Links Between Insulin Resistance, Adenosine A2B Receptors, and Inflammatory Markers in Mice and Humans.

1.2 MB·English

by  Figler, Robert A.

ORIGINALARTICLE
Links Between Insulin Resistance, Adenosine A
2B
Receptors, and Inflammatory Markers in Mice
and Humans
Robert A. Figler,1 Guoquan Wang,2 Susseela Srinivasan,1 Dae Young Jung,3 Zhiyou Zhang,3
James S. Pankow,4 Katya Ravid,5 Bertil Fredholm,6 Catherine C. Hedrick,1 Stephen S. Rich,7
Jason K. Kim,3 Kathryn F. LaNoue,5 and Joel Linden1
OBJECTIVE—Todeterminethemechanismsbywhichblockade A2BR signaling in diabetes increases insulin resistance in
part by elevating proinflammatory mediators. Selective A R
ofadenosineA receptors(A Rs)reduces insulinresistance. 2B
2B 2B blockers may be useful to treat insulin resistance. Diabetes
RESEARCH DESIGN AND METHODS—We investigated the 60:669–679,2011
effectsofdeletingorblockingtheA Roninsulinsensitivityusing
2B
glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic
clampsinmousemodelsoftype2diabetes.Theeffectsofdiabetes O
onA Rtranscriptionandsignalingweremeasuredinhumanand besity and insulin resistance are associated
2B
mousemacrophagesandmouseendothelialcells.Inaddition,tag with low-grade systemic inflammation. Proin-
singlenucleotidepolymorphisms(SNPs)in;42kbencompassing flammatory mediators produced in adipose
the A R gene, ADORA2B, were evaluated for associations with
2B tissue (adipokines) that increase insulin resis-
markersofdiabetesandinflammation.
tance include interleukin (IL)-6 (1), C-reactive protein
RESULTS—Treatment ofmice with thenonselective adenosine (CRP) (2), and plasminogen activator inhibitor 1 (PAI-1)
receptor agonist 59-N-ethylcarboxamidoadensoine (NECA) in- (3). In addition, insulin resistance due to a high-fat diet
creasedfastingbloodglucoseandslowedglucosedisposalduring causes macrophage accumulation in adipose tissue and
GTTs.TheseresponseswereinhibitedbyA Rdeletionorblock-
2B M2-like remodeling (4). Endothelial dysfunction is also
ade and minimally affected by deletion of A1Rs or A2ARs. Dur- a hallmark of diabetes because inflammatory mediators
ing hyperinsulinemic-euglycemic clamp of diabetic KKAY mice,
activate receptors and transcription factors such as nu-
A R antagonism increased glucose infusion rate, reduced he-
pa2Bticglucoseproduction,andincreasedglucoseuptakeintoskel- clear factor-kB, toll-like receptors, c-Jun NH2-terminal ki-
etalmuscleandbrownadiposetissue.Diabetescausedafour-to nase (JNK), and the receptor for advanced glycation end
sixfold increase in A R mRNA in endothelial cells and macro- products,whichcausesystemicendothelialdysfunction(5).
2B
phagesandresultedinenhancedinterleukin(IL)-6productionin Severalstudieshavelinkedadenosinereceptorblockade
responsetoNECAduetoactivationofproteinkinasesAandC. with reversal of insulin resistance. Challis et al. reported
Five consecutive tag SNPs in ADORA2B were highly correlated that adenosine receptor antagonists (6) or degradation of
with IL-6 and C-reactive protein (CRP). Diabetes had a highly
adenosine with adenosine deaminase (7) reverse insulin
significant independent effect on variation in inflammatory
markers.ThestrengthofassociationsbetweenseveralADORA2B resistanceinskeletalmuscleisolatedfromdiabeticanimals.
SNPsandinflammatorymarkerswasincreasedwhenaccounting After a lengthy delay before the development of bioavail-
fordiabetesstatus. able adenosine receptor antagonists, the A1/A2B orally ac-
tiveantagonist,BW-1433,wasfoundtopersistently reverse
CONCLUSIONS—Diabetesaffectstheproductionofadenosine
insulin resistance in obese insulin-resistant Zucker rats
atinodn,thineseuxlipnreresssiiostnanocfeA,2aBnRdstthheatasstsimocuialattioenILb-6etawnedeCnRAPDpOrRoAdu2cB- (8–10). In these early studies, the effects of adenosine re-
SNPs and inflammatory markers. We hypothesize thatincreased ceptorantagonistswereattributedtoblockadeofA1Rs.This
idea was corrected when moderately selective blockers
oftheA Rwerefoundtolowerglucose levelsindiabetic
2B
mice, an effect that could not be replicated with the se-
Fromthe1CardiovascularResearchCenter,UniversityofVirginia,Charlottes- lective A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine
ville, Virginia; the 2Adenosine Therapeutics Group of PGxHealth, Clinical (11). In mice rendered insulin resistant due to a high-fat
Data Incorporated, Charlottesville, Virginia; the 3Department of Cellular diet, ADORA2B gene deletion results in reduced adiposity,
andMolecularPhysiology,PennsylvaniaStateUniversityCollegeofMedi-
cine, Hershey, Pennsylvania; the 4Department of Epidemiology and Com- reduced liver glycogen, increased energy expenditure, and
munity Health, University of Minnesota, Minneapolis, Minnesota; the increased lean body mass (12).
5Department of Biochemistry, Boston University, Boston, Massachusetts; In the current study we confirm that A R activation
the 6Department of Physiology and Pharmacology, Karolinska Institutet, 2B
Stockholm,Sweden;andthe7CenterforPublicHealthGenomics,University stimulatesIL-6productioninmacrophagesandendothelial
ofVirginia,Charlottesville,Virginia. cells (ECs) and show that these effects are enhanced in
Correspondingauthor:JoelLinden,[email protected].
cells derived from diabetic animals. Blockade of A Rs in
Received28July2010andaccepted26October2010. 2B
DOI:10.2337/db10-1070 diabetic mice reduces hepatic glucose production (HGP)
This article contains Supplementary Data online at http://diabetes. and enhances glucose disposal into skeletal muscle and
J.Kd.iKa.biestecsujorruernntalyls.aofrfigl/ilaotoedkuwpi/tshupthpel/dPorio:1g0r.a2m33i7n/dMb1o0le-1c0u7la0r/-/MDeCd1i.cine,Univer- brown adipose tissue. In addition, diabetes influences the
sityofMassachusettsMedicalSchool,Worcester,Massachusetts. association of single nucleotide polymorphisms (SNPs) in
(cid:1)2011bytheAmericanDiabetesAssociation.Readersmayusethisarticleas ADORA2B with IL-6 and CRP. These findings suggest that
longastheworkisproperlycited,theuseiseducationalandnotforprofit, diabetes and one or more SNPs in ADORA2B influence
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/fordetails. proinflammatory A2BR signaling.
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 669
ADENOSINEA RECEPTORSANDINSULINRESISTANCE
2B
RESEARCHDESIGNANDMETHODS compared with that obtained from the simulations to define the empiric
RT-PCR. Total RNA was isolated from ECs or macrophages using TRIzol Pvalue.Thisapproachwasalsousedwithindiabetic/nondiabeticclustersfor
(Invitrogen, Carlsbad, CA)accordingtothemanufacturer’sprotocol.Sense/ evaluation of SNP-diabetes interaction. The permutations were performed
antisense mouse PCR primers were KC 59-cttgaaggtgttgccctcag-39/59-tgggga- withinPLINKusingthemax(T)option.Thiseffectivelyteststheappropriate
caccttttagcatc-39; IL-6 59-ctgatgctggtgacaaccac-39/59-tccacgatttcccagagaac-39; distributional assumptions of the analyses; should the distributions of the
A R 59-tggcttggtgacgggtatg-39/59-cgcaggtctttgtggagttc-39; and A R 59-ctgg- phenotypes deviate significantly from normality, we would expect the per-
ga2cAacgagcgagag-39/59-gctggtggcactgtctttac-39. Sense/antisense h2uBman PCR muted P values to be far from those observed. In our case, the permuted
primerswereA R59-agttccgccagaccttcc-39/59-acctgctctccgtcactg-39;A R59- Pvaluesareconsistentwiththoseobserved.
2A 2B
ggtcattgctgtcctctg-39/59-ttcattcgtggttccatcc-39.
Isolation and culture of human macrophages. Heparinized blood was RESULTS
collectedfromhealthyanddiabeticvolunteersinaccordancewithguidance
CharacterizationofanovelselectiveA Rantagonist
fromtheUniversity ofVirginiaInstitutionalReview Board.Monocyteswere 2B
isolatedusingRosetteSephumanmonocytesenrichmentcocktail(StemCell ATL-692. Several potent and selective antagonists of the
Technologies,Tukwila,WA)andplatedinatissueculturedishinDulbecco’s A R such as MRS-1754 have been described (17). In
2B
modifiedEagle’smedium(DMEM)with10%autologousserumand10ng/ml general these compounds have poor aqueous solubility,
humanmacrophagecolony-stimulatingfactorfor3days.TotalRNAwasiso- poor bioavailability, and are less potent and selective at
latedfromthedifferentiatedmacrophagesusingTRIzolreagent(Invitrogen).
rodent than at human A Rs. We have recently described
cDNAwassynthesizedwithIscriptcDNAsynthesiskit(Bio-Rad)using1mgof 2B
totalRNA.ExpressionofA2AR,A2BR,andb-actinmRNAlevelsweremeasured ATL-801 as a selective A2B blocker with improved water
byquantitativeRT-PCR. solubilityusefulforinvivostudies(18).Figure1showsthe
Transgenicmice.TheUniversityofVirginiaAnimalCareandUseCommittee chemical structure and binding characteristics of a new
approvedanimalstudies.Micewithadenosinereceptordeletionsusedinthis antagonist, ATL-692, with greater potency and selectivity
study were congenicto C57BL/6 andwere created asdescribed previously: than ATL-801. The synthesis and pharmacological char-
AR2/2(13),A R2/2(14),andA R2/2(15).SomestudiesuseddiabeticB6.
1 2A 2B acterization of ATL-692 is described in Supplementary
Cg-m+/+Leprdb/J(db/db)withnondiabeticC57BL/6JcontrolsordiabeticKK.
Cg-Ay/J(KK-Ay)withlessdiabeticKK/1HJ(KK-a/a)controlsasidentifiedin data. In competition for radioligand binding to recombi-
the figure legends. Feeding a high-fat diet (55% calories from fat, Harlan nant adenosine receptors, ATL-692 is .400-fold selective
TD93075 6 125 mg/kg ATL-801) for 10 weeks was used to produce insulin fortheA2BRovertheotherrecombinanthuman,mouse,or
resistanceinC57BL/6mice. ratadenosinereceptorsubtypes.However,relativetoATL-
Invivoassessmentofinsulinsensitivity.A2-hhyperinsulinemic-euglycemic 801, ATL-692 has 103 lower aqueous solubility (3 vs. 30
clampwasperformedinconsciousmicetoassessinsulinactionandglucose mg/ml) and 53 lower oral bioavailability in rats (13 vs.
metabolisminindividualorgans.At4to5daysbeforeclampexperiments,mice
73%).Hence,weusedATL-692asthepreferredcompound
wereanesthetized,andanindwellingcatheterwasinsertedintherightinternal
for in vitro studies, whereas ATL-801 as preferred for in
jugular vein. On the day of clamp experiments, a three-way connector was
attachedtothecathetertointravenouslydeliversolutions(e.g.,glucose,in- vivo studies.
sulin).Afterovernightfast(;15h),a2-hhyperinsulinemic-euglycemicclamp Effect of A R deletion or blockade on glucose
2B
was conducted in conscious mice with a primed (150 mU/kg body wt) and metabolism. In previous studies, 2-alkynyl-8-aryladenine
continuousinfusionofhumanregularinsulin(Humulin;EliLilly,Indianapolis, adenosine antagonists with selectivity for the A R sub-
IN)atarateof2.5mU/kg/mintoraiseplasmainsulinwithinaphysiological 2B
type have been reported to have hypoglycemic activity in
range. Blood samples (20 ml)were collected at 20-min intervals for theim-
the KK-AY mouse model of type 2 diabetes (11). We ex-
mediatemeasurementofplasmaglucoseconcentration,and20%glucosewas
infused at variable rates to maintain glucose at basal concentrations. Basal aminedtheeffectsofATL-801oninsulinsensitivityduring
and insulin-stimulated whole body glucose turnover were estimated with
acontinuousinfusionof[3H]glucose(PerkinElmer,Boston,MA)for2hbefore
theclamps(0.05mCi/min)andthroughouttheclamps(0.1mCi/min),respec-
tively. All infusions were performed using the microdialysis pumps (CMA/
Microdialysis,NorthChelmsford,MA).Toestimateinsulin-stimulatedglucose
uptake in individual tissues, 2-deoxy-D-[1–14C]glucose was administered as
abolus(10mCi)at75minafterthestartofclamps.Bloodsamplesweretaken
before,during,andattheendofclampsforthemeasurementofplasma[3H]
glucose,3HO,2-deoxy-D-[1–14C]glucoseconcentrations,and/orinsulinconcen-
2
trations.Attheendoftheclamps,micewerekilledandtissuesweretakenfor
biochemical andmolecular analysis. KKAY mice were treated with 20 mg/kg
ATL-801administeredbyoralgavagefourtimesat12-hintervalswiththelast
dosegiven90minbeforetheclamp.
Association of human ADORA2B SNPs with phenotypic markers. The
Multi-EthnicStudyofAtherosclerosis(MESA)isaprospectivecohortstudy
designedtostudytheprogressionofsubclinicalcardiovasculardisease,con-
sistingof6,814menandwomenaged45–85yearswhowerefreeofclinical
cardiovasculardiseaseatentry.TheparticipantswererecruitedfromsixU.S.
communities.Thesamplingprocedureshavebeendescribedpreviously(16),
andtheprotocolandresearchmethodsareavailableontheMESAWebsite
(http://www.mesa-nhlbi.org).Asubcohortof2,880MESAsubjects(720ineach
of the four ethnic groups) was randomly selected from subjects who gave
informed consent for genetic studies. All phenotypic data reported in this
studywerecollectedatthefirstMESAexaminationaccordingtoDeclaration
ofHelsinkiprinciples.Detailsofphenotyptingandgenotypingproceduresare
describedinSupplementaryData.
EvaluationofSNP-diabetesinteractions.ForallSNPs,genotype-specific
meansandvariancesforeachquantitativephenotypewereestimatedoverall FIG. 1. Structure and adenosine receptor binding characteristics of
andwithinstrata(ethnicgroupanddiabetesstatus).EmpiricPvalueswere ATL-692.KivaluesforATL-692atrat,mouse,andhuman(h)adenosine
receptorsareexpressedasmeannM6SE(N=3)andwerecalculated
determined by permutation. A label-swapping approach was used, in which
from the half-maximal inhibitory concentration (IC ) of ATL-692 to
eachSNPgenotypeispermutedforeachphenotype(homeostasismodelas- 50
compete for radioligand binding to recombinant receptors on human
sessment[HOMA],IL-6,CRP,solubleIL-2receptor[IL-2sR],andPAI-1)within embryonickidney(HEK)293cellmembranes.Theradioligandsusedwere
eachofthefourclustersdefinedbyethnicgroup.Atotalof5,000permutations 125I-ABA(ARandA R),125I-ZM241385(A R),and125I-ABOPX(A R).
1 3 2A 2B
were performed for each SNP-phenotype, and the observed statistic was Bindingisplottedasfractionofcontrolspecificbinding.
670 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
R.A.FIGLERANDASSOCIATES
hyperinsulinemic-euglycemic clamps in KKAY mice. Body We reasoned that if insulin resistance occurs as a con-
weight and basal plasma glucose levels were not affected sequenceofA Ractivation,injectingmicewiththestable
2B
by short-term (2 day) ATL-801 treatment (Fig. 2A and B). nonselective adenosine receptor agonist 59-N-ethyl-
During the clamp, plasma glucose levels were maintained carboxamidoadensoine(NECA)shouldactivateadenosine
at ;7 mmol/L (Fig. 2C). Steady-state rates of glucose receptor–mediated effects and also inhibit glucose dis-
infusion to maintain euglycemia during clamps were posal.Figure3AshowsthatoralgavagewithNECA35min
significantly elevated in ATL-801–treated KKAY mice as before an oral glucose tolerance test (GTT) in wild-type
comparedwithuntreatedKKAYcontrols(Fig.2D).Insulin- fasted mice results in a substantial delay in glucose dis-
stimulated glucose uptake in skeletal muscle and brown posal during GTT. These effects of NECA were somewhat
adiposetissuewereincreasedby20to;50%inKKAYmice attenuated in mice lacking A or A receptors but were
1 2A
(Fig.2EandF).BasalandclampHGPratesweremarkedly almost completely abolished in mice lacking the A R.
2B
reducedinATL-801–treatedKKAYmice,resultingina30% Moreover, NECA significantly increased fasting glucose
increaseinhepaticinsulinaction(Fig.2G–I).Insum,ATL- levels in C57BL/6 but not in A R2/2 mice (Fig. 3B). In-
2B
801 treatment of diabetic mice increased insulin action in sulin resistance in response to NECA was associated with
liver and increased glucose uptake in skeletal muscle and an increase in plasma IL-6 measured 4 h after NECA ad-
brown adipose tissue. ministration (Fig. 3C). Genetic ablation of the A R has
2B
FIG.2.ATL-801treatmentincreasesinsulinsensitivityinKKAYmice.ATL-801wasadministeredbyoralgavage4timesat12-hinternalswiththe
lastdosegiven4hbeforeclamp.A:BodyweightchangeduringATL-801administration.B:Basalplasmaglucoselevels.C:Plasmaglucoselevels
duringclamps.D:Steady-stateglucoseinfusionratesduringclamps.E:Insulin-stimulatedskeletalmuscleglucoseuptake.F:Insulin-stimulated
glucoseuptakeinbrownadiposetissue.G:BasalHGP.H:HGPduringclamps(insulin-stimulatedstate). I:Hepaticinsulinactionreflectedas
insulin-mediatedpercentsuppressionofbasalHGP.*P,0.05bytwo-tailedStudentttest;N=7.
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2B
FIG.3.A R-mediatedregulationofglucosemetabolism.A:C57BL/6Jandadenosinereceptorknockoutmice(male,8weeks,N=5)receivedan
2B
oralbolusofvehicleorNECA(0.3mg/kg)attime235.Attime0,miceweresubjectedtoanGTT(1.25g/kgip).Attheindicatedtimepoints,blood
glucoselevelsweredeterminedusingaOneTouchUltraglucometer(LifeScan,Milpitas,CA)andareaunderthecurve(AUC)wascalculatedusing
GraphPadPRISMsoftware.*P,0.0001.B:Fastingglucoselevelsweremeasuredinwild-typeandA R2/2mice35minafterNECAgavage(N=5).
2B
C:PlasmaIL-6wasmeasured4hafterC57BL/6JmicereceivedvehicleorNECA.D:C57BL/6micewerefedahigh-fatdiet610mg/kg/dayofATL-
801for10weeks.Bloodglucosewasmeasuredaftera4-hfast(N=5).Con,control.
previously been shown to dramatically reduce the ability increaseinIL-6mRNAandamoresustainedstimulationof
ofNECAtoraiseIL-6plasmalevelsinvivoandtoabrogate IL-6 production. NECA also stimulates the production of
NECA-induced IL-6 release from mouse peritoneal mac- themurineIL-8homolog KC(Fig.4CandD).As shown in
rophages (19). The addition of the orally active A R an- Fig. 4E, A R mRNA in ECs from diabetic mice(db/dbor
2B 2B
tagonist ATL-801 (10 mg/kg/day) to high-fat mouse diet KKAY) is increased six- to sevenfold compared with ECs
(55% of calories from lipid) for 10 weeks significantly re- derivedfromage-andsex-matchedcongeniccontrols.This
duced diet-induced elevated fasting blood glucose (Fig. induction is associated with a shift to the left in the dose
3D). The results indicate that the effects of adenosine re- responsecurveforNECA-inducedIL-6productionandan
ceptor antagonists to reduce insulin resistance are medi- increase in the maximal response (Fig. 4F).To determine
ated by blockade of the A R subtype. In addition, the whetherdiabetescausesinductionofA RmRNAinhuman
2B 2B
results suggest that endogenous levels of adenosine in di- tissues, we prepared macrophages from the monocytes of
abetic animals are sufficient to activate A Rs. diabetic and nondiabetic individuals. Monocyte pop-
2B
NECA causes induction of cytokine transcripts in ulations readily differentiate into macrophages in tissue
ECsand macrophages.Becauseendothelial activation is and,duringthedifferentiationprocess,retaintheirgenetic
a hallmark of insulin resistance, we sought to determine identity (20). These macrophages, and not their mono-
whetherNECAcausesinductionofcytokinetranscriptsin cyte precursors, are the culprits in inflammation and
ECs and whether the response is influenced by diabetes. disease (21). As shown in Fig. 4G, diabetes is associated
As shown in Fig. 4A and B, NECA triggers a transient with increased expression of A R mRNA inmacrophages
2B
672 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
R.A.FIGLERANDASSOCIATES
FIG.4.A RtranscriptionandfunctioninECsandmacrophages.Timecoursesofresponsesto1mmol/LNECAinmouseaorticECsareIL-6mRNA
2B
(A),IL-6protein(B),KCmRNA(C),andKCprotein(D).E:EffectofdiabetesintwostrainsofmiceonECA RmRNArelativetocongenic
2B
nondiabetic controls. *P , 0.05. F: Effect of diabetes on the dose-response curve of NECA to stimulate IL-6 production in ECs at 24 h. The
concentrationsofNECAthatproduce50%ofmaximalresponses(EC )are140nmol/LC57BL/6and46nmol/Ldb/db.G:A RmRNAinmonocyte-
50 2B
derivedmacrophagespreparedfromcontrolsordiabetichumandonors(N=6).H:IL-6productioninC57BL/6(N=3)orKKAY(N=6)mouse
peritonealmacrophagestreatedinvitrofor24hwithvehicle(Veh),1mmol/LNECA,orNECA+1mmol/LATL-692.*P,0.01.
derived from human monocytes. We also examined the ef- near0intheabsenceorpresenceofNECA.Thesefindings
fect of diabetes on NECA-stimulated IL-6 production in suggest that constitutive A R activity or constitutive pro-
2B
mouse peritoneal macrophages in vitro. As shown in Fig. duction of adenosine by ECs stimulates low-level cytokine
4H, diabetes significantly increases A R-mediated IL-6 productioninvitro.Thismayalsooccurinvivowherelocal
2B
production in mouse macrophages. adenosine production in response to shear stress, platelet
Characterization of adenosine receptors on mouse activation, or nerve activation likely stimulates endothelial
aortic ECs. There are differences in the adenosine recep- A Rs and cytokine production. As further confirmation
2B
tors found on ECs in various vascular beds. To pharmaco- that A Rs mediate the effects of NECA in mouse aortic
2B
logically evaluate the adenosine receptor subtype that ECs, agonists of adenosine receptors subtypes added at
mediatescytokinereleasefrommouseaorticECs,weused doses sufficiently low to exert receptor subtype selectivity
100 nmol/L FSPTP, a highlyselective A R antagonist(22), (CPA,A ;CGS21680,A ;andCl-IB-MECA,A )werefound
2A 1 2A 3
and 1 mmol/L ATL-692, a highly selective A R antagonist to be without effect on IL-6 production (Fig. 5C).
2B
(Fig.1).TheA RisprimarilycoupledtoG ,whiletheA R SNPs in ADORA2B. Having established a relationship
2A s 2B
is dually coupled to G and to G (23). Consistent with G between diabetes, A R mRNA induction, and cytokine
s q s 2B
coupling,NECAcausedarapidincreaseincyclicAMPthat production in mice, we examined SNPs in the A R gene,
2B
isblockedby1mmol/LATL-692butnotaffectedbyFSPTP ADORA2B, in 2,847 subjects from the MESA for associa-
(Fig. 5A). Thus, although A Rs are found on some ECs, tionsbetweenreceptorSNPsanddiabetesorinflammation.
2A
cyclicAMPaccumulationinmouseaorticECsisexclusively Table 1 lists by SNP genotypethe means for HOMA-insulin
mediatedbyA Rs.Weusedkinaseinhibitorstoinvestigate resistance (HOMA-IR) and inflammatory adipokines, ad-
2B
signaling downstream of A R activation in ECs. Both justedforcovariates. The minoralleles ofADORA2B SNPs
2B
Gö6976, an inhibitor of PKC, and KT5720, an inhibitor of (theallelewiththelowerfrequencyandthusconsideredthe
PKA,significantlyinhibitedIL-6mRNAinductionbyNECA, variant allele) are listed first in the table. For five consec-
and the combination of the two agents had an additive ef- utivetagSNPsnumbered2–5inthetable,thereisastriking
fect(Fig.5B).ThusbothPKAandPKCappeartocontribute association of allelic genotype (homozygous minor, het-
to induction of IL-6 in response to A R activation. In ECs erozygote, homozygous major)withplasmaconcentrations
2B
derived from A R2/2 mice, IL-6 release was reduced to ofIL-6andCRP.AmongthesametagSNPs,therelationship
2B
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2B
FIG.5.NECAincreasesIL-6productioninmouseaorticECsbyactivatingA Rs.A:TimecourseofNECAtoincreasecyclicAMPinECsfrom
2B
C57BL/6mice,andtheeffectsofantagonists,100nmol/LFSPTP,or1mmol/LATL-692(N=6).B:InductionofIL-6mRNAbyNECAinECsis
attenuatedbyinhibitorsofPKC(1mmol/LGö6976)orPKA(1mmol/LKT5720).#P,0.05vs.noinhibitors.Thecombinationofinhibitorsismore
effectivethaneitheralone(N=6,*P,0.01).C:ECsfromC57BL/6JandA 2/2micewerestimulatedfor16hwithNECA(100nmol/L)6ATL-692.
IL-6productionintheA R2/2ECsissignificantlylowerthanbasalC57BL2/B6JECs(#P,0.001).D:ECsweretreatedfor16hwith10nmol/LCPA
2B
(A Ragonist),100nmol/LCGS21680(A Ragonist),10nmol/LCl-IBMECA(A Ragonist),or100nmol/LNECA(nonspecificagonist).Mediawere
1 2A 3
collectedandassayedforIL-6byELISA(N=6).*P,0.01vs.othertreatments.
is inverted for IL-2sR. These findings indicate that minor ancestry informative markers). Among patients with di-
allele frequency in ADORA2B SNPs influences the expres- abetes,significantassociationsbetweenoneormoreSNPs
sion of inflammatory markers in the MESA population. andallfourmarkersofinflammationwerenoted.Inseven
EffectofdiabetesstatusonassociationofADORA2B instancesdenotedinthetable,thereisa.10-folddecrease
SNPs with inflammation markers. In models that in- in the P value of SNP associations with inflammatory
cludeddiabetesasanindependentpredictorofvariationin phenotypes in patients with diabetes compared with non-
inflammatory markers, the diabetes effect was highly sig- diabetics.
nificant (P (cid:1) 10217) for all ADORA2B SNPs IL-6, CRP, or
IL-2sR. We further evaluated the effect of diabetes on the
associations between individual ADORA2B SNPs and DISCUSSION
markers of inflammation. Within patients with diabetes IL-6, CRP, and PIA-1 are all adipokines, i.e., proin-
and nondiabetics, clusters were defined to test for homo- flammatory mediators produced in adipose tissue, that
geneity of SNP association with each phenotype using the have been associated with diabetes (24). Inflammation in
Cochrane-Mantel-Haenszel approach. Analyses of associ- diabetes may be triggered in part by elevated concen-
ation between ADORA2B SNPs with individual MESA trations of free fatty acids that increase CD11c+ macro-
phenotypes are shown in Table 2, adjusted for covariates phage accumulation and activation in adipose tissue (25).
(age, sex, ethnicity, site of ascertainment, smoking) and The results of this study suggest that adenosine signaling
populationadmixture(firstfiveprincipalcomponentsfrom through the A R also contributes to insulin resistance by
2B
674 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
R.A.FIGLERANDASSOCIATES
TABLE1
Genotypicmeans6SDsofADORA2BSNPsforMESAphenotypes,combinedethnicgroups
SNP Site Genotype HOMA-IR ln(IL-6)(pg/mL) ln(CRP)(pg/mL) ln(IL-2sR)(pg/mL) PAI-1(ng/mL)
rs7225585 59 A/A 2.22(2.69) 0.15(0.62) 0.68(0.93) 20.33(0.20) 2.73(0.92)
(1)* G/A 2.00(1.90) 0.33(0.66) 0.89(1.17) 20.24(0.39) 2.76(0.84)
G/G 1.89(2.09) 0.15(0.68) 0.52(1.18) 20.12(0.37) 2.93(0.92)
rs2779193 59 A/A 2.17(2.15) 0.31(0.65) 0.90(1.02) 20.34(0.37) 2.84(0.87)
(2) G/A 2.06(2.16) 0.29(0.72) 0.78(1.21) 20.25(0.34) 2.77(0.90)
G/G 1.87(2.07) 0.14(0.66) 0.52(1.17) 20.11(0.37) 2.93(0.91)
rs758857 intron1 A/A 2.02(2.14) 0.22(0.69) 0.63(1.15) 20.26(0.38) 2.74(0.85)
(3) G/A 1.97(2.05) 0.17(0.69) 0.59(1.22) 20.16(0.36) 2.92(0.91)
G/G 1.82(2.11) 0.15(0.66) 0.53(1.16) 20.09(0.37) 2.94(0.92)
rs758858 intron1 A/A 1.95(1.33) 0.36(0.68) 0.88(1.17) 20.34(0.32) 2.69(0.81)
(4) G/A 2.02(2.08) 0.27(0.71) 0.77(1.18) 20.26(0.35) 2.72(0.88)
G/G 1.89(2.11) 0.14(0.66) 0.52(1.18) 20.11(0.37) 2.94(0.91)
rs2041458 intron1 A/A 1.94(1.86) 0.31(0.66) 0.86(1.11) 20.32(0.32) 2.68(0.86)
(5) C/A 2.08(2.14) 0.28(0.69) 0.80(1.17) 20.22(0.35) 2.78(0.88)
C/C 1.87(2.10) 0.13(0.67) 0.48(1.18) 20.11(0.38) 2.95(0.92)
rs8069362 intron1 A/A 1.90(1.72) 0.30(0.67) 0.94(1.14) 20.33(0.37) 2.52(0.80)
(6) G/A 2.04(2.03) 0.30(0.65) 0.89(1.10) 20.27(0.34) 2.79(0.87)
G/G 1.89(2.11) 0.14(0.68) 0.51(1.19) 20.12(0.37) 2.93(0.92)
rs17715109 intron1 A/A 1.45(0.90) 20.08(0.40) 20.14(1.03) 20.30** 2.30**
(7) C/A 1.96(1.86) 0.12(0.70) 0.49(1.15) 20.23(0.28) 3.02(0.96)
C/C 1.91(2.12) 0.18(0.67) 0.59(1.19) 20.13(0.38) 2.89(0.90)
rs2015353 intron1 G/G 1.86(2.91) 0.21(0.64) 0.73(1.11) 20.05(0.41) 2.85(0.95)
(8) A/G 1.97(1.77) 0.23(0.66) 0.69(1.19) 20.14(0.35) 2.86(0.92)
A/A 1.88(2.00) 0.09(0.70) 0.40(1.18) 20.20(0.36) 3.00(0.86)
rs2779211 intron1 G/G 1.86(3.11) 0.19(0.65) 0.71(1.12) 20.02(0.41) 2.88(0.97)
(9) A/G 1.96(1.78) 0.23(0.66) 0.67(1.19) 20.12(0.36) 2.86(0.92)
A/A 1.89(1.95) 0.12(0.69) 0.46(1.18) 20.21(0.36) 2.96(0.87)
rs1045599 39 G/G 1.85(1.98) 0.06(0.70) 0.31(1.18) 20.15(0.37) 3.00(0.81)
(10) A/G 1.95(1.87) 0.19(0.67) 0.63(1.19) 20.15(0.36) 2.90(0.94)
A/A 1.92(2.51) 0.23(0.65) 0.74(1.14) 20.12(0.39) 2.84(0.93)
rs2286795 39 G/G 1.84(2.03) 0.04(0.69) 0.25(1.16) 20.15(0.38) 2.99(0.83)
(11) A/G 1.89(1.69) 0.17(0.66) 0.60(1.19) 20.13(0.36) 2.93(0.94)
A/A 2.00(2.51) 0.26(0.67) 0.77(1.14) 20.15(0.39) 2.82(0.92)
Minor(variant)allelesarelistedfirst.Thers7225585(1)throughthers758857(3)rowsoftheHOMA-IRcolumn,thers2779193(2)throughthe
rs8069362 (6) rows of the ln(IL-6) and ln(CRP) columns, and the rs2015353 (8) and rs2779211 (9) rows of the ln(CRP) column indicate
ADORA2BSNPsinwhichthehomozygousminor,heterozygous,andhomozygousmajorallelesareassociatedwithhightolow(high,medium,
andlowplasmainflammatorymarkermeansIL-6,CRP,orPAI-1).Inthers7225585(1)throughrs17715109(7)rowsoftheln(IL-2sR)column,
theorderofassociationisreversed,fromlowtohigh.*SequentiallynumberedtagSNPsreferredtointhetext.**Smallsamplesizeforthis
allele.
altering the production of IL-6 and other cytokines. IL-6 is a DNA region statistically associated with the SNP, influ-
produced primarily by macrophages and adipocytes and ences either the function or expression of the gene prod-
drivestheproductionofCRP,anacute-phasereactantthat uct. Because the current study was not a genome-wide
rises dramatically during inflammatory processes. We association study, it was not subject to large type 1 error,
demonstrate six types of associations between diabetes/ i.e., the false apparent associations that can occur when
insulinresistanceandA Rs:1)diabetesisassociatedwith largenumbersofgenesareanalyzed.Thegenotypicmeans
2B
elevatedA RmRNAexpressioninECsandmacrophages, (minorhomozygote,heterozygote,andmajorhomozygote)
2B
2) diabetes is associated with elevated A R-mediated of five adjacent ADORA2B SNPs (numbered 2–6 in Table
2B
cytokine production in ECs and macrophages, 3) A R 1)arecorrelatedwithincreasedlevelsofIL-6andCRPand
2B
activation in mice elevates fasted blood glucose levels, decreased levels of IL-2sR. This compelling pattern
4) A R activation in mice inhibits whole body glucose stronglysuggeststhatoneoftheseSNPsoranotherSNPin
2B
disposal, 5) A R blockade inhibits high-fat diet–induced linkage disequilibrium is involved in regulating the func-
2B
blood glucose elevation, and 6) A R blockade inhibits tion or expression of the A R. Our analysis does not en-
2B 2B
diabetes-induced insulin-resistance during hyperinsulinemic- able us to identify which SNP is responsible for altered
euglycemic glucose clamp. Our findings suggest that A R receptorexpressionorfunction.Therehavebeenprevious
2B
blockers may combat insulin resistance by impairing HGP attempts to associate particular SNPs in adenosine re-
and by attenuating the production of IL-6 and other ceptors with diseases. One such study failed to associate
cytokines that influence glucose and fat metabolism. coding SNPs in ADORA2B with cystic fibrosis (26). In an
AssociationofADORA2BSNPsandproinflammatiory investigationofalladenosinereceptorgenes,aSNPinthe
mediators. SNP analysis seeks to identify significant 39-UTR of ADORA1 was associated with increased sus-
associations between gene sequences and phenotypes. If ceptibility to aspirin-intolerant asthma (AIA), whereas
asignificantassociationisfound,itcanthenbeconcluded another SNP in the coding region was associated with
that the SNP polymorphism, or a nearby polymorphism in decreased susceptibility. The functional consequences of
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 675
ADENOSINEA RECEPTORSANDINSULINRESISTANCE
2B
TABLE2
Association ofADORA2B SNPsandinflammatoryphenotypesbydiabetesstatus
Pvalues
SNP Site ln(IL-6) ln(CRP) ln(IL-2sR) PAI-1
rs7225585
Nondiabetics 59 0.042‡(0.043)* 0.215(0.211) 0.629(0.627) 0.333(0.329)
Patients withdiabetes 0.294(0.288)† 0.526(0.529) 0.055(0.056) 0.315(0.322)
rs2779193
Nondiabetics 59 0.782(0.784) 0.844(0.841) 0.177(0.169) 0.785(0.787)
Patients withdiabetes 0.607(0.607) 0.690(0.688) 0.550(0.550) 0.108(0.108)
rs758857
Nondiabetics intron1 0.198(0.189) 0.189(0.189) 0.189(0.182) 0.033‡ (0.037)
Patients withdiabetes 0.936(0.935) 0.427(0.428) 0.535(0.539) 0.228(0.230)
rs758858
Nondiabetics intron1 0.859(0.853) 0.508(0.502) 0.043‡(0.050) 0.153(0.165)§
Patients withdiabetes 0.284(0.273) 0.950(0.942) 0.448(0.449) 0.015‡ (0.014)§
rs2041458
Nondiabetics intron1 0.766(0.761) 0.879(0.876) 0.185(0.194) 0.071(0.074)
Patients withdiabetes 0.241(0.231) 0.839(0.836) 0.051(0.048) 0.044‡ (0.040)
rs8069362
Nondiabetics intron1 0.755(0.759) 0.328(0.333) 0.317(0.324)§ 0.252(0.251)
Patients withdiabetes 0.079(0.079) 0.813(0.807) 0.009(0.010)§ 0.033‡ (0.034)
rs17715109
Nondiabetics intron1 0.709(0.714) 0.945(0.943) 0.760(0.755) 0.380(0.377)
Patients withdiabetes 0.835(0.843) 0.230(0.230) 0.779(0.770) 0.452(0.455)
rs2015353
Nondiabetics intron1 0.463(0.463)§ 0.721(0.716)§ 0.081(0.085) 0.443(0.456)
Patients withdiabetes 0.047‡(0.047)§ 0.001‡ (0.001)§ 0.154(0.148) 0.472(0.466)
rs2779211
Nondiabetics intron1 0.261(0.253) 0.469(0.456)§ 0.216(0.221) 0.731(0.729)
Patients withdiabetes 0.112(0.113) 0.003‡ (0.002)§ 0.825(0.825) 0.534(0.526)
rs1045599
Nondiabetics 39 0.499(0.495) 0.372(0.364) 0.584(0.590) 0.763(0.755)
Patients withdiabetes 0.059(0.058) 0.050‡ (0.049) 0.496(0.490) 0.454(0.454)
rs2286795
Nondiabetics 39 0.303(0.303) 0.390(0.388)§ 0.936(0.938) 0.886(0.889)§
Patients withdiabetes 0.235(0.239) 0.009‡ (0.008)§ 0.550(0.544) 0.038‡ (0.038)§
*P values from the additive (1 df) model in nondiabetic subjects, adjusted for age, sex, center of ascertainment, pack-years smoking, and
ancestry(thefirstfiveprincipalcomponentsfrom200AIMs),Bonferroniadjusted(numberinparenthesisrepresentstheempiricPvalue).
†Pvaluesfromtheadditive(1df)modelindiabeticsubjects,adjustedforage,sex,ethnicity,centerofascertainment,pack-yearssmoking,and
ancestry(thefirstfiveprincipalcomponentsfrom200AIMs),Bonferroniadjusted(numberinparenthesisrepresentstheempiricPvalue).
‡P,0.05.§Pvalueinnondiabeticsis.103patientswithdiabetes.
particular variants were not defined. Other SNPs in aden- production in response to endotoxin (15). Thus,insome
osine deaminase, ADORA2A, ADORA2B, and ADORA3, settings, signaling via the A R reduces inflammation. On
2B
were not significantly associated with AIA (27). Recently, the other hand, in this and several previous studies, acti-
therehasbeenanexplosionofgenome-wideandcandidate vation of A Rs increased IL-6 plasma levels in mice, and
2B
geneassociationofSNPswithbothdiseaseandquantitative by several types of isolated cells (28), including macro-
(associated) phenotypes. Despite early expectations that phages (19) and dendritic cells (19,29). IL-62/2 mice de-
SNPsincodingregionsofgeneswouldbemostsignificant, velop mature onset obesity accompanied by abnormal
most of the SNPs that have been shown to exhibit the glucose and fat metabolism (30). Although IL-6 is associ-
strongest associations have been either intronic or inter- ated with diabetes, its actions are complex. IL-6 impairs
genic(notinthecodingregions).Mutationsintheseregions insulin action in the liver and adipose tissue, but these
are most likely to regulate gene transcription. Hence it is effectsdependonitsconcentrationanddurationofaction
possible that a functional SNP in ADORA2B results in (31). In skeletal muscle IL-6 has a dual role, acutely pro-
modification of gene transcription. Based on the results of moting insulin sensitivity but chronically resulting in
the current study we conclude that in patients with diabe- insulin resistance through induction of JNK, suppressors
tes, signaling through A Rs is influenced by one or more of cytokine signaling-3, and protein tyrosine phosphatase
2B
SNPsthatmodifyproductionofIL-6andCRP,whichinturn 1b (32). IL-6 also is directly involved in stimulating the
influence insulin resistance. production of transcription factors that enhance CRP
Proinflammatory and anti-inflammatory signaling by production (33). It is interesting that SNP genotypes as-
A Rs. Deletion of the mouse A R resulted in a proin- sociated with IL-6 and CRP are inversely associated with
2B 2B
flammatory phenotype manifested as mild vascular in- another inflammatory marker, IL-2sR (Table 2). Unlike
flammation at baseline and exacerbation of cytokine IL-6, CRP, and PAI-1, IL-2sR is not an adipokine and is a
676 DIABETES,VOL.60,FEBRUARY2011 diabetes.diabetesjournals.org
R.A.FIGLERANDASSOCIATES
marker of T cell activation. The results suggest that A R been attributed to increased circulating free fatty acids,
2B
signalingcanresultininhibitionoflyphocyteactivation,at resultinginliverlipiddeposition.Anotherenzymeinvolved
least in some individuals. in adenosine production is the ecto-59-nucleotidase CD73,
Adenosine receptor signaling in diabetes. Previous which converts AMP to adenosine in the extracellular
studieshaveshownthatthestablenonselectiveadenosine space. It is notable that statins stimulate the induction of
analog NECA stimulates glucose production by hep- CD73 and have been shown in numerous studies to elicit
atocytes (34). In the current study we show that oral ga- insulinresistance.Statinsalsoenhanceischemia-mediated
vage with NECA acutely increases fasting glucose levels vasodilation in humans that is blocked by caffeine, con-
and strongly inhibits glucose disposal. Both deletion of sistent with an effect to enhance adenosine production
the A R gene and selective A R blockade with ATL-801 (44). We speculate that enhanced adenosine production,
2B 2B
implicate the A R as the primary mediator of these by activating A Rs, may contribute to the well-known
2B 2B
responses. These findings indicate that the previously effect of statins to provoke insulin resistance (45).
noted effects of adenosine receptor antagonists to reduce Diabetes and regulation of A R transcription. In the
2B
diabetes-induced insulin resistance (6–11) can be attrib- current study we demonstrate that diabetes triggers in-
uted to adenosine receptor blockade and not to off-target ductionofA RmRNAinmacrophagesandECs,resulting
2B
effects.WealsoobservedasmalleffectofdeletingtheA R in increased cytokine production in response to A R ac-
1 2B
to increase glucose disposalafter NECA administration to tivation. Analyses of the cloned human A R promoter
2B
mice, possibly due to the known effect of A R blockade to identified a functional binding site for hypoxia-inducible
1
increasepancreaticinsulinsecretion(35).Hyperinsulinemic- factor (46) and identified TNF-a and the oxidative stress-
euglycemic glucose clamps in KKAY mice demonstrate that promoting enzyme NAD(P)H oxidase as additional regula-
blockade of A R signaling enhances insulin sensitivity and torsofA Rgeneexpression(47).BecauseelevatedTNF-a
2B 2B
glucose metabolism in skeletal muscle, brown adipose tis- and oxidative stress are associated with diabetes (48,49),
sue,andliver.Thesedataareconsistentwiththehypothesis itisreasonabletospeculatethatthesefactorscontributeto
that activation of the A R causes insulin resistance that induction of A R mRNA in patients with diabetes. We no-
2B 2B
may be mediated in part by cytokine production. ticed a strong effect of diabetes on the probability of asso-
Associationofcoffeeconsumption withdiabetes.The ciations between ADORA2B SNPs and inflammatory
most potent activity of the methylxanthine caffeine is markers (Table 2). A R signaling in nondiabetics due to
2B
nonselective blockade of A , A , and A adenosine lowadenosinelevelsandlowA Rexpressioncouldrender
1 2A 2B 2B
receptors (36). It is notable, however, that ATL-692 is SNPs in ADORA2B that might influence A R signaling in-
2B
about5,000 times more potentthan caffeine asa competi- consequential in this population. In patients with diabetes,
tive antagonist of the human A R. In human epidemio- on the other hand, strong A R signaling may enhance
2B 2B
logic studies, long-term coffee consumption is strongly functional consequences of ADORA2B SNPs. The findings
associated with a reduction in the incidence of type 2 di- of this study, in particular the induction of A R mRNA in
2B
abetes. However,factors other than caffeinecontributeto ECsandmacrophagesfromdiabeticanimals,areconsistent
thiseffect,andthecontributionofcaffeineiscontroversial with the possibility that one or more SNPs in ADORA2B
(37,38). Moreover, blockade of A Rs acutely counteracts influences A R mRNA expression. It will be of interest in
1 2B
insulin actions by stimulating catecholamine release and future studies to determine whether ADORA2B genotypes
by counteracting the antilipolytic effect of A R activation are associated with A R mRNA expression in human
1 2B
in adipocytes. Perhaps due to the complex pharmacology monocytes.
of coffee and caffeine, it has not been possible in epide- In sum, the results of this study are consistent with the
miologicstudiestoclearlydemonstrateasignificanteffect idea that diabetes enhances signaling through A Rs both
2B
of caffeine as a contributor of coffee-induced protection by elevating adenosine levels and by increasing the ex-
from type 2 diabetes. However, in rigorously controlled pression of the A R. Our findings indicate that A R
2B 2B
studies in diabetic KKAY mice, consumption of high signaling can facilitate the release of proinflammatory
amounts of coffee or equivalent doses of pure caffeine cytokines from human macrophages and mouse ECs.
reduce hyperglycemia, decrease fat mass, reduce the ex- Blockade or deletion of the A R reverses the effects of
2B
pression of tumor necrosis factor-a (TNF-a) and IL-6 in diabetes on cytokine production and insulin resistance
whiteadiposetissue,andreducetheexpressionofhepatic assessed by GTT or hyperinsulinemic-euglycemic clamp.
genes involved in fatty acid synthesis (39). The results of Theminor(variant)alleleofseveral(sequential)tagSNPs
the current study suggest that at least some of the effects in ADORA2B are strongly correlated with IL-6 and CRP,
ofcaffeineindiabeticanimalsaremediatedbyblockadeof acute phase proteins that are highly associated with di-
A Rs. It is pertinent also that in human studies, genetic abetes.Wealsoobservedastrongeffectofdiabetesonthe
2B
variability in the activity of polymorphic forms of adeno- association between ADORA2B SNPs and markers of in-
sine deaminase is associated with obesity and type 2 di- flammation. These findings suggest that both diabetes and
abetes (40). An increase in the activity of adenosine ADORA2BgenotypecaninfluenceA Rexpression.Itwill
2B
deaminase, by reducing adenosine levels, has an effect be of interest to determine whether new potent and se-
similar to nonselective adenosine receptor blockade pro- lective A blockers that are currently in clinical de-
2B
duced by caffeine. velopment are effective in reducing obesity or insulin
Diabetes and adenosine metabolism. Human gesta- resistance in human disease.
tional diabetes is associated with elevated extracellular
adenosine (41). In rats, diabetes also enhances adenosine
accumulationandsignalinganddiminishestheexpression ACKNOWLEDGMENTS
of cytosolic adenosine kinase, the enzyme that converts ThisworkwassupportedbyNIHGrantsR01-HL-37942(to
adenosinetoAMP(42). Inmice,ablationoftheadenosine J.L.)andR01-DK-80756(toJ.K.K.)andbyMESAcontracts
kinase gene results in severe hepatic steatosis (43) that is N01-HC-95159 through N01-HC-95165 and N01-HC-95169
strongly associated with diabetes. Hepatic steatosis has fromtheNationalHeart,Lung,andBloodInstitute.Additional
diabetes.diabetesjournals.org DIABETES,VOL.60,FEBRUARY2011 677
ADENOSINEA RECEPTORSANDINSULINRESISTANCE
2B
support to J.K.K. was provided by the American Diabetes 15. YangD,ZhangY,NguyenHG,etal.TheA2Badenosinereceptorprotects
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