Abrogation of adenosine A 1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses

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1 Dibetologi (21) :11 12 DOI 1.17/s ARTICLE Abrogtion of denosine receptor signlling improves metbolic regultion in mice by modulting oxidtive stress nd inflmmtory responses Ting Yng 1 & Xing Go 2 & Monic Sndberg 2 & Christ Zollbrecht 1 & Xing-Mei Zhng & Michel Hezel 1 & Ming Liu 1 & Mri Peleli 1 & En-Yin Li & Robert A. Hrris & A. Erik G. Persson 2 & Bertil B. Fredholm 1 & Leif Jnsson 2 & Mttis Crlström 1, Received: 22 Jnury 21 /Accepted: 2 Mrch 21 /Published online: April 21 # Springer-Verlg Berlin Heidelberg 21 Abstrct Aims/hypothesis Adenosine is n importnt regultor of metbolism; however, the role of the receptor during geing nd obesity is uncler. The im of this study ws to investigte the effects of signlling in modulting metbolic function during geing. Methods Age-mtched young nd ged (lso known s Ador1)-knockout ( ) nd wild-type ( ) mice were used. Metbolic regultion ws evluted by body composition, nd glucose nd insulin tolernce tests. Isolted islets nd islet rterioles were used to detect islet endocrine nd vsculr function. Oxidtive stress nd inflmmtion sttus were mesured in metbolic orgns nd systemiclly. Results Advnced ge ws ssocited with both reduced glucose clernce nd insulin sensitivity, s well s incresed Ting Yng nd Xing Go re joint first uthors. Electronic supplementry mteril The online version of this rticle (doi:1.17/s ) contins peer-reviewed but unedited supplementry mteril, which is vilble to uthorised users. Mttis Crlström mttis.crlstrom@ki.se viscerl dipose tissue (VAT) in compred with mice. Islet morphology nd insulin content were similr between genotypes, but reltive chnges in in vitro insulin relese following glucose stimultion were reduced in ged compred with mice. Islet rteriolr responses to ngiotensin II were stronger in ged mice, this being ssocited with incresed NADPH oxidse ctivity. Ageing resulted in multiple chnges in compred with mice, including enhnced NADPH oxidse-derived O 2 formtion nd NADPH oxidse isoform 2 (Nox2) protein expression in pncres nd VAT; elevted levels of circulting insulin, leptin nd proinflmmtory cytokines (TNF-α, IL-1β, IL- nd IL-12); nd ccumultion of CD + T cells in VAT. This ws ssocited with impired insulin signlling in VAT from ged mice. Conclusions/interprettion These studies emphsise tht receptors regulte metbolism nd islet endocrine nd vsculr functions during geing, including vi the modultion of oxidtive stress nd inflmmtory responses, mong other things. Keywords Insulin sensitivity nd resistnce. Islets. Metbolic physiology in vivo. Metbolic syndrome. Oxidtive stress. Type 2 dibetes. Viscerl dipose tissue 1 2 Deprtment of Physiology nd Phrmcology, Krolinsk Institutet, Nnn Svrtz Väg 2, SE Stockholm, Sweden Deprtment of Medicl Cell Biology, Uppsl University, Uppsl, Sweden Deprtment of Clinicl Neuroscience, Krolinsk Institutet, Stockholm, Sweden Division of Nephrology & Hypertension, nd Hypertension, Kidney & Vsculr Reserch Center, Georgetown University, Wshington, DC, USA Abbrevitions ANG II Angiotensin II -Cyclopentyl-1,-dipropylxnthine DEXA Dul-emission x-ry bsorptiometry GLP-1 Glucgon-like peptide-1 IPGTT Intrperitonel glucose tolernce test IPITT Intrperitonel insulin tolernce test

2 Dibetologi (21) : Nox2 NADPH oxidse isoform 2 O 2 Superoxide nion VAT Viscerl dipose tissue Introduction Type 2 dibetes is chrcterised by bet cell dysfunction nd insulin resistnce [1 ] leding to endothelil dysfunction with devstting long-term vsculr impirment mnifested s numerous complictions [, ]. The incidence of type 2 dibetes increses with ge nd obesity, both of which re ssocited with oxidtive stress nd chronic inflmmtion. Mechnistic insights into the pthogenesis of type 2 dibetes nd novel therpeutic pproches re urgently needed. Severl clinicl nd epidemiologicl studies hve demonstrted tht coffee consumption, minly cffeine itself, is ssocited with reduced risk of developing type 2 dibetes [ ]. Cffeine inhibits the receptor-medited ctions of denosine [9], which exerts biologicl effects vi four types of receptors (,A 2A,A 2B nd A )[1]. Adenosine is n importnt regultor of metbolism; it modultes viscerl dipose tissue (VAT) function through receptor-medited ctions in decresing lipolysis nd incresing lipogenesis [11, 12]. Studies utilising gene-modified mice hve lso suggested tht the receptor intercts with insulin nd glucgon signlling [1, 1] or secretion [1]. Adenosine ws demonstrted to medite metboliclly induced vsodiltion in severl tissues [1, 17], nd we hve previously shown tht receptor ctivtion modultes in vivo islet blood flow in response to glucose [1]. These results, ll obtined in younger nimls, indicte tht denosine, vi signlling, could ffect glucose homeostsis in multiple wys. However, the role of the receptor in gerelted metbolic disorders, which is n independent risk fctor of type 2 dibetes [19 2], hs not been clerly studied. We hypothesised tht brogtion of receptor signlling ttenutes metbolic dysfunction ssocited with geing nd obesity, by modulting islet function, oxidtive stress nd inflmmtory responses. Indeed, our findings demonstrte this nd my hve therpeutic implictions. Methods This study ws pproved by the Institutionl Animl Cre nd Use Committee (IACUC) t Georgetown University, nd by equivlent IACUCs in Uppsl nd Stockholm, nd ws performed ccording to the Ntionl Institutes of Helth guidelines for the conduct of experiments in nimls. Animls Experiments were conducted on denosine receptor gene (lso known s Ador1)-deleted ( )nd wild-type mice ( ) from heterozygous breeding pirs. The strin ws developed by Johnsson nd co-workers [2] nd bckcrossed by the Jckson Lbortory (Br Hrbor, ME, USA) to C7BL/J bckground. Both sexes were used, with equl distribution for young ( months) nd ged (1 1 months) mice. Mice were housed in temperturecontrolled rooms with 12 h light/drk cycles nd received stndrd rodent chow (% ft, R, Lctmin AB, Kimstd, Sweden) nd tp wter d libitum. Food intke nd body composition nlysis Food intke ws ssessed (9 h period) nd dul-emission x-ry bsorptiometry (DEXA) studies were performed using Lunr PIXImus densitometer (GE Medicl-Lunr, Mdison, WI, USA) in isoflurne-nesthetised (Forene; Abbott Scndinvi AB, Soln, Sweden) nimls to determine ft nd len msses, s previously described [2]. Glucose nd insulin tolernce test Intrperitonel glucose (IPGTT) nd insulin tolernce tests (IPITT) were performed, nd the cute effects of phrmcologicl inhibition of the receptor with -cyclopentyl-1,-dipropylxnthine (), potent nd selective ntgonist for the receptor (.2 mg/kg body weight; Sigm-Aldrich; St Louis, MO, USA) or sline (1 mmol/l NCl; plcebo) were investigted. See Electronic Supplementry Mteril (ESM) Methods for further detils. Pncretic islet rterioles: vsculr rectivity studies Single islets with ttched rterioles were dissected nd perfused, s previously described [2]. Arteriolr responses to ngiotensin II (ANG II; 1 to 1 12 mol/l; Sigm-Aldrich) lone, or together with pocynin (1 mol/l; Sigm-Aldrich) were investigted during high (1.7 mmol/l) nd low glucose (2. mmol/l). Dose responses to denosine (1 to 1 11 mol/l; Sigm-Aldrich) were lso investigted. See ESM Methods for further detils. Pncretic islets: insulin relese nd contents Mouse pncretic islets were isolted through collgense digestion nd cultured in groups of 1 islets for dys. Insulin relese ws mesured in groups of ten islets from ech niml fter incubtion with low (1.7 mmol/l) nd high (1.7 mmol/l) glucose. Insulin content in the incubtion medi nd homogentes were determined using mouse insulin ELISA kit (Mercodi, Uppsl, Sweden) [27]. Lucigenin-dependent chemiluminescence of superoxide production NADPH oxidse-medited superoxide (O 2 )formtion ws detected by lucigenin-dependent chemiluminescence ssy [2]. Pncres, liver nd VAT were seprtely

3 112 Dibetologi (21) :11 12 homogenised nd used for subsequent ctivity mesurement. See ESM Methods for further detils. Plsm nlysis Metbolic mrkers nd cytokines were nlysed in mouse blood smples using MesoScle Discovery Multi Arry Technology (MSD, Rockville, MD, USA). See ESM Methods for further detils. Body weight (g) 2 2 b Food intke (g bw -1 2 h -1 ) Western blotting Pncres, liver nd VAT obtined from young nd ged mice under bsl conditions, fter pretretment with (i.p..2 mg/kg body weight) or 1 min fter stimultion with insulin (i.p..7 U/kg body weight), with nd without -pretretment, were homogenised. Tissue extrcts were prepred for SDS-PAGE followed by western blotting of NADPH oxidse isoform 2 (Nox2; BD Biosciences, Stockholm, Sweden), nd totl nd phosphorylted Akt (Ser7; Cell Signling/BioNordik, Stockholm, Sweden). Protein bnds were quntified using densitometry nd results re reported s reltive opticl density of the specific proteins. Flow cytometric nlysis Mouse tissues were incubted in digestion medium followed by erythrocyte lysis nd cells were stined with ntibodies specific for CD11b, F/, CD, MHC II, CD, CD, CDα nd respective isotype controls. Smples were nlysed in Gllios flow cytometer. See ESM Methods for further detils. Expression of denosine receptors mrna levels of denosine, A 2A, A 2B nd A receptors in whole pncres, VAT nd isolted pncretic islets together with islet rterioles were determined by quntittive PCR. See ESM Methods for further detils. Histology nd insulin stining Pncretic tissue ws fixed nd processed for evlution of islet morphology, volume nd insulin content. See ESM Methods for further detils. Dt nlysis Vlues re presented s mens±sem. Single comprisons between normlly distributed vribles were tested for significnce using the Student s pired or unpired t test, s pproprite. For multiple group comprisons, onewy ANOVA followed by Bonferroni s post hoc test ws used to llow for more thn one comprison with the sme vrible. Sttisticl significnce ws defined s p<.. Results Animl chrcteristics Body weight ws similr in young mice between genotypes. However, ged hd slightly lower body weight thn mice (Fig. 1), despite similr dily food intke (Fig. 1b). DEXA nlysis reveled n c Totl len mss (g) e Totl bdominl ft (g) Len mss frction (% of body weight) Abdominl ft frction (% of body weight) geing-relted len mss decrese (Fig. 1c, d) nd bdominl ft increse (Fig. 1e, f) in but not in mice. Glucose nd insulin tolernce tests To investigte the role of receptors in modulting the metbolic phenotype during geing, we performed glucose nd insulin tolernce tests in young nd ged mice. There ws no difference in bsl blood glucose levels or in glucose tolernce between genotypes in young mice (Fig. 2). However, in ged mice, fsting blood glucose ws somewht higher in thn in mice (.7 ±.2 vs.9±.2 mmol/l, p<.), nd IPGTT reveled significntly better glucose homeostsis in ged mice (Fig. 2b, c). The glucose lowering effect of insulin ws similr between genotypes t young ge (Fig. 2d). Ageing ws ssocited with reduced insulin sensitivity (Fig. 2e) nd incresed AUC (Fig. 2f) in, but not in mice. To vlidte the effect of receptor inhibition on glucose tolernce nd insulin sensitivity, pired crossover mesurements were conducted in ged mice. significntly improved both glucose (Fig. 2g) nd insulin (Fig. 2j) responses in mice compred with plcebo. These effects of the ntgonist were not observed in mice (Fig. 2h, k). CorrespondingAUCsreshowninFig.2i, l. Fig. 1 Body composition nd food intke. Body weight () ndfood intke (b) were mesured nd DEXA ws used to determine both totl len mss (c), len mss frction (d), totl bdominl ft (e) nd bdominl ft frction (f)inyoungndingedmice.vlues remen±sem,n= 2 2/group, except for (b) (n=1 2/group). p<. d f

4 Dibetologi (21) : b c d e f g h i j Fig. 2 IPGTT nd IPITT. Plsm glucose levels were mesured in IPGTT (, b) ndipitt(d, e) in young (, d; n=1 1) nd ged (b, e; n=2 2) mice, respectively. The AUC (mmol l 1 min 1 ) for the 12 min period ws clculted for both glucose (c) nd insulin(f) tolernce tests. To investigte the effects of receptor inhibition, pired crossover mesurements were conducted in ged nd mice given sline (plcebo) or the receptor ntgonist min prior to the IPGTT or the IPITT (n=1 1/group). Inhibition of receptor signlling significntly improved glucose clernce in (g), but hd k Totl AUC ( 12 min) Totl AUC ( 12 min) Totl AUC ( 12 min) l Totl AUC ( 12 min) 1, 1,2 1, 7 7 1, 1,2 1, 7 7 A 1 A 1 A 1 A 1 A 1 A 1 A 1 A 1 no effect in mice (h). The AUC IPGTT for the 12 min period ws clculted (i). Inhibition of receptor signlling lso improved the insulin response in (j), but hd no effect in mice (k). The AUC IPITT for the 12 min period ws clculted (l). Blck tringles, young ; white tringles, young ; blck squres, ged ; white squres, ged ; dshed line, tretment. Vlues re men±sem. p<. vs ged-mtched mice, or mong the indicted groups for AUC; p<. vs young mice of the sme genotype; p<. between plcebo nd Metbolic hormones in plsm Advnced ge ws ssocited with elevted levels of glucgon-like peptide-1 (GLP-1), glucgon, insulin nd leptin in mice (Fig. d). However, geing only induced smll leptin increse in mice. No significnt differences were observed in these hormone concentrtions between the genotypes t young ge. Insulin relese nd insulin content in isolted islets To investigte specificlly whether receptors modulte insulin relese upon glucose chllenge, we used isolted islets. High glucose concentrtions stimulted insulin relese in btchtype incubtions in both young nd ged nd mice, but this response ws ttenuted during geing (Fig. ). The reltive insulin relese response to glucose loding ws significntly reduced in ged compred with young mice nd lso compred with ge-mtched mice (Fig. b). This effect could not be explined by differences in totl insulin content mong the four groups investigted (Fig. c). In ddition, the islet morphology ws similr mong groups (ESM Fig. 1). The number of islets ws.±. nd.2±.7 per mg pncres in young nd mice, nd.±.11 nd.7±.12 in ged nd mice (p>.), respectively. Reltive islet volume (%) ws 1.1±. nd 1.2±.9 in young nd mice, nd 1.1±. nd 1.2±.1 in ged nd mice (p>.), respectively. There were lso no differences in pncretic weight between young nd mice (7.9±.2 vs.±. mg/g body weight) or between ged nd mice (.±. vs.2±. mg/g body weight). Isolted nd perfused islet rterioles To exmine how the receptor influences the microvsculture, isolted nd perfused pncretic islet rterioles (Fig. ) were used for vsculr rectivity studies. In ged mice, glucose-induced diltion

5 11 Dibetologi (21) :11 12 GLP-1 (ng/l) c Insulin (pmol/l) on fferent rterioles ws similr between genotypes, but the subsequent contrctile responses to ANG II were significntly ttenuted in rterioles from mice (Fig. c). However, in young mice these differences were much less pronounced (Fig. b). Simultneous incubtion with pocynin, to inhibit NADPH oxidse ctivity, ttenuted ANG II-medited contrctions in mice (2±1% vs 12±%), but hd no effect on rterioles from mice (1±% vs 12±1%). With pocynin, the rteriolr responses were similr between b Glucgon (ng/l) d Leptin (µg/l) Fig. Metbolic hormones in plsm. Plsm levels of GLP-1, glucgon, insulin nd leptin ( d) were determined using the MesoScle Discovery ssy in young nd ged mice. Vlues re men±sem, n=12 1/ group. p<. Insulin relese per islet (ng h -1 ) b Chnge in insulin (%) 2 1 LG 7 2 HG LG HG LG c Insulin content (ng/1 islets) HG LG 1, 9 7 HG Fig. Glucose-stimulted insulin relese nd insulin contents in islets. () Insulin relese fter low glucose (LG; 2. mmol/l) nd high glucose (HG; 1.7 mmol/l) stimultion in islets. Insulin responses were clculted s percentge chnge (b), nd the totl insulin content ws nlysed (c). Vlues re men± SEM, n=/group. p<. nd mice (Fig. d), suggesting tht the difference between genotypes could be relted to the regultion of NADPH oxidse function nd oxidtive stress. There ws no difference with regrd to the rteriolr dimeters mong groups (ESM Fig. 2). NADPH oxidse-medited superoxide production nd Nox2 level To investigte further whether oxidtive stress contributes to the pthogenesis of ge-relted metbolic disorders, O 2 formtion ws mesured in pncres, liver nd VAT. Ageing ws ssocited with incresed O 2 genertion in pncres (Fig. ) ndvat(fig.b) from mice. In mice, this ge-dependent chnge ws much less nd only occurred in pncres. In greement with this, the protein levels of Nox2 in both pncres (Fig. c, e)ndvat(fig.d, f)were significntly incresed in mice during geing while no significnt chnges were observed in mice. Moreover, the Nox2 level in VAT in ged ws significntly higher compred with tht in mice. No differences between genotypes were reveled in young mice. NADPH oxidse ctivity nd Nox2 levels in liver were similr between genotypes nd did not chnge with geing. Cytokines in plsm Considering the interction between oxidtive stress nd inflmmtion, we mesured systemic cytokine levels to investigte if brogtion of receptor signlling lso influenced the inflmmtory sttus. Advnced ge ws ssocited with elevted circulting TNF-α,IL-1β,IL-, IL-12 nd IL-1 levels in mice, but in ged mice only IL-1 ws elevted (Fig. 7 e). In greement with this, the proinflmmtory cytokines levels were significntly higher in ged mice compred with ge-mtched mice. No differences of the cytokines investigted were observed between genotypes in young mice. Popultions of mcrophges nd T cells in VAT To further understnd the potentil role of signlling in regulting inflmmtion during geing nd metbolic disorder, flow cytometric nlysis ws performed on single-cell suspensions extrcted from VAT (Fig. ). There were no significnt differences in totl mcrophge popultion in either young or ged mice between genotypes (Fig. b, d), but the number of CD + mcrophges ws significntly higher in ged mice compred with ge-mtched mice (Fig. c). mice showed n ge-relted enhncement of totl T cells nd specificlly of the CD + T cell popultion, this not being evident in mice (Fig. e, f). There were no differences regrding numbers of mcrophges nd T cells in the pncres or liver. Akt phosphoryltion in VAT To exmine if the difference in insulin sensitivity, oxidtive stress nd immune cell popultion ws mnifested in downstrem cellulr event, we

6 Dibetologi (21) : Fig. Islet rteriolr rectivity. The microgrph shows the experimentl set-up () with n isolted nd perfused islet rteriole (scle br, μm). (b d) Chnges in luminl dimeter of the rteriole were mesured in response to high glucose (HG; 1.7 mmol/l) nd cumultive doses of ANG II from 1 pmol/l ( )to1μmol/l (1 1 ) in young mice (b), ged mice (c) nd with simultneous inhibition of NADPH oxidse with pocynin (1 mol/l) (d). Blck tringles, young ; white tringles, young ; blck squres, ged ; white squres, ged ; dshed line, pocynin tretment. Vlues re men±sem, n=/group. p<. compred with ge-mtched mice; p<. compred with rterioles from ged-mtched mice without simultneous pocynin tretment (c) Superoxide production (totl CLU min -1 mg -1 ) c Nox2 β-actin e Nox2/β-ctin rtio,,, 2, 1, Superoxide production (totl CLU min -1 mg -1 ) b d Nox2 β-actin f Nox2/β-ctin rtio 2, 2, , 1,, Fig. Superoxide production nd Nox2 level in pncres nd VAT. NADPH oxidse-derived superoxide formtion in whole pncres () nd VAT (b) were mesured with lucigenin-dependent chemiluminescence signl (CLU, chemiluminescence unit). Western blot ws used to detect Nox2 level in pncres (c, e) ndvat(d, f). Prt (c, d) illustrtes two representtive smples per group obtined from different gels. Expression levels were normlised to β-ctin fter densitometric quntifiction. Vlues re men±sem, n=1 1/group. p<.; p=. determined Akt nd Akt phosphoryltion in VAT from young nd ged nd mice, nd in ged mice 1 min following i.p. injection with insulin (.7 U/kg body weight) (Fig. 9 d). There were no differences in bsl Akt nd p- Akt levels between genotypes (Fig. 9b, c), but geing ws ssocited with significntly higher p-akt/akt rtio in ged compred with young mice (Fig. 9d). Importntly, insulin-induced phosphoryltion of Akt ws mrkedly incresed in, but not significntly chnged in gemtched mice (Fig. 9d). In nother cohort, mice were treted with min prior to the insulin chllenge. lone did not chnge the expression nd phosphoryltion levels of Akt. However, cute inhibition of receptor signlling incresed p-akt nd p-akt/akt rtio following insulin chllenge in the mice, to the sme level s in the (Fig. 9e h). Expression of denosine receptors To clrify if the different metbolic phenotypes during geing nd between nd mice were ttributble to the possible differences of denosine receptors expression, we used quntittive PCR to determine the expression of ll four subtypes of denosine receptors in pncres (ESM Fig. d), islets together with their rterioles (ESM Fig. e h) nd in VAT (ESM Fig. i l). The receptor gene expression ws undetectble in mice. In ll tissues, no differences in A 2A, A 2B nd A receptor expression between genotypes were observed in either young or ged mice. The sme denosine receptor subtypes were lso similrly expressed in young nd ged mice of the sme genotype.

7 11 Dibetologi (21) :11 12 TNF- (ng/l) c IL- (ng/l) Discussion e IL-1 (ng/l) 2 1 Our mjor finding is tht brogtion of signlling improves the metbolic profile during geing. The underlying mechnisms re multifctoril: besides the known direct ctions on lipolysis nd lipogenesis [11, 12], we found ctions on peripherl insulin signlling, presumbly vi ttenution of NADPH oxidse function, s well s modultion of inflmmtory pthwys in VAT pprently being involved. Moreover, direct effects of signlling on the islet microvsculture, nd insulin relese, re lso involved. We observed n ge-dependent reduction in len mss nd n ccumultion of VAT in wild-type but not in mice. This is entirely comptible with previous studies showing tht endogenous denosine, through receptor signlling, reduces lipolysis nd enhnces lipogenesis [29]. We lso found tht dvnced ge ws ssocited with elevted blood glucose levels, impired glucose tolernce nd insulin responses or signlling in but not in mice. Phrmcologicl inhibition of the receptor improved glucose tolernce in ged wild-type mice, but hd no effect in knockouts. Our b IL-1 (ng/l) d IL-12 (ng/l) Fig. 7 Cytokines in plsm. Plsm levels of TNF-α, IL-1β, IL-, IL-12 nd IL-1 ( e) were determined using the MesoScle Discovery ssy in young nd ged mice. There were no differences of plsm IFN-γ nd kertinocyte-derived chemokine/growth-relted oncogene (KC/GRO) mong groups (dt not shown). Vlues re men±sem, n=12 1/ group. p<. dt indicte tht not only cute intervention of receptors will ffect the metbolic function, but tht receptors re lso prticipting in the metbolic derngement in mice during geing. Tken together, these findings demonstrte tht receptor signlling influences both VAT nd glucose regultion during geing nd emphsise the potentil therpeutic vlue of trgeting of receptors in type 2 dibetes. Adenosine is known to ffect the endocrine pncres per se [], nd previous studies suggested tht denosine receptors cn modulte insulin nd glucgon secretion [1]. We found dvnced ge to be ssocited with n elevtion of insulin, glucgon, GLP-1 nd leptin in mice, but in ged mice only leptin levels were incresed. Thus, the beneficil effect of eliminting receptor signlling ws extended to the hormone sttus. Incresed leptin concentrtion or resistnce were suggested s contributing to the inflmmtory sttus in dipose tissue [1] nd hve been linked to gessocited disorders including obesity, crdiovsculr diseses, the metbolic syndrome nd dibetes [2 ]. Elevted glucgon levels nd insulin resistnce re generlly thought to contribute to the pthophysiology of hyperglycemi in individuls with type 2 dibetes. Our islet studies demonstrted tht the bsence of receptors does not directly ffect islet morphology or insulin content. However, geing ws ssocited with reduced glucose-stimulted insulin relese in wild-type mice, but this ge-dependent reduction ws not observed in mice. Islet blood flow is normlly coupled to islet insulin relese [7, ]. To provide better possibility to study only the islet fferent rteriole, we used recently developed technique with isolted nd perfused single islets with ttched rterioles [2]. Similr to tht recently described for renl fferent rterioles [9], islet rterioles from mice displyed reduced contrctility to ANG II. This ws pprent during both normond hyperglycemic conditions, lthough much more profound during the ltter. Since the bseline dimeters of the islet rterioles were similr in ll the groups, one my speculte tht, t physiologicl ANG II concentrtions, ged mice would hve n incresed rteriolr resistnce. However, future studies with other techniques re required to confirm this hypothesis. Oxidtive stress, prticulrly O 2, hs been demonstrted to reduce islet blood flow []. ANG II stimultes NADPH oxidse-medited O 2 formtion, which contributes to its pronounced vsoctive properties. Interestingly, reduction of oxidtive stress by the NADPH oxidse inhibitor pocynin ttenuted ANG II-medited contrction in mice, but hd no effect on islet rterioles from mice. This suggests n importnt role of the denosine receptor in modulting O 2 production. This notion hs lso been described in models of ANG II-induced hypertension in which blood pressure elevtion nd oxidtive stress were mrkedly ttenuted in genedeleted mice [9, 1].

8 Dibetologi (21) : Fig. Mcrophges nd T cell popultions in VAT. Prt () depicts the gting strtegy. No significnt differences in totl mcrophges (CD11b + F/ + ) nd MHCII + mcrophges (CD11b + MHCII + ) were evident between genotypes (b, d). However, CD + mcrophges (CD11b + CD + ) were significntly higher in ged mice compred with ge-mtched mice (c). T cell popultion nlysis reveled n gedependent increse of totl T cells (CD + CD11b + ) nd CD + Tcells (CD + CD + )in mice, but this phenotype ws bsent in mice (e, f). No differences were evident in CD + Tcell popultions (CD + CD + ) between genotypes t different ges (g). Vlues re men±sem, n=/group. p<.; p=.1 F/ CD11b CD11b + F/ + cells (% totl cells) CD + cells (% totl cells) CD11b CD CD + cells (% totl mcrophges) CD + CD + cells (% totl cells) 2 MHCII + cells (% totl mcrophges) F/+ CD CD CD CD+ CDCD b c d e f g 1 2 MHCII CD CD + CD + cells (% totl cells) CDCD MHCII+ Incresing evidence from experimentl nd clinicl studies hs demonstrted tht oxidtive stress nd inflmmtion re key fctors tht contribute to the progression of metbolic disorders including type 2 dibetes [2, ]. In the present study we show tht geing is ssocited with incresed NADPH oxidse-derived O 2 formtion, together with higher Nox2 levels, in both pncres nd VAT from mice. This ge-dependent elevtion in O 2 formtion nd oxidtive stress ws clerly ttenuted, or even bsent, in gene-deleted nimls, which certinly my contribute to their better metbolic phenotype. Age-relted VAT ccumultion is ssocited with chronic, low-grde inflmmtion nd hs been incresingly recognised s n independent risk fctor of the pthogenesis of type 2 dibetes [ ]. Almost ll immune cell subsets re present in VAT. Their functions re still under discussion, lthough it is generlly ccepted tht totl T cell nd mcrophge popultions re incresed nd contribute to the metinflmmtion evident in obesity. We did not discern differences in totl mcrophges mong groups, but observed significnt enhncement of the CD + mcrophge popultion in ged mice, indicting elevted ntigen presenttion cpcity. Interestingly, nother recent study reported lipolysis-relted mcrophge infiltrtion in dipose tissue without presenting pro-inflmmtory chrcters [7]. As mice hve elevted lipolysis nd remin len, this enhncement of CD + mcrophges my be due to response to the continuous relese of non-esterified ftty cids. As young mice hve much lower lipolysis levels, no differences (e.g. in ft mss) were evident between genotypes t n erly ge. However, geing resulted in significnt increse of totl TcellsndCD + T cells in VAT in mice, while this ws not the cse in mice. In ddition, levels of circulting proinflmmtory cytokines (including TNF-α, IL-1β, IL-

9 11 Dibetologi (21) :11 12 Fig. 9 Phosphoryltion of Akt in VAT. Prts (, e) illustrte three representtive smples per group obtined from different gels. Expression levels were normlised to β-ctin, nd results fter densitometric quntifiction re shown for p-akt (b), totl Akt (c) nd reltive p-akt-to-akt levels (d). Acute inhibition with, followed by insulin chllenge, incresed p-akt nd p-akt/akt rtio in the mice to the sme level s in the mice (e h). Vlues re men±sem, n=/group. p<. + insulin Akt p-akt β-actin b c d A 1 A 1 A 1 A 1 A 1 A 1 A 1 p-akt/β-ctin rtio e Akt/β-ctin rtio p-akt/akt rtio A 1 A 1 +insulin +insulin +insulin insulin Akt p-akt β-actin f g h p-akt/β-ctin rtio insulin Akt/β-ctin rtio insulin p-akt/akt rtio insulin nd IL-12) were incresed in ged but not in mice. These findings indicte tht sustined inflmmtion occurs during geing nd obesity, nd my led to subsequent metbolic disorders, s hs been previously suggested [, 9]. Moreover, ccumultion of CD + T cells in VAT my be key contributor to this systemic inflmmtion nd cn be modulted vi receptor signlling. Further investigtions on the immunomodultion effects of the receptor during geing nd the metbolic syndrome re needed nd will provide dditionl insights in developing therpeutic strtegies for type 2 dibetes. It seems likely tht decresed VAT ccumultion nd inflmmtion my contribute to the better metbolic regultion in the mice during geing, including better insulin sensitivity. Indeed, our dt show significntly better insulin signlling in ged mice, s demonstrted by incresed phosphoryltion of Akt kinse in VAT upon insulin Fig. 1 Proposed effects of the denosine receptor in modulting metbolic derngement during geing Abrogtion of receptor signlling Ageing VAT ccumultion Oxidtive stress Leptin relese Insulin resistnce CD + T cells Circulting inflmmtory cytokines Insulin relese Glucgon relese ANG II rectivity Oxidtive stress

10 Dibetologi (21) : stimultion. This insulin signlling pthwy ws mrkedly reduced in ged-mtched wild-type mice nd my contribute to their impired glucose clernce function. Besides the receptor, lso denosine receptor A 2A nd A 2B ply role in modulting glucose homeostsis nd obesity [, 1]. We did not revel ny significnt differences regrding the A 2 nd A receptor expression mong the groups, suggesting no compenstory chnges following receptor deletion. Thus, we believe the improved metbolic phenotypes in the A 1 mice in our study were due to the brogtion of signlling nd our findings further suggest pivotl role of the receptor in modulting especilly the function of VAT, which my ffect the metbolic homeostsis. In conclusion, the present study demonstrtes n importnt role of the denosine receptor in modulting glucose nd insulin homeostsis, s well s islet endocrine nd rteriolr function, during geing. Mechnisticlly, our findings suggest tht brogtion of signlling my protect from gedependent oxidtive stress nd production of proinflmmtory cytokines, nd hence improve insulin relese nd signlling (Fig. 1). Thus, brogtion of receptor signlling cn both hve cute effects nd give long-term prevention of the development or progression of dibetes. Future studies in humns should be imed t determining the therpeutic vlue of modulting signlling. Acknowledgements We thnk E. Lindgren (Krolinsk Institutet) nd M. Quch (Uppsl University) for technicl ssistnce nd helpful discussions. Funding This work ws supported by grnts from the Swedish Reserch Council ( to MC nd to LJ), Swedish Hert nd Lung Foundtion (21, 2119), Jenssons Foundtion (JS21-), Swedish Dibetes Foundtion, n EXODIAb grnt, Swedish Society of Medicl Reserch (SSMF), the Wenner-Gren Foundtion, the Fmily Ernfors Fund, the Swedish Society of Medicine nd Novo Nordisk Foundtion Excellence Project. 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