Vol 457 26 Ferury 29 oi:1.138/nture7617 Deficiency of -rrestin-2 signl complex contriutes to insulin resistnce Bing Lun 1, Jin Zho 1, Hiy Wu 3, Boyu Dun 1, Gungwen Shu 1, Xioying Wng 4, Dngsheng Li 2, Weiping Ji 3, Jiuhong Kng 1 & Gng Pei 1,5 resistnce, hllmrk of type 2 ietes, is efect of insulin in stimulting insulin receptor signlling 1,2, which hs ecome one of the most serious pulic helth threts. Upon stimultion y insulin, insulin receptor recruits n phosphoryltes insulin receptor sustrte proteins 3, leing to ctivtion of the phosphtiylinositol-3-oh kinse (PI(3)K) pthwy. Activte phosphoryltes ownstrem kinses n trnscription fctors, thus meiting most of the metolic ctions of insulin 46. -rrestins meite iologicl functions of G-proteincouple receptors y linking ctivte receptors with istinct sets of ccessory n effecter proteins, therey etermining the specificity, efficiency n cpcity of signls 711. Here we show tht in ietic mouse moels, -rrestin-2 is severely ownregulte. Knockown of -rrestin-2 excertes insulin resistnce, wheres ministrtion of -rrestin-2 restores insulin sensitivity in mice. Further investigtion revels tht insulin stimultes the formtion of new -rrestin-2 signl complex, in which -rrestin-2 scffols n to insulin receptor. Loss or ysfunction of -rrestin-2 results in eficiency of this signl complex n isturnce of insulin signlling in vivo, therey contriuting to the evelopment of insulin resistnce n progression of type 2 ietes. Our finings provie new insight into the moleculr pthogenesis of insulin resistnce, n implicte new preventive n therpeutic strtegies ginst insulin resistnce n type 2 ietes. We first investigte the expression pttern of -rrestins in the / mouse moel of type 2 ietes. There ws ecrese in -rrestin-2 protein n messenger RNA levels in liver n skeletl muscle of / mice (Fig. 1, ). Consistently, in ietry-inuce insulin-resistnt moel mice (high-ft iet (HFD) mice) 12, expression of -rrestin-2 lso ecrese in liver n skeletl muscle (Fig. 1c, ). Expression levels of -rrestin-2 in ipose tissue (Fig. 1), rin or lung of / or HFD mice i not chnge (t not shown). -rrestin-1 ws ownregulte in liver n skeletl muscle, ut to lesser extent thn -rrestin-2 (t not shown). We lso foun similr ownregultion of -rrestins in liver from clinicl smples of type 2 ietes (eight pirs) (Supplementry Fig. 1). Chnges in -rrestin levels in these insulin-resistnt mice n clinicl smples rise the possiility tht -rrestins might hve role in insulin signlling n tht its eficiency might contriute to insulin resistnce n type 2 ietes. We then ssesse the potentil roles of -rrestin-2 in whole-oy insulin ction n glucose metolism. -rrestin-2 knockout (-rr2-ko) mice re vile n fertile, with norml oy weight n foo intke (Supplementry Fig. 2). Their fste loo glucose n insulin levels were norml, ut re-fe loo glucose n insulin levels were much higher thn wil-type littermtes (Fig. 2, ), suggesting potentil ecrese in systemic insulin sensitivity. In fct, glucose tolernce tests (GTTs) revele significnt eteriortion in glucose metolism in -rr2-ko mice (Fig. 2c). secretion in response to glucose lo uring GTTs ws lso higher in -rr2-ko mice (Supplementry Fig. 2c), consistent with insulin tolernce tests (ITTs) which showe significntly ecrese insulin sensitivity in -rr2-ko mice (Fig. 2). To efine the role for -rrestin-2 in moulting whole-oy insulin sensitivity further, we performe hyperinsulinemiceuglycemic clmp stuies. We foun no significnt ifference in sl heptic glucose prouction etween -rr2-ko mice n wil-type littermtes, ut clmp heptic glucose prouction ws significntly higher in -rr2-ko mice (Supplementry Fig. 2). Whole-oy glucose isposl n infusion rtes were significntly ecrese in -rr2-ko mice (Supplementry Fig. 2e). These results together emonstrte tht -rr2-ko mice exhiite impire insulin sensitivity. c Len + + + RD + + + + + + / + + + HFD + + + + + + Tuulin Tuulin Reltive protein level Reltive mrna level.2.2 Len / Len / Reltive protein level Reltive mrna level.2.2 RD HFD RD HFD Figure 1 Downregultionof-rrestin-2inieticmice.,, Immunolot () n quntittive RTPCR ()of-rrestin expression in ipose tissue, liver n skeletl muscle of len (n 5 5) n / mice (n 5 5). Densitometric nlysis is shown. c,, Immunolot (c) n quntittive PCR with reverse trnscription (RTPCR) () of -rrestin expression in ipose tissue, liver n skeletl muscle of C57BL/6 mice fe with regulr iet (RD) (n 5 5) or HFD (n 5 5). Densitometric nlysis is shown. Dt re presente s men n s.e.m. P,.5, P,.5, versus control. 1 Lortory of Moleculr Cell Biology, Institute of Biochemistry n Cell Biology, n Grute School of the Chinese Acemy of Sciences, 2 Shnghi Informtion Center for Life Sciences, Shnghi Institutes for Biologicl Sciences, Chinese Acemy of Sciences, 231, Shnghi, Chin. 3 Deprtment of Enocrinology n Metolism, Shnghi Jiotong University Affilite Sixth People s Hospitl; Shnghi Dietes Institute; Shnghi Clinicl Center of Dietes, 2233, Shnghi, Chin. 4 Fun University Affilite Zhongshn Hospitl, 232, Shnghi, Chin. 5 School of Life Science n Technology, Tongji University, 292, Shnghi, Chin. 1146 29 Mcmilln Pulishers Limite. All rights reserve

NATURE Vol 457 26 Ferury 29 c -KO -KO 3 6 2 5 4 3 15 2 2 1 Fste Re-fe Fste Re-fe 15369 12 e f g h -Tg 3 -Tg 2.5 2 2. 15 1.5 2.5 5 Fste Re-fe Fste Re-fe 15 3 69 12 (ng ml 1 ) (ng ml 1 ) i Bloo glucose (mg l 1 ) 7 6 Glucose (mg l 1 ) Glucose (mg l 1 ) 5 4 3 2 4 6 8 Time (ys) l 5 Bloo glucose (mg l 1 ) 4 3 2 1536912 j Bloo glucose (mg l 1 ) 6 5 Bloo glucose (mg l 1 ) Bloo glucose (mg l 1 ) 4 3 2 15 3 6 912 -KO + -KO + 2 15 3 6 912 Trnsgenic mice expressing humn -rrestin-2 riven y the cytomeglovirus (CMV) promoter (-rr2-tg) h n pproximte twofol increse in liver -rrestin-2 expression compre with control mice (Supplementry Fig. 2). -rr2-tg mice evelop normlly with norml foo intke n oy weight (Supplementry Fig. 2). Their serum insulin n loo glucose levels were similr to wiltype mice in fste stges ut much lower in re-fe stges (Fig. 2e, f). GTTs n ITTs showe ugmente glucose metolism n insulin sensitivity in -rr2-tg mice (Fig. 2g, h, n Supplementry Fig. 2c). Similr results were otine using -rr2-ko, -rr2-tg n their wil-type littermtes tht h een fe on HFD (Supplementry Fig. 3), further suggesting potentil nti-ietic role of -rrestin-2. We next explore the possiility tht ministrtion of -rrestin-2 my hve therpeutic potentil ginst insulin resistnce n type 2 ietes. To this en, we ministere -rrestin-2 in / mice y using enovirus. Intrvenous injection of recominnt enovirus expressing -rrestin-2 le to n pproximte threefol increse of -rrestin-2 protein levels in liver, without ltering the foo intke n oy weight (Supplementry Fig. 2f, g). The loo glucose levels uner fe conitions were reuce in / mice tht receive the -rrestin-2 enovirus, compre with control mice (Fig. 2i). Injection of -rrestin-2 enovirus meliorte glucose tolernce m k 14 12 8 6 4 2 12 8 6 4 12 8 6 -KO -Tg 4 15 3 6 912 15 3 6 912 -KO + 12 -KO + 8 6 4 2 15 3 6 912 Figure 2 -rrestin-2 ffects the evelopment of insulin resistnce.,, Serum insulin () n loo glucose () levels in -rr2-ko mice (n 5 8) n wil-type () littermtes (n 5 8) uner fste n re-fe stges. c,, Glucose levels uring GTTs (1 g kg 21 )(c) n ITTs (1 U kg 21 ) ()in-rr2-ko mice (n 5 1) n wil-type littermtes (n 5 8). e, f, Serum insulin (e) n loo glucose (f) levels in -rr2-tg mice (n 5 8) n wiltype littermtes (n 5 8) uner fste n re-fe stges. g, h, GTTs (1 g kg 21 ) (g) n ITTs (.75 U kg 21 )(h) in-rr2-tg mice (n 5 11) n wil-type littermtes (n 5 8). i, Bloo glucose concentrtion of / mice (n 5 8) injecte with inicte enovirus. j, k, GTTs (1.5 g kg 21 )(j) n ITTs (1.5 U kg 21 )(k)in/ mice t 7 ys fter enovirus injection. l, m, GTTs (1.5 g kg 21 )(l) n ITTs (1.5 U kg 21 )(m)in-rr2-ko mice fe on HFD t 7 ys fter enovirus injection. Dt re presente s men 6 s.e.m. P,.5, P,.5, versus control. 29 Mcmilln Pulishers Limite. All rights reserve n insulin sensitivity, s shown in GTTs n ITTs (Fig. 2j, k). Similrly, rescue of -rrestin-2 expression y enovirus injection in -rr2-ko mice fe on HFD mene glucose intolernce n insulin resistnce in these mice (Fig. 2l, m). These results inicte tht -rrestin-2 is positive regultor of insulin sensitivity. At the moleculr level, insulin resistnce results from efects in insulin signlling in peripherl tissues 4. Interestingly, lthough ctivtion of PI(3)K y insulin ws similr in mice of ll genotypes (Supplementry Fig. 4), we oserve ifferent ctivities of in liver of -rr2-ko, -rr2-tg n wil-type mice, s monitore y in vitro kinse ssy (Supplementry Fig. 4). Further, insulin-stimulte phosphoryltion of, - n were rmticlly reuce in liver of -rr2-ko mice ut lrgely increse in -rr2-tg mice (Fig. 3,, n Supplementry Fig. 4c). Similr results were lso oserve in skeletl muscle n ipose tissue (Supplementry Fig. 4g). These results show tht -rrestin-2 promotes insulinstimulte ctivtion of ut oes not ffect PI(3)K ctivity. Recent stuies hve shown tht phosphoryltion of t Tyr 315/ 326 y enhnces serine/threonine phosphoryltion n is prerequisite of full ctivtion 1317. We hve lso oserve tht, in the presence of inhiitor PP2, tyrosine phosphoryltion n serine/ threonine phosphoryltion of were remrkly reuce in Hep3B heptocytes (Supplementry Fig. 5). Furthermore, ctivity ws reuce when Tyr 315/326 were sustitute y Al (Supplementry Fig. 5). In mouse liver, we oserve tht insulin stimultion le to n pproximte twofol increse in tyrosine phosphoryltion of in wil-type mice (Fig. 3c n Supplementry Fig. 5c), which ws reuce y out 4% in -rr2-ko mice n increse y out 1.5-fol in -rr2-tg mice (Fig. 3c n Supplementry Fig. 5c). Interestingly, knockown of y short interfering RNA (sirna) (Fig. 3) in primry heptocytes isolte from -rr2-ko mice n their wil-type littermtes olishe the ifference in ctivities (Fig. 3e) n moertely epresse the ifferentil reuction on g6p/pepck expression (Supplementry Fig. 5, e). The suppressive effect on heptocytic glucose prouction conferre y -rrestin-2 ws lso olishe in the presence of sirna (Fig. 3f). Together, these results suggest tht -rrestin-2 promotes ctivtion n glucose metolism through. -rrestins hve een reporte to function s ptors n to promote the ctivtion of vrious mitogen-ctivte protein kinses such s ERK1/2 n JNK3 (ref. 18). A similr scffoling mechnism might lso operte in insulin signlling. We foun tht in liver of C57BL/6 mice, enogenous -rrestin-2, n were co-purifie with ech other (Fig. 3g). However, the ssocition etween n ws rmticlly reuce in liver smples from -rr2-ko mice (Fig. 3h n Supplementry Fig. 6). Conversely, n interction ws remrkly enhnce in -rr2-tg mice (Fig. 3i n Supplementry Fig. 6). These results strongly suggest tht -rrestin-2, n form complex in vivo n tht -rrestin-2 is essentil for meiting the ssocition of with. Next, we exmine whether the formtion of this /rrestin-2/ signlling complex epens on insulin stimultion. Intrperitonel ministrtion of insulin triggere mrke increse in / interction s well s /-rrestin-2 ssocition in livers of C57BL/6 mice (Fig. 3j n Supplementry Fig. 6c), implicting the promotion of /-rrestin-2/ interction y insulin stimultion. Furthermore, we foun -rrestin-2 intercts irectly with insulin receptor (Supplementry Fig. 6). -rrestin-2 s well s n ssocite with insulin receptor in similr time-epenent mnner (Fig. 3k n Supplementry Fig. 6e). Moreover, interction of n with insulin receptor epens on the expression level of -rrestin-2 (Supplementry Fig. 6f, g), supporting the ie tht -rrestin-2 is essentil for the ssocition of / with insulin receptor. We exmine series of trunction mutnts of -rrestin-2 y immunoprecipittion ssy. A -rrestin-2 frgment comprising mino cis intercts with s efficiently s wil-type -rrestin-2 ut completely loses the interction with (Fig. 4). 1147

nti- nti-ir-β nti-ir-β nti- nti- nti- nti- NATURE Vol 457 26 Ferury 29 p(thr 38) p(ser 473) p KO Tg + + + + p(thr 38) p(ser 473) p c nti- p-tyr p-tyr KO + + Tg + + HA + HA + HA + HA + + + + + + + HA p(thr 38) p(ser 473) p + + + + sirna sirna +sirna e KO KO + + + + p(thr 38) h p(ser 473) nti- j nti- β-rrestin KO + i nti- nti- TG 1 3 6 9 12 Overexpression of -rrestin-2 in Hep3B heptocytes inhiite / interction (t not shown) n rmticlly reuce insulin-stimulte ctivtion (Fig. 4), presumly y competing with enogenous -rrestin-2. Moreover, overexpression of -rrestin-2, truncte mutnt tht intercts with insulin receptor ut not with or (Fig. 4c) in Hep3B heptocytes, ominnt-negtively suppresse insulin-stimulte ctivtion (Fig. 4). These results clerly emonstrte the pivotl role of -rrestin-2 in scffoling the ctive insulin receptor// -rrestin-2/ signlling complex fter insulin stimultion. f Heptocyte glucose output k 1.6 KO ++ sirna ++ +sirna IR-β β-rrestin g IgG nti-β-rrestin IgG nti- 1 3 6 9 12 Figure 3 stimulte the formtion of Receptor//-rrestin-2/ signl complex.,, Activtion of in livers of -rr2-ko mice (n 5 6) (), -rr2-tg mice (n 5 6) () n their wil-type littermtes (n 5 6). Mice were injecte with either sline or insulin (1 U kg 21 ) for 1 min. c, Tyrosine phosphoryltion of immunopurifie from livers of -rr2-ko mice (n 5 6), -rr2-tg mice (n 5 6), n their wil-type littermtes (n 5 6)., Suppression of in primry heptocytes y sirna. e, ctivities in primry heptocytes shown in (n 5 3). f, Glucose prouction trete with or without 1 nm insulin for 3 h in primry heptocytes shown in (n 5 3). g, Interctions of /-rrestin-2/ ssye y immunoprecipittion from livers of C57BL/6 mice (n 5 5). h, i, Interction of n in liver extrcts of -rr2-ko mice (n 5 6) (h), -rr2-tg mice (n 5 6) (i), n their wil-type littermtes (n 5 6) were ssye y immunoprecipittion. j, inuces formtion of /rrestin-2/ complex. Immunoprecipittion ws conucte in liver extrcts of C57BL/6 mice (n 5 6) injecte with 1 U kg 21 insulin for the inicte times. k, stimulte interction of /-rrestin-2/ with insulin receptor (n 5 6). Dt re presente s men n s.e.m. P,.5, P,.5, versus control. 1148 c HA + HA e f g 3 2 Bloo glucose (mg l 1 ) IR-β 175 15 125 2 4 6 8 Dy 29 Mcmilln Pulishers Limite. All rights reserve + Bloo glucose (mg l 1 ) 2 HA + HA 15 3 6 9 12 We further teste whether expression of these -rrestin-2 mutnts woul contriute to insulin resistnce in vivo. Aenoviruses encoing -Gl, -rrestin-2 or -rrestin-2 were injecte intrvenously into C57BL/6 or / ietic mice. C57BL/6 mice infecte with enoviruses expressing -rrestin-2 n -rrestin-2 showe higher loo glucose levels thn those of control mice (Fig. 4e). -rrestin-2 n enovirus injection lso eteriorte glucose tolernce n insulin sensitivity, s shown in GTTs n ITTs (Fig. 4f, g). Similr results were otine in / ietic mice (Supplementry Fig. 7). Tken together, we hve shown tht ssignment of with n ctivte insulin receptor y -rrestin-2 is essentil for proper insulin signlling n whole-oy insulin ction. In contrst to the clssicl known pthwy, -rrestin-2 meites ctivtion of through, which oes not ffect PI(3)K. However, this -rrestin-2- pthwy n PI(3)K pthwy re not necessrily seprte. One possiility coul e tht insulin receptor sustrtes n PI(3)K, s well s PDKs, my e involve in the signl complex, integrting these two pthwys into one insulin-signlling network for efficient signl trnsuction (Supplementry Fig. 7). Current n future investigtions of the function n mechnism of this + + HA p(thr 38) p(ser 473) p + + + 1.1.9.7.5 15 3 6 9 12 Figure 4 Muttion of -rrestin-2 contriutes to insulin resistnce in vivo., -rrestin-2 intercts with ut not. Immunoprecipittion ws conucte from Hep3B heptocytes trnsfecte with inicte plsmis (n 5 3)., -rrestin-2 inhiits ctivtion of in Hep3B heptocytes (n 5 3). c, -rrestin-2 intercts with insulin receptor ut not or in Hep3B heptocytes (n 5 3)., -rrestin-2 inhiits ctivtion of in Hep3B heptocytes (n 5 3). e, Bloo glucose concentrtion of C57BL/6 mice (n 5 8) injecte with inicte enovirus. f, g, GTTs (1 g kg 21 )(f) n ITTs (1 U kg 21 )(g) in C57BL/6 mice t 7 ys fter enovirus injection. Dt re presente s men 6 s.e.m. P,.5, P,.5, versus control.

NATURE Vol 457 26 Ferury 29 novel signl complex will provie new insight into the unerstning of insulin resistnce n type 2 ietes, n uncover potentil moleculr trgets for treting metolic iseses. METHODS SUMMARY Immunoprecipittion n immunolotting. Mouse tissues were quickly excise n frozen in liqui nitrogen. Tissue lyste ws prepre n use for immunoprecipittion n immunolotting s escrie 19,2. Blots were incute with IRDyeTM8CW-conjugte seconry ntioy. The imge ws cpture n nlyse y the Oyssey infrre imging system n Scion Imge (Li-Cor Bioscience). mrna nlysis. We nlyse -rrestin mrna levels y rel-time PCR fter reverse trnscription s escrie 21. Hypoxnthinegunine phosphoriosyltrnsferse (HPRT) mrna levels were use for normliztion etween smples. GTTs n ITTs. For GTTs, mice were injecte intrperitonelly with glucose fter strvtion for 6 h. Bloo glucose ws mesure t inicte time points. We injecte insulin intrperitonelly to mice uner fe conitions for ITTs. We collecte loo n etermine the glycemi using glucometer (Roche Accu-chek). Serum insulin levels were mesure using rt/mouse enzymelinke immunosorent ssy (ELISA) kit. Sttisticl nlysis. All t re shown s men 6 s.e.m. Mesurements t single time points were nlyse y nlysis of vrince (ANOVA) or, if pproprite, y Stuent s t-test. Time courses were nlyse y repete-mesurements (mixemoel) ANOVA with Bonferroni post-tests. Full Methos n ny ssocite references re ville in the online version of the pper t www.nture.com/nture. Receive 25 Jnury; ccepte 7 Novemer 28. Pulishe online 4 Jnury 29; correcte 26 Ferury 29 (etils online). 1. Mtthei, S., Stumvoll, M., Kellerer, M. & Hring, H. U. Pthophysiology n phrmcologicl tretment of insulin resistnce. Enocr. Rev. 21, 585618 (2). 2. Tniguchi, C. M., Emnuelli, B. & Khn, C. R. Criticl noes in signlling pthwys: insights into insulin ction. Nture Rev. Mol. Cell Biol. 7, 8596 (26). 3. Sun, X. J. et l. 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An /-rrestin 2/PP2A signling complex meites opminergic neurotrnsmission n ehvior. Cell 122, 261273 (25). 11. Beulieu, J. M. et l. A -rrestin 2 signling complex meites lithium ction on ehvior. Cell 132, 125136 (28). 12. Yng, Q. et l. Serum retinol ining protein 4 contriutes to insulin resistnce in oesity n type 2 ietes. Nture 436, 356362 (25). 13. Chen, R. et l. Regultion of /PKB ctivtion y tyrosine phosphoryltion. J. Biol. Chem. 276, 3185831862 (21). 14. Jing, T. & Qiu, Y. Interction etween n C-terminl proline-rich motif of is require for ctivtion. J. Biol. Chem. 278, 1578915793 (23). 15. Crxton, A., Jing, A., Kuroski, T. & Clrk, E. A. Syk n Bruton s tyrosine kinse re require for B cell ntigen receptor-meite ctivtion of the kinse. J. Biol. Chem. 274, 3644365 (1999). 16. Wong, B. R. et l. TRANCE, TNF fmily memer, ctivtes /PKB through signling complex involving TRAF6 n c-. Mol. Cell 4, 141149 (1999). 17. Dtt, K., Bellcos, A., Chn, T. O. & Tsichlis, P. N. is irect trget of the phosphtiylinositol 3-kinse. Activtion y growth fctors, v-src n v-h-rs, in Sf9 n mmmlin cells. J. Biol. Chem. 271, 38353839 (1996). 18. DeWire, S. M., Ahn, S., Lefkowitz, R. J. & Shenoy, S. K. -rrestins n cell signling. Annu. Rev. Physiol. 69, 48351 (27). 19. Go, H. et l. Ientifiction of -rrestin2 s G protein-couple receptorstimulte regultor of NF-kB pthwys. Mol. Cell 14, 33317 (24). 2. Lun, B., Zhng, Z., Wu, Y., Kng, J. & Pei, G. -rrestin2 functions s phosphoryltion-regulte suppressor of UV-inuce NF-kB ctivtion. EMBO J. 24, 42374246 (25). 21. Kng, J. et l. A nucler function of -rrestin1 in GPCR signling: regultion of histone cetyltion n gene trnscription. Cell 123, 833847 (25). Supplementry Informtion is linke to the online version of the pper t www.nture.com/nture. Acknowlegements We re grteful to R. J. Lefkowitz for proviing us with -rr2-ko mice. We thnk J.-L. Gun for iscussions n comments on the mnuscripts. We thnk ll memers of the lortory for shring regents n vice. This reserch ws supporte y the Ministry of Science n Technology (25CB52246, 26CB9439, 27CB94794, 27CB947, 27CB948 n 29CB941), Ntionl Nturl Science Fountion of Chin (362191, 362514, 36233, 3871285 n 971347), Shnghi Municipl Commission for Science n Technology (7PJ1499 n 6DZ2232), Chinese Acemy of Sciences (KSCX2-YW-R-56 n 27KIP24). Author Contriutions This stuy ws esigne y B.L., J.Z. n G.P. The experiments were performe y B.L., B.D. n G.S. H.W. n W.J. contriute to the hyperinsulinemiceuglycemic clmp experiments. X.W. provie type 2 ietes clinic smples. G.P. supervise the project. B.L. n J.Z. contriute to the writing of the pper. D.L. helpe with the mnuscript. Author Informtion Reprints n permissions informtion is ville t www.nture.com/reprints. Corresponence n requests for mterils shoul e resse to G.P. (gpei@sis.c.cn). 29 Mcmilln Pulishers Limite. All rights reserve 1149

oi:1.138/nture7617 METHODS Mice. -rr2-ko mice were provie y R. J. Lefkowitz (Duke University Meicl Center). -rr2-tg mice were generte s escrie 22. All other mice were from Shnghi Lortory Animl Center, Chinese Acemy of Sciences. Mice were fe with regulr iet (Formul 58, Liet 553) or high-ft iet (55% ft clories) (Hrln-Tekl 9375) n h free ccess to wter n iet. We injecte enovirus (7 3 1 9 virl prticles per ml sline) into the til vein of / n C57BL/6 mive specificlly to trget the liver. Boy weight n foo intke were mesure s escrie 23. Len mice were 8 weeks ol n fe with regulr iet (oy weight 2.3 6 g, foo intke 3.5 6.5 g), / mice were 8 weeks ol n fe with regulr iet (oy weight 38.5 6 1.4 g, foo intke 4.8 6.7 g). Six-week-ol C57BL/6 mice were fe on regulr iet (oy weight 22.6 6 2.1 g, foo intke 3.6 6.3 g) or HFD (oy weight 36.7 6 1.9 g, foo intke 4.1 6.2 g) for n itionl 8 weeks. All mice except for HFD feeing were 8 weeks ol when experiments were conucte; / mice were 8 weeks ol when they receive enovirus injection. GTTs n ITTs were conucte t 7 ys fter enovirus injection. Cell trnsfection n plsmis. Humn heptocyte Hep3B cells were trnsfecte y LipofectAMINE (Invitrogen). For ll trnsfection experiments, CMV--Gl ws use to compenste the totl DNA input. Full lengths of n were clone into moifie pcdna3 vector in-frme with HA or Flg t the mino (N) terminus. Tyr315/326Al ws lso clone into moifie pcdna3 vector in-frme with Flg t the N terminus. Plsmis contining complementry DNA (cdna) encoing -rrestin-2 n its trunction mutnts were generte s escrie 19. The uthenticity of the DNA sequences ws confirme y sequencing. Mterils n regents. Anti--rrestin rit polyclonl ntioy (A1CT n A2CT) ws gift from R. J. Lefkowitz (Duke University Meicl Center). Antioies irecte ginst (totl), (phosphorylte Thr38), (phosphorylte Ser473), -/ (phosphorylte Ser21/9), (totl), (phosphorylte Ser256),, phosphorylte Tyr n insulin receptor were otine from Cell Signlling. -/ ntioy ws from Snt Cruz. PP2 n wortmnnin were from Sigm. The Rt/Mouse ELISA Kit ws from Linco Reserch. The PI3-Kinse ELISA Kit ws from Echelon Biosciences. The Rt/Mouse Glucgon ELISA Kit, NEFA, triglyceries, n cholesterol etection kit were from WAKO Chemicls USA. The Mouse Epinephrine ELISA Kit ws from USCN Life Science. The Kinse Assy Kit ws from Cell Signlling. Aenovirus preprtion n injection. We generte enoviruses encoing Gl, -rrestin-2, -rrestin-2 n -rrestin-2 using the Esy system ccoring to the mnufcturer s instructions (Strtgene). Quntittive PCR couple with RTPCR. The primer pirs use were: mouse -rrestin-1 sense, 59- AAGGGACACGAGTGTTCAAGA-39; ntisense, 59-CCC GCT TTC CCA GGT AGA C-39; mouse -rrestin-2 sense, 59-GGC AAG CGC GAC TTT GTA G-39; ntisense, 59- GTG AGG GTC ACG AAC ACT TTC-39; mouse HPRT sense, 59-CCT GCT GGA TTA CAT TAA AGC ACT G-39; ntisense, 59-TTC AAC ACT TCG AGA GGT CCT-39. Primry heptocyte culture n glucose prouction. Primry heptocytes were isolte n culture fter perfusion n collgense igestion of the liver 24. Glucose prouction in primry heptocytes ws mesure s escrie 12, n Glucose (GO) Assy Kit from Sigm ws use to etect glucose concentrtion. Hyperinsulinemiceuglycemic clmps. Hyperinsulinemiceuglycemic clmps were performe in 8-week-ol mice s escrie 25,26. ws infuse t 2.5 mu kg 21 min 21. 22. Shi, Y. et l. Criticl regultion of CD4 1 T cell survivl n utoimmunity y -rrestin 1. Nture Immunol. 8, 817824 (27). 23. Nete, M. G. et l. Deficiency of interleukin-18 in mice les to hyperphgi, oesity n insulin resistnce. Nture Me. 12, 65656 (26). 24. Arkn, M. C. et l. IKK- links inflmmtion to oesity-inuce insulin resistnce. Nture Me. 11, 191198 (25). 25. Shen, H. Q., Zhu, J. S. & Bron, A. D. Dose-response reltionship of insulin to glucose fluxes in the wke n unrestrine mouse. Metolism 48, 96597 (1999). 26. Hluzik, M. M. et l. Improvement of insulin sensitivity fter peroxisome prolifertor-ctivte receptor-lph gonist tretment is ccompnie y proxicl increse of circulting resistin levels. Enocrinology 147, 45174524 (26). 29 Mcmilln Pulishers Limite. All rights reserve