cloning of canine gpr120 : total rna sample , which was extracted from 
 canine jejunum tissue for other study and had been stored at 80c , was used for cloning of 
 gpr120 . the yield and quality of the rna sample the rna was reverse - transcribed using quantiscript reverse transcriptase 
 ( qiagen , hilden , germany ) to produce cdna templates . polymerase chain reaction ( pcr ) was 
 performed with cdna templates and the primers designed for 5- and 3- untranslated regions 
 of canine gpr120 ( orf - fwd and orf - rev , table 
 1table 1.primers for the canine gpr120 sequencesprimersequence ( 53)product size ( bp)orf - fwdggcatgtcccctgagtgcg1,193orf - revggctacattgatgtcatgcacctggexon 1-fwdaggtgtcgcaaccgcctc810exon 1-revacagcgacacagcgggcaexon 2-fwdagccagagccaaacaagca223exon 2-revgctgattccaagcctaagtggexon 3-fwdtagctgctttgctcctccaa502exon 3-revatcatggctttccagagggtbeta - actingccaaccgtgagaagatgact90cccagagtccatgacaataccag ) based on canine genomic database ( nc_006610.3 ) . pcr was conducted for 30 
 cycles at 98c for 10 sec and 70c for 90 sec with primestar hs dna polymerase ( takara , 
 otsu , japan ) . portion of the pcr products was electrophoresed , and the product size was 
 confirmed to be theoretical . the pcr products were purified using purification kit ( qiagen ) 
 and sequenced ( eurofins genomics , tokyo , japan ) . tissue distribution of canine gpr120 : commercially obtained rna samples 
 extracted from canine tissues ( ams biotechnology , abingdon , u.k . ) were used for the tissue 
 distribution analysis of gpr120 in dogs . the samples included 16 kinds of organs , such as 
 lung , stomach , duodenum , jejunum , ileum , colon , liver , kidney , cerebrum , hypothalamus , 
 hippocampus , spinal cord , bone marrow , skeletal muscle , dermis and adipose tissue . reverse 
 transcription , cdna purification , pcr and electrophoresis were carried out in the same 
 manner as cloning of gpr120 . pcr for beta - actin gene was demonstrated as an internal 
 control , too . exploration of genetic variants of gpr120 in dogs : genetic variations of 
 gpr120 in client - owned dogs visiting the veterinary medical teaching hospital in nippon 
 veterinary and life science university and aikouishida animal hospital since 2012 through 
 2013 were investigated . the patient dogs were selected with no regard to diseases , and their 
 body condition scores were evaluated in a 5-point scale . ultimately , blood samples were 
 collected from 141 dogs ( 6 months14 years old , 69 males and 72 females , consisting of 21 
 breeds ) . whole blood was collected into an edta tube , and genome dna was extracted using 
 flexigene dna kit ( qiagen ) . pcr was performed with genome templates and the primers designed 
 for intron sequences around three gpr120 exons ( 3 pairs of exon - fwd and rev , table 1 ) based on canine genome database 
 ( nc_006610.3 ) . pcr was conducted for 30 cycles at 98c for 10 sec and 72c for 90 sec ( exon 
 1 ) , or 98c for 10 sec , 66c for 15 sec and 72c for 90 sec ( exons 2 and 3 , respectively ) 
 with mightyamp dna polymerase ( takara ) . electrophoresis and sequencing were carried out in 
 the same manner as cloning of gpr120 . the determined gpr120 exon sequences were compared to 
 the genomic data , and single nucleotide polymorphisms ( snps ) were explored . the study was 
 carried out according to the regulation of nippon veterinary and life science university . molecular structure of canine gpr120 : the canine gpr120 cdna included an 
 open reading frame ( orf ) consisting of 1,086 nucleotides ( registered in dna data bank of 
 japan , lc019015 ) . the nucleotide sequence was 8495% identical to those of other species 
 including humans , mice , rats , cats , horses , pigs and white bears ( table 2table 2.molecular identities of canine gpr120 with other speciesnucleotidesamino acidsaccessioncat95%96%xm_003994220.2white bear93%94%xm_008696357.1pig90%89%nm_001204766.1horse89%86%xm_001500867.2human89%84%bc_101175.2mouse84%85%nm_181748.2rat84%83%nm_001047088.1 ) . 1.primary structure of canine gpr120 and the sequence alignment with feline , swine , 
 human and murine gpr120 . the sequence is presented in single letter code , and 
 identical amino acid residues are expressed in dots . ) , which was 7896% identical to those of other species we compared ( table 2 ) . primary structure of canine gpr120 and the sequence alignment with feline , swine , 
 human and murine gpr120 . the sequence is presented in single letter code , and 
 identical amino acid residues are expressed in dots . tissue distributions of canine gpr120 : tissue distributions of canine 
 gpr120 were examined by rt - pcr . gpr120 mrna was detected in the lung , jejunum , ileum , colon , 
 hypothalamus , hippocampus , spinal cord , bone marrow , dermis and white adipose tissue of the 
 samples tested ( fig . total rna extracted from various organs of 
 dogs was reverse - transcribed and detected by pcr . beta - actin gene was amplified using 
 the same samples as an internal control . ) . total rna extracted from various organs of 
 dogs was reverse - transcribed and detected by pcr . beta - actin gene was amplified using 
 the same samples as an internal control . genetic variants of gpr120 in dogs : nine types of mutations were found in 
 gpr120 genes from the 141 patient dogs , in which 5 mutations were synonymous substitutions 
 and 4 mutations were non - synonymous substitutions . the synonymous variants were c.252c > g 
 ( p.ala84ala ) , c.282c > g ( p.asp94asp ) , c.702a > g ( p.thr234thr ) , c.726g > a ( p.thr242thr ) 
 and c.984t > c ( p.asn328asn ) . the non - synonymous variants were c.287t > g ( p.leu96arg ) , 
 c.307g > a ( p.ala103thr ) , c.446g > c ( p.gly149ala ) and c.595c > a ( p.pro199thr ) . counts 
 and frequencies of the variants are summarized in table 
 3table 3.identified variants in canine gpr120 ( n=141)variantsmutationn ( hetero)n ( homo)frequencysynonymousp.ala84alac.252c > g1090.099p.asp94aspc.282c > g940.060p.thr234thrc.702a > g010.007p.thr242thrc.726g > a630.043p.asn328asnc.984t > c40190.277non - synonymousp.leu96argc.287t > g1190.103p.ala103thrc.307g > a100.004p.gly149alac.446g > c500.018p.pro199thrc.595c > a28120.184 , and the detail of the variants in different breeds is summarized in table 4table 4.the detail of the gpr120 variants in different breeds ( n=141 , hetero variants 
 were calculated as 0.5)breedshead - countsc.287t > gc.307g > ac.446g > cc.595c > aminiature dachshund36000.54.5beagle3614.5004.5welsh corgi170004yorkshire terrier120001miniature schnauzer60003.5toy poodle40000.5chihuahua30000shetland sheepdog30010borzoi30012border collie30001rough collie300.501.5shih tzu30000mix20000.5labrador retriever20000.5golden retriever20000.5flat - coated retriever10000.5ckcs10000.5pomeranian10000siberian husky10000irish setter10001belgian shepherd10000total14114.50.52.526a ) ckcs , cavalier king charles spaniel .. frequency of the c.287t > g ( p.leu96arg ) variant in all dogs ( n=141 ) was 
 0.125 and 0.500 in the beagle population ( n=36 ) . the c.307g > a ( p.ala103thr ) variant was 
 found only in one dog ( rough collie ) . the c.446g > c ( p.gly149ala ) variant was found in 5 
 dogs ( miniature dachshund , shetland sheepdog and borzoi ) . gene frequencies of c.595c > a in dogs with different bcss 
 were separately calculated , resulting in 0.312 in obese ( bcs=5/5 ) , 0.324 in overweight 
 ( 4/5 ) , 0.175 in optimum ( 3/5 ) and 0.000 in underweight ( 2/5 ) dogs ( table 5table 5.gene frequency of the c.595c > a ( p.pro199thr ) mutation in dogs with different 
 body condition scores ( n=141)bcsnc / cc / aa / afrequency4 and 5 ( obese)2513840.3203 ( optimum)103752080.1752 ( underweight)1313000.000a ) p=0.022 vs bcs=3 . ) . the gene frequency of c.595c > a mutation in the summation of bcs4 and bcs5 
 dogs ( 0.320 ) was significantly higher than that in the bcs3 dogs 
 ( p=0.022 ) . the cloned open reading frame of canine gpr120 was included in the canine chromosome 28 
 ( nc_006610.3 ) and consisted of 3 exons , such as exon 1 ( genome78017137802280 , 568 bp ) , exon 
 2 ( genome78127527812876 , 125 bp ) and exon 3 ( 78196297819989 , 420 bp ) . primary structure of 
 canine gpr120 was highly homologous to those of other species , especially cats and white 
 bears which are carnivores . seven transmembrane domains were conserved in canine gpr120 
 ( tm1 , amino acid residue ( aa ) 4565 ; tm2 , aa7398 ; tm3 , aa113137 ; tm4 , aa156177 ; tm5 , 
 aa209225 ; tm6 , aa266289 ; and tm7 , aa296316 ) like other species , so it was extrapolated 
 that canine gpr120 is a seven - transmembrane receptor like human and murine ones . two 
 n - glycosylation sites , the asparagine residues in the n- ( aa21 ) and c- ( aa322 ) terminals 
 were also conserved [ 3 , 13 ] . gpr120 was expressed in various organs also in dogs , which was basically similar to those 
 in humans and mice [ 5 , 6 ] . in gastrointestinal system , gpr120 mrna was expressed in jejunum , ileum and 
 colon , suggesting that canine gpr120 might be expressed in lower intestines intensively . gpr120 is associated with glp-1 secretion , and glp-1 is secreted from ileum , so the current 
 results harmonize the physiology of glp-1 . oh et al . have reported that gpr120 is expressed in monocytes and 
 macrophages , and ligand fatty acids cause anti - inflammatory effects via beta - arrestin 2 
 pathway in mice . it evokes the possibility that 
 anti - inflammatory effects of unsaturated fatty acids , which are medicated also in dogs 
 , depend on gpr120 . in a human study , the p.arg270his variant is found more in obese people , and this mutation 
 is considered obese - related variation . in the current study , the 270th amino acid was 
 arginine in all dogs tested , and the same mutation was not found . instead , we have found 9 
 genetic variants consisting of 5 synonymous mutations and 4 non - synonymous mutations in 141 
 dogs . since synonymous mutations do not affect the phenotype , we focused on the 
 non - synonymous variants . the variant c.287t > g ( p.leu96arg ) was detected only in beagles 
 ( 18 hetero and 9 homo mutations in 36 beagles ) . the gene frequency was 0.500 in beagles and 
 0.000 in other breeds ( 0.125 in all dogs ) . the 
 variant c.307g > a ( p.ala103thr ) was found only in one dog which was rough collie ( hetero ) . the 
 variant c.446g > c ( p.gly149ala ) was found in 5 dogs , in which 2 dogs were borzoi , 2 dogs 
 were shetland sheepdog , and the other one was miniature dachshund . although only 3 borzoi 
 and 4 shetland sheepdog were included in the study population , this variant was detected in 
 these breeds preferentially . it is necessary to add more numbers of cases to determine 
 clinical significance of this variant . the variant c.595c > a ( p.pro199thr ) was found in 40 dogs , so we analyzed the gene 
 frequency of this variant in different bcs groups . the gene frequency was significantly 
 higher in the groups with higher bcss ( bcs=4/5 and 5/5 ) , and it was suggested that this 
 variant would be associated with decreased metabolic rate . to examine the possibility that 
 some specific breeds have this mutation regardless of their bcss , we investigated the 
 relationship between the breed and variant frequencies ( table 4 ) . in the result , most breeds consisted of 13 dogs , and only 4 breeds 
 ( miniature dachshund , beagle , welsh corgi and yorkshire terrier ) consisted of more than 10 
 dogs . when we compared the gene frequencies of the c.595c > a variant among these 4 breeds , 
 the comparison showed no significant differences . on the other hand , the bcs4 - 5 group 
 included 8 breeds , i.e. the obese dogs were not concentrated in a few breeds . collectively , 
 the c.595c > a variant seemed to be linked to the bcss , rather than the breeds . limitation 
 of this study is that epidemiological data do not directly indicate the phenotypic 
 significance of the variants . it is difficult to prove the functional significance of the 
 gene variants by epidemiological research using clinical cases , because patient dogs have 
 widely different backgrounds including dietary habit , activity and other genetic factors , so 
 the focus of the current study was on finding candidate variants for further functional 
 analyses . if the gene variant is proved to associate with obesity , gene tests to determine 
 the genetic variance will be helpful for nutritional management of dogs . food dosage for 
 dogs will be justified depending on their genetic variations , as that in human medicine , in 
 which this technique has been already established . the purpose of this study was to investigate which types of genetic variants exist in 
 canine gpr120 and find candidate ones which relate to obesity in dogs . we conclude that 
 there are 4 non - synonymous variations in canine gpr120 , and the c.595c > a ( p.pro199thr ) 
 variant is a candidate one which relates to obesity . the next step is to confirm the 
 functional divergence of this mutant using the cell expression system . the ultimate purpose 
 is to establish the genetic tests to reveal metabolic characteristics of individual dogs and 
 apply the results to their nutritional management .
g protein - coupled receptor ( gpr ) 120 is an unsaturated fatty acid receptor , which is 
 associated with various physiological functions . 
 it is reported that the genetic variant 
 of gpr120 , p.arg270his , is detected more in obese people , and this genetic variation 
 functionally relates to obesity in humans . 
 obesity is a common nutritional disorder also 
 in dogs , but the genetic factors have not ever been identified in dogs . in this study , we 
 investigated the molecular structure of canine gpr120 and searched for candidate genetic 
 variants which may relate to obesity in dogs . 
 canine gpr120 was highly homologous to those 
 of other species , and seven transmembrane domains and two n - glycosylation sites were 
 conserved . 
 gpr120 mrna was expressed in lung , jejunum , ileum , colon , hypothalamus , 
 hippocampus , spinal cord , bone marrow , dermis and white adipose tissues in dogs , as those 
 in mice and humans . 
 genetic variants of gpr120 were explored in client - owned 141 dogs , 
 resulting in that 5 synonymous and 4 non - synonymous variants were found . 
 the variant 
 c.595c > a ( p.pro199thr ) was found in 40 dogs , and the gene frequency was significantly 
 higher in dogs with higher body condition scores , i.e. 0.320 in bcs45 dogs , 0.175 in bcs3 
 dogs and 0.000 in bcs2 dogs . 
 we conclude that c.595c > a ( p.pro199thr ) is a candidate 
 variant relating to obesity , which may be helpful for nutritional management of dogs .