Association of PPAR Alpha Intron 7 G/C, PPAR Gamma 2 …downloads.hindawi.com/journals/ppar/2017/7636019.pdf · 2019-07-30 · Cohort1 (𝑛=244) Cohort2 (𝑛=327) Referenceindividuals

Research ArticleAssociation of PPAR Alpha Intron 7 GCPPAR Gamma 2 Pro12Ala and C161T Polymorphisms withSerum Fetuin-A Concentrations

Bernadett Maacuterkus1 Krisztiaacuten Voumlroumls1 Dorina Supaacutek2 Zsolt Melczer2

Kaacuteroly Cseh3 and Laacuteszloacute Kalabay1

1Department of Family Medicine Semmelweis University Budapest Hungary22nd Department of Obstetrics and Gynecology Semmelweis University Budapest Hungary3Institute of Public Health Semmelweis University Budapest Hungary

Correspondence should be addressed to Laszlo Kalabay kalabaylaszlomedsemmelweis-univhu

Received 3 March 2017 Revised 24 May 2017 Accepted 6 June 2017 Published 11 July 2017

Academic Editor Marcin Baranowski

Copyright copy 2017 Bernadett Markus et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Both peroxisome activator proteins (PPARs) and fetuin-A play a role in lipid and glucose metabolism Aims Weinvestigated whether PPAR120572 intron 7 G2468C and PPAR1205742 Pro12Ala and PPAR120574 exon 6 C161T polymorphisms are associatedwith serum fetuin-A concentrations Patients and Methods The PPAR120572 intron 7 GC polymorphism was studied in cohort 1 (79reference individuals 165 postinfarction patients) The two PPAR120574 polymorphisms were investigated in cohort 2 (162 referenceindividuals 165 postinfarction patients) Fetuin-A levels and PPAR polymorphisms were determined by radial immunodiffusionand polymerase chain reaction-restriction fragment length polymorphism techniquesResultsTheC allele variant of PPAR120572 intron7 G2467C was associated with higher fetuin-A levels (119901 = 0018) Postinfarction status (119901 = 0001) PPAR120572 intron 7 GGGCCCgenotypes (119901 = 0032) and the C allele (119901 = 0021) were the strongest determinants of fetuin-A concentration in a multipleregression model Higher fetuin-A levels were associated with the Pro variant of PPAR1205742 (119901 = 0047) Postinfarction status (119901 =0041) and BMI (119901 lt 0001) but not PPAR1205742 Pro were the strongest determinants of fetuin-A concentrations PPAR120574 exon 6 C161Tgenotypes were not associated with fetuin-A levels Conclusions Fetuin-A was determined mainly by the PPAR120572 intron 7C alleleand postinfarction status in cohort 1 and the BMI and postinfarction in cohort 2 The PPAR120572 intron 7C and PPAR1205742 Pro variantsare associated with fetuin-A levels

1 Introduction

Human fetuin-A is a multifunctional hepatic glycoproteinthat has been involved in the development of obesity [1ndash3] insulin resistance [4] metabolic syndrome [1 5] type 2diabetes [6ndash8] adipocyte dysfunction [9] and fatty liver [4]

Peroxisome proliferator-activated receptors (PPARs) aremembers of the nuclear hormone receptor superfamily ofligand-activated transcription factors The PPAR subgroupsPPAR120572 PPAR120573120575 and PPAR120574 (1205741 and 1205742) play an importantrole in the pathogenesis of these processes which has beenextensively reviewed [10ndash12]

There are several observations suggesting a relationshipbetween serum fetuin-A levels and activities of different

PPARs For example the direct inhibitory effect of pioglita-zone on hepatic fetuin-A expression has been observed in rats[13] and in humans [14] the formerwas reversed byGW9602direct PPAR120574 inhibitor

Polymorphisms of PPAR120574 and PPAR120572 have also beendescribed and found to be associated with disorders ofhyperlipidemia glucose homeostasis and diabetes Thus theC allele of the PPAR120572 intron 7 polymorphism was found tobe more frequent in patients with myocardial infarction anddyslipidemia [15] The T allele of the PPAR120574 exon 6 C161Tpolymorphism was supposed to have protective role againstcoronary artery disease in Chinese population [16] whereasothers found that this allele was associated with an increased

HindawiPPAR ResearchVolume 2017 Article ID 7636019 8 pageshttpsdoiorg10115520177636019

2 PPAR Research

risk for coronary heart disease [12] The association betweenpolymorphic variants of PPAR and serum fetuin-A levelshowever has not been investigated yet

In our study we aimed to investigate whether polymor-phisms of PPAR120574 (PPAR1205742 Pro12Ala and exon 6 C161T) andPPAR120572 (intron 7 G2467C) are associated with or may affectserum fetuin-A levels in two cohorts

2 Patients and Methods

Three-hundred and forty-two patients were originallyinvolved in this study Exclusion criteria were as followsclinical or laboratory signs of acute vascular disease(myocardial infarction stroke) acute infection malignanttumor hepatic disease renal failure immune suppressionsevere medical or surgical conditions and trauma Finallywe had 327 patients (cohort 2) who had all comparabledata (including successful genotyping for both PPAR120574polymorphisms) We were able to perform successful PPAR120572genotyping in a smaller number of patients (cohort 1119899 = 244) The genotyping success rate was greater than 99in both cohorts The genotypes were in the Hardy-Weinbergequilibrium

PPAR120572 polymorphism was studied in cohort 1 Thiscohort comprised 244 individuals (120 men and 124 womenage 601 plusmn 112 years mean plusmn SD) Cohort 1 consisted of79 reference individuals (15 men 64 women age 610 plusmn 94years) and 165 patients surviving myocardial infarction (105men 60 women age 596 plusmn 122 years)

PPAR120574 polymorphisms were studied in cohort 2 Thiscohort consisted of 327 individuals (161 men 166 womenage 579 plusmn 130 years) This cohort consisted of 162 referencesubjects (61 men 101 women age 561 plusmn 138 years) and thesame 165 postinfarction patients as in cohort 1 (105 men 60women age 596 plusmn 122 years)

Postinfarction patients had a history of STEMI myocar-dial infarction (6ndash24 months prior to the start of the study)Diabetes was diagnosed based on fasting plasma glucose gt70mmoll or the 2-hr OGTT gt 111mmoll Patients withdiabetes were treated with diet metformin and bedtimeinsulin

All persons gave their informed consent prior to theirinclusion in the study The study was approved by thelocal Ethics Committee of the Karolyi Sandor MunicipalityHospital

21 Determination of PPAR Polymorphic Variants The deter-mination of the PPAR120574 and PPAR120572 variants was performedby PCR-RFLP technique

The PPAR120572G2467C intron 7 polymorphism (rs 4253778)was studied by PCR-RFLP technique using a 51015840 forwardprimer of ACA ATC ACT CCT TAA ATA TGG TGGand a 31015840 reverse primer of AAG TAG GGA CAG ACAGGA CCA GTA The PCR product was digested with Taq1(New England Biolabs Boston MA USA) resulting in onefragment of 266 bp of the carriers of the wild-type allele andtwo fragments of 216 and 50 bp in the carriers of the mutantallele (thermocycles 94∘C 15min 30 times 94∘C 30 sec 50∘C20 sec and 72∘C 30 sec) [17]

For PPAR120574Pro12Ala (rs 1801282) polymorphism we useda 51015840 forward primer of GCC AAT TCA AGC CCA GTC anda mutagenic 31015840 reverse primer of GAT ATG TTT GCA GACAGT GTA TCA GTG AAG GAA TCG CTT TCC G ThePCR product was digested with Bst U1 enzyme (NewEnglandBiolabs Boston MA USA) resulting in one fragment of270 bp in the carriers of wild-type and two fragments of 227and 43 bp in carriers of mutant allele (thermocycles 95∘C15min 35 times 94∘C 30 sec 65∘C 45 sec and 72∘C 1min) [18]

The exon 6 polymorphism C161T of PPAR120574 (rs 3856806)was investigated by PCR-RFLP technique using a 51015840 forwardprimer of CAA GAC AAC CTG CTA CAA GC and a 31015840reverse primer of TCC TTG TAG ATC TCC TGC AG ThePCR product was digested with Pml1 enzyme (New EnglandBiolabs Boston MA USA) resulting in two fragments of120 and 80 bp in carriers of the wild-type allele and onlyone fragment of 200 bp in the carriers of the mutant allele(thermocycles 94∘C 15min 30times 94∘C 30 sec 56∘C 30 sec and72∘C 30 sec) [19]

22 Determination of Serum Fetuin-A Concentration Serumfetuin-A concentrations were determined by radial immun-odiffusion using the commercially available product (anti-fetuin-A IgG fraction Incstar cat number 81931 137mgmlin a final concentration of 84 120583l115ml gel) as previouslydescribed [20]

23 Determination of Insulin Resistance Parameters Plasmaglucose and insulin were determined by the routine HK-G6P-DH and ELCIAmethods respectivelyTheHomeostasisModel Assessment-Insulin Resistance (HOMA-IR) modelwas calculated according to Matthews et al [21]

24 Statistical Analysis Statistical analysis was carried outusing the SPSS v21 statistical software (SPSS Inc ChicagoIL USA) Nonparametric methods including the Bonferroni(Dunn) post hoc test were used 119901 values lt 005 wereconsidered as significant

3 Results

31 Subject Characteristics The characteristics of the studyparticipants are shown in Table 1

Sixty-five per cent of the postinfarction patients receivedstatins and 70 of them aspirin Serum fetuin-A concentra-tions did not differ statistically between patients treated andnot treated with these two medications (687 plusmn 122 versus 636plusmn 81mgl 119901 = 0204 for statins and 665 plusmn 0120 versus 672 plusmn124mgl 119901 = 0795 for aspirin resp)

32 PPAR120572 Intron 7 GC PPAR1205742 Pro12Ala and PPAR120574Exon 6 C161TAllele Distribution in Postinfarction Patients andReference Individuals Thedistribution of PPAR120574 andPPAR120572alleles is shown in Table 2 PPAR120574 Pro12Ala and PPAR120572intron 7 GC alleles did not differ significantly betweenpostinfarction patients and reference subjects Postinfarctionpatients however had a significantly higher T allele fre-quency of the PPAR120574 C161T compared to reference subjects

PPAR Research 3

Table 1 Subject characteristics

Cohort 1(119899 = 244)

Cohort 2(119899 = 327)

Reference individuals(119899 = 79)

Postinfarction patients(119899 = 165)



Gender (malefemale) 1564 10560 61101 10560Age (years mean plusmn SD) 610 plusmn 94 596 plusmn 122 561 plusmn 138 596 plusmn 122BMI (kgm2) 241 plusmn 16 281 plusmn 42lowastlowast 274 plusmn 04 281 plusmn 42lowast

Obesity (noyes) 6811 46119lowastlowast 7884 46119lowastlowast

Diabetes status (noyes) 790 11253lowastlowast 13923 11253lowastlowast

HOMA-IR 10 plusmn 02 62 plusmn 46lowastlowast 15 plusmn 15 61 plusmn 46lowastlowast

lowast119901 lt 001 and lowastlowast119901 lt 0001 compared to reference individuals BMI body mass index HOMA-IR Homeostasis Model Assessment-Insulin ResistanceMannndashWhitney test

Table 2ThePPAR120572 intron 7GC PPAR1205742 Pro12Ala andPPAR120574 exon 6C161T allele distribution amongpostinfarction patients and referenceindividuals

Allelic frequency 1205942RR

(95 CI)OR

(95 CI) 119901

PPAR120572 intron 7 GC (rs4253778)

G P 08273R 08165 00861 1013

(09271ndash1107)1077

(06571ndash1764) 0769

C P 01727R 01835

PPAR1205742 Pro12Ala (rs1801282)

Pro12 P 08618R 08789 04279 09804

(09241ndash1040)08583

(05428ndash13570) 0513

Ala12 P 01382R 01211

PPAR120574 exon 6 C161T (rs3856806)

C P 08735R 09459 1025 09235

(08799ndash09693)03953

(02204ndash07091) 0001

T P 01265R 00541

P postinfarction patients R reference subjects 119901 1205942 test

33 Analysis of Association between PPAR120572 Intron 7 G2467CVariants and Serum Fetuin-A Concentrations Serum fetuin-A levels of individuals with the CC genotype were higherthan those of GG genotype (Table 3) In the dominantmodel (C versus non-C nucleotide) individuals with theminor variant C allele had significantly higher serum fetuin-A concentrations than those with the non-C In a recessivemodel (G versus non-G nucleotide) there was no differencebetween the two variants (651 plusmn 107mgl 119899 = 238 versus 662plusmn 171mgl 119899 = 6 119901 = 0702)

Except for age serum fetuin-A concentrations showedsignificant correlations with parameters listed in Table 4Serum fetuin-A levels associatedweakly but significantly withPPAR120572 intron 7 GGGCCC genotypes and the C allele butnot with the G allele During partial correlation analysishowever the correlation between fetuin-A concentrationsand PPAR120572 intron 7 GGGCCC genotype lost significancewhen corrected for BMI (119903 = 0100 119901 = 0125) diabetesstatus (119903 = 0108 119901 = 0092) HOMA-IR (119903 = 0116

119901 = 0072) and postinfarction status (119903 = 0122 119901 =0058) Correlation between fetuin-A levels and the PPAR120572intron 7C allele also became insignificant when corrected forBMI (119903 = 0107 119901 = 0095) diabetes status (119903 = 0115119901 = 0072) and HOMA-IR (119903 = 0123 119901 = 0056) butremained significant after correction for postinfarction status(119903 = 0131 119901 = 0041)

The results of the univariate linear regression analy-sis between independent variables (predictors) and serumfetuin-A concentration (dependent variable) are shown inTable 5 Serum fetuin-A levels showed weak but statisticallysignificant data with all investigated potential predictorsincluding PPAR120572 intron 7 GC genotypes and the C allele butnot with age Thus age was excluded from further analysis

Next we investigated whether PPAR120572 intron 7 GCgenotype and the C allele may determine serum fetuin-A concentration in a multiple regression model (Table 6)In the model containing all independent parameters weinvestigated the PPAR120572 intron 7 GGGCCC genotype and

4 PPAR Research

Table 3 Serum fetuin-A concentrations in individuals with different PPAR120572 intron 7 G2467C SNP polymorphisms and alleles

(a) PPAR120572 intron 7 G2467C polymorphisms

GG GC CC119901

Fetuin-A mgl 119899 Fetuin-A mgl 119899 Fetuin-A mgl 119899

641 plusmn 150 164 671 plusmn 110 74 662 plusmn 170 6 0040

(b) PPAR120572 intron 7 G2467C alleles

C allele non-C119901

Fetuin-A mgl 119899 Fetuin-A mgl 119899

670 plusmn 114 80 641 plusmn 105 164 0018sectKruskal-Wallis test sectMannndashWhitney test

Table 4 Correlation between serum fetuin-A and investigatedparameters (119899 = 244)

Parameter Correlation coefficient 119901

BMI 0167 0009Diabetes status (noyes) 0133 0038HOMA-IR 0205 0001Age minus0109 0188Gender minus0168 0017Postinfarction status (noyes) 0277 lt0001PPAR120572 GGGCCC 0144 0025PPAR120572 C allele 0151 0018PPAR120572 G allele 0025 0700BMI body mass index HOMA-IR Homeostasis Model Assessment-InsulinResistance Spearman correlation

Table 5 Univariate regression analysis between serum fetuin-Aconcentrations and metabolic parameters (119899 = 244)

Predictor Standardized 120573 119901

BMI 0146 0023Diabetes status (noyes) 0136 0034HOMA-IR 0163 0011Postinfarction status (noyes) 0212 0001Age minus0137 0098Gender minus0149 0027PPAR120572 GGGCCC 0131 0042PPAR120572 C allele 0140 0029BMI body mass index HOMA-IR Homeostasis Model Assessment-InsulinResistance

the C allele were the only statistically significant predictorsof serum fetuin-A levels In a backward stepwise regres-sion model the postinfarction status the PPAR120572 intron 7GGGCCC genotypes and the C allele proved to be thestrongest determinants of fetuin-A concentration

34 Analysis of Association between PPAR1205742 Pro12Ala Vari-ants and Serum Fetuin-A Concentrations Patients withProPro and ProAla genotype had significantly higher serumfetuin-A concentrations than those with the AlaAla geno-type (ProPro 681 plusmn 131mgl 119899 = 247 ProAla 706 plusmn

131mgl 119899 = 75 and AlaAla 565 plusmn 116mgl 119899 = 5119901 = 0043 Kruskal-Wallis test) In the recessive model (Proversus non-Pro) the fetuin-A level associated with the alleledetermining Pro exceeded that of the non-Pro variant (687 plusmn131mgl 119899 = 322 versus 565 plusmn 116mgl 119899 = 5 119901 = 0047MannndashWhitney test) Fetuin-A concentrations did not differin the recessive model (Ala versus non-Ala) (698 plusmn 143mgl119899 = 80 versus 681 plusmn 131mgl 119899 = 247 119901 = 0287)

Serum fetuin-A concentration was significantly associ-ated with BMI HOMA-IR gender and the Pro allele but notwith the diabetes and postinfarction status PPAR120574 ProProProAla and AlaAla genotypes or the Ala allele (Table 7)Thus these two latter parameters were left out from furtheranalysisThe correlation between fetuin-A concentration andthe Pro allele was lost following correction for BMI andgender but not with HOMA-IR (Table 8)

Univariate regression analysis showed that serum fetuin-A (dependent variable) weakly but significantly correlatedwith BMI and the PPAR120574 Pro allele (independent variableTable 9) This latter independent variable lost its predictorrole when BMI was included in the regression model

In the multiple backward stepwise regression modelpostinfarction status (120573 = 0111 119901 = 0041) and BMI (120573 =0426 119901 lt 0001) proved to be the strongest determinants offetuin-A concentrations

35 Analysis of Association between PPAR120574 Exon 6 C161TVariants and Serum Fetuin-A Concentrations We found nosignificant differences among serum fetuin-A concentrationsof individuals with different PPAR120574 exon 6 C161T genotypesnor between the C and non-C or T and non-T groups(Table 10) Fetuin-A levels did not correlate with the PPAR120574C161T genotypes C and T alleles either (data not shown)

There were 3 minor variant homozygotes (TT) in thepostinfarction but none in the reference group Thus thegenotype distribution of postinfarction patients markedlydiffered from that of reference individuals (CCCTTT130373 versus 140170 119901 = 0006) The T allele wassignificantly more frequent among postinfarction patientscompared to reference group (40170 = 235 versus 17157 =108 119901 = 0002) Accordingly postinfarction patients withthe CC genotype had lower fetuin-A levels than referencesubjects (668 plusmn 113mgl 119899 = 130 versus 710 plusmn 146mgl119899 = 140 119901 = 0037)

PPAR Research 5

Table 6 Multiple regression analysis of PPAR120572 intron 7 GC genotypes and C allele and serum fetuin-A concentration (119899 = 244)

Predictor PPAR120572 intron 7 GGGCGG PPAR120572 intron 7 C alleleStandardized 120573 119901 Standardized 120573 119901

All predictors includedBMI 0027 0708 minus0024 0743Diabetes status (noyes) 0054 0474 0045 0551HOMA-IR 0037 0651 0035 0666Postinfarction status (noyes) 0130 0127 0132 0121Gender minus0079 0260 minus0083 0242PPAR120572 intron 7 genetics 0134 0036 0144 0024

Model fit 119901 = 0006 Model fit 119901 = 0005Stepwise backward regression

Postinfarction status (noyes) 0214 0001 0215 0001PPAR120572 intron 7 genetics 0135 0032 0144 0021

Model fit 119901 lt 0001 Model fit 119901 lt 0001Regression parameters of the PPAR120572 intron 7 GGGCGG genotypes are indicated in the second and the third and those of the PPAR120572 intron 7 C allele inthe fourth and fifth columns of the table respectively BMI body mass index HOMA-IR Homeostasis Model Assessment-Insulin Resistance



BMI 0426 lt0001Diabetes status (noyes) 0102 0064HOMA-IR 0129 0027Gender minus0178 0008Postinfarction status(noyes) 0098 0078

PPAR120574 ProPro ProAlaand AlaAla 0050 0365

PPAR120574 Pro allele 0130 0018PPAR120574 Ala allele 0052 0349BMI body mass index HOMA-IR Homeostasis Model Assessment-InsulinResistance Spearman correlation

Table 8 Partial correlation between serum fetuin-A concentrationsand PPAR120574 Pro allele (119899 = 327)

Corrected for Correlation coefficient 119901

Uncorrected 0130 0018BMI 0069 0215HOMA-IR 0129 0027Gender 0129 0055

4 Discussion

In our study we investigated whether PPAR120572 intron 7 GCPPAR1205742 Pro12Ala and PPAR120574 C161T variants are associatedwith serum fetuin-A concentration Since subjects in ourgroups had several parameters that are known to affect fetuin-A levels such as age gender BMI parameters of insulinresistance (diabetes status HOMA-IR) and postinfarctionstatus [22] we chose regression model to estimate the impactof these variables

Table 9 Univariate regression analysis between serum fetuin-Aconcentrations and predictor parameters (119899 = 327)


BMI 0520 lt0001Diabetes status (noyes) 0065 0239HOMA-IR 0059 0291Postinfarction status (noyes) 0137 0013PPAR120574 Pro allele 0127 0022PPAR120574 Pro allele+ BMI

00590512

0215lt0001

BMI body mass index HOMA-IR Homeostasis Model Assessment-InsulinResistance

PPAR120572 has been termed as a lipid sensor and is involvedin microsomal 120596-oxidation and mitochondrial and peroxi-somal 120573-oxidation resulting in energy burning and reducedfat storage [11] The minor variant of the PPAR120572 intron 7Chas been considered to have a decreased activity comparedto G the major variant The C haplotype promotes the earlydevelopment of type 2 diabetes [17] and is more frequentamong postinfarction patients [12 15] Doney et al havefound that the risk of myocardial infarction is higher in thepresence of the C allele [23] We also have found that theC allele is associated with higher fetuin-A levels and themultiple regression revealed that fetuin-A levels are stronglydetermined by the postinfarction status and remarkably bythe PPAR120572 intron 7 GC polymorphism as well Althoughthe C allele was not more frequent among our postinfarctionpatients the higher fetuin-A concentration may also havedeleterious effects in them Elevated fetuin-A concentration isamarker of fatty liver characterized by decreased120573-oxidationof fatty acids [4]

Fetuin-A is synthesized almost exclusively by the hepa-tocytes in adults [24] and the PPAR120572 is mainly expressed inthe liver as well Fetuin-A is known as an endogenous ligand

6 PPAR Research

Table 10 Serum fetuin-A concentrations in individuals with different PPAR120574 exon 6 C161T polymorphisms and alleles (119899 = 327)

(a) PPAR exon 6 C161T polymorphisms

CC CT TT119901



(b) PPAR120574 exon 6 C161T Ala Allele

Fetuin-A mgl 119899 Fetuin-A mgl 119899 119901

C non-C685 plusmn 132 324 641 plusmn 104 3 0674sect

T non-T659 plusmn 123 57 690 plusmn 133 270 0138sectKruskal-Wallis test sectMannndashWhitney test

that binds to free fatty acids and functions as an endoge-nous ligand for the Toll-like receptor TLR-4 thereby linkingmetabolic diseases (hyperlipidemia insulin resistance) andsubclinical inflammation [25] This ldquomissing linkrdquo characterof fetuin-A has been further supported by the clinical studiesof Stefan and Haring [26] These findings are in line withour observation that postinfarction status an endpoint ofsubclinical inflammation and PPAR120572 intron 7C allele werethe strongest determinants of fetuin-A in our subjects

Compared to Ala metabolically disadvantageous char-acteristics are attributed to the Pro variant of PPAR1205742Pro12Ala [27] Indeed we found only the Pro allele amongindividuals with BMI over 25 kgm2 and only lean (BMIle 25 kgm2) subjects had the AlaAla homozygous variantNevertheless even among individuals with the Ala variantobesity was associated with higher fetuin-A levels comparedto lean ones Patients with diabetes had higher fetuin-Alevels the difference being significant in ProPro major allelehomozygotes (704 plusmn 124mgl 119899 = 64 versus 673 plusmn 132mgl119899 = 183 119901 = 0020) Since fetuin-A is known to be thenatural inhibitor of the insulin receptor tyrosine kinase thePro allele may convey increased insulin resistance Indeedwe found minor variant Ala to be more frequent amongnondiabetics (68251 = 271) compared to diabetics (1276= 158 119901 = 0044)This finding is in accordance with that ofVergotine who observed that the Pro allele increases insulinresistance alongwith IRS1Gly972 [28] Conversely theminorallele Ala seemed to be protective in Iranian and Chinesepopulations [29 30] In our model however the relationshipof fetuin-A levels with BMI and postinfarction status wasmuch stronger than the one with insulin resistance (diabetesstatus or HOMA) The Pro allele was associated with higherfetuin-A in the nondiabetic group as well which is reflectedby the weak correlation with diabetes status and HOMA-IRGiven the property of fetuin-A to increase insulin resistanceits weak association with the Pro allele can contribute to thedeleterious effects of this allele

Analysis of the effect of the PPAR120574 C161T variants onserum fetuin-A concentrations yielded the least consistentresults We had 3 minor homozygotes (TT) among thepostinfarction patients but not among the reference subjects

This finding is in accord with the observation of Qian et alwho found that coronary heart disease was not associatedwith the T-carrier state but these individuals had higher riskfor acute coronary heart syndrome [12] Wu et al found mildprotective effect of the T allele only in the Chinese but not inother populations [16] We found no marked associations ofC161T genotypes and alleles neither in postinfarction patientsnor in the reference group and not during the analysis ofnonobese nondiabetic individuals This suggests that theC161T has the weakest association with fetuin-A levels outof the three PPAR polymorphisms we studied Since PPAR120574is expressed mainly in the fat tissue its association with thelevels of the liver secretory protein fetuin-A cannot be as closeas that of PPAR120572

Although not yet entirely clarified several observationssuggest the molecular basis of the association betweenPPAR variants and fetuin-A synthesis The PPAR120572 agonistfibrates decrease fetuin-A expression in obese patients withor without type 2 diabetes mellitus [31] The PPAR120574 agonistpioglitazone strongly inhibits fetuin-A expression [14] Theupregulation of both PPAR120572 and PPAR120574 results in thedownregulation of fetuin-A and NF120581B and upregulation ofthe AMPK kinase activities Palmitate the oxidation of whichis highly induced by PPAR120572 has been shown to stimulateNF120581B binding to the fetuin-A promoter [9] Thus a lessfunctional variant of PPAR120572 could finally result in enhancedfetuin-A expression

Our study has its limitations First the sample size is notbig enough to allow for analysis of a comparable number ofminor variants Second our cohorts were not controlled forenvironmental factors and prescribedmedication and dietarysaturated and polyunsaturated fat as it has been suggested[32]

5 Conclusion

In summary our results indicate a relatively close relationshipbetween PPAR120572 intron 7 GC and PPAR1205742 Pro12Ala variantsand serum fetuin-A concentrations reflecting higher levelsin the presence of the C allele of the former and the Proallele of the latter one It is very likely that these associations

PPAR Research 7

are obscured by obesity andor diabetes Larger scale studiesare needed to further determine the biological and clinicalsignificance of the PPAR polymorphisms on fetuin-A levels

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Bernadett Markus was responsible for the preparation ofthe manuscript Krisztian Voros was responsible for thestatistical analysis Dorina Supak and Zsolt Melczer wereresponsible for patient and data management Karoly Csehwas responsible for the determination of PPAR polymor-phisms and critical review of the manuscript Laszlo Kalabaywas responsible for conceiving the idea of the study andcritical review of the manuscript

Acknowledgments

The authors thank V M Nagyne for the determination ofserum fetuin-A concentration This work was supported bygrant of the Hungarian Ministry of Health ETT 3682009

References

[1] T Reinehr and C L Roth ldquoFetuin-A and its relation tometabolic syndrome and fatty liver disease in obese childrenbefore and after weight lossrdquo Journal of Clinical Endocrinologyand Metabolism vol 93 no 11 pp 4479ndash4485 2008

[2] C Lavebratt S Wahlqvist L Nordfors J Hoffstedt and PArner ldquoAHSG gene variant is associated with leanness amongSwedish menrdquo Human Genetics vol 117 no 1 pp 54ndash60 2005

[3] P Suchanek I Kralova-Lesna R Poledne V Lanska and J AHubacek ldquoAn AHSG gene variant modulates basal metabolicrate and body composition development after a short-timelifestyle interventionrdquo Neuroendocrinology Letters vol 32 no2 pp 32ndash36 2011

[4] N Stefan A M Hennige H Staiger et al ldquo1205722-Heremans-Schmid glycoproteinfetuin-A is associated with insulin resis-tance and fat accumulation in the liver in humansrdquo DiabetesCare vol 29 no 4 pp 853ndash857 2006

[5] J H Ix M G Shlipak V M Brandenburg S Ali M Kettelerand M A Whooley ldquoAssociation between human fetuin-A andthe metabolic syndrome Data from the heart and soul studyrdquoCirculation vol 113 no 14 pp 1760ndash1767 2006

[6] N Stefan A Fritsche C Weikert et al ldquoPlasma fetuin-A levelsand the risk of type 2 diabetesrdquoDiabetes vol 57 no 10 pp 2762ndash2767 2008

[7] N Stefan H-U Haring and M B Schulze ldquoAssociation offetuin-A level and diabetes riskrdquo JAMA - Journal of theAmericanMedical Association vol 300 no 19 p 2247 2008

[8] J H Ix C L Wassel A M Kanaya et al ldquoFetuin-A andincident diabetes mellitus in older personsrdquo JAMA - Journal ofthe American Medical Association vol 300 no 2 pp 182ndash1882008

[9] S Dasgupta S Bhattacharya A Biswas et al ldquoNF-120581B mediateslipid-induced fetuin-A expression in hepatocytes that impairs

adipocyte function effecting insulin resistancerdquo BiochemicalJournal vol 429 no 3 pp 451ndash462 2010

[10] C P Dong L He J N Li F Ye M He and Y WangldquoAssociation of the Pro12Ala and C1431T polymorphism of thePPAR gamma2 gene and their haplotypes with obesity and type2 diabetesrdquo Chinese Journal of Medical Genetics vol 25 no 4pp 447ndash451 2008

[11] B Grygiel-Gorniak ldquoPeroxisome proliferator-activated recep-tors and their ligands nutritional and clinical implicationsmdashareviewrdquo Nutrition Journal vol 13 article 17 2014

[12] Y Qian P Li J Zhang et al ldquoAssociation between peroxisomeproliferator-activated receptor-alpha delta and gamma poly-morphisms and risk of coronary heart disease A case-controlstudy and meta-analysisrdquo Medicine (United States) vol 95 no32 Article ID e4299 2016

[13] A Ochi K Mori M Emoto et al ldquoDirect inhibitory effects ofpioglitazone on hepatic fetuin-A expressionrdquo PLoS ONE vol 9no 2 Article ID e88704 2014

[14] K Mori M Emoto T Araki et al ldquoEffects of pioglitazone onserum fetuin-A levels in patients with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 57 no 9 pp 1248ndash1252 2008

[15] S Purushothaman V K Ajitkumar and R Renuka Nair ldquoAsso-ciation of PPARalpha Intron 7 Polymorphism with CoronaryArtery Disease A Cross-Sectional Studyrdquo ISRN Cardiology vol2011 Article ID 816025 2011

[16] Z Wu Y Lou W Jin Y Liu L Lu and G Lu ldquoThe C161Tpolymorphism in the peroxisome proliferator-activated recep-tor gamma gene (PPAR120574) is associated with risk of coronaryartery disease a meta-analysisrdquoMolecular Biology Reports vol40 no 4 pp 3101ndash3112 2013

[17] D M Flavell H Ireland J W Stephens et al ldquoPeroxisomeproliferator-activated receptor 120572 gene variation influences ageof onset and progression of type 2 diabetesrdquo Diabetes vol 54no 2 pp 582ndash586 2005

[18] L-M Chuang C Hsiung Y-D Chen et al ldquoSibling-basedassociation study of the PPAR1205742 Pro12Ala polymorphism andmetabolic variables in Chinese and Japanese hypertensionfamilies A SAPPHIRe studyrdquo Journal of Molecular Medicinevol 79 no 11 pp 656ndash664 2001

[19] X L Wang J Oosterhof and N Duarte ldquoPeroxisomeproliferator-activated receptor 120574 C161rarrT polymorphism andcoronary artery diseaserdquo Cardiovascular Research vol 44 no3 pp 588ndash594 1999

[20] L Kalabay L Jakab Z Prohaszka et al ldquoHuman fetuin1205722HS-glycoprotein level as a novel indicator of liver cell function andshort-term mortality in patients with liver cirrhosis and livercancerrdquo European Journal of Gastroenterology and Hepatologyvol 14 no 4 pp 389ndash394 2002

[21] D R Matthews J P Hosker A S Rudenski B A Naylor DF Treacher and R C Turner ldquoHomeostasis model assessmentinsulin resistance and 120573-cell function from fasting plasmaglucose and insulin concentrations in manrdquo Diabetologia vol28 no 7 pp 412ndash419 1985

[22] K Voros L Graf Jr Z Prohaszka et al ldquoSerum fetuin-A inmetabolic and inflammatory pathways in patients withmyocar-dial infarctionrdquo European Journal of Clinical Investigation vol41 no 7 pp 703ndash709 2011

[23] A S F Doney B Fischer S Lee ADMorris G P Leese andCN A Palmer ldquoAssociation of common variation in the PPARAgene with incident myocardial infarction in individuals with

8 PPAR Research

type 2 diabetes A Go-DARTS studyrdquo Nuclear Receptor vol 3article no 4 2005

[24] J T Triffitt U Gebauer B A Ashton M E Owen andJ J Reynolds ldquoOrigin of plasma 1205722HS-glycoprotein and itsaccumulation in bonerdquo Nature vol 262 no 5565 pp 226-2271976

[25] D Pal S Dasgupta R Kundu et al ldquoFetuin-A acts as anendogenous ligand of TLR4 to promote lipid-induced insulinresistancerdquo Nature Medicine vol 18 no 8 pp 1279ndash1285 2012

[26] N Stefan andH-U Haring ldquoCirculating fetuin-A and free fattyacids interact to predict insulin resistance in humansrdquo NatureMedicine vol 19 no 4 pp 394-395 2013

[27] A S F Doney B Fischer J E Cecil et al ldquoAssociation of thePro12Ala and C1431T variants of PPARG and their haplotypeswith susceptibility to Type 2 diabetesrdquo Diabetologia vol 47 no3 pp 555ndash558 2004

[28] Z Vergotine Y Y Yako A P Kengne R T Erasmus andT E Matsha ldquoProliferator-activated receptor gamma Pro12Alainteracts with the insulin receptor substrate 1 Gly972Arg andincrease the risk of insulin resistance and diabetes in the mixedancestry population from South Africardquo BMC Genetics vol 15article no 10 2014

[29] A Motavallian S Andalib G Vaseghi H Mirmohammad-Sadeghi and M Amini ldquoAssociation between PRO12ALApolymorphismof the PPAR-1205742 gene and type 2 diabetesmellitusin Iranian patientsrdquo Indian Journal of Human Genetics vol 19no 2 pp 239ndash244 2013

[30] X Wang J Liu Y Ouyang M Fang H Gao and L LiuldquoThe association between the Pro12Ala variant in the PPAR1205742gene and type 2 diabetes mellitus and obesity in a Chinesepopulationrdquo PLoS ONE vol 8 no 8 Article ID e71985 2013

[31] M H Noureldein R S Abd El-Razek M H El-Hefnawyand H O El-Mesallamy ldquoFenofibrate reduces inflammationin obese patients with or without type 2 diabetes mellitus viasirtuin 1fetuin A axisrdquo Diabetes Research and Clinical Practicevol 109 no 3 pp 513ndash520 2015

[32] A V Contreras N Torres and A R Tovar ldquoPPAR-120572 asa key nutritional and environmental sensor for metabolicadaptationrdquo Advances in Nutrition vol 4 no 4 pp 439ndash4522013

Submit your manuscripts athttpswwwhindawicom

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

2 PPAR Research

risk for coronary heart disease [12] The association betweenpolymorphic variants of PPAR and serum fetuin-A levelshowever has not been investigated yet

In our study we aimed to investigate whether polymor-phisms of PPAR120574 (PPAR1205742 Pro12Ala and exon 6 C161T) andPPAR120572 (intron 7 G2467C) are associated with or may affectserum fetuin-A levels in two cohorts

2 Patients and Methods

Three-hundred and forty-two patients were originallyinvolved in this study Exclusion criteria were as followsclinical or laboratory signs of acute vascular disease(myocardial infarction stroke) acute infection malignanttumor hepatic disease renal failure immune suppressionsevere medical or surgical conditions and trauma Finallywe had 327 patients (cohort 2) who had all comparabledata (including successful genotyping for both PPAR120574polymorphisms) We were able to perform successful PPAR120572genotyping in a smaller number of patients (cohort 1119899 = 244) The genotyping success rate was greater than 99in both cohorts The genotypes were in the Hardy-Weinbergequilibrium

PPAR120572 polymorphism was studied in cohort 1 Thiscohort comprised 244 individuals (120 men and 124 womenage 601 plusmn 112 years mean plusmn SD) Cohort 1 consisted of79 reference individuals (15 men 64 women age 610 plusmn 94years) and 165 patients surviving myocardial infarction (105men 60 women age 596 plusmn 122 years)

PPAR120574 polymorphisms were studied in cohort 2 Thiscohort consisted of 327 individuals (161 men 166 womenage 579 plusmn 130 years) This cohort consisted of 162 referencesubjects (61 men 101 women age 561 plusmn 138 years) and thesame 165 postinfarction patients as in cohort 1 (105 men 60women age 596 plusmn 122 years)

Postinfarction patients had a history of STEMI myocar-dial infarction (6ndash24 months prior to the start of the study)Diabetes was diagnosed based on fasting plasma glucose gt70mmoll or the 2-hr OGTT gt 111mmoll Patients withdiabetes were treated with diet metformin and bedtimeinsulin

All persons gave their informed consent prior to theirinclusion in the study The study was approved by thelocal Ethics Committee of the Karolyi Sandor MunicipalityHospital

21 Determination of PPAR Polymorphic Variants The deter-mination of the PPAR120574 and PPAR120572 variants was performedby PCR-RFLP technique

The PPAR120572G2467C intron 7 polymorphism (rs 4253778)was studied by PCR-RFLP technique using a 51015840 forwardprimer of ACA ATC ACT CCT TAA ATA TGG TGGand a 31015840 reverse primer of AAG TAG GGA CAG ACAGGA CCA GTA The PCR product was digested with Taq1(New England Biolabs Boston MA USA) resulting in onefragment of 266 bp of the carriers of the wild-type allele andtwo fragments of 216 and 50 bp in the carriers of the mutantallele (thermocycles 94∘C 15min 30 times 94∘C 30 sec 50∘C20 sec and 72∘C 30 sec) [17]

For PPAR120574Pro12Ala (rs 1801282) polymorphism we useda 51015840 forward primer of GCC AAT TCA AGC CCA GTC anda mutagenic 31015840 reverse primer of GAT ATG TTT GCA GACAGT GTA TCA GTG AAG GAA TCG CTT TCC G ThePCR product was digested with Bst U1 enzyme (NewEnglandBiolabs Boston MA USA) resulting in one fragment of270 bp in the carriers of wild-type and two fragments of 227and 43 bp in carriers of mutant allele (thermocycles 95∘C15min 35 times 94∘C 30 sec 65∘C 45 sec and 72∘C 1min) [18]

The exon 6 polymorphism C161T of PPAR120574 (rs 3856806)was investigated by PCR-RFLP technique using a 51015840 forwardprimer of CAA GAC AAC CTG CTA CAA GC and a 31015840reverse primer of TCC TTG TAG ATC TCC TGC AG ThePCR product was digested with Pml1 enzyme (New EnglandBiolabs Boston MA USA) resulting in two fragments of120 and 80 bp in carriers of the wild-type allele and onlyone fragment of 200 bp in the carriers of the mutant allele(thermocycles 94∘C 15min 30times 94∘C 30 sec 56∘C 30 sec and72∘C 30 sec) [19]

22 Determination of Serum Fetuin-A Concentration Serumfetuin-A concentrations were determined by radial immun-odiffusion using the commercially available product (anti-fetuin-A IgG fraction Incstar cat number 81931 137mgmlin a final concentration of 84 120583l115ml gel) as previouslydescribed [20]

23 Determination of Insulin Resistance Parameters Plasmaglucose and insulin were determined by the routine HK-G6P-DH and ELCIAmethods respectivelyTheHomeostasisModel Assessment-Insulin Resistance (HOMA-IR) modelwas calculated according to Matthews et al [21]

24 Statistical Analysis Statistical analysis was carried outusing the SPSS v21 statistical software (SPSS Inc ChicagoIL USA) Nonparametric methods including the Bonferroni(Dunn) post hoc test were used 119901 values lt 005 wereconsidered as significant

3 Results

31 Subject Characteristics The characteristics of the studyparticipants are shown in Table 1

Sixty-five per cent of the postinfarction patients receivedstatins and 70 of them aspirin Serum fetuin-A concentra-tions did not differ statistically between patients treated andnot treated with these two medications (687 plusmn 122 versus 636plusmn 81mgl 119901 = 0204 for statins and 665 plusmn 0120 versus 672 plusmn124mgl 119901 = 0795 for aspirin resp)

32 PPAR120572 Intron 7 GC PPAR1205742 Pro12Ala and PPAR120574Exon 6 C161TAllele Distribution in Postinfarction Patients andReference Individuals Thedistribution of PPAR120574 andPPAR120572alleles is shown in Table 2 PPAR120574 Pro12Ala and PPAR120572intron 7 GC alleles did not differ significantly betweenpostinfarction patients and reference subjects Postinfarctionpatients however had a significantly higher T allele fre-quency of the PPAR120574 C161T compared to reference subjects

PPAR Research 3


Cohort 1(119899 = 244)

Cohort 2(119899 = 327)












(95 CI)OR

(95 CI) 119901


G P 08273R 08165 00861 1013

(09271ndash1107)1077

(06571ndash1764) 0769

C P 01727R 01835

PPAR1205742 Pro12Ala (rs1801282)

Pro12 P 08618R 08789 04279 09804

(09241ndash1040)08583

(05428ndash13570) 0513

Ala12 P 01382R 01211

PPAR120574 exon 6 C161T (rs3856806)

C P 08735R 09459 1025 09235

(08799ndash09693)03953

(02204ndash07091) 0001

T P 01265R 00541







4 PPAR Research



GG GC CC119901





















PPAR Research 5















4 Discussion





00590512

0215lt0001




6 PPAR Research



CC CT TT119901













5 Conclusion


PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 3


Cohort 1(119899 = 244)

Cohort 2(119899 = 327)












(95 CI)OR

(95 CI) 119901


G P 08273R 08165 00861 1013

(09271ndash1107)1077

(06571ndash1764) 0769

C P 01727R 01835

PPAR1205742 Pro12Ala (rs1801282)

Pro12 P 08618R 08789 04279 09804

(09241ndash1040)08583

(05428ndash13570) 0513

Ala12 P 01382R 01211

PPAR120574 exon 6 C161T (rs3856806)

C P 08735R 09459 1025 09235

(08799ndash09693)03953

(02204ndash07091) 0001

T P 01265R 00541







4 PPAR Research



GG GC CC119901





















PPAR Research 5















4 Discussion





00590512

0215lt0001




6 PPAR Research



CC CT TT119901













5 Conclusion


PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 PPAR Research



GG GC CC119901





















PPAR Research 5















4 Discussion





00590512

0215lt0001




6 PPAR Research



CC CT TT119901













5 Conclusion


PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 5















4 Discussion





00590512

0215lt0001




6 PPAR Research



CC CT TT119901













5 Conclusion


PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 PPAR Research



CC CT TT119901













5 Conclusion


PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 7






Acknowledgments


References

























8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 PPAR Research
















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Association of PPAR Alpha Intron 7 G/C, PPAR Gamma 2 …downloads.hindawi.com/journals/ppar/2017/7636019.pdf · 2019-07-30 · Cohort1 (𝑛=244) Cohort2 (𝑛=327) Referenceindividuals