cloning and characterization of spliced variants of the porcine g … · 2018. 10. 25. ·...

Research ArticleCloning and Characterization of Spliced Variants ofthe Porcine G Protein Coupled Receptor 120

Tongxing Song12 Jie Peng12 Jiao Ren12 Hong-kui Wei12 and Jian Peng12

1Department of Animal Nutrition and Feed Science College of Animal Science and TechnologyHuazhong Agricultural University Wuhan 430070 China2The Cooperative Innovation Center for Sustainable Pig Production Wuhan 430070 China

Correspondence should be addressed to Hong-kui Wei weihongkuimailhzaueducn

Received 14 February 2015 Revised 2 May 2015 Accepted 2 May 2015

Academic Editor Amelie Bonnefond

Copyright copy 2015 Tongxing Song et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in humanand rodents However the porcineGPR120 (pGPR120) has not beenwell characterized In the current study we found that pGPR120had 3 spliced variants Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WTwhich shared 88homologywith humanshort formGPR120 Transcript 1 was themainly expressed transcript of pGPR120 It was expressed predominantly in ileum jejunumduodenum spleen and adipose Transcript 3 (coding 320-aa isoform) was detected in spleen while the transcript 2 (coding 310-aaisoform) was only slightly expressed in spleen A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-lucand NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2 However 320-aaisoform was not a dominant negative isoform The extracellular signal-regulated kinase 12 (ERK12) phosphorylation levels incells transfected with construct for pGPR120-WT were well activated by PUFAs especially n-3 PUFA These results showed thatalthough pGPR120 had 3 transcripts transcript 1 which encoded pGPR120-WT was the mainly expressed transcript TUG-891 andPUFAs especially n-3 PUFA well activated pGPR120-WT The current study contributed to dissecting the molecular regulationmechanisms of n-3 PUFA in pigs

1 Introduction

Free fatty acids (FFAs) are well known in serving as importantnutrients to provide energy for the body and play impor-tant roles in various physiological regulation as signalingmolecules [1ndash3] Recently a group of G-protein coupledreceptors (GPCRs) have been identified as FFA receptorswhich mediate FFA-induced signaling in various tissues [2ndash9] In particular GPR120 which is described as an orphanreceptor originally functions as a polyunsaturated fatty acids(PUFAs) receptor and plays critical roles in systemic nutrienthomeostasis regulation [8 10]

In human mouse and rat GPR120 is expressed abun-dantly in intestinal tract where GPR120 mediates FFA-induced the secretion of the gut hormones (glucagon-likepeptide-1 and cholecystokinin) thereby regulating appetite[5 11] Additionally the expression of GPR120 is also sig-nificantly abundant in adipose tissue where it participates

in lipid and glucose metabolism as well as adipogenesisregulation [12ndash14] Ligands of GPR120 including n-3 PUFAsand synthetic GW9508 reduce inflammatory and increaseinsulin sensitivityin a GPR120-dependent manner in mice[8] Moreover GPR120 is found to mediate the taste of fattyacids and FFAs-induced antiapoptosis effects [15 16]

G120572q11 is well known as a coupled G protein which canimprove the cytoplasmic calcium released from endoplasmicreticulum and result in activation of pathways dependent onthe calcium [5 8] In addition several studies have showedthat GPR120 activates G120572q11 signaling in various cell types[5 8 17] The ligands of GPR120 activate the mitogen-activated protein kinase (MAPKA) pathway leading tophosphorylation of extracellular signal-regulated kinase 12(ERK12) [5] Hence both [Ca2+]i and ERK12 activation areused as indexes to reflect the function of GPR120 [5 13 18]

Human GPR120 has two isoforms a long (L) form anda short (S) form [19] However in mouse and rat only

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 813816 10 pageshttpdxdoiorg1011552015813816

2 BioMed Research International

Table 1 Primers used for polymerase chain reaction

Primeridentification Primer sequences (51015840-31015840)

Productsize forWT (bp)

Product sizefor WT (bp)


Annealingtemperature

(∘C)

GPR120 full F GAATTCGCCACCATGGGAATGTCCCTTGAGTGCR TCTAGACTAGCTGGAAATAACAGACAGATC 1086 930 960 62

GPR120-407 F AAGGAGGAGGCTCACGATGR TGACAAATAGATGCCGATAGAC 407 407 407 59

GPR120-Variant F GCTCTTCTACGTGATGACCCTAR CGTGAGCCTCCTCCTTGAT 388 232 262 60

120573-actin F CCAGGTCATCACCATCGGR CCGTGTTGGCGTAGAGGT 158 mdash mdash 60

one isoform of GPR120 is identified [11 20] Most recentlines of evidence show that the hGPR120-L and hGPR120-S have different tissue distributions and pharmacologicalcharacteristics [16 21] Compared with human and rodentsmuch less is known about the porcine GPR120 (pGPR120)In database of mRNA sequence pigs have 3 splice variants(NM 001204766 JQ437360 and JQ437361) However thereare no relative reports to characterize pGPR120 Recentlyit has been reported that feeding n-3 PUFA-enriched dietscould improve the intramuscular fat content and inhibit theexpression of inflammation related genes leading to increas-ing the mass of skeletal muscle of pigs [22 23] Howeverthe detailed mechanism is largely unknown In the currentstudy we presented the first evidence for tissue expression ofsplice variants of the pGPR120 and pharmacology of differentpGPR120 isoforms These results may provide a way to helpunderstand the nutrichemical role of n-3 PUFA in pigs

2 Materials and Methods

21 Cloning of pGPR120s into pcDNA31 The coding seq-uence (CDS) of pGPR120swas obtained by PCR amplificationusing mixed porcine complimentary DNA (cDNA) extractedfrom the spleen tissue of 3 Landrace barrows as templatesThe CDS of pGPR120 were inserted between restriction sitesEcoRI (51015840) and XbaI (31015840) in pcDNA31 (+) (Clontech PatoAlto CA USA) [21] The primer sequences and optimal PCRannealing temperatures are listed in Table 1 The identities ofall constructs were confirmed by sequencing

22 Phylogenetic and Comparative Analysis The amino acidssequences of GPR120s from various species were retrievedfrom GenBank Then the retrieved amino acids sequencesand the deduced amino acid sequences from depositedporcine CDS sequences of pGPR120 were subjected to mul-tiple sequence alignment using ClustalW program (httpwwwebiacukToolsclustalw2) using default settings TheMEGA 50 programwas used to reconstruct the phylogenetictrees by the Neighbor-Joining (NJ) method (httpwwwmegasoftwarenet)

23 Cell Culture and DNA Transfection Isolation mainte-nance and differentiation of porcine adipose derived stem

cells (ADSC) and dedifferentiated fat cells (DFAT) wereperformed as described [8 12 24] The 3D42 cells (ATCC)were grown in Roswell Park Memorial Institute (RPIM) 1640(Gibco San Diego CA USA) supplemented with 2mM L-glutamine and adjusted to contain 15 gL sodium bicarbon-ate 45 gL glucose 10mMHEPES 10mM sodium pyruvatesupplemented with 01mM nonessential amino acids (GibcoSan Diego CA USA) 90 fetal bovine serum (FBS) (GibcoSanDiego CAUSA) 10 PK15 IPECJ2 andHEK293T cellswere grown in Dulbeccorsquos Modified Eaglersquos Medium (45 gLglucose) (Gibco) supplemented with 2mM L-glutamine10 FBS (Gibco) penicillin (100 IUmL) and streptomycin(100 pgmL) in a humidified atmosphere 5 CO

295 air at

37∘C Cells were grown in 75-cm2 flasksThe cells were transfected at 60ndash70 confluence in

culture plates using Lipofectamine 2000 (Invitrogen Carls-bad CA USA) following Invitrogen protocols Briefly bothLipofectamine 2000 (1120583L) and plasmid (05 120583g) were dilutedusing Opti-MEM (Invitrogen) to a final volume of 50120583Lper well and then mixed together for 15 minutes at roomtemperature Then the lipo-DNA complex was added intowells Cells were assayed 48 h after transfection

24 RNA Isolation Semiquantitative RT-PCR and Quantita-tive PCR (qPCR) Total RNA was extracted from adiposeskeletal muscle ileum jejunum duodenum kidney lungspleen liver and heart of 6 Landrace barrows with Trizol(Invitrogen) following the manufacturerrsquos instructions TotalRNA from 3D42 PK15 IPECJ2 ADSC and DFAT as wellas differentiated ADSC and DFAT cells was also isolatedand extracted using Trizol reagent The 25 120583g of RNA wasreverse transcribed using First-Strand cDNA Synthesis Kit(TOYOBO Japan) and cDNAs were stored at minus20∘C

Total GPR120 expression in various tissues was deter-mined by semiquantitative RT-PCR The cross intron-exonboundaries primers (GPR120-407) were designed accordingto the common sequence of the pGPR120 transcripts as shownin Figure 3(a) The standard PCR program for amplificationconsisted of an initial denaturation step at 94∘C for 4minfollowed by 35 cycles of 30 sec at 94∘C 30 sec at the indicatedannealing temperature 119899min at 72∘C (119899 = size in kilobases ofamplified product) and a final elongation at 72∘C for 5min

BioMed Research International 3

The PCR products were separated by 2 agarose gel elec-trophoresis (Sigma St Louis MO USA) After staining netintensity of each band was scanned using GeneSnap system(Syngene Cambridge UK) The expression of pGPR120s wasquantized after normalization of samples using intensity of120573-actin To further check the tissue expression of total pGPR120expression we also determine the expression of totalGPR120in culture cells which corresponded to pGPR120 highlyexpressed tissues by qPCR using a Bio-Rad CFX ConnectReal-Time PCR Detection System (Bio-Rad Richmond CAUSA) The PK15 and IPEC-J2 are cell lines isolated fromthe porcine kidney and small intestine respectively The3D42 cell is the porcine macrophage which is one of thecommon cell types in spleen ADSC and DFAT cells arepreadipocytes whereas differentiated ADSC and DFAT cellsrepresented mature adipocytes Gene expression levels werecalculated after normalization to the standard housekeepinggene 120573-actin using the ΔΔCT method In brief the meanof the triplicate cycle thresholds (CT) of the pGPR120 wasnormalized to the mean of triplicate CT of the reference 120573-actin using the calculation formula ldquo2CT120573-actinminusCTpGPR120rdquo whichindicated a relative value as a fraction of pGPR120

The relative expression levels of the 3 spliced variantsin pGPR120 highly expressed tissues were also detectedby semiquantitative RT-PCR The primer pair (GPR120-Variants) designed for amplification of 3 spliced variants isshown in Figure 4(a) By using this primer pair amplificationwill produce 3 different fragments from3 spliced variantsThePCRproducts were separated by 2 agarose gel electrophore-sis or 10 polyacrylamide gel electrophoresis and detectedby using ethidium bromide (Sigma) to measure the levels ofpGPR120s mRNA from various tissues and cell lines All theprimers sequences were listed in Table 1

25 Luciferase Reporter Assays For luciferase reporter assaysthe HEK293T cells were plated into 24-well plates ThenSRE-luc orNFAT-luc vector combinedwith Renilla luciferaseexpression plasmid (pTK) was cotransfected with expressionvectors for various isoform of pGPR120 or pcDNA31 controlplasmid Then 24 h after transfection the cells were changedto the medium without FBS and maintained for at least 18 hfor SRE-luc or NFAT-luc analysis Then cells were treatedwith the appropriate ligand for 6 h After treatment cells wereharvested and luciferase activity in cell extracts was deter-mined using a luciferase assay system according to standardmethods in a Dual-GLO reporter assay system (ProgemaMadison USA) Luciferase values were normalized by theRenilla values Transfection experiments were performed induplicate and repeated at least three times [25]

26 ERK12 Phosphorylation Assay HEK293T cells wereplated into 24-well plates and transfected with pGPR120-WTexpression vector or pcDNA31 using Lipofectamine 2000After 24 h of transfection cells were washed 2 times andmaintained in the medium without FBS for at least 18 hThen cells were treated with 10 120583M docosahexaenoic acid(DHA) eicosapentaenoic acid (EPA)120572-linolenic acid (ALA)linoleic acid (LA) palmitic acid (PLA) or 10 120583M TUG-891

(Sigma) for 10min After treatment cells were washed withPBS and lysed in lysis buffer (Beyotime Shanghai China)Lysed samples were centrifuged for 10min at 10000 rpm at4∘C and the supernatant was subjected to SDS-PAGE andimmunoblotting Twenty mg of proteinslane was separatedon a 10 polyacrylamide and precast SDS gel (Bio-Rad)followed by transfer on polyvinylidene difluoride membrane(Millipore Billerica MA USA) The membrane was blockedfor 2 h with 5 skim milk powder (Sigma) and incubatedovernight with the anti-phosphorylated ERK12 and anti-tubulin at a 1 2000 dilution After 3 washes the secondaryantibody was added at a 1 10000 dilution and incubated atroom temperature for 1 h After 3 washes the membranewas exposed by using WesternBrigh Peroxide (AdvanstaCalifornia USA) in imaging system (Carestream New YorkUSA) The protein amount was normalized with the amountof tubulin as internal controls

27 Statistical Analyses Data were presented as means plusmnSD and the difference between groups was determined byStudentrsquos 119905-test

3 Results

31 Molecular Characterization and Evolution of PorcineGPR120 The pGPR120 had 3 alternative transcripts includ-ing a 1086 bp transcript (transcript 1) a 930 transcript (tran-script 2) and a 960 bp transcript (transcript 3)The transcript1 encoded wild type pGPR120 (pGPR120-WT) of 362 aminoacids (aa) whereas transcript 2 and transcript 3 encoded 310-aa and 320-aa isoforms of GPR120 respectively The 310-aaisoform (encoded by pGPR120-V2) and the 320-aa isoform(encoded by pGPR120-V3) were the truncated isoformswhich did not contain fourth or fifth transmembrane domainrespectively (Figure 1(a)) Multiple sequence alignments andphylogenic trees comparisons of the CDS and deduced aminoacid sequences of GPR120s from different species are shownin Figures 1 and 2 The pGPR120 shared higher homology(88 in CDS and 894 in amino acid sequence) with thehuman receptor sequences than they are to mouse and rat(85 and 85 in CDS 884 and 870 in amino acidsequence) no matter for CDS or the amino acid sequence

32 Transcriptional Expression of Total pGPR120 The primerpair called GPR120-407 was used to detect the expression oftotal pGPR120 in various tissues as Figure 3(a) shows Semi-quantitative RT-PCR analysis showed that total pGPR120waswell detected in jejunum duodenum spleen and adipose andnegligibly expressed in other tissues (Figure 3(b)) To furthercheck the expression of pGPR120 in the correspondingestablished cell lines related to the highly expressed tissueswe subsequently adopted a qPCR strategy to examine theexpression of total pGPR120 in various cell lines and primarycultured cells GPR120 was highly expressed in adipose tissueas well as adipogenic differentiated ADSC and DFAT atday 7 In addition pGPR120 was also expressed in 3D42IPECJ2 and PK15 (Figure 3(c)) which represent the porcinemacrophage kidney and small intestine cells respectively


Pig N-terminus 1 M S L E C A Q A A C T G P A R S L E S A N R T R F P F F S D V K G D H R L 37Pig-V2 1 M S L E C A Q A A C T G P A R S L E S A N R T R F P F F S D V K G D H R L 37Pig-V3 1 M S L E C A Q A A C T G P A R S L E S A N R T R F P F F S D V K G D H R L 37Human 1 M S P E C A R A A G D A P LR S L E Q A N R T R F P F F S D V K G D H R L 37Rat 1 M S P E C A Q T TG P G P S R T P D Q V N R T H F P F F S D V K G D H R L 37Mouse 1 M S P E C A Q T TG P G P S H T LD Q V N R T H F P F F S D V K G D H R L 37Pig TM1 38 V L S T V E T V V L A L I F V V S LL G N V C A L V L V - A R R R R G G 72 IL1Pig-V2 38 V L S T V E T V V L A L I F V V S LL G N V C A L V L V - A R R R R G G 72Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

38 V L S T V E T V V L A L I F V V S LL G N V C A L V L V - A R R R R G G 72Human 38 V L A A V E T T V L V L I F A V S LL G N V C A L V L V - A R R R R R G 72Rat 38 V L S V L E T T V L G L I F V V S LL G N V C A L V L V - V R R R R R G 72Mouse 38 V L S V V E T T V L G L I F V V S LL G N V C A L V L V - A R R R R R G 72Pig TM2 73 T T A S L V LN L F C A D L L F T S A I P L V L A V R W T E AW L LG 107 EL1

73 T T A S L V LN L F C A D L L F T S A I P L V L A V R W T E AW L LG 10773 T T A S L V LN L F C A D L L F T S A I P L V L A V R W T E AW L LG 107

Human 73 A T A C L V LN L F C A D L L F I S A I P L V L A V R W T E AW L LG 107Rat 73 A T V S L V LN L F C A D L L F T S A I P L V L V V R W T E AW L LG 107Mouse 73 A T A S L V LN L F C A D L L F T S A I P L V L V V R W T E AW L LG 107Pig TM3 108 P V A C H L L F Y V M T L SG S V T I L T L A A V S L E RM V C IV R L Q R G V RG P G R 152 IL2

108 P V A C H L L F Y V M T L SG S V T I L T L A A V S L E RM - - -- - - - - - - -- - - - 152108 P V A C H L L F Y V M T L SG S V T I L T L A A V S L E RM V C IV R L Q R G V RG P G R 152

Human 108 P V A C H L L F Y V M T L SG S V T I L T L A A V S L E RM V C IV H L Q R G V RG P G R 152Rat 108 P V V C H L L F Y V M T M SG S V T I L T L A A V S L E RM V C IV R L R R G L SG P G R 152Mouse 108 P V V C H L L F Y V M T M SG S V T I L T L A A V S L E RM V C IV R L R R G L SG P G R 152Pig TM4 153 R A R A A L L A L I W G Y S A L A A L P LC I F F R V V PQ R F PG A D Q E I P IC T L IW P S V A G 203 EL2

- - - - - - - - -- - -- - - - - -- - - -- - - - - - - - - - -- - - - E I P IC T L IW P S V A G 203153 R A R A A L L A L I W G Y S A L A A L P LC I F F R V V PQ R F PG A D Q - - - -- - - -- - - - - - 203

Human 153 R A R A V L L A L I W G Y S A V A A L P LC V F F R V V PQ R L PG A D Q E I S IC T L IW P T I P G 203Rat 153 R TQ A A L L A F I W G Y S A L A A L P LC I L F R V V PQ R L PG G D Q E I P IC T L DW P N R I G 203Mouse 153 R TQ A A L L A F I W G Y S A L A A L P LC I L F R V V PQ R L PG G D Q E I P IC T L DW P N R I G 203Pig TM5 204 E I S WD V S F V T LN F L V PG LL I V IS Y S K 229

204 E I S WD V S F V T LN F L V PG LL I V IS Y S K 229204 - - - - - - - - - - - -- - - - - -- - - -- - - - 229

Human 204 E I S WD V S F V T LN F L V PG LV I V IS Y S K 229Rat 204 E I S WD V F F V T LN F L V PG LV I V IS Y S K 229Mouse 204 E I S WD V F F V T LN F L V PG LV I V IS Y S K 229Pig 230 I L Q I T K A S R R R L TM S L A Y S E S H Q I R V SQ Q 258 IL3

230 I L Q I T K A S R R R L TM S L A Y S E S H Q I R V SQ Q 258230 - - - I T K A S R R R L TM S L A Y S E S H Q I R V SQ Q 258

Human 230 I L Q I T K A S R K R L T V S L A Y S E S H Q I R V SQ Q 258Rat 230 I L Q I T K A S R K R L T L S L A Y S E S H Q I R V SQ Q 258Mouse 230 I L Q I T K A S R K R L T L S L A Y S E S H Q I R V SQ Q 258Pig TM6 259 D V R L F R T L F L LM I S F FI M W S P I I I T I L L IL I Q N FK Q N L V I W P 300 EL3

259 D V R L F R T L F L LM I S F FI M W S P I I I T I L L IL I Q N FK Q N L V I W P 300259 D V R L F R T L F L LM I S F FI M W S P I I I T I L L IL I Q N FK Q N L V I W P 300

Human 259 D F R L FR T L F L LM V S F FI M W S P I I I T I L L IL I Q N FK Q D L V I W P 300Rat 259 D Y R L FR T L F L LM V S F FI M W S P I I I T I L L IL I Q N FR Q D L V I W P 300Mouse 259 D Y R L FR T L F L LM V S F FI M W S P I I I T I L L IL I Q N FR Q D L V I W P 300Pig TM7 301 S L F FW VM A F T F A N S A V N P I L Y N M S L F R N EW R K F FH C 336

301 S L F FW VM A F T F A N S A V N P I L Y N M S L F R N EW R K F FH C 336301 S L F FW VM A F T F A N S A V N P I L Y N M S L F R N EW R K F FH C 336

Human 301 S L F FW V V A F T F A N S A LN P I L Y N M T L C R N EW K K I FC C 336Rat 301 S L F FW V V A F T F A N S A LN P I L Y N M S L F R S EW R K I FC C 336Mouse 301 S L F FW V V A F T F A N S A LN P I L Y N M S L F R N EW R K I FC C 336

H8Pig C-terminus 337 F F F P E K G AM F T D T S V R R N D L S VI S S 361337 F F F P E K G AM F T D T S V R R N D L S VI S S 361337 F F F P E K G AM F T D T S V R R N D L S VI S S 361

Human 337 FW F P E K G AI L T D T S V K R N D L S II S G 361Rat 337 F F F P E K G AI F T E T S I R R N D L S VI S T 361Mouse 337 F F F P E K G AI F T D T S V R R N D L S VI S S 361

(a)

Pig Human-S Rat Mouse

Pig 1000 894 873 889Human-S 1000 870 884Rat 1000 979Mouse 1000

(b)

RatMousePig

Human-S

105105

554

554

100

549

(c)

Figure 1 Comparison of GPR120 amino acid sequences among human mouse rat and pig (a) Alignment of the amino acid sequencesamong human mouse rat and pig The ELs and ILs are boxed The most conserved residues in each TM are shaded in gray (b) Homologyand (c) phylogenetic analysis of GPR120-S amino acid sequences from human mouse rat and pig NCBI reference numbers for the proteinsare human NP 0011826841 mouse NP 8614131 rat NP 0010405531 pig HQ6625641


Pig Human-S Rat MousePig 1000 880 840 830Human-S 1000 850 850Rat 1000 950Mouse 1000

(a)

Mouse

RatHuman-S

Pig

203

203617

617157

571

(b)

Figure 2Homology (a) andphylogenetic (b) analysis ofGPR120mRNAsequences fromhumanmouse rat andpigNCBI reference numbersfor the proteins are human NP 0011826841 mouse NP 8614131 rat NP 0010405531 pig HQ6625641

pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-407

Products

407bp

(a)

M 1 2 3 4 5 6 7 8 9 10

pGPR120

120573-actin

(b)

020406080

100

500

1000

1500

Rela

tive G

PR120

expr

essio

n

00

02

04

06

08

10

12

14

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d

GPR1

20120573-actin

Adip

ose

Skel

etal

mus

cle

Ileum

Jeju

num

Duo

denu

m

Kidn

ey

Lung

Sple

en

Live

r

Hea

rt

(c)

Figure 3 Tissue and cell expression of total pGPR120 Primer GPR120-407 was designed for the common sequence of three variants whichcould produce the 407 bp fragment (a) The blue domain represents the common sequence while the red domain represents the missedsequence of V2 and the green domain represents the missed sequence of V3 PCR products of total pGPR120 and 120573-actinwere detected by 2agarose gel electrophoresis (b) Lane 1 adipose Lane 2 skeletal muscle Lane 3 ileum Lane 4 jejunum Lane 5 duodenum Lane 6 kidneyLane 7 lung Lane 8 spleen Lane 9 liver Lane 10 heart M DL2000 DNA marker Samples were extracted from the cell lines 3D42 PK15and IPEC-J2 and the primary cells isolated from pigs ADSC and DFAT The expression of total pGPR120 was determined by q-PCR (c)

33 Transcriptional Expression of pGPR120 Spliced VariantsTo investigative the tissue expression of 3 spliced transcriptsin the highly expressed tissues we designed a primer calledGPR120-Variant which can detect all of the transcripts andproduce different length products for 3 spliced transcripts asFigure 4(a) shows In Figures 4(b) and 4(c) the transcriptcoding pGPR120-WTwas expressed predominantly in ileumjejunum duodenum spleen and adipose The pGPR120-V2was found to be coexpressed in 3D42 IPECJ2 and PK15and detected in ileum jejunum and duodenum especially in

spleen but not in adipose Another alternatively spliced tran-script pGPR120-V3 was only slightly expressed in spleen

34 Signaling Properties of Isoforms Encoded by pGPR120 WTand V2 To identify the molecular signaling of pGPR120spGPR120-WT and pGPR120-V2 expression plasmid weretransiently transfected into HEK293T cells with the Ca2+or ERK12 reporter system [26] As seen in Figure 5 underthe treatment of the synthetic human and mouse GPR120agonist TUG-891 signaling properties of pGPR120-WT and


pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-Variant

Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)

pGPR120-WTpGPR120-V2

100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)

Figure 4 Tissue and cell expression of pGPR120 spliced variants Primer GPR120-Variant was designed for the spliced sequence which couldproduce three different fragments from the variants (a) The blue domain represents the common sequence while the red domain representsthe missed sequence of V2 and the green domain represents the missed sequence of V3The pGPR120 spliced variants were separated by 10polyacrylamide gel electrophoresis in adipose ileum jejunum duodenum and spleen (b) Samples were extracted from the cell lines 3D42PK15 IPEC-J2 and the primary cells isolated from pigs ADSC andDFATThe expression of spliced variants was detected by semiquantitativeRT-PCR (c)

pGPR120-V2 encoded isoforms were detected as described inSection 2 The TUG-891 was used at the range of concentra-tion from 10minus8 to 10minus4M [27]The pGPR120-WT showed wellresponse to the TUG-891 (EC50 = 188 plusmn 052 120583M by NFATreporter system and EC50 = 125 plusmn 036 120583M by SRE reportersystem) Besides theWT receptor also could bewell activatedby endogenous free fatty acids including EPA DHA andALA but not LA or PLA However the pGPR120-V2 encodedisoform remained inactivated either in the NFAT or in theSRE reporter systems

35 Isoform Encoded by pGPR120-V2 Was Not a Dominant-Negative Isoform of pGPR120 The alternative splicing cangenerate truncated GPCRs which may play a dominant-negative role in retarding the wild type receptor in the endo-plasmic reticulum (ER) [28] To explore whether pGPR120-V2 can act as a dominant-negative isoform of pGPR120-WTwe coexpressed pGPR120-V2 and pGPR120-WT at variousratios The results showed that stimulation of TUG-891significantly increased NFAT-Luc activity not only in cellscotransfected with pGPR120-WT and pcDNA31 but also

in that cotransfected with pGPR120-WT and pGPR120-V2(Figure 6) The similar result was observed by using the SRE-driven reporter system

36 PUFAs Activates ERK12 Pathway via pGPR120-WT Tofurther evaluate the effects of pGPR120-WT on ERK12signaling by nutrients pGPR120-WT expression plasmid orpcDNA31 control was transiently transfected into HEK293Tcells As Figure 7 shows TUG-891 increased the phosphory-lation level of ERK12 compared with the basal level as wellas various PUFAs In Figure 7 the phosphorylation level ofERK12 activated by TUG-891 acted as a positive group andthe basal group showed no response for activation of ERK12Compared with TUG-891 n-3 PUFAs had the equivalentability to increase phosphorylation of ERK12 whereas n-6PUFAs (LA) had a lower ability to increase phosphorylationof ERK12 It was worth noting that though PLA increasedthe levels of ERK12 activity in cell transiently transfectedwith pGPR120-WT expression plasmid PLA also showed theability to induce the phosphorylation level of ERK12 in thecells transiently transfected with empty plasmid in which


minus10 minus9 minus8 minus7 minus6 minus5 minus4

1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)

Figure 5 Signaling properties of pGPR120 WT and V2 isoforms HEK293T cells were transfected with pGPR120-WT or pGPR120-V2expression plasmid together with the NFAT ((a) (c)) and SRE ((b) (d)) reporter systems plasmids for 24 h TUG-891 and endogenous fattyacids (EPADHAALA LA and PLA)were used at the range of concentration from 10minus9 to 10minus4Mto treat the transfected cells with serum-freestarvation for 6 h The cells were harvested to measure the luciferase activity normalized to the Renilla values

other PUFAs were unable to induce the phosphorylation ofERK12

4 Discussion

As a receptor of n-3 PUFA GPR120 is involved in a widerange of biological events including but not limited to anti-inflammation and antiapoptosis in human and mouse [5 8]In the current study we cloned the pGPR120 and showedthat pGPR120 had 3 transcripts We showed that pGPR120-WT was well matched with human receptor sequence In thepresent study compared to the mouse and rat GPR120 thepGPR120-WT mRNA encodes a 362-aa protein that sharedhigher homology to the hGPR120 In addition comparedwith human and mice the similar expression patterns ofpGPR120 in intestinal tissues maybe show the concordantfunctions such as inducing GLP-1 secretion [5 12 20] and

inhibiting NF-120581B activity in intestinal cells [10] The notableexpressions of pGPR120 in 3D42 cells maybe imply thesignificant role of pGPR120 in macrophages such as anti-inflammation in mouse [8] However pGPR120 showed thelow expression in lung and the abundant expression in spleenwhile the hGPR120 and mGPR120 were expressed highly inlung and showed low expression level in spleen [5]

Like the hGPR120 the pGPR120 also had other alterna-tively spliced transcripts which were called pGPR120-V2 andpGPR120-V3 in the current study These 3 spliced transcriptshave been published in database of mRNA sequence asNM 001204766 JQ437360 and JQ437361 However in thecurrent study we showed that transcript 2 which corre-sponds to JQ437360 only had a slight expression levelin spleen Compared with the pGPR120-V3 the notableexpression of pGPR120-V2 in the spleen as well as the porcinemacrophage cell 3D42 was observed naturally suggesting


0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

WT + pcDNA31 V2 + pcDNA31 WT + V2

lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)

Figure 6 Characterization of dominant-negative pGPR120-V2 HEK293T cells were transfected as the method described The dominant-negative pGPR120-V2 was measured by the NFAT (a) and SRE (b) reporter systems under the treatment of 10 120583MTUG-891 Results representmean plusmn SD and lowast119875 lt 005 lowastlowast119875 lt 001 compared with the control

Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or

Figure 7 Effect of fatty acids on phosphorylation level of ERK12in HEK293T cells transfected with pGPR120 expression plasmidor empty plasmid HEK293T cells were transfected with pGPR120-WT expression plasmid or empty plasmid for 24 h The cells werechanged to the medium without FBS and maintained for at least18 h After a 10min incubation of TUG-891 (10 120583M) ALA (10 120583M)EPA (10 120583M) DHA (10120583M) LA (10 120583M) or PLA (10120583M) cells wereharvested to measure the phosphorylation level of ERK12

that this alternative spliced isoform might act a role ofinflammation in that tissue Thus the role of pGPR120-V2 inimmunologic tissues and cells may be of great concern

The distinction between the same GPCRs from variousspecies might cause the pharmacological variation [28]Therefore we study the pharmacology of 2 main isoformsof pGPR120s by assaying both [Ca2+]i and ERK12 signalingAs expected the specific agonist for human and mouse

GPR120 TUG-891 can activate pGPR120-WT However thepGPR120-V2 almost showed negligible or no response tothe small molecular agonist It has been showed that uponthe stimulation of another human agonist GW9508 thehGPR120L also showed the poor response to the treatmentwhile the hGPR120S can be well activated by this agonist[21] As shown in the human GPR120 splice variants theGPR120L inserts a splice in the key juxtamembrane betweenthe TM domain 5 and IC3 regions which leads to the lesssensitivity of the longer receptors [21] Compared with thepGPR120-WT the 52 amino acids residues were deletedfrom the wild type amino acid sequence with the fourthtransmembrane domains truncated which may affect thenormal functionality Therefore the truncated fragmentsof pGPR120 thus suggest that the splice variant receptormay influence its receptor activity Compared with resultsobserved from other reports Oh 2010 12 the fold changesof the NFAT and SRE luciferase assay in the current studywere lower [25]This differencemight be caused by the subtlealterations in sequence of GPR120 within different speciesHowever the EC50 values of treatments in the current studyalso range between the reliable scopes (1ndash10120583M)

Alternative splicing acts as a main mechanism for reg-ulating the expression of GPCR genes and allowing for theprotein synthesis of truncated isoforms [28] The truncatedisoforms of some GPCRs play a pronounced role in associat-ing with the detaining of theWT isoform in the endoplasmicreticulum (ER) which makes the attenuated expression ofthe WT isoform on the cell surface [28 29] Thus anothergoal of this research was to explore whether the pGPR120-V2is dominant-negative isoform of pGPR120 However in ourresearch results showed that pGPR120-V2 had no inhibitoryeffects on pGPR120-WT signaling Hence we suggested thatpGPR120-V2 was not a dominant-negative isoform Basedon the tissues and cells distributions of pGPR120-V2 thetruncated isoforms showed remarkable expression in 3D42


and PK15 cell lines The high expression of pGPR120 maybeimplies that pGPR120 functions in these tissues and celllines therefore these cell lines could be models for furtherresearch which we did not study in depth in the presentstudiesMoreover whether alternative splicing of pGPR120 inspleen contributes to regulation of pGPR120-WT expressionand thereby affects the pGPR120 signalingmight be interestedto study

As previously mentioned in human and mouse acti-vation of GPR120 under the stimulus of the PUFAs couldstimulate the ERK12 phosphorylation signal transductioncascade [5 8 10 17] Thus GPR120 is named as the PUFAsensor and receptor [8] In our studies a series of n-3 (ALADHA and EPA) and the agonist TUG-891 had similar abilityin activation of ERK12 However n-6 PUFA (LA) improvedthe phosphorylation of ERK12 at a lower level comparedwith n-3 PUFA and TUG-891 suggesting that the pGPR120-WT could also be activated by PUFAs especially n-3 PUFAOther reports have showed that n-6 PUFAs increase ERK12phosphorylation level and even improve the mRNA expres-sion of GPR120 [10 30] It is worth noting that both the n-3and n-6 PUFAs increase ERK12 phosphorylation through aGPR120-dependent manner However PLA cannot activateeither NFAT or SRE reporter gene but can increase ERK12phosphorylation in cells transfected with GPR120 or controlplasmid therefore PLA might activate ERK12 signaling in aGPR120-independent manner These results match well withthe results reported by Oh et al [8] In the study of Oh et althey showed that the PLA increased phosphorylation level ofERK12 in GPR120-knockdown RAW2647 cells and the PLAalso showed no response to the SRE-luc activity in transientlytransfected HEK293 cells

Additionally pGPR120-WT was also highly expressedin the adipose tissue and mature adipocyte but not inthe preadipocyte This result matched well with the murinephenotype [12 13] Knockdown of GPR120 by siRNA couldreduce the expression of adipogenicmarker genes and impairthe triglyceride accumulation [12] Consequently it impliesthat GPR120 may be involved in porcine adipogenesis regu-lation

5 Conclusion

In the current study we cloned and characterized pGPR120for the first time We showed that pGPR120 was expressedpredominantly in ileum jejunum duodenum spleen andadipose in pigs Although pGPR120 has 3 spliced variantstranscript 1 which encoded pGPR120-WT was the mainlyexpressed transcript of pGPR120 In addition pGPR120 wasalso the only functional isoform which was activated by syn-thetic agonist (TUG-891) and PUFAs especially n-3 PUFAOur study contributed to understanding the molecular regu-lation mechanisms of PUFA in pigs

Abbreviations

ALA a-Linolenic acid (C183n-3)BSA Bovine serum albuminDHA Docosahexaenoic acid (C226n-3)

EPA Eicosapentaenoic acid (C205n-3)ERK12 Extracellular signal-regulated kinase 12GPCR G protein coupled receptorLA Linoleic acid (C182n-6)NFAT-RE The nuclear factor of activated T-cell

response elementPLA Palmitic acid (C160)PUFA Polyunsaturated fatty acidSRE The serum response elementTM domain Transmembrane domain

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jian Peng and Hong-kui Wei conceived and designed theexperiments Tongxing Song Jie Peng and Jiao Ren carriedout the experiments Tongxing Song and Hong-kui Weianalyzed the data and wrote the paper All authors read andapproved the final paper

Acknowledgments

This work was supported by the Fundamental ResearchFunds for the Central Universities (2013QC004 and2013PY047) Hubei Provincial Creative Team Project ofAgricultural Science and Technology (no 2007-620)and Hubei Provincial Natural Science Foundation (no2013CFA010)

References

[1] E P Haber HM A Ximenes J Procopio C R O Carvalho RCuri and A R Carpinelli ldquoPleiotropic effects of fatty acids onpancreatic 120573-cellsrdquo Journal of Cellular Physiology vol 194 no 1pp 1ndash12 2003

[2] Y Itoh Y Kawamata M Harada et al ldquoFree fatty acids regulateinsulin secretion from pancreatic beta cells through GPR40rdquoNature vol 422 no 6928 pp 173ndash176 2003

[3] A Ichimura A Hirasawa T Hara and G Tsujimoto ldquoFreefatty acid receptors act as nutrient sensors to regulate energyhomeostasisrdquo Prostaglandins and Other LipidMediators vol 89no 3-4 pp 82ndash88 2009

[4] C P Briscoe M Tadayyon J L Andrews et al ldquoThe orphanG protein-coupled receptor GPR40 is activated by medium andlong chain fatty acidsrdquoThe Journal of Biological Chemistry vol278 no 13 pp 11303ndash11311 2003

[5] A Hirasawa K Tsumaya T Awaji et al ldquoFree fatty acidsregulate gut incretin glucagon-like peptide-1 secretion throughGPR120rdquo Nature Medicine vol 11 no 1 pp 90ndash94 2005

[6] J Wang X Wu N Simonavicius H Tian and L LingldquoMedium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84rdquo Journal of Biological Chemistry vol281 no 45 pp 34457ndash34464 2006

[7] K M Maslowski A T Vieira A Ng et al ldquoRegulation ofinflammatory responses by gutmicrobiota and chemoattractantreceptorGPR43rdquoNature vol 461 no 7268 pp 1282ndash1286 2009


[8] D Y Oh 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

[9] I Kimura D Inoue T Maeda et al ldquoShort-chain fatty acidsand ketones directly regulate sympathetic nervous system viaG protein-coupled receptor 41 (GPR41)rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 19 pp 8030ndash8035 2011

[10] KMobraten TMHaug C R Kleiveland and T Lea ldquoOmega-3 and omega-6 PUFAs induce the same GPR120-mediatedsignalling events but with different kinetics and intensity inCaco-2 cellsrdquo Lipids in Health and Disease vol 12 article 1012013

[11] T Tanaka S Katsuma TAdachi T-A Koshimizu AHirasawaand G Tsujimoto ldquoFree fatty acids induce cholecystokininsecretion through GPR120rdquo Naunyn-Schmiedebergrsquos Archives ofPharmacology vol 377 no 4ndash6 pp 523ndash527 2008

[12] C Gotoh Y H Hong T Iga et al ldquoThe regulation of adipo-genesis through GPR120rdquo Biochemical and Biophysical ResearchCommunications vol 354 no 2 pp 591ndash597 2007

[13] S Miyauchi A Hirasawa T Iga et al ldquoDistribution andregulation of protein expression of the free fatty acid receptorGPR120rdquoNaunyn-Schmiedebergrsquos Archives of Pharmacology vol379 no 4 pp 427ndash434 2009

[14] A Ichimura A Hirasawa O Poulain-Godefroy et al ldquoDys-function of lipid sensor GPR120 leads to obesity in both mouseand humanrdquo Nature vol 483 no 7389 pp 350ndash354 2012

[15] C Cartoni K Yasumatsu T Ohkuri et al ldquoTaste preference forfatty acids is mediated by GPR40 and GPR120rdquo The Journal ofNeuroscience vol 30 no 25 pp 8376ndash8382 2010

[16] M M Galindo N Voigt J Stein et al ldquoG protein-coupledreceptors in human fat taste perceptionrdquo Chemical Senses vol37 no 2 pp 123ndash139 2012

[17] S Katsuma N Hatae T Yano et al ldquoFree fatty acids inhibitserum deprivation-induced apoptosis through GPR120 in amurine enteroendocrine cell line STC-1rdquoThe Journal of Biolog-ical Chemistry vol 280 no 20 pp 19507ndash19515 2005

[18] X-L Mo H-K Wei J Peng and Y-X Tao ldquoFree fatty acidreceptor GPR120 and pathogenesis of obesity and type 2 dia-betes mellitusrdquo Progress in Molecular Biology and TranslationalScience vol 114 pp 251ndash276 2013

[19] K Moore Q Zhang N Murgolo T Hosted and R DuffyldquoCloning expression and pharmacological characterization ofthe GPR120 free fatty acid receptor from cynomolgus monkeycomparison with human GPR120 splice variantsrdquo ComparativeBiochemistry and Physiology Part B Biochemistry andMolecularBiology vol 154 no 4 pp 419ndash426 2009

[20] T Tanaka T Yano T Adachi T-A Koshimizu A Hirasawaand G Tsujimoto ldquoCloning and characterization of the rat freefatty acid receptor GPR120 in vivo effect of the natural ligandon GLP-1 secretion and proliferation of pancreatic beta cellsrdquoNaunyn-Schmiedebergrsquos Archives of Pharmacology vol 377 no4ndash6 pp 515ndash522 2008

[21] S J Watson A J H Brown and N D Holliday ldquoDifferentialsignaling by splice variants of the human free fatty acid receptorGPR120rdquo Molecular Pharmacology vol 81 no 5 pp 631ndash6422012

[22] H-F Luo H-K Wei F-R Huang Z Zhou S-W Jiang andJ Peng ldquoThe effect of linseed on intramuscular fat content andadipogenesis related genes in skeletalmuscle of pigsrdquoLipids vol44 no 11 pp 999ndash1010 2009

[23] H-K Wei Y Zhou S Jiang Y-X Tao H Sun and J PengldquoFeeding aDHA-enriched diet increases skeletal muscle proteinsynthesis in growing pigs association with increased skeletalmuscle insulin action and local mRNA expression of insulin-like growth factor 1rdquo British Journal of Nutrition vol 110 no 4pp 671ndash680 2013

[24] H Ono Y Oki H Bono and K Kano ldquoGene expression profil-ing inmultipotent DFAT cells derived frommature adipocytesrdquoBiochemical and Biophysical Research Communications vol 407no 3 pp 562ndash567 2011

[25] D Y Oh J A Song J S Moon et al ldquoMembrane-proximalregion of the carboxyl terminus of the gonadotropin-releasinghormone receptor (GnRHR) confers differential signal trans-duction between mammalian and nonmammalian GnRHRsrdquoMolecular Endocrinology vol 19 no 3 pp 722ndash731 2005

[26] Z Cheng D Garvin A Paguio P Stecha K Wood and FFan ldquoLuciferase reporter assay system for deciphering GPCRpathwaysrdquo Current Chemical Genomics vol 4 pp 84ndash91 2010

[27] B D Hudson B Shimpukade A E Mackenzie et al ldquoThepharmacology of TUG-891 a potent and selective agonist of thefree fatty acid receptor 4 (FFA4GPR120) demonstrates bothpotential opportunity and possible challenges to therapeuticagonismrdquo Molecular Pharmacology vol 84 no 5 pp 710ndash7252013

[28] H Wise ldquoThe roles played by highly truncated splice variantsof G protein-coupled receptorsrdquo Journal of Molecular Signalingvol 7 article 13 2012

[29] A CHanyaloglu andM vonZastrow ldquoRegulation ofGPCRs byendocytic membrane trafficking and its potential implicationsrdquoAnnual Review of Pharmacology andToxicology vol 48 pp 537ndash568 2008

[30] F Rodriguez-Pacheco S Garcia-Serrano E Garcia-Escobar etal ldquoEffects of obesityfatty acids on the expression of GPR120rdquoMolecular Nutrition amp Food Research vol 58 no 9 pp 1852ndash1860 2014


Table 1 Primers used for polymerase chain reaction

Primeridentification Primer sequences (51015840-31015840)

Productsize forWT (bp)



Annealingtemperature

(∘C)

GPR120 full F GAATTCGCCACCATGGGAATGTCCCTTGAGTGCR TCTAGACTAGCTGGAAATAACAGACAGATC 1086 930 960 62

GPR120-407 F AAGGAGGAGGCTCACGATGR TGACAAATAGATGCCGATAGAC 407 407 407 59

GPR120-Variant F GCTCTTCTACGTGATGACCCTAR CGTGAGCCTCCTCCTTGAT 388 232 262 60

120573-actin F CCAGGTCATCACCATCGGR CCGTGTTGGCGTAGAGGT 158 mdash mdash 60

one isoform of GPR120 is identified [11 20] Most recentlines of evidence show that the hGPR120-L and hGPR120-S have different tissue distributions and pharmacologicalcharacteristics [16 21] Compared with human and rodentsmuch less is known about the porcine GPR120 (pGPR120)In database of mRNA sequence pigs have 3 splice variants(NM 001204766 JQ437360 and JQ437361) However thereare no relative reports to characterize pGPR120 Recentlyit has been reported that feeding n-3 PUFA-enriched dietscould improve the intramuscular fat content and inhibit theexpression of inflammation related genes leading to increas-ing the mass of skeletal muscle of pigs [22 23] Howeverthe detailed mechanism is largely unknown In the currentstudy we presented the first evidence for tissue expression ofsplice variants of the pGPR120 and pharmacology of differentpGPR120 isoforms These results may provide a way to helpunderstand the nutrichemical role of n-3 PUFA in pigs

2 Materials and Methods

21 Cloning of pGPR120s into pcDNA31 The coding seq-uence (CDS) of pGPR120swas obtained by PCR amplificationusing mixed porcine complimentary DNA (cDNA) extractedfrom the spleen tissue of 3 Landrace barrows as templatesThe CDS of pGPR120 were inserted between restriction sitesEcoRI (51015840) and XbaI (31015840) in pcDNA31 (+) (Clontech PatoAlto CA USA) [21] The primer sequences and optimal PCRannealing temperatures are listed in Table 1 The identities ofall constructs were confirmed by sequencing

22 Phylogenetic and Comparative Analysis The amino acidssequences of GPR120s from various species were retrievedfrom GenBank Then the retrieved amino acids sequencesand the deduced amino acid sequences from depositedporcine CDS sequences of pGPR120 were subjected to mul-tiple sequence alignment using ClustalW program (httpwwwebiacukToolsclustalw2) using default settings TheMEGA 50 programwas used to reconstruct the phylogenetictrees by the Neighbor-Joining (NJ) method (httpwwwmegasoftwarenet)

23 Cell Culture and DNA Transfection Isolation mainte-nance and differentiation of porcine adipose derived stem

cells (ADSC) and dedifferentiated fat cells (DFAT) wereperformed as described [8 12 24] The 3D42 cells (ATCC)were grown in Roswell Park Memorial Institute (RPIM) 1640(Gibco San Diego CA USA) supplemented with 2mM L-glutamine and adjusted to contain 15 gL sodium bicarbon-ate 45 gL glucose 10mMHEPES 10mM sodium pyruvatesupplemented with 01mM nonessential amino acids (GibcoSan Diego CA USA) 90 fetal bovine serum (FBS) (GibcoSanDiego CAUSA) 10 PK15 IPECJ2 andHEK293T cellswere grown in Dulbeccorsquos Modified Eaglersquos Medium (45 gLglucose) (Gibco) supplemented with 2mM L-glutamine10 FBS (Gibco) penicillin (100 IUmL) and streptomycin(100 pgmL) in a humidified atmosphere 5 CO

295 air at

37∘C Cells were grown in 75-cm2 flasksThe cells were transfected at 60ndash70 confluence in

culture plates using Lipofectamine 2000 (Invitrogen Carls-bad CA USA) following Invitrogen protocols Briefly bothLipofectamine 2000 (1120583L) and plasmid (05 120583g) were dilutedusing Opti-MEM (Invitrogen) to a final volume of 50120583Lper well and then mixed together for 15 minutes at roomtemperature Then the lipo-DNA complex was added intowells Cells were assayed 48 h after transfection

24 RNA Isolation Semiquantitative RT-PCR and Quantita-tive PCR (qPCR) Total RNA was extracted from adiposeskeletal muscle ileum jejunum duodenum kidney lungspleen liver and heart of 6 Landrace barrows with Trizol(Invitrogen) following the manufacturerrsquos instructions TotalRNA from 3D42 PK15 IPECJ2 ADSC and DFAT as wellas differentiated ADSC and DFAT cells was also isolatedand extracted using Trizol reagent The 25 120583g of RNA wasreverse transcribed using First-Strand cDNA Synthesis Kit(TOYOBO Japan) and cDNAs were stored at minus20∘C

Total GPR120 expression in various tissues was deter-mined by semiquantitative RT-PCR The cross intron-exonboundaries primers (GPR120-407) were designed accordingto the common sequence of the pGPR120 transcripts as shownin Figure 3(a) The standard PCR program for amplificationconsisted of an initial denaturation step at 94∘C for 4minfollowed by 35 cycles of 30 sec at 94∘C 30 sec at the indicatedannealing temperature 119899min at 72∘C (119899 = size in kilobases ofamplified product) and a final elongation at 72∘C for 5min








3 Results





Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3


















(a)



(b)

RatMousePig

Human-S

105105

554

554

100

549

(c)




(a)

Mouse

RatHuman-S

Pig

203

203617

617157

571

(b)


pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-407

Products

407bp

(a)

M 1 2 3 4 5 6 7 8 9 10

pGPR120

120573-actin

(b)

020406080

100

500

1000

1500

Rela

tive G

PR120

expr

essio

n

00

02

04

06

08

10

12

14

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d

GPR1

20120573-actin

Adip

ose

Skel

etal

mus

cle

Ileum

Jeju

num

Duo

denu

m

Kidn

ey

Lung

Sple

en

Live

r

Hea

rt

(c)






pGPR120-V2

pGPR120-V3

pGPR120-WT


Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)


100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)








1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References







































3 Results





Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3


















(a)



(b)

RatMousePig

Human-S

105105

554

554

100

549

(c)




(a)

Mouse

RatHuman-S

Pig

203

203617

617157

571

(b)


pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-407

Products

407bp

(a)

M 1 2 3 4 5 6 7 8 9 10

pGPR120

120573-actin

(b)

020406080

100

500

1000

1500

Rela

tive G

PR120

expr

essio

n

00

02

04

06

08

10

12

14

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d

GPR1

20120573-actin

Adip

ose

Skel

etal

mus

cle

Ileum

Jeju

num

Duo

denu

m

Kidn

ey

Lung

Sple

en

Live

r

Hea

rt

(c)






pGPR120-V2

pGPR120-V3

pGPR120-WT


Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)


100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)








1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References


































Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3

Pig-V2Pig-V3


















(a)



(b)

RatMousePig

Human-S

105105

554

554

100

549

(c)




(a)

Mouse

RatHuman-S

Pig

203

203617

617157

571

(b)


pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-407

Products

407bp

(a)

M 1 2 3 4 5 6 7 8 9 10

pGPR120

120573-actin

(b)

020406080

100

500

1000

1500

Rela

tive G

PR120

expr

essio

n

00

02

04

06

08

10

12

14

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d

GPR1

20120573-actin

Adip

ose

Skel

etal

mus

cle

Ileum

Jeju

num

Duo

denu

m

Kidn

ey

Lung

Sple

en

Live

r

Hea

rt

(c)






pGPR120-V2

pGPR120-V3

pGPR120-WT


Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)


100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)








1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References


































(a)

Mouse

RatHuman-S

Pig

203

203617

617157

571

(b)


pGPR120-V2

pGPR120-V3

pGPR120-WT

Primer GPR120-407

Products

407bp

(a)

M 1 2 3 4 5 6 7 8 9 10

pGPR120

120573-actin

(b)

020406080

100

500

1000

1500

Rela

tive G

PR120

expr

essio

n

00

02

04

06

08

10

12

14

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d

GPR1

20120573-actin

Adip

ose

Skel

etal

mus

cle

Ileum

Jeju

num

Duo

denu

m

Kidn

ey

Lung

Sple

en

Live

r

Hea

rt

(c)






pGPR120-V2

pGPR120-V3

pGPR120-WT


Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)


100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)








1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References

































pGPR120-V2

pGPR120-V3

pGPR120-WT


Products

388bp

232bp

262bp

(a)

pGPR120-WT

pGPR120-V2

pGPR120-V3

100 bp

250bp

400bp

350bp

200 bp

300bp

Adip

ose

Ileum

Jeju

num

Duo

denu

m

MSple

en

(b)


100 bp

250bp

250bp

500bp

3D4

2

IPEC

-J2

PK15

AD

SC0

d

AD

SC7

d

DFA

T0

d

DFA

T7

d120573-actin

(c)








1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References


































1

2

3

4 pGPR20-WTN

FAT-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(a)


10

15

20

25

30 pGPR20-WT

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

Log[agonist] (M)

(b)


TUG-891EPADHA

ALALAPLA

05

10

15

20 pGPR120-V2

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)

Log[agonist] (M)

(c)


TUG-891EPADHA

ALALAPLA

Log[agonist] (M)

05

10

15

20 pGPR120-V2

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)

(d)



4 Discussion





0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References

































0

1

2

3

4

Basal

NFA

T-lu

c act

ivity

(fol

d in

duct

ion)


lowastlowast

lowastlowast

10120583M TUG-891

(a)

00

05

10

15

20

25

Basal

SRE-

luc a

ctiv

ity (f

old

indu

ctio

n)


lowast

lowastlowast

10120583M TUG-891

(b)


Tubulin

pERK12

Tubulin

pERK12

LAPLATUG-891 Basal

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

DHAEPATUG-891 ALA

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

GPR1

20

Vect

or

Basal

GPR1

20

Vect

or










5 Conclusion


Abbreviations








Acknowledgments


References




































5 Conclusion


Abbreviations








Acknowledgments


References
































cloning and characterization of spliced variants of the porcine g … · 2018. 10. 25. ·...

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