research article inverse relationship of the cmklr1...

Research ArticleInverse Relationship of the CMKLR1 Relative Expression andChemerin Serum Levels in Obesity with Dysmetabolic Phenotypeand Insulin Resistance

Fernanda-Isadora Corona-Meraz123 Rosa-Elena Navarro-Hernaacutendez1234

Sandra-Luz Ruiacutez-Quezada34 Perla-Monserrat Madrigal-Ruiacutez123

Jorge Castro-Albarraacuten23 Efraiacuten Chavarriacutea-Aacutevila235 Milton-Omar Guzmaacuten-Ornelas23

Eduardo Goacutemez-Bantildeuelos236 Marcelo-Heroacuten Petri7 Joel-Isidro Ramiacuterez-Cedano34

Mariacutea-Elena Aguilar-Aldrete3 Clara Riacuteos-Ibarra2 and Moacutenica Vaacutezquez-Del Mercado1268

1Departamento de Biologıa Molecular y Genomica Centro Universitario de Ciencias de la Salud Universidad de GuadalajaraSierra Mojada No 950 Colonia Independencia CP 44340 Guadalajara JAL Mexico2Instituto de Investigacion en Reumatologıa y del Sistema Musculo Esqueletico Centro Universitario de Ciencias de la SaludUniversidad de Guadalajara Sierra Mojada No 950 Colonia Independencia CP 44340 Guadalajara JAL Mexico3UDG-CA-701 Grupo de Investigacion Inmunometabolismo en Enfermedades Emergentes (GIIEE)Centro Universitario de Ciencias de la Salud Universidad de GuadalajaraSierra Mojada No 950 Colonia Independencia CP 44340 Guadalajara JAL Mexico4Departamento de Farmacobiologıa Centro Universitario de Ciencias Exactas e Ingenierıas Universidad de GuadalajaraBoulevard Marcelino Garcıa Barragan No 1421 CP 44430 Guadalajara JAL Mexico5Departamento de Disciplinas Filosofico Metodologico e Instrumentales Centro Universitario de Ciencias de la SaludUniversidad de Guadalajara Sierra Mojada No 950 Colonia Independencia CP 44340 Guadalajara JAL Mexico6UDG-CA-703 Grupo de Investigacion en Inmunologıa y Reumatologıa Centro Universitario de Ciencias de la SaludUniversidad de Guadalajara Sierra Mojada No 950 Colonia Independencia CP 44340 Guadalajara JAL Mexico7Translational Cardiology Centre for Molecular Medicine Department of MedicineKarolinska Institutet L803 17176 Stockholm Sweden8Servicio de Reumatologıa Division de Medicina Interna Hospital Civil ldquoDr Juan I Menchacardquo Universidad de GuadalajaraSalvador de Quevedo y Zubieta No 750 CP 44340 Guadalajara JAL Mexico

Correspondence should be addressed to Monica Vazquez-Del Mercado dravmehotmailcom

Received 10 February 2016 Accepted 10 April 2016

Academic Editor Jose C Rosa

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

Background In obesity there is a subclinical chronic low-grade inflammatory response where insulin resistance (IR) may developChemerin is secreted in white adipose tissue and promotes low-grade inflammatory process where it expressed CMKLR1 receptorThe role of chemerin and CMKLR1 in inflammatory process secondary to obesity is not defined yetMethods Cross-sectional studywith 134 individuals classified as with and without obesity by body mass index (BMI) and IR Body fat storage measurements andmetabolic and inflammatory markers were measured by routine methods Soluble chemerin and basal levels of insulin by ELISAand relative expression of CMKLR1 were evaluated with qPCR and 2minusΔΔCT method Results Differences (119875 lt 005) were observedbetween obesity and lean individuals in body fat storage measurements and metabolic-inflammatory markers Both CMKLR1expression and chemerin levels were increased in obesity without IR Soluble chemerin levels correlate with adiposity andmetabolicmarkers (119903 = 88 to 385) 119875 lt 005 Conclusion The increment of CMKLR1 expression was associated with insulin productionIncreased serum levels of chemerin in obesity were observed favoring a dysmetabolic response The results observed in this studysuggest that both chemerin and CMKLR1 have opposite expression in the context of low-grade inflammatory response manifestedin the development of IR

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 3085390 9 pageshttpdxdoiorg10115520163085390

2 Mediators of Inflammation

1 Introduction

Obesity which is the excess storage of white adipose tissue(WAT) and low-grade inflammation are the key factors fordevelopment of insulin resistance (IR) [1ndash4]

In WAT primed immune cells are recruited as adiposityincreases and these cells became resident cells (mainlymacrophages) and secrete proinflammatory adipokines thatpromote further recruitment of circulating monocytes [4ndash7] Later they polarize towards M1 macrophages favoring aninflammatory subclinical chronic state [6 8ndash10]

Chemerin is an adipokine secreted by adipocytes it isclosely associated with amount of fat and distribution Asa chemoattractant protein chemerin acts as a ligand forthe coupled G-receptor protein (ChemR23) and participatesin both adaptive and innate immunity [11] In humanschemerin gene (RRARES2) is highly expressed in WAT andto a lesser extent in liver and lungs On immune cellschemerin is known to stimulate chemotaxis of dendriticcells macrophages and NK cells Meanwhile its recep-tor ChemR23 gene (CMKLR1) is expressed in dendriticcells monocytemacrophages and endothelial cells [11ndash14]ChemR23 is involved in the differentiation of adipocytesand increased intracellular glucose or lipids promote itsexpression [14]

The interaction of chemerinChemR23 has been shownto reduce cytokines chemokines and phagocytosis provingto be important in the inflammatory process associated withobesity [12 14 15]

In this context chemerinChemR23 axis has been shownto impact IR development which influences the clinicalcourse and severity of obesity-related diseases [10] How-ever the association with immunometabolic markers andchemerin and its receptor ChemR23 is scarce Therefore theaim of this study was to characterize the inflammatory andmetabolic phenotype of subjects with obesity-IR state basedon the chemerin soluble levels and its receptor CMKLR1expression

2 Material and Methods

21 Study Design In this cross-sectional study a total of134 adults aged 20 to 59 years were recruited from generalpopulation in the west of Mexico We included individualswho at the time of enrollment did not present glucose intol-erance infectious diseases hypertension pregnancy anemiacardiovascular disease malignancy and renal and metabolicdiseases such as type 2 diabetes mellitus (T2DM) Subjectswith current medication use were excluded

Subjects were classified in two forms based on obesityand then by IR First they were classified according to therecommendationsrsquo of World Health Organization by bodymass index (BMI) waist circumference (WC) waist-hip ratio(WHR) and waist to height ratio (WHtR) in two groupssubjects with obesity if any of the following conditions werepresent BMI ge 300 kgm2 WC ge 900 cmWHR ge 090 andWHtR ge 525 in Men and WC ge 800 cm WHR ge 080 andWHtR ge 530 in women and lean subjects that are lowerin these measurements For detection of IR in subjects with

obesity they were secondly classified according to Gayosocriteria in two groups with and without IR [16]

For ethical purposes participants were informed aboutthe study and signed a consent form following the Helsinkideclaration guidelines [17] and the institutional (GuadalajaraUniversity) review boardsrsquo committees

22 Subjectsrsquo Medical History and Physical Examination Allindividuals who satisfied inclusion criteria were clinicallyevaluated by a physician who performed a complete medicalhistory Assessment of general health status and vital signswere included blood pressure (measured 3 times with thesubject in the sitting position for 15 minutes before theevaluation) heart and respiratory rate and body temperature

23 Subjectsrsquo Body Fat Storage Measurements We evaluatedheight which was measured to the nearest 1mm by using astadiometer (Seca GmbH amp Co KG Hamburg Germany)body weight and total and trunk body fat mass (abso-lute kg and relative ) were determined by bioelectricalimpedance analysis (TANITA BC-418 Segmental Body Com-position Analyzer Tokyo JPN) to the nearest 01 kg WChip circumference (HC) and coronal abdominal diameterwere measured by using an anthropometric fiberglass tape(GULICK length 0ndash180 cm precision plusmn01 USA) At thelevel of the iliac crest (L4-5) sagittal abdominal diameterwas measured using a sliding-beam abdominal caliper (pre-cision plusmn01 cm Holtain Ltd Crosswell Crymych PembsSA41 3UF UK) with the patient lying in a supine positionon the examination table [18] Five measures of skinfoldthickness (ie abdominal bicipital tricipital subscapularand suprailiac) were obtained on the right side of the bodyby using a Harpenden skinfold caliper (opened 80mm withprecision of plusmn02mm constant pressure 10 gmm2 HoltainLtd Crosswell Crymych Pembs SA41 3UF UK) All thesemeasurements were carried out by the same Physician induplicate following the procedures recommended by anthro-pometric indicators measurement guide [19 20]

To determine obesity and adiposity indexes thefollowing calculations were used BMI kgm2 = weight(kg)height2 (m) WHtR = WC (cm)height (cm) [21]WHR = WC (cm)HC (cm) conicity index (CI) = WC(cm)0109radic(weight (kg)height (cm)) BFR = total bodyfat mass (kg)height (cm) total adipose area (TAA cm2) =WC24120587 visceral area (VA cm2) = 120587(WC2120587 minus abdominalskinfold)2 subcutaneous abdominal area (cm2) = TAA minusVA [18] visceral adiposity index (VAI) for males VAI =(WC3658 + (1896BMI))6(TG081)6(152HDLc) andfemales VAI = (WC3968 + (1886BMI)) 6(TG103)6(131HDLc) [22] homeostasis model assessment-insulin resist-ance (HOMA-IR) = [basal glucose mgdL times (basal insulin120583UImL)405] [23 24]

24 Metabolic Inflammatory Markers and Chemerin LevelsMeasurements Individuals included in the study were fast-ing 12 hours before the blood samples were taken allowingthem to clot at room temperature and then were centrifugedat 1509 RCF (Rotanta 460R Andreas Hettich GmbH amp Co

Mediators of Inflammation 3

Table 1 Status of body fat storage in individuals in the study group

Study group Lean Obesity without IR Obesity with IRBMI (kgm2) [216 (185ndash247)] [320 (301ndash347)] [326 (302ndash349)]119899 = 134 49 32 53MeasurementHeight (cm) 1660 plusmn 84 1628 plusmn 78 1638 plusmn 90Body weight (kg) 600 plusmn 99 901 plusmn 128a 973 plusmn 189a

Total body fat mass () 203 plusmn 59 403 plusmn 68a 406 plusmn 69a

Total body fat mass (kg) 120 plusmn 36 366 plusmn 88a 398 plusmn 124a

Trunk body fat mass () 238 plusmn 106 381 plusmn 114a 354 plusmn 105a

Trunk body fat mass (kg) 59 plusmn 24bc 184 plusmn 39ac 194 plusmn 61ab

Waist circumference (cm) 783 plusmn 66 1096 plusmn 119a 1137 plusmn 132a

Hip circumference (cm) 933 plusmn 48 1157 plusmn 124a 1188 plusmn 108a

Coronal abdominal diameter (cm) 302 plusmn 64bc 380 plusmn 87ac 449 plusmn 120ab

Sagittal abdominal diameter (cm) 167 plusmn 21 246 plusmn 34a 259 plusmn 44a

Total adipose area (cm2) 4912 plusmn 823 9679 plusmn 2166a 10425 plusmn 2496a

Visceral area (cm2) 2274 plusmn 3730 2927 plusmn 5164 5997 plusmn 8361a

Subcutaneous abdominal area (cm2) 3142 plusmn 3240 6968 plusmn 5608a 5626 plusmn 9087BMI body mass index [119909 (minndashmax)] Data are shown in 119909 plusmn SD aDifference versus lean bDifference versus obesity without IR cDifference versus obesitywith IR (P lt 005 ANOVA Tukey post hoc) IR insulin resistance

KG) for 10 minutes at 20∘C Serum was collected and storedat minus86∘C until further analysis

We quantified serum concentration of glucose and nones-terified fatty acids (NEFA) with routine enzymatic methodstriglycerides (TG) and total cholesterol (TC) with routinecolorimetric methods high and low density lipoproteincholesterol (HDLc and LDLc resp) apolipoproteins A1(Apo-A1) and B (Apo-B) and high sensitivity C reactiveprotein (CRP) with immunoturbidimetry methods (RandoxLaboratories 55DiamondRoad CrumlinCo AntrimNorth-ern Ireland UK) and erythrocyte sedimentation rate (ESR)withWintrobe method [25] And the low density lipoproteincholesterol (VLDLc) was obtained with the Friedewald for-mula [26]

Through using commercial enzyme-linked immunoab-sorbent assays (ELISA) soluble levels of insulin were deter-mined (sensitivity of 0399 120583UImL) (ALPCO 26-G Keeway-din Drive Salem NH 03079) and chemerin was determinedwith a limit of detection of 108ndash78 ngmL (RampD SystemsMinneapolis USA)

25 CMKLR1 Relative Expression Analysis Mononuclearcells from the subjects studied were isolated by density gra-dient media with separating solution Lymphoprep (AXIS-SHIELDPOBox 6863 Rodelokka 0504Oslo Norway) TotalRNA was isolated from purified mononuclear cells usingTRIzol LS Reagent (Ambion RNA Life Technologies 5791Van AllenWay Carlsbad CA 92008) based on the single-stepRNA isolation modified method reported by Chomczynski[27] ComplementaryDNA synthesis (cDNA)was performedwith 2120583g of each total RNA sample using a reaction sizeof 20120583L with oligo (dT) 18 primer (100 ng120583L) RNase freeDEPC treated water and SuperScript Reverse TranscriptaseIII kit (Applied Biosystems 850 Lincoln Centre Drive Foster

City CA 94404) and stored atminus20∘Cuntil used for expressionanalyses

Real-Time Quantitative Polymerase Chain Reaction(qPCR)was conducted using the StepOnedetection systemEXPRESS SYBR GreenER and ROX qPCR SuperMixUniversal and sequence detector software v23 (AppliedBiosystems 850 Lincoln Centre Drive Foster City CA94404) was used for data analysis A threshold cycle (CT)value was determined from each amplification plot

In brief CMKLR1 mRNA expression was performedin a final reaction volume of 20120583L (10 120583M forward andreverse primer 500 nM ROX 1X SYBR Green qPCR mastermix and cDNA 1000 ng) The conditions of the reactionwere as follows holding 95∘C10min cycling (35 cycles of95∘C15 s 60∘C60 s) and melt curves 95∘C15 s 60∘C60 sand 95∘C15 s Expression of target genes was normalizedby the endogenous reference gen RPS28 sequence specificprimers were forward 51015840-GGTCTGTCACAGTCTGCTCC-31015840 and reverse 51015840-CATCTCAGTTACGTGTGGCG-31015840 andfor CMKLR1 target gen forward 51015840-GTGGTGGTCTAC-AGCATCGT-31015840 and reverse 51015840-ATGGCGGCATAGGTG-ATATGG-31015840

The relative expression fold change of target gene wascalculated using the comparative CT method with 2minusΔΔCT

equation [28] To ensure accuracy of data experiments weredone in duplicate blank internal controls andmelt curve datawere collected with applications of StepOne detector software(Cat 4376357)

26 Statistical Analysis Data were analyzed with statisticssoftware SPSS v21 (IBM Inc Chicago IL USA) and Graph-Pad Prism v601 (2014 Inc 2236 Beach Avenue Jolla CA92037) Results are given as mean plusmn standard deviation (SD)The data distribution of clinical and laboratory variables


was evaluated with 119885 Kolmogorov-Smirnov test The clinicaland laboratory characteristics of study group were comparedwith one way ANOVA with Tukey post hoc Data fromserum concentrations of chemerin and insulin laboratorialassessment and adiposity variables were subjected to Pearsoncorrelation tests A two-tailed 119875 value lt 005 was consideredstatistically significant

3 Results

31 Status Assessment of Body Fat Showed High Adiposity andIR In this study the observed frequency of obesity and IRwas 63 and 39 respectively while IR was not presentin lean individuals (with average BMI of 216 kgm2 180 to247) For individuals classified with obesity IR prevalencewas 62 The adiposity measurements are shown in Table 1

The fat distribution assessed by the skinfold thicknessesshowed magnitudes as follows the group with obesity andIR gt obesity without IR group gt lean group (Figure 1(a))Adiposity indexes were increased in both groups with obesityversus lean subjects except VAI whose increase is observed inindividuals with IR versus lean subjects (Figure 1(b))

32 Individuals with Obesity and IR Presented a SubclinicalInflammatory State and Dyslipidemia Lean individuals pre-sented lower levels of glucose insulin and HOMA-IR whencompared with individuals with and without IR whereasNEFA presented no changes in different groups (Figure 1(c))lean individuals showed lower levels of triglycerides andLDLc compared to subjects with and without IR also leanindividuals presented lower levels of total cholesterol andVLDLc when compared to obesity group with IR Leansubjects also presented lower levels of Apo-B compared withindividuals without IR whereas HDLc and Apo-A1 presentedno statistical changes (Figure 1(d)) Levels of CRP and ESRwere higher in obesity with IR versus lean (683plusmn565 1746plusmn1167 119875 lt 0001 resp)

33 Obesity without IR Showed a Contrasting Context onChemerin Levels Increased levels of soluble chemerin wereobserved in obese individuals without IR compared to obeseindividuals with IR and lean subjects groups (Figure 2(a))

34 Chemerin and Insulin Levels Were Associated withDysmetabolic Phenotype and Body Fat Adiposity MarkersPositive correlations of chemerin and insulin were observedwith the increase in the status of body fat and subcutaneousfat accumulation with lipid profile (Table 2)

35 Higher CMKLR1 Relative Expression Was Associated withObesity without IR Increased expression levels of CMKLR1receptor were observed in individuals with obesity withoutIR versus lean and obesity with IR individuals (Figure 2(b))

A detailed test by tertiles was performed (describing thefirst tertile as lower expression second tertile as intermediateexpression and third tertile as higher expression) we foundincreased accumulation of abdominal fat mass andmetabolicmarkers between individuals with high expression versusindividuals with low expression of CMKLR1 independent of

Table 2 Correlation of chemerin and insulin serum levels with thedysmetabolic phenotype and body fat adipositymarkers status in thestudy groups

MeasurementsChemerin(ngmL)

Insulin(120583UImL)

Correlation ()Body weight (kg) minus28 347lowastlowast

BMI (kgm2) 153 462lowastlowast

Body fat mass () 385lowastlowast 415lowastlowast

Total body fat mass (kg) 261lowastlowast 467lowastlowast

Waist circumference (cm) 188lowast 364lowastlowast

Hip circumference (cm) 216lowastlowast 415lowastlowast

Waist-hip ratio 58 169lowastlowast

Body fat ratio 315lowastlowast 471lowastlowast

Waist to height ratio 266lowastlowast 388lowastlowast

Conicity index 307lowastlowast 183lowastlowast

Coronal abdominal diameter (cm) 84 130lowast

Sagittal abdominal diameter (cm) 157 464lowastlowast

Total adipose area (cm2) 188lowast 364lowastlowast

Visceral area (cm2) 192lowast 370lowastlowast

Subcutaneous abdominal area (cm2) 176 194lowastlowast

Glucose (mgdL) minus63 225lowastlowast

Skinfold thickness (mm)Abdominal minus103 173lowastlowast

Bicipital 327lowastlowast 458lowastlowast

Tricipital 304lowastlowast 390lowastlowast

Subscapular 111 388lowastlowast

Suprailiac 161lowast 365lowastlowast

Triglycerides (mgdL) 204lowast 261lowastlowast

LDLc (mgdL) 130 125lowast

VLDLc (mgdL) 204lowast 209lowastlowast

HDLc (mgdL) 174lowast 101NEFA (mmolL) 302lowastlowast 90CRP (mgL) 141 315lowastlowast

ESR (mmh) 302lowastlowast 18IR insulin resistance HDLc LDLc and VLDLc (lipoproteins of high lowand very lowdensity cholesterol resp) NEFA nonesterified fatty acids CRPC reactive protein ESR erythrocyte sedimentation rate lowastlowast119875 lt 0001 lowastP lt005 Pearson correlation test

the presence of IR andor obesity Other relevant informationprovided in this analysis is that there is an inverse correspon-dence in insulin levels HOMA-IR and NEFA regarding theexpression of CMKLR1 (Table 3)

4 Discussion

Twomain findings emerge from this study first theCMKLR1receptor expression was associated with obesity and itsfeatures and second its ligand chemerin was associated notonly with obesity but also with metabolic dysfunction suchas dyslipidemia and IR


LeanObesity without IRObesity with IR

lowast

lowast

0

10

20

30

40

50M

agni

tude

(mm

)

Bicipital Tricipital Subescapular SuprailiacAbdominalSkinfold thickness

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast


lowastlowast

lowastlowast

(a) Subcutaneous fat mass distribution


WHR CI BFR VAIWHtR00

05

10

15

15

20

25

Mag

nitu

de





lowastlowast

(b) Adiposity indexes


0

1

2

25

80

120

160

Leve

ls

HOMA-IR(mmolL)

NEFA(mgdL)Glucose Insulin

(120583UImL)

lowast

lowastlowast



(c) Metabolic markers


TC HDLc LDLc VLDLc Apo-A1 Apo-BTG0

100

200

300Se

rum

conc

entr

atio

n (m

gdL

)

lowastlowastlowast

lowast


(d) Lipid profile

Figure 1 Body fat mass distribution adiposity indexes and metabolic markers in the study groups Lean [BMI 214 (185ndash247) kgm2]119899 = 49 obesity without IR [BMI 320 (301ndash347) kgm2] 119899 = 32 obesity with IR [BMI 326 (302ndash349) kgm2] 119899 = 53 IR insulinresistanceWHtR waist to height ratioWHRwaist-hip ratio CI conicity index VAI visceral adiposity index BFR body fat ratio HOMA-IRhomeostatic model assessment of insulin resistance NEFA none esterified fatty acids TG triglycerides TC total cholesterol HDLc LDLcand VLDLc (lipoproteins of high low and very low density cholesterol resp) Data are shown in 119909 plusmn SD lowastlowast119875 lt 0001 lowast119875 lt 005 (ANOVATukey post hoc)

Adipose tissue has been suggested to be an importantsource of low-grade inflammation based on three biolog-ical aspects quantity (total mass and relative proportion)anatomical distribution and phenotype of resident cells [1029]

Regarding the quantity and site our study supportedthat dysfunctional WAT may play an important role in low-grade inflammation because the individuals with obesityshown increased accumulation of fat manly in the abdominalregion along with low-grade inflammation and dysmetabolic

phenotype (represented by dyslipidemic state and increasedadiposity indexes) although not all individuals with obesitywere IR

Another important point from this study is that it sup-ports the fact that the IR is a component for developinga chronic-degenerative disease The IR first promotes dys-lipidemia (as an intermediate event) leading to metabolicsyndrome and subsequently development of T2DM [1 3 30]

The dysmetabolic phenotype observed in individualswith obesity in our study can be explained in the context


Table 3 Adiposity and metabolic markers in the relative expression levels of CMKLR1

Tertile First Second ThirdCMKLR1 relative expression [0546 (0034ndash1060)] [4148 (1148ndash8934)] [69787 (9009ndash473260)] obesity 487 441 517 IR 590 529 483Measurement


Coronal abdominal diameter 3515 plusmn 120 4218 plusmn 105a 4097 plusmn 89VAI 244 plusmn 26 333 plusmn 23 454 plusmn 241a

Insulin (120583UImL) 1522 plusmn 114 1308 plusmn 84 874 plusmn 51a

HOMA-IR 349 plusmn 25 305 plusmn 19 213 plusmn 14a

NEFA 0725 plusmn 02 0618 plusmn 026 0500 plusmn 025a

The expression levels of CMKLR1 are in relative units [119909 (minndashmax)] Data are shown in 119909 plusmn SD and were classified by tertiles aDifference versus first tertile(119875 lt 005 ANOVA Tukey post hoc) IR insulin resistance VAI visceral adiposity index HOMA-IR homeostatic model assessment of insulin resistanceNEFA nonesterified fatty acids

lowast

Obesity without IR Obesity with IRLean

lowastlowast

0

5

10

15

Relat

ive e

xpre

ssio

n of

CMKL

R1(m

etho

d2minusΔΔ

C T)

(a)

P = 0032P = 0002

Obesity without IR Obesity with IRLean0

50

100

150

Solu

ble l

evels

of c

hem

erin

(ng

mL)

(b)

Figure 2 Levels of soluble chemerin and relative expression of CMKLR1 in the study groups (a) Relative expression of CMKLR1 Method2minusΔΔCT lowastdifference between obesity without IR and lean (79-fold 119875 = 0003) and lowastlowastobesity with IR versus lean (38-fold 119875 lt 0001) (b)Serum levels of chemerin (ANOVA Tukey post hoc) IR insulin resistance

of immune system dysregulation that exists in IR by thedevelopment of two alternative mechanisms not exclusivein one the WAT of individuals with obesity have increasedresident M1 macrophages able to produce chemokines and inadditional one it has been found that high concentrations offatty acids induce expression of TNF120572 and Toll-like receptor-4 signaling [4 30] This pathway converges with insulinsignaling in adipocytes affecting positively glucose transportglycogen synthesis and cell differentiation while negativelyaffecting the lipolysis and gluconeogenesis [8 31 32]

The polarization of monocytesmacrophages to M1 inWAT is favored by coupling of chemerin through ChemR23that in turn displayed a proinflammatory profile wherechemotactic ligand-receptor interaction regulates continuedmigration of monocytes to WAT [13 14] Soluble chemerin

produced by adipocytes and resident macrophages M1 inWAT binds to ChemR23 with high affinity and its levelsdecrease as recruited circulating monocytes differentiate intomacrophages [12ndash14 33 34]

This study evaluated the gene expression of CMKLR1receptor in circulating monocytes It presented 79- or 38-fold higher expression in individuals with obesity without IRor obesity with IR respectively than lean individuals Inter-estingly the increase in expression levels was directly asso-ciated with the proportion of abdominal fat mass and bodydimensions whereas an inverse association was observedwith the production of insulin nonesterified free fatty acidsand HOMA-IR

In this regard previous study showed that the chemerinChemR23 signaling does not affect the inflammatory


response in ex vivo humanmacrophages [35]The expressionlevels of CMKLR1 found in our study suggest that the level ofexpression is higher in early activation of primed circulatingmonocytes but decreases at later stages which can beexplained based on the reports in other studies in animalmodels [31 36] although changes of CMKLR1 expression inhuman macrophage differentiationpolarization still remainto be established

The association of CMKLR1 expression levels observedin our study can be explained due to IR being closelyassociated with excess in abdominal accumulation of WAT[30] and based on other reports in which an adipocyte cellline expression of CMKLR1 was analyzed during differen-tiation process where upregulation was in the early stagewhereas a downregulation was observed in late stages [3738] Proinflammatory stimulus such as TNF120572 or adiponectinhas been shown to upregulate gene expression of CMKLR1in differentiated adipocytes [39] This shows the complexityof the CMKLR1 expression and functionality that is notdependent on the expression of its ligand but also dependenton the cell type presented on the disease

Nevertheless the limitations of this study were thatprotein levels of theChemR23 inmonocytewere not assessedlimiting complementary studies about the functional recep-tor Another limitation that should be taken in considerationis that this same receptor is known to bind with the sameaffinity to the lipid mediator resolving E1 which has anti-inflammatory properties The quantification of such ligandwas not done

One of the main findings was the increased serum levelsof chemerin in individuals with obesity without IR versusindividuals with IR and lean In parallel correlation withindicators of adiposity and metabolic markers was observed

In this regard previous studies report conflicting resultson chemerin levels in different diseases with an inflam-matory component such as chronic pancreatitis (940 ngmL) [40] T2DM individuals (1790 ngmL) [41] lipodys-trophy (2343 ngmL) [42] and obesity without diabetes(59008 ngmL) [43] Chemerin levels were increased exceptfor the levels reported in rheumatoid arthritis (350 ngmL)[44] (an inflammatory disease per se) although this canbe explained based on treatment however other studieshave suggested that chemerin may be the functional linkbetween chronic inflammation and obesity-related T2DMand cardiovascular disease [45]

Our results can be explained because monocytesma-crophages are decisive in the pathogenic process of IRbased on the fact that they are an important source ofproinflammatory markers (TNF120572 IL-6 chemerin and Creactive protein) along with increased levels of expressionof adipokines chemokines and proinflammatory cytokinesassociated with an equivalent increase in hyperplastic andhypertrophic adipocytes [13 30 46]

One important observation from this study is that in thecontext of obesity soluble chemerin levels enhanced both theinflammation and dysmetabolic phenotype showing how anopposite change in the expression of CMKLR1 takes placeonce IR is established

In previous reports it is postulated that chemerin pro-duction has a dual profile proanti-inflammatory [9 15] Inthis context we suggest that in the initial stage of IR theincrease in chemerin levels promotes dysmetabolic profilearising from the dysfunctional adipose tissue [36] Althoughin the setting of obesity during the process of establishment ofIR the levels of chemerin can be decreased the dysmetabolicprofile is maintained Its scenery might be explained dueto that action of chemerin where it regulates the insulinmetabolism [6]

5 Conclusions

This is the first study that links the increment of CMKLR1expression with insulin production showing an associationwith fat mass and corporal dimensions while the increasedserum levels of chemerin in obesity were observed favoringa dysmetabolic response

Taking together the results observed in this study suggestthat both chemerin andCMKLR1 have opposite expression inthe context of low-grade inflammatory response manifestedin the development of IR

Functional studies are necessary to clarify the biologicalfunctions of chemerin signaling in the pathogenesis of IR

Competing Interests

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

Authorsrsquo Contributions

Fernanda-Isadora Corona-Meraz and Rosa-Elena Navarro-Hernandez equally contributed to this work

Acknowledgments

This work was supported by Grant PRODEP no 220214 toUDG-CA-701 Academic group ldquoAging Immunometabolismand Oxidative Stressrdquo

References

[1] K Makki P Froguel and I Wolowczuk ldquoAdipose tissuein obesity-related inflammation and insulin resistance cellscytokines and chemokinesrdquo ISRN Inflammation vol 2013Article ID 139239 12 pages 2013

[2] C de Luca and J M Olefsky ldquoInflammation and insulinresistancerdquo FEBS Letters vol 582 no 1 pp 97ndash105 2008

[3] J Ye ldquoMechanisms of insulin resistance in obesityrdquo Frontiers ofMedicine vol 7 no 1 pp 14ndash24 2013

[4] Z Wang and T Nakayama ldquoInflammation a link betweenobesity and cardiovascular diseaserdquoMediators of Inflammationvol 2010 Article ID 535918 17 pages 2010

[5] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011


[6] A Chawla K D Nguyen and Y P S Goh ldquoMacrophage-mediated inflammation in metabolic diseaserdquo Nature ReviewsImmunology vol 11 no 11 pp 738ndash749 2011

[7] V Bourlier and A Bouloumie ldquoRole of macrophage tissueinfiltration in obesity and insulin resistancerdquo Diabetes ampMetabolism vol 35 no 4 pp 251ndash260 2009

[8] J McNelis and J Olefsky ldquoMacrophages immunity andmetabolic diseaserdquo Immunity vol 41 no 1 pp 36ndash48 2014

[9] L Yao O Herlea-Pana J Heuser-Baker Y Chen and J Barlic-Dicen ldquoRoles of the chemokine system in development ofobesity insulin resistance and cardiovascular diseaserdquo Journalof Immunology Research vol 2014 Article ID 181450 11 pages2014

[10] H S Schipper B Prakken E Kalkhoven and M BoesldquoAdipose tissue-resident immune cells key players inimmunometabolismrdquo Trends in Endocrinology andMetabolismvol 23 no 8 pp 407ndash415 2012

[11] B A Zabel M Kwitniewski M Banas K Zabieglo K Murzynand J Cichy ldquoChemerin regulation and role in host defenserdquoAmerican Journal of Clinical and Experimental Immunology vol3 no 1 pp 1ndash19 2014

[12] M C Ernst and C J Sinal ldquoChemerin at the crossroadsof inflammation and obesityrdquo Trends in Endocrinology andMetabolism vol 21 no 11 pp 660ndash667 2010

[13] B Bondue V Wittamer and M Parmentier ldquoChemerinand its receptors in leukocyte trafficking inflammation andmetabolismrdquo Cytokine and Growth Factor Reviews vol 22 no5-6 pp 331ndash338 2011

[14] F Mariani and L Roncucci ldquoChemerinchemR23 axis ininflammation onset and resolutionrdquo Inflammation Researchvol 64 no 2 pp 85ndash95 2014

[15] T Yoshimura and J J Oppenheim ldquoChemokine-like recep-tor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2(CCRL2) two multifunctional receptors with unusual proper-tiesrdquo Experimental Cell Research vol 317 no 5 pp 674ndash6842011

[16] P Gayoso-Diz A Otero-Gonzalez M X Rodriguez-Alvarezet al ldquoInsulin resistance (HOMA-IR) cut-off values and themetabolic syndrome in a general adult population effect ofgender and age EPIRCE cross-sectional studyrdquo BMC EndocrineDisorders vol 13 article 47 2013

[17] Council for International Organizations of Medical Sciencesand World Health Organization International Ethical Guide-lines for Epidemiological Studies CIOMS Geneva Switzerland2009

[18] M Garaulet J J Hernandez-Morante F J Tebar S Zamoraand M Canteras ldquoTwo-dimensional predictive equation toclassify visceral obesity in clinical practicerdquo Obesity vol 14 no7 pp 1181ndash1191 2006

[19] NHANES Anthropometry Procesures Manual Edited byNCfH Statistics CDCNational Center for Health Statistics2012

[20] R Ness-Abramof and C M Apovian ldquoWaist circumferencemeasurement in clinical practicerdquoNutrition in Clinical Practicevol 23 no 4 pp 397ndash404 2008

[21] Q Zeng Y He S Dong et al ldquoOptimal cut-off values of BMIwaist circumference and waist height ratio for defining obesityin Chinese adultsrdquo British Journal of Nutrition vol 112 no 10pp 1735ndash1744 2014

[22] M C Amato C Giordano M Galia et al ldquoVisceral adiposityindex a reliable indicator of visceral fat function associatedwith

cardiometabolic riskrdquoDiabetes Care vol 33 no 4 pp 920ndash9222010

[23] 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

[24] T M Wallace and D R Matthews ldquoThe assessment of insulinresistance in manrdquoDiabetic Medicine vol 19 no 7 pp 527ndash5342002

[25] MMWintrobe and JW Landsberg ldquoA standardized techniquefor the blood sedimentation test 1935rdquoThe American Journal ofthe Medical Sciences vol 346 no 2 pp 148ndash153 2013

[26] W T Friedewald R I Levy and D S Fredrickson ldquoEstimationof the concentration of low-density lipoprotein cholesterol inplasma without use of the preparative ultracentrifugerdquoClinicalChemistry vol 18 no 6 pp 499ndash502 1972

[27] P Chomczynski ldquoA reagent for the single-step simultaneousisolation of RNA DNA and proteins from cell and tissuesamplesrdquo BioTechniques vol 15 no 3 pp 532ndash537 1993

[28] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[29] M-J Lee Y Wu and S K Fried ldquoAdipose tissue heterogeneityimplication of depot differences in adipose tissue for obesitycomplicationsrdquoMolecular Aspects of Medicine vol 34 no 1 pp1ndash11 2013

[30] E Fuentes F Fuentes G Vilahur L Badimon and I PalomoldquoMechanisms of chronic state of inflammation asmediators thatlink obese adipose tissue andmetabolic syndromerdquoMediators ofInflammation vol 2013 Article ID 136584 11 pages 2013

[31] S Guo ldquoInsulin signaling resistance and metabolic syndromeinsights from mouse models into disease mechanismsrdquo Journalof Endocrinology vol 220 no 2 pp T1ndashT23 2014

[32] A M F Johnson and J M Olefsky ldquoThe origins and drivers ofinsulin resistancerdquo Cell vol 152 no 4 pp 673ndash684 2013

[33] M Herova M Schmid C Gemperle and M HersbergerldquoChemR23 the receptor for chemerin and resolvin E1 isexpressed and functional on M1 but not on M2 MacrophagesrdquoThe Journal of Immunology vol 194 no 5 pp 2330ndash2337 2015

[34] M C Ernst I D Haidl L A Zunıga et al ldquoDisruption of thechemokine-like receptor-1 (CMKLR1) gene is associated withreduced adiposity and glucose intolerancerdquo Endocrinology vol153 no 2 pp 672ndash682 2012

[35] B Bondue O de Henau S Luangsay et al ldquoThechemerinChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages exvivordquo PLoS ONE vol 7 no 6 Article ID e40043 2012

[36] M Takahashi Y Okimura G Iguchi et al ldquoChemerin regulates120573-cell function in micerdquo Scientific Reports vol 1 article 1232011

[37] Y Suzuki Y H Hong S H Song et al ldquoThe regula-tion of chemerin and CMKLR1 genes expression by TNF-120572 adiponectin and chemerin analog in bovine differentiatedadipocytesrdquo Asian-Australasian Journal of Animal Sciences vol25 no 9 pp 1316ndash1321 2012

[38] M Takahashi Y Takahashi K Takahashi et al ldquoChemerinenhances insulin signaling and potentiates insulin-stimulatedglucose uptake in 3T3-L1 adipocytesrdquo FEBS Letters vol 582 no5 pp 573ndash578 2008


[39] K B Goralski T C McCarthy E A Hanniman et alldquoChemerin a novel adipokine that regulates adipogenesis andadipocyte metabolismrdquoThe Journal of Biological Chemistry vol282 no 38 pp 28175ndash28188 2007

[40] K Adrych M Stojek M Smoczynski T Sledzinski S-WSylwia and J Swierczynski ldquoIncreased serum chemerin con-centration in patients with chronic pancreatitisrdquo Digestive andLiver Disease vol 44 no 5 pp 393ndash397 2012

[41] M J Neuparth J B Proenca A Santos-Silva and S CoimbraldquoThe positive effect of moderate walking exercise on chemerinlevels in portuguese patients with type 2 diabetes mellitusrdquoJournal of Investigative Medicine vol 62 no 2 pp 350ndash3532014

[42] K Miehle T Ebert S Kralisch et al ldquoCirculating serumchemerin levels are elevated in lipodystrophyrdquo ClinicalEndocrinology 2015

[43] H Lorincz M Katko M Harangi et al ldquoStrong correlationsbetween circulating chemerin levels and lipoprotein subfrac-tions in nondiabetic obese and nonobese subjectsrdquo ClinicalEndocrinology vol 81 no 3 pp 370ndash377 2014

[44] P H Dessein L Tsang A J Woodiwiss G R Norton and ASolomon ldquoCirculating concentrations of the novel adipokinechemerin are associated with cardiovascular disease risk inrheumatoid arthritisrdquo Journal of Rheumatology vol 41 no 9pp 1746ndash1754 2014

[45] P Fulop I Seres H Lorincz M Harangi S Somodi andG Paragh ldquoAssociation of chemerin with oxidative stressinflammation and classical adipokines in non-diabetic obesepatientsrdquo Journal of Cellular andMolecularMedicine vol 18 no7 pp 1313ndash1320 2014

[46] C Cheng and C Daskalakis ldquoAssociation of adipokines withinsulin resistance microvascular dysfunction and endothelialdysfunction in healthy young adultsrdquo Mediators of Inflamma-tion vol 2015 Article ID 594039 9 pages 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


1 Introduction

Obesity which is the excess storage of white adipose tissue(WAT) and low-grade inflammation are the key factors fordevelopment of insulin resistance (IR) [1ndash4]

In WAT primed immune cells are recruited as adiposityincreases and these cells became resident cells (mainlymacrophages) and secrete proinflammatory adipokines thatpromote further recruitment of circulating monocytes [4ndash7] Later they polarize towards M1 macrophages favoring aninflammatory subclinical chronic state [6 8ndash10]

Chemerin is an adipokine secreted by adipocytes it isclosely associated with amount of fat and distribution Asa chemoattractant protein chemerin acts as a ligand forthe coupled G-receptor protein (ChemR23) and participatesin both adaptive and innate immunity [11] In humanschemerin gene (RRARES2) is highly expressed in WAT andto a lesser extent in liver and lungs On immune cellschemerin is known to stimulate chemotaxis of dendriticcells macrophages and NK cells Meanwhile its recep-tor ChemR23 gene (CMKLR1) is expressed in dendriticcells monocytemacrophages and endothelial cells [11ndash14]ChemR23 is involved in the differentiation of adipocytesand increased intracellular glucose or lipids promote itsexpression [14]

The interaction of chemerinChemR23 has been shownto reduce cytokines chemokines and phagocytosis provingto be important in the inflammatory process associated withobesity [12 14 15]

In this context chemerinChemR23 axis has been shownto impact IR development which influences the clinicalcourse and severity of obesity-related diseases [10] How-ever the association with immunometabolic markers andchemerin and its receptor ChemR23 is scarce Therefore theaim of this study was to characterize the inflammatory andmetabolic phenotype of subjects with obesity-IR state basedon the chemerin soluble levels and its receptor CMKLR1expression

2 Material and Methods

21 Study Design In this cross-sectional study a total of134 adults aged 20 to 59 years were recruited from generalpopulation in the west of Mexico We included individualswho at the time of enrollment did not present glucose intol-erance infectious diseases hypertension pregnancy anemiacardiovascular disease malignancy and renal and metabolicdiseases such as type 2 diabetes mellitus (T2DM) Subjectswith current medication use were excluded

Subjects were classified in two forms based on obesityand then by IR First they were classified according to therecommendationsrsquo of World Health Organization by bodymass index (BMI) waist circumference (WC) waist-hip ratio(WHR) and waist to height ratio (WHtR) in two groupssubjects with obesity if any of the following conditions werepresent BMI ge 300 kgm2 WC ge 900 cmWHR ge 090 andWHtR ge 525 in Men and WC ge 800 cm WHR ge 080 andWHtR ge 530 in women and lean subjects that are lowerin these measurements For detection of IR in subjects with

obesity they were secondly classified according to Gayosocriteria in two groups with and without IR [16]

For ethical purposes participants were informed aboutthe study and signed a consent form following the Helsinkideclaration guidelines [17] and the institutional (GuadalajaraUniversity) review boardsrsquo committees

22 Subjectsrsquo Medical History and Physical Examination Allindividuals who satisfied inclusion criteria were clinicallyevaluated by a physician who performed a complete medicalhistory Assessment of general health status and vital signswere included blood pressure (measured 3 times with thesubject in the sitting position for 15 minutes before theevaluation) heart and respiratory rate and body temperature

23 Subjectsrsquo Body Fat Storage Measurements We evaluatedheight which was measured to the nearest 1mm by using astadiometer (Seca GmbH amp Co KG Hamburg Germany)body weight and total and trunk body fat mass (abso-lute kg and relative ) were determined by bioelectricalimpedance analysis (TANITA BC-418 Segmental Body Com-position Analyzer Tokyo JPN) to the nearest 01 kg WChip circumference (HC) and coronal abdominal diameterwere measured by using an anthropometric fiberglass tape(GULICK length 0ndash180 cm precision plusmn01 USA) At thelevel of the iliac crest (L4-5) sagittal abdominal diameterwas measured using a sliding-beam abdominal caliper (pre-cision plusmn01 cm Holtain Ltd Crosswell Crymych PembsSA41 3UF UK) with the patient lying in a supine positionon the examination table [18] Five measures of skinfoldthickness (ie abdominal bicipital tricipital subscapularand suprailiac) were obtained on the right side of the bodyby using a Harpenden skinfold caliper (opened 80mm withprecision of plusmn02mm constant pressure 10 gmm2 HoltainLtd Crosswell Crymych Pembs SA41 3UF UK) All thesemeasurements were carried out by the same Physician induplicate following the procedures recommended by anthro-pometric indicators measurement guide [19 20]

To determine obesity and adiposity indexes thefollowing calculations were used BMI kgm2 = weight(kg)height2 (m) WHtR = WC (cm)height (cm) [21]WHR = WC (cm)HC (cm) conicity index (CI) = WC(cm)0109radic(weight (kg)height (cm)) BFR = total bodyfat mass (kg)height (cm) total adipose area (TAA cm2) =WC24120587 visceral area (VA cm2) = 120587(WC2120587 minus abdominalskinfold)2 subcutaneous abdominal area (cm2) = TAA minusVA [18] visceral adiposity index (VAI) for males VAI =(WC3658 + (1896BMI))6(TG081)6(152HDLc) andfemales VAI = (WC3968 + (1886BMI)) 6(TG103)6(131HDLc) [22] homeostasis model assessment-insulin resist-ance (HOMA-IR) = [basal glucose mgdL times (basal insulin120583UImL)405] [23 24]

24 Metabolic Inflammatory Markers and Chemerin LevelsMeasurements Individuals included in the study were fast-ing 12 hours before the blood samples were taken allowingthem to clot at room temperature and then were centrifugedat 1509 RCF (Rotanta 460R Andreas Hettich GmbH amp Co



























3 Results










Insulin(120583UImL)




























4 Discussion




lowast

lowast

0

10

20

30

40

50M

agni

tude

(mm

)


lowastlowast





lowastlowast

lowastlowast




05

10

15

15

20

25

Mag

nitu

de





lowastlowast



0

1

2

25

80

120

160

Leve

ls

HOMA-IR(mmolL)


(120583UImL)

lowast

lowastlowast






100

200

300Se

rum

conc

entr

atio

n (m

gdL

)

lowastlowastlowast

lowast


(d) Lipid profile
















lowast


lowastlowast

0

5

10

15

Relat

ive e

xpre

ssio

n of

CMKL

R1(m

etho

d2minusΔΔ

C T)

(a)

P = 0032P = 0002


50

100

150

Solu

ble l

evels

of c

hem

erin

(ng

mL)

(b)
















5 Conclusions




Competing Interests




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































3 Results










Insulin(120583UImL)




























4 Discussion




lowast

lowast

0

10

20

30

40

50M

agni

tude

(mm

)


lowastlowast





lowastlowast

lowastlowast




05

10

15

15

20

25

Mag

nitu

de





lowastlowast



0

1

2

25

80

120

160

Leve

ls

HOMA-IR(mmolL)


(120583UImL)

lowast

lowastlowast






100

200

300Se

rum

conc

entr

atio

n (m

gdL

)

lowastlowastlowast

lowast


(d) Lipid profile
















lowast


lowastlowast

0

5

10

15

Relat

ive e

xpre

ssio

n of

CMKL

R1(m

etho

d2minusΔΔ

C T)

(a)

P = 0032P = 0002


50

100

150

Solu

ble l

evels

of c

hem

erin

(ng

mL)

(b)
















5 Conclusions




Competing Interests




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowast

lowast

0

10

20

30

40

50M

agni

tude

(mm

)


lowastlowast





lowastlowast

lowastlowast




05

10

15

15

20

25

Mag

nitu

de





lowastlowast



0

1

2

25

80

120

160

Leve

ls

HOMA-IR(mmolL)


(120583UImL)

lowast

lowastlowast






100

200

300Se

rum

conc

entr

atio

n (m

gdL

)

lowastlowastlowast

lowast


(d) Lipid profile
















lowast


lowastlowast

0

5

10

15

Relat

ive e

xpre

ssio

n of

CMKL

R1(m

etho

d2minusΔΔ

C T)

(a)

P = 0032P = 0002


50

100

150

Solu

ble l

evels

of c

hem

erin

(ng

mL)

(b)
















5 Conclusions




Competing Interests




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























lowast


lowastlowast

0

5

10

15

Relat

ive e

xpre

ssio

n of

CMKL

R1(m

etho

d2minusΔΔ

C T)

(a)

P = 0032P = 0002


50

100

150

Solu

ble l

evels

of c

hem

erin

(ng

mL)

(b)
















5 Conclusions




Competing Interests




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusions




Competing Interests




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article inverse relationship of the cmklr1...

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