLPS+FKThe results are expressed asmRNA levels (fold stimulationover CTL set to 1) and are the means plusmn SEM of 3 independentexperiments Data were analyzed using 119905-test for unique sample andpaired 119905-test lowast119901 lt 005 versus CTL 119901 lt 001 versus LPS

0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast

Figure 5 mRNA expression of 120573-Arrestin 2 in differentiated3T3-L1 cells treated with LPS in the presence or absence of FKDifferentiated 3T3-L1 cells were treated as described inMaterials andMethods under the following conditions CTL 10120583M FK 1 120583gmLLPS and LPS + FK The results are expressed as mRNA levels(fold stimulation over CTL set to 1) and are the means plusmn SEMof 3 independent experiments Data were analyzed using 119905-test forunique sample and paired 119905-test lowast119901 lt 005 versus CTL

NF120581B nuclear translocation [32] This hypothesis relies onthe fact that only 10 of NF120581B is translocated to the nucleusat a steady state Therefore even a 2-fold increase in I120581B120572protein concentration might be sufficient to retain the entireNF120581B activity in the cytoplasm [32] Other studies haveshown that Forskolin inhibits NF120581B-mediated transcription


0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)

Figure 6NF120581B1mRNAand I120581B120572 protein levels in differentiated 3T3-L1 cells treatedwith LPS in the absence or presence of FKDifferentiated3T3-L1 cells were treated as described in Materials and Methods under the following conditions CTL 10 120583M FK 1 120583gmL LPS and LPS +FK (a) The results are expressed as mRNA levels (fold stimulation over CTL set to 1) (b) Phospho-I120581B120572 and total I120581B120572 protein levels weredetermined in DC by Western blot analysis (c) Semiquantitative determination of protein expression was performed as described underMaterials andMethodsThe results are expressed as protein expression (ratio of total I120581B120572 or P-I120581B120572 band density over 120573-actin band density)expressed as percent of the CTL value (CTL set to 100) and are the mean plusmn SEM (119899 = 3) (in of CTL) Data were analyzed using 119905-test forunique sample and paired 119905-test lowast119901 lt 005 versus CTL 119901 lt 005 versus LPS

of several genes and that various doses of Forskolin did notsignificantly reduce the proteolytic degradation of I120581B120572 inHUVEC and THP-1 cells [39] On the contrary Forskolinincreased the cytoplasmic levels of I120581B120572 in Jurkat T-cellswhich selectively decreases the nuclear translocation of p65[40] while another report [41] has shown that 120573-agonistsexert their anti-inflammatory effects by increasing the cyto-plasmic concentrations of I120581B120572 in monocytic cells It shouldbe noted that these authors did not investigate the productionof I120581B120572 at the same time points and in the same cell typesThe

free I120581B120572 has a short half-life in vivo [42 43] supporting theidea that dissociation from NF-kB leads to rapid degradationof I120581B120572 In contrast to its remarkable stability when boundto NF120581B free I120581B120572 is intrinsically very unstable its half-lifeis lt10min and it is rapidly degraded in a process that doesnot require phosphorylation or ubiquitination [44 45] Thisrapid degradation of I120581B120572 could explain why in our studyno changes in I120581B120572 protein levels (total and phosphorylated)could be detected following 24 h incubation with Forskolinin the absence or presence of LPS Even if the effects of


0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

CTL LPS CLI + BAY +LPS LPS

lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)

Figure 7mRNAexpression ofMCP-1 (a) TLR-4 (b) GPR120 (c) andNF120581B1 (d) in differentiated 3T3-L1 cells treatedwith LPS in the presenceor absence of inhibitors Differentiated 3T3-L1 cells were treated as described inMaterials andMethods under the following conditions CTL1 120583gmL LPS 3 120583MCLI-095 + 1 120583gmL LPS (CLI + LPS) and 10 120583M of BAY11-7082 + 1 120583gmL LPS (BAY + LPS) The results are expressed asmRNA levels (fold stimulation over CTL set to 1) and are the means plusmn SEM of 3 independent experiments Data were analyzed using 119905-testfor unique sample and paired 119905-test lowast119901 lt 005 lowastlowast119901 lt 001 versus CTL 119901 lt 005 119901 lt 001 versus LPS

Forskolin on the I120581B120572 protein level differ between studies[39ndash41] including ours we showed that Forskolin exerts itsanti-inflammatory effects by inhibiting the expression ofNF120581B-dependent genes In our study we observed thatLPS-induced increases in MCP-1 TLR-4 and NF120581B mRNAlevels were downregulated by Forskolin On the other handGPR120 mRNA levels were upregulated in the presenceof Forskolin GPR120 plays an anti-inflammatory role byactivating anti-inflammatory pathways [14] Our data clearlyreveal an interaction between NF120581B and cAMP pathways inadipocytes as Forskolin exerts its anti-inflammatory role in

adipocytes by inhibiting NF120581B-mediated gene transcriptionSuch inhibition could result from the binding competitionbetween cAMP response element-binding protein (CREB)and NF120581B to the coactivator CREB-binding protein (CBP)a protein necessary for efficient gene transcription [46]Indeed upon activation of the protein kinase A (PKA)pathway increased phosphorylated CREB-CBP complexeswill form inducing an inhibition of NF120581B (p65) [46] PKAactivation reduces the induction of a distinct set of genesin monocytes and endothelial cells by inhibiting NF120581B-mediated gene transcription [47] Therefore to account for


NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNAdarr GPR120 mRNA

minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA

(b) Effect of FK on the LPS response

Figure 8 Data summary (a) When adipocytes are stimulated with LPS GPR120 mRNA levels are decreased while MCP-1 mRNA andprotein levels are upregulated via TLR-4 signaling pathway and NF120581B activation (b) When adipocytes are treated with LPS in the presenceof FK the LPS-induced effects on both MCP-1 and GPR120 are inhibited via PKA and CREB activation necessitating CBP binding ACadenylyl cyclase ATP adenosine triphosphate cAMP cyclic adenosinemonophosphate CBPCREB-binding protein CREB cAMP responseelement-binding protein FFA free fatty acids FK Forskolin G-3-P glycerol-3-phosphate GK glycerol kinase GPR120 G-protein coupledreceptor 120 H catecholamines or hormones HSL hormone sensitive lipase I120581B120572 inhibitor of kappa B LPS lipopolysaccharide MCP-1monocyte chemotactic protein-1 NF120581B nuclear factor-120581B p phosphorylated PKA protein kinase A TAG triacylglycerol TLR-4 Toll-likereceptor 4

the data observed in our study we hypothesize that Forskolinexerts its anti-inflammatory effects on LPS-induced mod-ulation of NF120581B target genes in adipocytes by increasingcAMP levels PKA activity CREB phosphorylation andCREB interaction with CBP (Figure 8) Additional studieswill be required to assess the involvement of PKA and CREBactivation as well as interaction between CBP CREB andNF120581B in such process

In conclusion pharmacological agents that elevate intra-cellular levels of cAMP such as Forskolin may be useful forthe treatment of inflammation associated with obesity

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this article

Acknowledgments

The authors thank Pierre Cullus for his statistical assistanceand Violette Dirix for helpful discussion This work wassupported by grants from Van Buuren Fund and DefayFund (Universite Libre de Bruxelles) Jeanne Durendale


Chiadakwas a recipient of aDoctoral Fellowship fromldquoFondsXenophiliardquo (Universite Libre de Bruxelles) and a travel grantfrom the ldquoFederation Wallonie Bruxellesrdquo

References

[1] E E Kershaw and J S Flier ldquoAdipose tissue as an endocrineorganrdquoThe Journal of Clinical Endocrinology ampMetabolism vol89 no 6 pp 2548ndash2556 2004

[2] K E Wellen and G S Hotamisligil ldquoObesity-induced inflam-matory changes in adipose tissuerdquo The Journal of ClinicalInvestigation vol 112 no 12 pp 1785ndash1788 2003

[3] S P Weisberg D McCann M Desai M Rosenbaum RL Leibel and A W Ferrante Jr ldquoObesity is associated withmacrophage accumulation in adipose tissuerdquo Journal of ClinicalInvestigation vol 112 no 12 pp 1796ndash1808 2003

[4] H Xu G T Barnes Q Yang et al ldquoChronic inflammation in fatplays a crucial role in the development of obesity-related insulinresistancerdquoThe Journal of Clinical Investigation vol 112 no 12pp 1821ndash1830 2003

[5] K Takahashi S Mizuarai H Araki et al ldquoAdiposity elevatesplasma MCP-1 levels leading to the increased CD11b-positivemonocytes in micerdquo The Journal of Biological Chemistry vol278 no 47 pp 46654ndash46660 2003

[6] P Sartipy and D J Loskutoff ldquoMonocyte chemoattractantprotein 1 in obesity and insulin resistancerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 12 pp 7265ndash7270 2003

[7] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006

[8] S E Shoelson and A B Goldfine ldquoGetting away from glucosefanning the flames of obesity-induced inflammationrdquo NatureMedicine vol 15 no 4 pp 373ndash374 2009

[9] E Manickam A J Sinclair and D Cameron-Smith ldquoSuppres-sive actions of eicosapentaenoic acid on lipid droplet formationin 3T3-L1 adipocytesrdquo Lipids inHealth andDisease vol 9 article57 2010

[10] AMullen C E Loscher and HM Roche ldquoAnti-inflammatoryeffects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophagesrdquo Journal of Nutritional Biochemistry vol21 no 5 pp 444ndash450 2010

[11] H W Hsueh Z Zhou J Whelan et al ldquoStearidonic and eicos-apentaenoic acids inhibit interleukin-6 expression in obobmouse adipose stem cells via toll-like receptor-2-mediatedpathwaysrdquo Journal of Nutrition vol 141 no 7 pp 1260ndash12662011

[12] R K Murumalla M K Gunasekaran J K Padhan et al ldquoFattyacids do not pay the toll effect of SFA and PUFA on humanadipose tissue and mature adipocytes inflammationrdquo Lipids inHealth and Disease vol 11 article 175 2012

[13] E Oliver F C McGillicuddy K A Harford et al ldquoDocosahex-aenoic acid attenuates macrophage-induced inflammation andimproves insulin sensitivity in adipocytes-specific differentialeffects between LC n-3 PUFArdquo Journal of Nutritional Biochem-istry vol 23 no 9 pp 1192ndash1200 2012

[14] D YOh S Talukdar E J Bae et al ldquoGPR120 is an omega-3 fattyacid receptor mediating potent anti-inflammatory and insulin-sensitizing effectsrdquo Cell vol 142 no 5 pp 687ndash698 2010

[15] R L Bradley F M Fisher and E Maratos-Flier ldquoDietary fattyacids differentially regulate production of TNF-120572 and IL-10 by

murine 3T3-L1 adipocytesrdquo Obesity vol 16 no 5 pp 938ndash9442008

[16] C YeopHan A Y Kargi M Omer C K Chan M Wabitschand K D OrsquoBrien ldquoDifferential effect of saturated and unsat-urated free fatty acids on the generation of monocyte adhe-sion and chemotactic factors by adipocytes dissociation ofadipocyte hypertrophy from inflammationrdquo Diabetes vol 59no 2 pp 386ndash396 2010

[17] J Zhang L S Barak K E Winkler M G Caron and S SG Ferguson ldquoA central role for 120573-arrestins and clathrin-coatedvesicle-mediated endocytosis in 120573

2-adrenergic receptor resen-

sitization Differential regulation of receptor resensitization intwo distinct cell typesrdquoThe Journal of Biological Chemistry vol272 no 43 pp 27005ndash27014 1997

[18] H Shi M V Kokoeva K Inouye I Tzameli H Yin and JS Flier ldquoTLR4 links innate immunity and fatty acid-inducedinsulin resistancerdquo Journal of Clinical Investigation vol 116 no11 pp 3015ndash3025 2006

[19] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in Toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[20] T A McNicholas J D Dean H Mulder C Carnegie andN A Jones ldquoA novel testosterone gel formulation normalizesandrogen levels in hypogonadal men with improvements inbody composition and sexual functionrdquo BJU International vol91 no 1 pp 69ndash74 2003

[21] M P Godard B A Johnson and S R Richmond ldquoBody com-position and hormonal adaptations associated with forskolinconsumption in overweight and obese menrdquo Obesity Researchvol 13 no 8 pp 1335ndash1343 2005

[22] S E Atkinson E SMaywood J E Cheshamet al ldquoCyclicAMPsignaling control of action potential firing rate and molecularcircadian pacemaking in the suprachiasmatic nucleusrdquo Journalof Biological Rhythms vol 26 no 3 pp 210ndash220 2011

[23] S T Ballard L Trout J Garrison and S K Inglis ldquoIonicmechanism of forskolin-induced liquid secretion by porcinebronchirdquo American Journal of Physiology-Lung Cellular andMolecular Physiology vol 290 no 1 pp L97ndashL104 2006

[24] J-L Cao V F Vialou M K Lobo et al ldquoEssential role ofthe cAMP-cAMP response-element binding protein pathwayin opiate-induced homeostatic adaptations of locus coeruleusneuronsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 39 pp 17011ndash17016 2010

[25] I V Sokolova H A Lester and N Davidson ldquoPostsynapticmechanisms are essential for forskolin-induced potentiation ofsynaptic transmissionrdquo Journal of Neurophysiology vol 95 no4 pp 2570ndash2579 2006

[26] T Arsenijevic F Gregoire V Delforge C Delporte and JPerret ldquoMurine 3T3-L1 Adipocyte cell differentiation modelvalidated reference genes for qPCR gene expression analysisrdquoPLoS ONE vol 7 no 5 Article ID e37517 2012

[27] T Arsenijevic F Gregoire J Chiadak et al ldquoPituitaryAdenylateCyclase Activating Peptide (PACAP) participates in adipogene-sis by activating ERK signaling pathwayrdquo PLoS ONE vol 8 no9 Article ID e72607 2013

[28] J Vandesompele K De Preter F Pattyn et al ldquoAccurate nor-malization of real-time quantitative RT-PCR data by geometricaveraging of multiple internal control genesrdquo Genome Biologyvol 3 no 7 2002

[29] K M Ajuwon and M E Spurlock ldquoAdiponectin inhibitsLPS-induced NF-120581B activation and IL-6 production and


increases PPAR1205742 expression in adipocytesrdquo American Journalof PhysiologymdashRegulatory Integrative and Comparative Physiol-ogy vol 288 no 5 pp R1220ndashR1225 2005

[30] 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

[31] 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

[32] S Miyamoto P J Chiao and I M Verma ldquoEnhanced I120581B120572degradation is responsible for constitutive NF-120581B activity inmaturemurine B-cell linesrdquoMolecular and Cellular Biology vol14 no 5 pp 3276ndash3282 1994

[33] A J Alonso-Castro R Zapata-Bustos G Gomez-Espinozaand L A Salazar-Olivo ldquoIsoorientin reverts TNF-120572-inducedinsulin resistance in adipocytes activating the insulin signalingpathwayrdquo Endocrinology vol 153 no 11 pp 5222ndash5230 2012

[34] J E Davis N K Gabler J Walker-Daniels and M E SpurlockldquoTlr-4 deficiency selectively protects against obesity induced bydiets high in saturated fatrdquoObesity vol 16 no 6 pp 1248ndash12552008

[35] G M Cooper ldquoProtein degradationrdquo in The Cell A MolecularApproach Sinauer Associates Sunderland Mass USA 2ndedition 2000

[36] K Eguchi I Manabe Y Oishi-Tanaka et al ldquoSaturated fattyacid and TLR signaling link 120573 cell dysfunction and isletinflammationrdquo Cell Metabolism vol 15 no 4 pp 518ndash533 2012

[37] K Takeda T Kaisho and S Akira ldquoToll-like receptorsrdquoAnnualReview of Immunology vol 21 pp 335ndash376 2003

[38] MM Cranmer-Byng D M Liddle A A De Boer J MMonkand L E Robinson ldquoProinflammatory effects of arachidonicacid in a lipopolysaccharide-induced inflammatory microen-vironment in 3T3-L1 adipocytes in vitrordquo Applied PhysiologyNutrition and Metabolism vol 40 no 2 pp 142ndash154 2015

[39] P Ghersa R H Van Huijsduijnen J Whelan Y CambetR Pescini and J F DeLamarter ldquoInhibition of E-selectingene transcription through a cAMP-dependent protein kinasepathwayrdquo Journal of Biological Chemistry vol 269 no 46 pp29129ndash29137 1994

[40] M Neumann T Grieshammer S Chuvpilo et al ldquoRelAp65 isa molecular target for the immunosuppressive action of proteinkinase Ardquo EMBO Journal vol 14 no 9 pp 1991ndash2004 1995

[41] P Farmer and J Pugin ldquoBeta-adrenergic agonists exerttheir lsquoanti-inflammatoryrsquo effects in monocytic cells throughthe IkappaBNF-kappaB pathwayrdquo American Journal ofPhysiologymdashLung Cellular and Molecular Physiology vol 279no 4 pp L675ndashL682 2000

[42] S Ghosh and D Baltimore ldquoActivation in vitro of NF-120581B byphosphorylation of its inhibitor I120581BrdquoNature vol 344 no 6267pp 678ndash682 1990

[43] S-C Sun P AGanchi DW Ballard andWCGreene ldquoNF-120581bcontrols expression of inhibitor i120581B120572 evidence for an inducibleautoregulatory pathwayrdquo Science vol 259 no 5103 pp 1912ndash1915 1993

[44] D Krappmann F G Wulczyn and C Scheidereit ldquoDifferentmechanisms control signal-induced degradation and basalturnover of the NF-120581B inhibitor I120581B120572 in vivordquo EMBO Journalvol 15 no 23 pp 6716ndash6726 1996

[45] E Mathes E L OrsquoDea A Hoffmann and G Ghosh ldquoNF-120581Bdictates the degradation pathway of I120581B120572rdquoThe EMBO Journalvol 27 no 9 pp 1357ndash1367 2008

[46] G C N Parry and N Mackman ldquorole of cyclic AMP responseelement-binding protein in cyclic AMP inhibition of NF-120581B-mediated transcriptionrdquo Journal of Immunology vol 159 no 11pp 5450ndash5456 1997

[47] V Ollivier G C Parry R R Cobb D de Prost and NMackman ldquoElevated cyclic AMP inhibits NF-120581B-mediatedtranscription in human monocytic cells and endothelial cellsrdquoJournal of Biological Chemistry vol 271 no 34 pp 20828ndash20835 1996

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


which can blunt lipopolysaccharide- (LPS-) stimulated ele-vations in adipokines [9ndash13] Furthermore activation of G-protein coupled receptor 120 (GPR120) by DHA antagonizesthe proinflammatory effects of tumor necrosis factors-alpha(TNF-120572) and lipopolysaccharide in a macrophage cell line[14]The inhibitory effects of DHA on LPS-Toll-like receptor-4- (TLR-4-) induced activation of nuclear factor-kappa B(NFkB) activity [15 16] might be mediated by a GPR120 and120573-Arrestin 2-dependent mechanism [14] 120573-Arrestin proteinsplay an important role in regulating the responsiveness ofG-protein coupled receptors (GPCRs) by contributing tomechanisms involved in both GPCR desensitization andresensitization [17]

Previous studies suggest that saturated fatty acids pro-mote inflammation by activating TLR-4 on adipocytes andmacrophages [18] Indeed mice lacking TLR-4 are pro-tected against high-fat diet-induced obesity inflammationand insulin resistance because they are resistant to thesuppression of insulin signaling during lipid infusion andexhibit reduced insulin-mediated changes in systemic glu-cose metabolism [19] Forskolin (FK) is a labdane diterpeneisolated from the roots of the Coleus forskohlii plant aperennial herb with fleshy fibrous roots belonging to themintfamily of plants In the early-to-mid-1980s Forskolin wasprimarily used as an agent to help a number of cardiovasculardisease conditions mainly through a vasodilator effect [20]This effect resulted from increased adenylate cyclase activitywithin the body Forskolin causes positive changes in bodycomposition in overweight and obese adult men [21] Oneof the potential explanations for the decrease in fat massand body fat percentage may be adenylate cyclase activa-tion and thus cyclic adenosine monophosphate (cAMP)accumulation within adipose tissue which stimulates freefatty acid release and lipolysis Indeed Forskolin has beenwidely used as a potent activator of adenylate cyclase incellular preparations to study cAMP-dependent transductionpathways [22ndash25] As circulating MCP-1 levels are increasedin rodent obesity and the role of Forskolin in fat massreduction is clearly established the aim of this study wasto determine on one hand the expression of MCP-1 TLR-4 GPR120 120573-Arrestin 2 and NF120581B1 expression in DC andon the other hand the signaling pathway by which Forskolinaffects MCP-1 and GPR120 expression in the LPS-inducedinflammatory response in 3T3-L1 cells differentiated intoadipocytes

2 Materials and Methods

21 Reagents Dulbeccorsquos modified Eaglersquos medium (DMEM45 gL glucose) streptomycinpenicillin fetal bovine serumhorse serum and calf serum were provided by Invitrogen(Carlsbad CA USA) Bovine insulin 3-isobutyl-1-methyl-xanthine (IBMX) dexamethasone lipopolysaccharides andForskolin were purchased from Sigma (St Louis MO USA)TLR-4 signaling inhibitor (CLI-095) and kappa B (I120581B120572)inhibitor (BAY11-7082) were purchased from InvivoGen (SanDiego CA USA) Anti-120573-actin and anti-I120581B120572 came fromMillipore (Temecula CA USA) and anti-phospho-I120581B120572

(Ser3236) (5A5)was purchased fromCell Signaling Technol-ogy Inc (Danvers MA USA)

22 Cell Culture and Treatments 3T3-L1 murine preadipo-cyte cells were grown in DMEM supplemented with 10 calfserum 200UmL penicillin and 200UmL streptomycin in8 CO

2humidified atmosphere at 37∘C until confluence

Two days after confluence to induce adipocyte differentia-tion cells were incubated for 60 h in DMEM supplementedwith 10 fetal bovine serum and containing 500 120583M IBMX025 120583Mdexamethasone and 10 120583gmL insulinThe cells werethen maintained in the culture medium supplemented withinsulin only and this media was changed every 2 days (day 5and day 7) until complete differentiation (monitored by lipiddroplet accumulation under the microscope and confirmedby Oil Red Coloration) had occurred (day 9) On day 9 thedifferentiated 3T3-L1 cells (DC) were treated for 4 h withwater followed by 4 h with water and ethanol (CTL) or 4 hwith water and 4 h with 10 120583M Forskolin (FK) or 4 h with1 120583gmL LPS followed by 4 h with LPS and ethanol (LPS) or4 h with LPS followed by 4 h LPS and FK (LPS + FK) Bothcells and culture media from cultured undifferentiated cells(UDC at day 0) and from DC (at day 9 following treatment)were harvested To determine how the expressions of thegenes of interest are modulated by NF120581B activation the DCwere treated with I120581B120572 inhibitor BAY11-7082 at 10120583M TheTLR-4 signaling inhibitor CLI-095 at 3 120583M was also usedFor inhibitor studies DC were pretreated with the inhibitorfor 1 hour before exposure to LPS stimulation and thencoincubated with LPS and the inhibitor for 4 h prior to RNAextraction and 24 h prior to protein extraction

23 RNA Isolation Isolation of RNA as well as assessmentof RNA concentration and purity and RNA integrity wereperformed as previously described [26 27]

24 RT-qPCR Design of qPCR primers cDNA synthesisand qPCR reactions were performed as previously described[26]The primer pairs which were used are shown in Table 1

25 Analysis of Gene Expression Stability Gene expressionstability analysis and matching statistics were performedusing Biogazelle qBASE Plus software [28] Data were nor-malized using the references genes tyrosine 3-monoox-ygenasetryptophan 5-monooxygenase activation proteinzeta polypeptide (mmYwhaz) non-POU-domain contain-ing octamer binding protein (mmNONO) and 120573-actin(mmACTB) which were previously validated for this cellularand experimental system [26]

26 MCP-1 ELISA Assay MCP-1 protein levels of the culturemedia were determined by ELISA using a Duoset ELISA kitfrom RampD Diagnostics (Minneapolis MN USA)

27 Protein Extraction and Western Blot Analysis DC werewashed with calcium- and magnesium-free PBS and lysedin 1mL of lysis buffer containing 50mM TrisHCl (pH75) 150mM NaCl 05 Nonidet P40 50mM NaF 1mM






3 Results









0

02

04

06

08

1

12

UDC DC

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowastlowastlowast

(a)

0

1

2

3

4

5

6

7

UDC DC

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(b)

0

20

40

60

80

100

120

140

160

UDC DC

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

2

UDC DC

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

(d)







0

5

10

15

20

25

30

35

40

45

50

CTL FK LPS LPS + FK

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(a)

0

10000

20000

30000

40000

50000

60000

70000

CTL FK LPS LPS + FK

Secr

eted

MCP

-1 p

rote

in (p

gm

L)

lowast

(b)


4 Discussion






0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast



0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast


0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast




0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results









0

02

04

06

08

1

12

UDC DC

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowastlowastlowast

(a)

0

1

2

3

4

5

6

7

UDC DC

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(b)

0

20

40

60

80

100

120

140

160

UDC DC

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

2

UDC DC

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

(d)







0

5

10

15

20

25

30

35

40

45

50

CTL FK LPS LPS + FK

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(a)

0

10000

20000

30000

40000

50000

60000

70000

CTL FK LPS LPS + FK

Secr

eted

MCP

-1 p

rote

in (p

gm

L)

lowast

(b)


4 Discussion






0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast



0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast


0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast




0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

UDC DC

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowastlowastlowast

(a)

0

1

2

3

4

5

6

7

UDC DC

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(b)

0

20

40

60

80

100

120

140

160

UDC DC

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)

lowastlowastlowast

(c)

0

02

04

06

08

1

12

14

16

18

2

UDC DC

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

(d)







0

5

10

15

20

25

30

35

40

45

50

CTL FK LPS LPS + FK

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(a)

0

10000

20000

30000

40000

50000

60000

70000

CTL FK LPS LPS + FK

Secr

eted

MCP

-1 p

rote

in (p

gm

L)

lowast

(b)


4 Discussion






0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast



0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast


0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast




0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

35

40

45

50

CTL FK LPS LPS + FK

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

(a)

0

10000

20000

30000

40000

50000

60000

70000

CTL FK LPS LPS + FK

Secr

eted

MCP

-1 p

rote

in (p

gm

L)

lowast

(b)


4 Discussion






0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast



0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast


0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast




0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

14

16

18

CTL FK LPS LPS + FK

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast



0

05

1

15

2

25

3

CTL FK LPS LPS + FK

GPR

120

mRN

A (f

old

stim

ulat

ion)

lowast

lowast


0

02

04

06

08

1

12

CTL FK LPS LPS + FK

120573-A

rres

tin 2

mRN

A le

vels

(fold

stim

ulat

ion)

lowast




0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

8

9

10

CTL FK LPS LPS + FK

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

lowast

lowast

(a)

CTL FKLPS LPS + FK

p-I120581B120572

I120581B120572

120573-Actin

(b)

0

20

40

60

80

100

120

140

CTL LPS FK LPS + FK

Prot

ein

expr

essio

n (

of C

TL)

p-I120581B120572I120581B120572

(c)





0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70

80

90

100

MCP

-1 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowastlowast

lowastlowast

lowast

(a)

0

05

1

15

2

25

3

35

TLR-

4 m

RNA

leve

ls (fo

ld st

imul

atio

n)


lowast

lowast

(b)

0

02

04

06

08

1

12

GPR

120

mRN

A le

vels

(fold

stim

ulat

ion)


lowast

lowastlowast

lowastlowast

(c)

0

05

1

15

2

25

CTL LPS CLI + BAY +

NF120581

B1 m

RNA

leve

ls (fo

ld st

imul

atio

n)

LPS LPS

lowast

lowastlowast

(d)





NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NF120581B-p

NF120581B-p

I120581B120572-p

uarr MCP-1 mRNA

darr GPR120 mRNA

CBP

LPS

TLR-4

+

+

NF120581B-I120581B120572

(a) Response to LPS

LPS

CREB-p

CREB-p

CREB

NF120581B-p

NF120581B-p

I120581B120572-p


minus

+ +

++

+

CBP

Glycerol

Glycerol

G-3-P

HSL

PKAGK

TAG

FK

TLR-4 AC

ATP cAMP

NF120581B-I120581B120572

FFA





Competing Interests


Acknowledgments




References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Forskolin Inhibits Lipopolysaccharide-Induced...

Documents

Transcript of Research Article Forskolin Inhibits Lipopolysaccharide-Induced...