Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice

Transcription

1 ORIGINL RESERCH Physiologicl Reports ISSN X Erythroferrone is not required for the glucoregultory nd hemtologic effects of chronic erythropoietin tretment in mice Richrd Coffey 1, Ugo Srdo,Leon Kutz, Victori Gyn 1, Elizet Nemeth 1 & Toms Gnz 1 1 Deprtment of edicine, Dvid Geffen School of edicine, University of Cliforni, Los ngeles, Cliforni IRSD, Universite de Toulouse, INSER U1, INR U116, ENVT, UPS, Toulouse, rnce Keywords Erythroferrone, erythropoietin, glucose, hepcidin, myonectin. Correspondence Toms Gnz, Deprtment of edicine, Dvid Geffen School of edicine, University of Cliforni, Los ngeles, C. Tel: +1 (31) x: +1 (31) E-mil: TGnz@mednet.ucl.edu Received: 16 Septemer 18; ccepted: 18 Septemer 18 doi: 1.181/phy.1389 Physiol Rep, 6 (19), 18, e1389, strct Erythropoietin (EPO) cts on erythroid progenitor cells to promote their survivl nd differentition to mture erythrocytes. long with this cnonicl role, EPO is lso reported to modulte energy metolism, resulting in improved glucose tolernce nd insulin sensitivity. EPO lso stimultes the production of the hormone erythroferrone (ERE) which cts to suppress hepcidin production, thus incresing dietry iron sorption nd moilizing stored iron for use in erythropoiesis. ERE (initilly termed myonectin) ws lso reported hve n effect on systemic lipid metolism y promoting the clernce of nonesterifed ftty cids (NE) from circultion. s incresed levels of circulting NE lunt insulin sensitivity nd impir glucose tolernce, ERE-induced clernce of NE fter EPO dministrtion would hve eneficil effect on glucose metolism. The im of this study ws to determine if the known metolic effect of EPO tretment on glucose homeostsis is medited y ERE, produced in response to EPO. ice lcking Erfe did not differ from wild-type mice in lood lipid prmeters, lood glucose, nd glucose or insulin tolernce t seline or fter chronic EPO tretment. dditionlly, hepcidin suppression nd the response of erythrocyte prmeters to chronic EPO tretment were unffected y the sence of Erfe. These findings suggest tht the known eneficil effects of EPO on glucose metolism re not ttriutle to n ccompnying increse in ERE production, nd tht Erfe is dispensle for norml glucose homeostsis. urthermore, our dt indicte tht ERE-independent mechnisms cn suppress hepcidin in response to chroniclly elevted EPO levels. Introduction In vertertes, most of the ody s iron is contined within hemogloin in erythrocytes, where it inds oxygen for trnsport to tissues. Stedy-stte erythropoiesis consumes more thn 8% of circulting iron (inch et l. 197) to replce senescent erythrocytes tht re removed from circultion y mcrophges of the reticuloendothelil system (Knutson nd Wessling-Resnick 3). The production of new erythrocytes cn e sustntilly incresed in response to erythropoietic stress fter dverse events such s lood loss or hemolysis (Colemn et l. 1953; Hillmn nd Henderson 1969), which further increse iron requirements. Indequte iron delivery to the erythron impirs erythropoiesis nd cn mnifest s hypochromic nd microcytic nemi (rton nd ottomley ). Erythropoiesis is regulted y erythropoietin (EPO), glycoprotein hormone produced y the kidney in response to hypoxi. EPO increses erythrocyte production y preventing the progrmmed cell deth of erythroid precursors nd promoting their mturtion (Koury nd ondurnt 1988, 199). eyond this cnonicl effect of EPO in regulting erythropoiesis, EPO lso indirectly supports erythropoiesis y incresing the production of the hormone erythroferrone (ERE). ERE, memer of the C1q/TN-relted protein fmily, is encoded y the ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. This is n open ccess rticle under the terms of the Cretive Commons ttriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited. 18 Vol. 6 Iss. 19 e1389 Pge 1

2 Effect of ERE on Glucose etolism R. Coffey et l. gene ERE, formerly nmed 13, nd is secreted into circultion y erythrolsts in response to elevted plsm EPO concentrtions (Kutz et l. 1). ERE functions to enhnce iron vilility to mtch incresed erythropoiesis: ERE cts directly on heptocytes to suppress the production of hepcidin (Kutz et l. 1), the mster regultor of orgnisml iron homeostsis tht inhiits iron sorption nd moiliztion from stores (Nemeth et l. ). ice lcking Erfe fil to cutely suppress hepcidin fter leeding nd tke longer to recover from nemi (Kutz et l. 1). In ddition to promoting erythropoiesis, EPO signling lso elicits systemic metolic effects. Tretment with EPO increses insulin sensitivity nd glucose tolernce in humns (llegr et l. 1996; k 1998) nd mice (Ktz et l. 1; oskett et l. 11; lneeli et l. 1). The protein encoded y the ERE gene ws initilly termed myonectin, myokine secreted y skeletl muscle nd linked to lipid metolism nd nutrient sensing (Seldin et l. 1, 13). In those studies, injection of recominnt ERE reduced serum nonesterified ftty cid (NE) levels in mice, nd in vitro ERE tretment incresed NE uptke y heptocyte- nd dipocyte-derived cell lines (Seldin et l. 1). Elevted NE levels re known to decrese insulin-stimulted glucose uptke (oden et l. 199; oden nd Chen 1995) so ERE-medited reduction of circulting NE levels would e expected to increse glucose tolernce nd insulin sensitivity (Sntomuro et l. 1999). We therefore surmised tht some of the metolic effects of EPO could e medited y its ility to induce ERE secretion y erythrolsts. In this study, we used Erfe knockout mice (Erfe / )to determine whether ERE t physiologic concentrtions modultes circulting NE levels, nd to wht extent incresed glucose tolernce nd insulin sensitivity fter prolonged tretment with EPO is ttriutle to its stimultion of ERE production. Our findings indicte tht, t physiologic concentrtions, ERE does not lter plsm lipid homeostsis nd tht incresed ERE production does not medite the effect of EPO on lood glucose homeostsis. dditionlly, we report tht the sence of ERE does not mesurly restrict erythropoiesis during chronic EPO tretment. terils nd ethods Experimentl nimls Erfe +/ mice on C57L/6J ckground, descried previously (Kutz et l. 15), were red to generte littermte Erfe / mice nd wild-type (WT) controls used for metolic testing, serum NE mesurements, complete lood counts (CC), nd iron prmeter nlysis. or chronic tretment experiments, mice were injected intrperitonelly with either U recominnt mouse EPO (iolegend) or sterile sline 3x per wk, on ondy, Wednesdy, nd ridy, eginning t 6 weeks nd ending t 8 weeks of ge. The finl injection ws performed 15 h prior to termintion y isoflurne inhltion, nd mice were fsted during those 15 h. ecuse metolic testing requires frequent lood smpling, seprte groups of mice were used for metolic testing from those tht were used for the nlysis of serum NE, CC, nd iron prmeters. WT C57L/6J mice used in cute EPO tretment experiments were otined from the Jckson Lortory t 5 weeks of ge nd housed t UCL until testing. t 8 weeks of ge, mice were injected intrperitonelly with either single dose of U recominnt mouse EPO or sterile sline 15 h prior to scrifice, nd were lso fsted during this time. ice used in these experiments were mintined on low-ft, low-sucrose (3.5%) nturl ingredient diet contining pproximtely 185 ppm iron (Ldiet, #553) nd housed in specific pthogen-free rrier fcility t UCL. Erfe / mice used in the chrcteriztion of lood lipid nd lood glucose prmeters t 1 nd 18 weeks of ge were generted y reeding Erfe / mice on C57L/6J ckground. WT C57L/6J mice used s controls were otined from Jnvier Lortories t 5 weeks of ge. ice were fed low-ft, low-sucrose (7%) purified diet (Ssniff Spezildi ten GmH, #E15753) nd scrificed y retro-oritl exsnguintion fter either unrestricted ccess to food (the 1-week ge group) or fter 16 h fst (the 18-week ge group). ice used in these experiments were housed in specific pthogen-free rrier fcility in the niml fcilities of INSER US6. ll experimentl protocols involving mice reported in this mnuscript were crried out with pprovl from the University of Cliforni, Los ngeles nd the Universite de Toulouse. Glucose nd insulin tolernce testing t 8 weeks of ge, mice were injected with either EPO or sterile sline nd fsted 15 h overnight prior to glucose tolernce testing. ice were injected intrperitonelly with 1.5 g/kg odyweight glucose diluted in sterile sline, nd lood glucose levels were mesured immeditely prior to injection nd t 15, 3, 6, nd 1 min. Insulin tolernce testing (ITT) ws performed dys fter glucose tolernce testing. ice were injected with n dditionl dose of either EPO or sterile sline 1 h prior to, nd fsted for 6 h prior to, the strt of ITT. ice were injected intrperitonelly with.75 U/kg humn insulin (Novolin R, Novo Nordisk) diluted in sterile sline nd 18 Vol. 6 Iss. 19 e1389 Pge ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society.

3 R. Coffey et l. Effect of ERE on Glucose etolism lood glucose levels were mesured immeditely prior to injection, nd t 15, 3, 5, nd 6 min. Glucose mesurements were otined y using n lphtrk hnd-held glucometer (Zoetis). During oth glucose nd insulin tolernce testing, lood ws otined from the distl til tip. Iron prmeter nlysis nd complete lood counts Liver nonheme iron levels were mesured, following cid digestion, y colorimetric ssy ccording to the mnufcturer s protocol (Sekisui Dignostics). Livers were homogenized prior to smpling for nonheme iron nlysis, to prevent vriility resulting from differences in the regionl distriution of iron. Complete lood counts were otined using HemVet lood nlyzer (Drew Scientific). Quntifiction of serum ERE, hepcidin, nd lood lipid prmeters Serum ERE levels were determined s descried previously (Kutz et l. 15) using ntiodies developed y Silrus Therpeutics, L Joll, C. Serum hepcidin concentrtions were determined y ELIS s previously detiled (Kutz et l. 1) using nti-hepcidin ntiodies developed y mgen. Serum NE levels were mesured y using the HR Series NE-HR kit (Wko Dignostics). lood lipid nd glucose prmeters were nlyzed in mice t 1 nd 18 weeks of ge y using n X Pentr clinicl chemistry nlyzer (Hori edicl). RN isoltion nd gene expression nlysis Totl RN ws isolted from tissues y using Trizol (Thermoisher Scientific). cdn ws synthesized y using the iscript cdn Synthesis Kit (io-rd) ccording to the mnufcturer s protocol. Reltive mrn levels for genes of interest were quntified y using qrt-pcr nd Ssodvnced Universl SYR Green Supermix (io-rd) run on CX96 Rel-Time PCR Detection System. The following primer sequences were designed to detect ll known trnscript isoforms. mhprt (hypoxnthine gunine phosphoriosyl trnsferse):,; CTG-GTT-G-CG-TC-GC-CCC- R; CG-GG-GTC-CTT-TTC-CC-GC, merfe: ; TG- GGG-CTG-GG-C-GC R; TGG-CT-TGT-CC-G- G-C. Sttisticl nlysis Sttisticl nlysis ws performed y using the SigmPlot 1.5 pckge (Systt Softwre). Groups of mice were compred with other groups of the sme sex, to eliminte vriility resulting from sex differences in prmeters relted to iron or glucose homeostsis (cotel et l. 9; clchln et l. 17). Group mens were compred y using the student s t-test, one-wy NOV, twowy repeted mesures NOV, or three-wy NOV where indicted. In response to differences etween group mens, Tukey s (for one nd three-wy NOV) or Holm Sidk (for two-wy repeted mesures NOV) multiple comprisons testing ws performed to determine which groups differed significntly. Differences etween groups were considered significnt t P vlue of <.5. Results The effect of ERE on serum lipid levels To determine whether the sence of ERE results in ltered serum lipid homeostsis under seline physiologic conditions, we mesured serum NE, triglyceride, totl cholesterol, HDL-C, nd LDL-C levels in mle WT nd Erfe / mice under oth fed nd fsted conditions (ig. 1 E). We detected no sttisticlly significnt difference in lood lipid prmeters etween genotypes during either nutritionl sttes. lood glucose levels were lso not different etween WT nd Erfe / mice during either fed or fsted conditions (ig. 1). Circulting ERE levels re low under seline conditions nd increse in response to erythropoietic stimuli (Kutz et l. 15). Therefore, we chroniclly treted WT nd Erfe / mice with either high-dose EPO or sline on lternting dys for weeks to determine if incresed levels of ERE lter serum lipid homeostsis. We focused on NE levels s tretment with ERE hs een reported to modulte serum NE concentrtions (Seldin et l. 1). In response to chronic tretment with EPO, one mrrow mrn expression of Erfe ws elevted compred with tht of sline-treted controls nd serum ERE levels incresed from elow the threshold of detection (ig. nd ). Despite the upregultion of ERE expression y EPO tretment, serum NE levels were not different etween WT nd Erfe / mice during EPO-stimulted conditions (ig. C). To determine whether n initil effect of elevted serum ERE on NE levels ws lunted y tchyphylxis fter chronic exposure to ERE, we mesured serum NE concentrtions in WT mice fter the dministrtion of single dose of EPO. Erfe mrn expression in the one mrrow nd serum ERE incresed to levels comprle to those detected in mice chroniclly treted with EPO (ig. 3 nd ). However, serum NE levels were not different in mice tht received cute EPO tretment compred with sline-treted controls (ig. 3C). ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. 18 Vol. 6 Iss. 19 e1389 Pge 3

4 Effect of ERE on Glucose etolism R. Coffey et l. 5 ed sted. ed sted Serum NE (mg/dl) 3 1 Serum triglycerides (mmol/l) C 5 ed sted D ed sted Serum Cholesterol (mmol/l) 3 1 Serum HDL-C (mmol/l) 3 1 E. ed sted 5 ed sted Serum LDL-C (mmol/l).3..1 Serum glucose (mg/dl) igure 1. Erfe / mice hve norml lood lipid levels during oth fed nd fsted conditions. Serum NE (), triglyceride (), totl cholesterol (C), HDL-C (D), LDL-C (E), nd serum glucose levels () in mle WT nd Erfe / knockout mice tht hd ccess to food or were fsted for 16 h prior to scrifice. Prmeters were mesured t 1 weeks of ge in fed mice nd t 18 weeks in fsted mice (n = 8 1 per group). Group mens etween WT nd Erfe-/- mice were independently compred under either fed or fsted conditions y using the student s t-test. Different superscripts indicte sttisticl significnce (P <.5). Vlues re presented s group mens SE. The effect of ERE on lood glucose homeostsis To determine the influence of ERE on lood glucose homeostsis we performed glucose tolernce testing (GTT) under sl conditions or fter chronic EPO tretment in WT nd Erfe / mice. fter n overnight fst, seline lood glucose levels trended to e lower in EPO-treted compred with sline-treted mice of the sme genotype, s reported efore (oskett et l. 11). This difference ws 18 Vol. 6 Iss. 19 e1389 Pge ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society.

5 R. Coffey et l. Effect of ERE on Glucose etolism 1 ΔCt (Hprt-Erfe) * *** Serum erythroferrone (ng/ml) 8 6 P =.5 ** 1 C 1 NCl EPO NCl EPO NCl EPO NCl EPO 8 Serum NE (mg/dl) 6 NCl EPO NCl EPO NCl EPO NCl EPO igure. Serum levels of nonesterified ftty cids re not ffected y physiologic concentrtions of ERE. one mrrow mrn expression of Erfe () nd serum ERE levels () in WT mice fter chronic tretment with either EPO or sline (NCl) (n = 5 6 per group for ech sex). (C) Serum nonesterified ftty cid (NE) concentrtions in WT nd Erfe / mice fter chronic tretment with either EPO or sline (NCl) (n = 5 7 per group for ech sex). In pnels nd C dt re presented s individul vlues from experimentl nimls with line indicting the group men. Group mens etween mice of the sme sex were compred y using either the student s t-test ( nd ) or one-wy NOV (C). sterisks indicte sttisticlly significnt difference etween groups s determined y t-test (*P <.5, **P <.1, ***P <.1) nd mens without common lpheticl superscript differ significntly s determined y one-wy NOV (P <.5). Dt re shown s the men SE or s individul dt points. sttisticlly significnt in mle Erfe / nd femle WT mice ut filed to rech significnce in other groups. ERE did not ffect fsting lood glucose levels, s we oserved no difference etween WT nd Erfe / mice under either sl or EPO-stimulted conditions. nlysis of lood glucose levels during GTT determined tht, for oth mle nd femle mice, tretment with EPO ltered glucose tolernce compred to sline tretment, ut there ws no effect of Erfe genotype (ig., C; twowy repeted mesures NOV followed y multiple comprisons testing). re under the curve (UC) nlysis of lood glucose vlues during testing indicted tht chronic EPO tretment improved glucose tolernce to similr degree in oth WT nd Erfe / mice (ig., D). UC vlues during testing were significntly lower in mle WT, mle Erfe /, nd femle WT mice fter EPO tretment. In femle Erfe / mice the difference in the UC during testing in EPO-treted mice compred with slinetreted controls did not rech sttisticl significnce. The effect of ERE on insulin tolernce To detect whether the lck of difference in glucose tolernce etween WT nd Erfe / mice ws the result of ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. 18 Vol. 6 Iss. 19 e1389 Pge 5

6 Effect of ERE on Glucose etolism R. Coffey et l. ΔCt (Hprt-Erfe) *** *** Serum erythroferrone (ng/ml) 3 1 *** *** 8 NCl EPO NCl EPO NCl EPO NCl EPO C Serum NE (mg/dl) NCl EPO NCl EPO igure 3. Serum levels of nonesterified ftty cids re not ffected y cute EPO tretment. one mrrow mrn expression of Erfe (), serum erythroferrone levels (), nd serum nonesterified ftty cid (NE) concentrtions in wild-type mice 15 h fter tretment with single dose of either U EPO or sline (NCl). In pnels nd C, dt re presented s individul vlues from experimentl nimls with line indicting the group men. Group mens etween mice of the sme sex were compred y using the student s t-test. sterisks indicte sttisticlly significnt difference etween groups (***P <.1). Dt re shown s the men SE or s individul dt points (n = 5 per group for ech sex). glucose-induced compenstory insulin secretion, we performed insulin tolernce testing (ITT) in mice used previously for GTT nlysis. fter 6 h fst prior to testing, there ws trend towrd lower lood glucose levels in mice chroniclly treted with EPO compred with slinetreted mice, regrdless of genotype. This difference ws sttisticlly significnt in mle mice ut filed to rech significnce in femle mice. nlysis of lood glucose levels during insulin tolernce testing determined tht in mle mice EPO-treted groups were significntly different from sline-treted controls (ig. 5; two-wy repeted mesures NOV followed y multiple comprisons testing). Groups differing in genotype, ut not tretment, were not significntly different. UC nlysis of lood glucose levels during ITT lso determined tht lood glucose levels did not differ significntly etween genotypes in either sline or EPOtreted mle mice (ig. 5). ITT of femle mice filed to demonstrte ny difference in insulin sensitivity etween WT nd Erfe / mice under sl or EPO-stimulted conditions (ig. 5C), s determined y two-wy repeted mesures NOV followed y multiple comprisons testing. UC vlues from EPO-treted femle mice during testing, from either genotype, were not different from sline-treted controls (ig. 5D). Contriution of ERE to erythropoiesis in response to chronic EPO tretment No differences in hemtologicl prmeters were detected etween WT nd Erfe / mice during either sl, nonstimulted conditions or fter chronic tretment with 18 Vol. 6 Iss. 19 e1389 Pge 6 ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society.

7 R. Coffey et l. Effect of ERE on Glucose etolism lood Glucose (mg/dl) WT NCl WT EPO Erfe / NCl Erfe / EPO UC [glucose (-1 min)]/ Time (minutes) NCl EPO NCl EPO C lood Glucose (mg/dl) WT NCl WT EPO Erfe / NCl Erfe / EPO Time (minutes) D UC [glucose (-1 min)]/1 3 1 NCl EPO NCl EPO igure. Erythroferrone does not ffect glucose tolernce under sl conditions or fter chronic tretment with EPO. lood glucose levels during glucose tolernce testing in mle () nd femle (C) mice. Curves tht re not shring letter re significntly different s determined y two-wy repeted mesures NOV (P <.5). re under the curve (UC) nlysis of lood glucose vlues for mle () nd femle (D) mice during glucose tolernce testing. Groups of the sme sex without common lpheticl superscript differ significntly s determined y one-wy NOV (P <.5). Dt re shown s the men SE (n = 6 8 per group for ech sex). EPO (ig. 6 D). s expected, mice treted with EPO displyed elevted hemogloin, red lood cell, nd hemtocrit levels compred with sline-treted mice, lthough the trend towrd incresed red lood cell levels with EPO tretment did not rech sttisticl significnce for femle WT mice. en corpusculr volume ws unffected y either chronic EPO tretment or genotype. To determine if lck of ERE ffects the moiliztion of stored iron in response to chronic erythropoietic stimultion, we mesured indices of iron sttus in WT nd Erfe / mice treted with either EPO or sline. Serum hepcidin concentrtions were significntly lower in mice treted chroniclly with EPO compred with those tht received sline, s determined y three-wy NOV, ut neither genotype nor sex hd detectle effect on serum hepcidin concentrtions (ig. 7). In ddition, no interction etween tretment, genotype, or sex ws detected. In greement with the lower hepcidin levels mesured in EPO-treted compred with sline-treted mice of the sme genotype, we detected trend towrd lower liver nonheme iron concentrtions in mice tht received EPO, lthough these differences did not rech sttisticl significnce (ig. 7). Discussion The erythroid hormone ERE medites cute hepcidin suppression in response to erythropoietic stimuli to mtch iron supply with erythropoietic demnd (Kutz et l. 1). However, previous study suggested tht ERE plys role in the regultion of lood lipid homeostsis (Seldin et l. 1). Tretment with ERE ws reported to ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. 18 Vol. 6 Iss. 19 e1389 Pge 7

8 Effect of ERE on Glucose etolism R. Coffey et l C lood glucose (mg/dl) lood glucose (mg/dl) Time (minutes) WT NCl WT EPO Erfe / NCl Erfe / EPO 6 WT NCl WT EPO Erfe / NCl Erfe / EPO 6 Time (minutes) 1 UC [glucose (-6 min)]/1 8 6 NCl EPO NCl EPO D NCl EPO NCl EPO UC [glucose (-6 min)]/1 igure 5. Erythroferrone does not ffect insulin tolernce under sl conditions or fter chronic tretment with EPO. lood glucose levels during insulin tolernce testing in mle () nd femle (C) WT nd Erfe-/-mice chroniclly treted with either U EPO or sline (NCl). Curves not shring letter re significntly different s determined y two-wy repeted mesures NOV (P <.5). re under the curve (UC) nlysis of lood glucose vlues for mle () nd femle (D) mice during insulin tolernce testing. Groups without common lpheticl superscript differ significntly from other groups of the sme sex s determined y one-wy NOV (P <.5). Dt re shown s the men SE (n = 6 8 per group for ech sex). increse NE uptke y heptocytes nd dipocytes in vitro nd decrese serum NE levels in mice in vivo (Seldin et l. 1). s reducing circulting levels of NE improves insulin sensitivity nd glucose tolernce (Sntomuro et l. 1999), we imed to test the possiility tht eneficil effects of EPO tretment on glucose homeostsis (llegr et l. 1996; k 1998; Ktz et l. 1; oskett et l. 11; lneeli et l. 1) re medited y incresed ERE signling. This study is the first to investigte the influence of physiologiclly relevnt concentrtions of ERE on lood lipid nd glucose homeostsis. In contrst to the previous indiction tht ERE my modulte serum lipid homeostsis, specificlly serum NE levels, we detected no influence of ERE on lood lipid prmeters. Discrepncies etween the previous nd current study my e ttriutle to differences in the ERE concentrtions reched fter exogenous ERE dministrtion s compred with those from endogenously produced ERE. trnsient decrese in serum NE ws reported fter the intrperitonel dministrtion of suprphysiologicl dose, 5 lg/grm ody weight, of purified ERE (Seldin et l. 1). However, circulting levels of ERE in mice rnge from elow the threshold of detection, t seline, to the low ng/ml rnge, in response to erythropoietic stimultion (Kutz et l. 15). Therefore, chnges oserved in response to concentrtions of ERE exceeding those detected in vivo y orders of mgnitude my not ccurtely represent the effect of ERE in the context of physiologicl responses. It is lso possile tht t high doses recominnt ERE cts s 18 Vol. 6 Iss. 19 e1389 Pge 8 ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society.

9 R. Coffey et l. Effect of ERE on Glucose etolism Hemogloin (g/dl) RC (/μl) NCl EPO NCl EPO NCl EPO NCl EPO NCl EPO NCl EPO NCl EPO NCl EPO C 8 D 8 Hemtocrit (%) CV (fl) NCl EPO NCl EPO NCl EPO NCl EPO WT NCl WT Erfe / Erfe / EPO NCl EPO NCl EPO NCl EPO igure 6. Stimultion of erythropoiesis y chronic erythropoietin tretment is preserved in mice lcking erythroferrone. Hemogloin (), red lood cell (), hemtocrit (C), nd men corpusculr volume (D) levels mesured in mle nd femle mice chroniclly treted with either U EPO or sline (NCl). Groups without common lpheticl superscript differ significntly from other groups of the sme sex s determined y one-wy NOV (P <.5). Dt re shown s the men SE (n = 5 6 per group for ech sex). noncognte lignd for receptors involved in the regultion of lipid homeostsis, ccounting for the previously reported effect of ERE on lood lipids. s tretment with EPO simultneously improves glucose tolernce nd insulin sensitivity while stimulting the production of ERE (Kutz et l. 1, 15), we investigted the possiility tht improvements in lood glucose homeostsis in response to chronic EPO tretment re influenced y incresed ERE signling. We found no effect of ERE on the enhncement of glucose tolernce in response to prolonged tretment with EPO, s mice lcking ERE demonstrted comprle glucose tolernce to tht of WT mice fter tretment with EPO. Determintion of insulin sensitivity y ITT in this study lso indictes tht ERE does not modulte insulin-stimulted glucose clernce. Lower fsting lood glucose vlues in EPO-treted mice hinder the determintion of whether EPO tretment improved insulin sensitivity compred with sline-treted controls in this study, ecuse lower glucose concentrtions in EPO-treted mice my enhnce counterregultory responses (Jcoson et l. 6) tht could llevite hypoglycemi during testing. However, the sence of ERE did not ffect insulin tolernce under sl or EPO stimulted conditions, s the response in Erfe / mice ws similr to tht oserved in WT mice, suggesting tht physiologic levels of ERE do not ffect insulin sensitivity, in greement with the oserved lck of effect on glucose tolernce. EPO my ffect glucose metolism y direct, EPO-receptor medited signling, s mice lcking the EPO receptor in dipocytes develop glucose intolernce nd insulin resistnce (Wng et l. 13). In this study, erythropoiesis, hepcidin suppression, nd depletion of iron stores in response to chronic EPO tretment were unffected y the sence of ERE. These findings suggest tht ERE, while cting s n cute regultor of iron homeostsis in response to erythropoietic stimultion, is dispensle for hepcidin suppression during prolonged moderte erythropoietic ugmenttion. One signl likely contriuting to hepcidin suppression in this model is iron depletion induced y chronic EPO tretment (ig. 7). The ide tht mechnisms independent of ERE could contriute to the iron homeosttic response to ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. 18 Vol. 6 Iss. 19 e1389 Pge 9

10 Effect of ERE on Glucose etolism R. Coffey et l. Serum hepcidin (ng/ml) Genotype, =.395, P =.533 Tretment, =.6, P =. Sex, =., P =.98 1 Liver non-heme iron (μg/g) NCl EPO NCl EPO NCl EPO NCl EPO WT NCl c c WT Erfe / Erfe / EPO NCl EPO incresed erythropoiesis is lso rised y the H th3/+ mouse, model of thlssemi intermedi (Yng et l. 1995), chrcterized y persistent nemi, incresed EPO levels, nd suppressed hepcidin expression, resulting in tissue iron ccumultion (De rnceschi et l. 6; Kutz et l. 15). lthough the hepcidin suppression is reversed y Erfe ltion, the iron overlod phenotype of H th3/+ mice is only prtilly corrected (Kutz et l. 15), suggesting either tht hepcidin is still inppropritely low for the degree of iron overlod or tht hepcidin-resistnt mechnism contriutes to iron overlod in these thlssemic mice. The demonstrle modultion of hepcidin y ERE in H th3/+ mice, ut not in this study, my e ttriutle c NCl EPO NCl EPO igure 7. Liver iron content nd hepcidin expression re not different etween WT nd Erfe / mice under sl conditions or fter chronic EPO tretment. () Serum hepcidin concentrtions in WT nd Erfe / mice chroniclly treted with either EPO or sline (NCl). Groups were nlyzed y three-wy NOV (genotype, sex, tretment), nd there ws no significnt interction etween fctors (P <.5). () Liver nonheme iron concentrtions in WT nd Erfe / mice chroniclly treted with either EPO or sline (NCl). Groups without common lpheticl superscript differ significntly from other groups of the sme sex s determined y one-wy NOV (P <.5). Dt re shown s the men SE (n = 5 7 per group for ech sex). to higher ERE expression in H th3/+ mice (Kutz et l. 15) compred with tht mesured fter chronic EPO tretment. t modertely elevted levels of ERE, s in this study, the influence of ERE signling on hepcidin suppression my e msked y other regultory mechnisms ut would still e discernle in models with more roust ERE production, such s the H th3/+ mouse. In conclusion, our dt indicte tht physiologic concentrtions of ERE do not ffect lood lipid or glucose homeostsis nd tht the glucometolic responses oserved in response to chronic EPO tretment re ERE-independent. oreover, chronic EPO therpy suppresses hepcidin y mechnism tht does not require ERE. These findings dd to the evidence tht the primry physiologic function of ERE is s stress hormone in the setting of cute lood loss or other cute nemi where EPO-stimulted ERE suppresses hepcidin nd therey ccelertes the supply of iron to compenstory erythropoiesis. s is the cse with other hormones, ERE my hve noncnonicl effects when its concentrtions re unphysiologiclly incresed to very high levels, s in mny ptients with -thlssemi or other disorders with ineffective erythropoiesis, or fter the dministrtion of exogenous ERE. cknowledgments The uthors thnk Le or Chrrot from the Phenotypge-nexplo pltform (US6-CRERE) for iochemicl ssys. Conflicts of Interest E.N. nd T.G. re consultnts nd shreholders of Intrinsic LifeSciences nd Silrus Therpeutics. The remining uthors declre no competing finncil interests. References llegr, V., G. engozzi, L. rtiminco, nd. Vsile Erly nd lte effects of erythropoietin on glucose metolism in mintennce hemodilysis ptients. m. J. Nephrol. 16:3 38. lneeli,.,.. Rk, O. Gvrilov, R. Teng, T. Chnturiy, nd C. T. Noguchi. 1. Erythropoietin signling: novel regultor of white dipose tissue inflmmtion during diet-induced oesity. Dietes 63: rton, J. C., nd S. S. ottomley.. Iron deficiency due to excessive therpeutic phleotomy in hemochromtosis. m. J. Hemtol. 65:3 6. oden, G., nd X. Chen Effects of ft on glucose uptke nd utiliztion in ptients with non-insulin-dependent dietes. J. Clin. Invest. 96: Vol. 6 Iss. 19 e1389 Pge 1 ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society.

11 R. Coffey et l. Effect of ERE on Glucose etolism oden, G., X. Chen, J. Ruiz, J. V. White, nd L. Rossetti echnisms of ftty cid-induced inhiition of glucose uptke. J. Clin. Invest. 93:38 6. Colemn, D. H.,. R. Jr Stevens, H. T. Dodge, nd C.. inch Rte of lood regenertion fter lood loss. rch. Intern. ed. 9: De rnceschi, L.,. Drio,. ilippini, S. Crturn, E.. uchitsch,. Roetto, et l. 6. Liver expression of hepcidin nd other iron genes in two mouse models of et-thlssemi. Hemtologic 91: inch, C.., K. Deueleiss, J. D. Cook, J. W. Eschch, L.. Hrker, D. D. unk, et l errokinetics in mn. edicine (ltimore) 9: oskett,.,. lneeli, L. Wng, R. Teng, nd C. T. Noguchi. 11. The effects of erythropoietin dose titrtion during high-ft diet-induced oesity. J. iomed. iotechnol. 11: Hillmn, R. S., nd P.. Henderson Control of mrrow production y the level of iron supply. J. Clin. Invest. 8:5 6. Jcoson, L., T. nsri, nd O. P. cguinness. 6. Counterregultory deficits occur within h of single hypoglycemic episode in conscious, unrestrined, chroniclly cnnulted mice. m. J. Physiol. Endocrinol. et. 9: E678 E68. Ktz, O.,. Stuile, N. Golishevski, L. Lifshitz,. L. Tremly,. Gssmnn, et l. 1. Erythropoietin tretment leds to reduced lood glucose levels nd ody mss: insights from murine models. J. Endocrinol. 5: Kutz, L., G. Jung, E. V. Vlore, S. Rivell, E. Nemeth, nd T. Gnz. 1. Identifiction of erythroferrone s n erythroid regultor of iron metolism. Nt. Genet. 6: Kutz, L., G. Jung, X. Du, V. Gyn, J. Chpmn,. Nsoff, et l. 15. Erythroferrone contriutes to hepcidin suppression nd iron overlod in mouse model of etthlssemi. lood 16: Knutson,., nd. Wessling-Resnick. 3. Iron metolism in the reticuloendothelil system. Crit. Rev. iochem. ol. iol. 38: Koury,. J., nd. C. ondurnt intennce y erythropoietin of viility nd mturtion of murine erythroid precursor cells. J. Cell. Physiol. 137:65 7. Koury,. J., nd. C. ondurnt Erythropoietin retrds DN rekdown nd prevents progrmmed deth in erythroid progenitor cells. Science 8: cotel, Y., J. oucher, T. T. Trn, nd C. R. Khn. 9. Sex nd depot differences in dipocyte insulin sensitivity nd glucose metolism. Dietes 58: k, R. H etolic effects of erythropoietin in ptients on peritonel dilysis. Peditr. Nephrol. 1: clchln, S., K. E. Pge, S.. Lee,. Loguinov, E. Vlore, S. T. Hui, et l. 17. Hmp1 mrn nd plsm hepcidin levels re influenced y sex nd strin ut do not predict tissue iron levels in inred mice. m. J. Physiol. Gstrointest. Liver Physiol. 313:G511 G53. Nemeth, E.,. S. Tuttle, J. Powelson,.. Vughn,. Donovn, D.. Wrd, et l.. Hepcidin regultes cellulr iron efflux y inding to ferroportin nd inducing its internliztion. Science 36:9 93. Sntomuro,. T., G. oden,. E. Silv, D.. Roch, R.. Sntos,. J. Ursich, et l Overnight lowering of free ftty cids with cipimox improves insulin resistnce nd glucose tolernce in oese dietic nd nondietic sujects. Dietes 8: Seldin,.., J.. Peterson,. S. yerly, Z. Wei, nd G. W. Wong. 1. yonectin (CTRP15), novel myokine tht links skeletl muscle to systemic lipid homeostsis. J. iol. Chem. 87: Seldin,.., X. Lei, S. Y. Tn, K. P. Stnson, Z. Wei, nd G. W. Wong. 13. Skeletl muscle-derived myonectin ctivtes the mmmlin trget of rpmycin (mtor) pthwy to suppress utophgy in liver. J. iol. Chem. 88: Wng, L., R. Teng, L. Di, H. Rogers, H. Wu, J.. Kopp, et l. 13. PPRlph nd Sirt1 medite erythropoietin ction in incresing metolic ctivity nd rowning of white dipocytes to protect ginst oesity nd metolic disorders. Dietes 6: Yng,., S. Kiry, J. Lewis, P. J. Detloff, N. ed, nd O. Smithies mouse model for et -thlssemi. Proc. Ntl. cd. Sci. U S 9: ª 18 The uthors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of The Physiologicl Society nd the mericn Physiologicl Society. 18 Vol. 6 Iss. 19 e1389 Pge 11