Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system.

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1 Aging Cell (216) 15, pp28 38 Doi: /el.1245 Alterntive rpmyin tretment regimens mitigte the impt of rpmyin on gluose homeostsis nd the immune system Aging Cell Sestin I. Arriol Apelo, 1,2 Joshu C. Neumn, 2,3 Emm L. Br, 1,2 Fizn A. Syed, 1,2 Niole E. Cummings, 1,2,4 Hrpreet K. Brr, 1,2 Cssidy P. Pumper, 1,2 Mihelle E. Kimple 1,2,3,4 nd Dudley W. Lmming 1,2,3,4,5 1 Deprtment of Mediine, University of Wisonsin-Mdison, Mdison, WI, USA 2 Willim S. Middleton Memoril Veterns Hospitl, Mdison, WI, USA 3 Interdisiplinry Grdute Progrm in Nutritionl Sienes, University of Wisonsin-Mdison, Mdison, WI, USA 4 Endorinology nd Reprodutive Physiology Grdute Trining Progrm, University of Wisonsin-Mdison, Mdison, WI, USA 5 University of Wisonsin Crone Cner Center, Mdison, WI, USA Summry Inhiition of the mehnisti trget of rpmyin (mtor) signling pthwy y the FDA-pproved drug rpmyin hs een shown to promote lifespn nd dely ge-relted diseses in model orgnisms inluding mie. Unfortuntely, rpmyin hs potentilly serious side effets in humns, inluding gluose intolerne nd immunosuppression, whih my prelude the long-term prophylti use of rpmyin s therpy for ge-relted diseses. While the enefiil effets of rpmyin re lrgely medited y the inhiition of mtor omplex 1 (mtorc1), whih is utely sensitive to rpmyin, mny of the negtive side effets re medited y the inhiition of seond mtor-ontining omplex, mtorc2, whih is muh less sensitive to rpmyin. We hypothesized tht different rpmyin dosing shedules or the use of FDApproved rpmyin nlogs with different phrmokinetis might expnd the therpeuti window of rpmyin y more speifilly trgeting mtorc1. Here, we identified n intermittent rpmyin dosing shedule with miniml effets on gluose tolerne, nd we find tht this shedule hs redued impt on pyruvte tolerne, fsting gluose nd insulin levels, et ell funtion, nd the immune system ompred to dily rpmyin tretment. Further, we find tht the FDApproved rpmyin nlogs everolimus nd temsirolimus effiiently inhiit mtorc1 while hving redued impt on gluose nd pyruvte tolerne. Our results suggest tht mny of the negtive side effets of rpmyin tretment n e mitigted through intermittent dosing or the use of rpmyin nlogs. Key words: ging; mehnisti trget of rpmyin; mie; rpmyin. Correspondene Dudley W. Lmming, Willim S. Middleton Memoril Veterns Hospitl, Overlook Terre, Room C3127 Reserh 151, Mdison, WI 53, USA. Tel.: x12861; fx: ; e-mil: dlmming@mediine.wis.edu Aepted for pulition August 215 Introdution Rpmyin is n FDA-pproved ompound tht roustly extends lifespn in yest, worms, flies, nd mie (Johnson et l., 213). Rpmyin is n ute inhiitor of the mehnisti trget of rpmyin (mtor) omplex 1 (mtorc1), protein kinse whih regultes numerous ellulr proesses inluding riosoml iogenesis, protein trnsltion, nd utophgy through the phosphoryltion of sustrtes tht inlude S6K1, 4E-BP1, nd Ulk1. Mie lking S6K1 or with deresed mtorc1 tivity hve extended longevity, demonstrting tht deresed mtorc1 signling is suffiient to promote longevity, espeilly in femles (Selmn et l., 29; Lmming et l., 212; Wu et l., 213). Unfortuntely, the potentil for serious side effets in humns, inluding immunosuppression nd gluose intolerne, my prelude the long-term prophylti use of rpmyin s therpy for ge-relted diseses (Lmming et l., 213). While investigting the mehnisti sis for the effet of rpmyin on gluose tolerne, we disovered tht long-term tretment with rpmyin lso inhiits seond mtor omplex, mtorc2, in vivo, resulting in hepti insulin resistne (Lmming et l., 212, 213). mtorc2 hs lso reently een shown to hve n importnt role in promoting immune funtion, whih suggests tht the immunosuppressive effets of rpmyin my e due in prt to the inhiition of mtorc2 signling (Powell et l., 212; Byles et l., 213; Festui et l., 214). Finlly, we reently ompleted lifespn study, finding tht geneti inhiition of mtorc2 ws severely deleterious to survivl of mle, ut not femle, mie (Lmming et l., 214). The evidene to dte suggests tht the inhiition of mtorc1 will promote longevity nd retrd ge-relted diseses, while the inhiition of mtorc2 is likely deleterious to helth nd impirs gluose homeostsis nd the immune system. While rpmyin inhiits oth omplexes, it is potent nd ute inhiitor of mtorc1, nd inhiits mtorc2 signling only fter prolonged tretment (Srssov et l., 26). Interestingly, rpmyin dministrtion for 2 weeks out of every four n signifintly extend lifespn (Anisimov et l., 21, 211), demonstrting tht rpmyin dministrtion does not hve to e ontinuous to extend lifespn. However, the impt of rpmyin on gluose homeostsis persists for 2 weeks following esstion of rpmyin (Yng et l., 212; Liu et l., 214), suggesting tht this dosing strtegy my not minimize side effets. Gluose intolerne ws lso oserved in reent study of mie reeiving rpmyin three times per week (Leontiev et l., 214). In this study, we tested severl intermittent rpmyin tretment shedules to identify the most frequent rpmyin dosing shedule tht is still omptile with gluose tolerne in C57BL/6J mie. We then ompred the impt of dily rpmyin tretment with the impt of the seleted intermittent dosing shedule on gluose tolerne, pyruvte tolerne, nd in vivo nd ex vivo et ell funtion, nd exmined the impt on T-ell popultions in splenoytes. Importntly, we find tht mtorc1 inhiition is sustined in mny tissues despite intermittent dosing. Finlly, we ompred the impt of dily rpmyin tretment on gluose homeostsis nd the immune system with the impt of two FDA-pproved rpmyin nlogs, everolimus nd temsirolimus. Both everolimus nd temsirolimus effiiently inhiited mtorc1 signling, ut hd redued impt on gluose homeostsis ompred to rpmyin. 28 ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd. This is n open ess rtile under the terms of the Cretive Commons Attriution Liense, whih permits use, distriution nd reprodution in ny medium, provided the originl work is properly ited.

2 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. 29 Results Intermittent tretment with rpmyin hs redued effet on gluose tolerne We first sought to identify rpmyin dosing shedule tht would seletively inhiit mtorc1 while minimizing deleterious, mtorc2- medited effets on gluose metolism nd immune system. We treted 9-week-old mle C57BL/6J mie for 2 weeks with vehile or 2 mg/kg rpmyin every dy (19/dy) or weekly (19/7 dys), nd nlyzed the effet on gluose tolerne. We performed fsting gluose tolerne test 7 dys fter the most reent rpmyin tretment of the weekly (19/7 dys) group; the effets of hroni rpmyin tretment on gluose tolerne persist for 2 weeks (Yng et l., 212; Liu et l., 214). As expeted, we found tht dily rpmyin tretment signifintly impirs the performne of mie during gluose tolerne test (GTT), with 2 116% inrese in lood gluose levels t every time point (Fig. 1A, left), nd 71% inrese in totl gluose urden over the time ourse of the ssy s mesured y re under the urve (AUC) (Fig. 1A, right). In omprison, weekly rpmyin tretment did not impir gluose tolerne, whether mesured t eh time point or y AUC (Fig. 1A). We oserved similr results in GTT performed fter 5 weeks of tretment, on the 4th dy fter the most reent tretment of the weekly (19/7 dys) group (Fig. S1A). In greement with our previous findings using diet-delivered rpmyin (Lmming et l., 213), neither dily nor weekly rpmyin tretment inresed the level of glyted hemogloin (Fig. S1B). In order to understnd how these results relted to the phrmokinetis of rpmyin tretment, we determined the rpmyin ontent of lood from mie treted either dily or weekly with rpmyin for 8 weeks. Sixteen hours following dministrtion of rpmyin, we oserved similr rpmyin levels in mie treted dily with rpmyin (19/dy) nd mie treted weekly (19/7 dys) (Fig. 1B, dy 1). As expeted, the rpmyin ontent of lood deresed shrply with time in the weekly treted mie, rehing the detetion threshold (1 ng/ml) 7 dys fter the injetion. We lulte tht the hlf-life of rpmyin in mouse lood is pproximtely 15 h, with lood levels of rpmyin rehing 4.9 nm 3 dys fter injetion, onentrtion ple of inhiiting mtor signling in tissue ulture ell lines (Srssov et l., 26). We oserved very similr rpmyin kinetis in liver (Fig. S1C). We nlyzed mtor signling in the musles of mie treted with vehile, dily rpmyin, or weekly rpmyin for 2 months (Fig. 1C). As we previously reported (Lmming et l., 212), dily tretment with 2 mg/kg rpmyin effiiently inhiited the phosphoryltion of oth S6 S24/244, redout of mtorc1 signling, nd AKT S473, n mtorc2 sustrte. Rpmyin ws eqully effiious in inhiiting phosphoryltion of S6 in mie treted dily with rpmyin s in mie treted weekly (19/7d) nd srified on the dy following tretment (D1). We oserved similr effet in liver (Fig. S1D). However, AKT S473 phosphoryltion in musle ws only inhiited in mie treted dily with rpmyin (Fig. 1C). Mie treted weekly with rpmyin ut srified on the third dy (D3) following tretment hd deresed men phosphoryltion of S6 (38% derese) nd AKT (29% derese), ut these results did not reh sttistil signifine. By 7 dys following tretment, we did not oserve ny inhiition of S6 phosphoryltion in either liver or musle (Fig. S1E). We proeeded to test the effets of two more frequent rpmyin dosing shedules, rpmyin dosed one every three (19/3d) or five (19/ 5d) dys, on gluose tolerne (Fig. 1D). While rpmyin delivered 19/3 dys signifintly impired gluose tolerne, rpmyin delivered 19/5 dys hd no effet on gluose tolerne (Fig. 1D), or in performne during n insulin tolerne test (Fig. S1F). All of the intermittent dosing regimens hd deresed impt on AUC ompred to dily rpmyin tretment (Fig. 1E). Rpmyin tretment one every 5 dys (19/5 dys) hd the smllest impt on gluose tolerne, nd we therefore seleted this dosing shedule for further nlysis. Rpmyin tretment one every 5 dys hs deresed impt on gluose homeostsis reltive to dily rpmyin We treted new ohort of C57BL/6J mles with vehile, rpmyin dosed intermittently one every 5 dys (19/5 dys), or rpmyin dosed dily (19/dy) t 2 mg/kg strting t 9 weeks of ge. After 3 weeks, we performed fsting gluose tolerne test on the dy immeditely following dministrtion of rpmyin to the 19/5 dys mie. We oserved no effet of intermittent rpmyin on gluose tolerne, while dily rpmyin tretment used roust derese in gluose tolerne (Fig. 2A). One tretment yle lter, we performed pyruvte tolerne test (PTT); pyruvte n e utilized s sustrte for gluoneogenesis y the liver, permitting us to ssess hepti gluoneogenesis (Houde et l., 21; Lmming et l., 212). As expeted, dily rpmyin tretment indued signifint pyruvte intolerne (Fig. 2B), inditing inresed levels of hepti gluoneogenesis. Mie treted intermittently with rpmyin were lso pyruvte intolernt, lthough the inrese in the AUC indued y dily rpmyin tretment ws twie tht oserved in the AUC of mie treted intermittently (Fig. 2B). Chroni rpmyin results in fsting hyperglyemi in oth humns nd mie (Lmming et l., 213). After 5 weeks of tretment, mie Fig. 1 Intermittent tretment with rpmyin minimizes gluose intolerne nd mtorc2 inhiition. Gluose tolerne test on mle C57BL/6J mie (A) treted with vehile or with 2 mg/kg rpmyin (19/dy or 19/7 dys) for 2 weeks, performed 7 dys fter the lst tretment of the rpmyin 19/7 dys group (D7) [n = 1 vehile, n = 9 19/ dy rpmyin, nd n = 11 19/7 dys rpmyin; for GTT, P <.5, P <.1 vs. ll groups, Tukey Krmer test following two-wy repeted-mesures ANOVA; for AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (B) Rpmyin onentrtion in lood from mle C57Bl/6J mie treted with 2 mg/kg rpmyin (19/dy or 19/7 dys) for 8 weeks; lood from 19/7 dys mie ws olleted 1 dy (D1), 3 dys (D3), or 7 dys (D7) fter the more reent rpmyin injetion (n = 3 6/group; mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5). (C) Western lotting nlysis nd quntifition of phosphorylted S6 (S24/244) nd Akt (S473) phosphoryltion in skeletl musle [n = 9 vehile, 7 19/dy rpmyin, 3 rpmyin 19/7d D1, 6 rpmyin 19/7d D3; mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (D) Gluose tolerne test on mie treted intermittently with either vehile or with 2 mg/kg rpmyin (19/3 or 5 dys) for 2 weeks, performed 3 dys fter the lst tretment of the rpmyin 19/3 dys group nd 5 dys fter the lst tretment of the 19/5 dys group [n = 11/, for GTT, P <.5 rpmyin 19/3 dys vs. rpmyin 19/5 dys, P <.2 rpmyin 19/3 dys vs. ll groups, Tukey Krmer test following two-wy repeted-mesures ANOVA; for AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (E) Are under the urve for the gluose tolerne tests in Fig. 1A nd 1D, expressed s perent of the AUC for vehile-treted mie in the orresponding experiment (P <.1 vs. vehile in the orresponding experiment, two-tiled t-test). Error rs represent stndrd error. ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

3 3 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. (A) Blood gluose (mg dl 1 ) GTT (D7) Rpmyin 1x/7 dys Rpmyin 1x/dy Are under the urve Rp 1x/7 d Rp 1x/d (B) Blood rpmyin (ng ml 1 ) Rpmyin 1x/7 d 1x/d Dys post rpmyin (C) p-s24/244 S6, AKT p-s24/244 S6 S6 Rpmyin 1x/d 1x/7 d D1 1x/7 d D3 Protein phosphoryltion (% of ) 1 5, Protein phosphoryltion (% of ) 1 5, Rp 1x/d Rp 1x/7 d D1 Rp 1x/7 d D3 Rp 1x/d Rp 1x/7 d D1 Rp1x/7 d D3 (D) Blood gluose (mg dl 1 ) GTT (D3/D5) Rpmyin 1x/5 dys Rpmyin 1x/3 dys Are under the urve 2 1 Rp 1x/5 d Rp 1x/3 d (E) GTT AUC (% ) Dosing intervl (dys) ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

4 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. 31 (A) Blood gluose (mg dl 1 ) 3 2,,, GTT (D1), 1 Rpmyin 1x/5 dys Rpmyin 1x/dy , Are under the urve Rp 1x/5 d Rp 1x/d (B) Blood gluose (mg dl 1 ) 3 2 1, PTT (D1),,, Rpmyin 1x/5 dys Rpmyin 1x/dy, Are under the urve Rp 1x/5 d Rp 1x/d (C) Blood gluose (mg dl 1 ) Rpmyin 1x/5 dys Rpmyin 1x/dy, Insulin (ng ml 1 ) (D) 1.2 HOMA2-IR (E) HOMA2 %B 1 5 Fsting Gluose stimulted Fsting Gluose stimulted Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d (F) Sereted insulin (G) Islet insulin (H) Insulin seretion Insulin (ng ml 1 ) mm gluose Rp 1x/5 d Rp 1x/d Insulin (ng per islet) 5 Rp 1x/5 d Rp 1x/d Seretion (%) 3 2 1, mm gluose Rp 1x/5 d Rp 1x/d Fig. 2 Redued impt of intermittent rpmyin dministrtion on gluose homeostsis. (A) Gluose nd (B) pyruvte tolerne test on mle C57BL/6J mie treted with vehile or with 2 mg/kg rpmyin (19/dy or 19/5 dys) for 2 or 3 weeks, respetively [(n = 9 per tretment; for GTT/PTT, Tukey Krmer test following two-wy repetedmesures ANOVA, = P <.5 vs. vehile, = P <.5 vs. rpmyin 19/5 dys; for AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (C) Fsting nd gluose-stimulted insulin seretion (GSIS) were mesured y fsting mie treted for 5 weeks overnight, olleting serum, injeting 1 g/kg gluose, nd olleting serum 15 min fter injetion [n = 9/group (gluose), 4/group (insulin), mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (D, E) HOMA2-IR nd HOMA2%B were lulted using the fsting insulin dt in C nd fsting gluose dt from the sme mie [n = 4/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (F H) Islets were isolted from vehile nd rpmyin (19/dy or 19/5 dys) mie treted for 8 weeks nd were nlyzed to determine insulin seretion in response to low (1.7 mm) nd high (16.7 mm) gluose [n = 6 mie per tretment, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. Error rs represent stndrd error. treted dily with rpmyin, ut not mie treted intermittently, showed fsting hyperglyemi nd hyperinsulinemi (Fig. 2C). We used these mesures to lulte insulin resistne nd et ell sensitivity using homeostsis model ssessment (HOMA2-IR) (Levy et l., 1998). The HOMA2-IR model ws derived empirilly from humn insulin gluose lmp dt, ut remins useful surrogte mesure of insulin resistne in mie (Mther, 29). Mie treted dily with rpmyin hd signifintly higher HOMA2-IR vlue thn mie treted intermittently with rpmyin or mie treted with vehile (Fig. 2D). We oserved trend towrd deresed et ell funtion (HOMA2%B, surrogte mesurement of et ell funtion) in mie treted dily with rpmyin (Fig. 2E), lthough this ws not sttistilly signifint. With signifint evidene tht rpmyin impirs et ell funtion (Brlow et l., 213), we deided to investigte the impt of rpmyin dosing on islet funtion more losely. We isolted ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

5 32 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. (A) AKT p-s24/244 S6 S6 Musle Rpmyin 1x/dy 1x/5 d D5 Protein phosphoryltion (% of ) 1 5 p-s24/244 S6 Protein phosphoryltion (% of ) 1 5 (C) Testes weight (mg) 2 1 Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d (B) AKT p-s24/244 S6 S6 Adipose Rpmyin 1x/dy 1x/5 d D5 Protein phosphoryltion (% of ) 1 5 p-s24/244 S6 Protein phosphoryltion (% of ) Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Fig. 3 Sustined impt of intermittent rpmyin on dipose mtorc1 nd testes weight. (A) Musle lyste nd (B) dipose tissue lyste were nlyzed y Western lotting, nd the phosphoryltion of S6 24/244 nd AKT S473 reltive to their respetive totl protein ws quntified. Tissues were olleted from mie treted with vehile or rpmyin (19/dy or 19/5 dys) for 8 weeks, with the tissue olletion sheduled suh tht the intermittent rpmyin tretment group ws srified 5 dys fter the previous rpmyin injetion. Mie were fsted overnight nd srified following stimultion with. U/kg insulin for 15 min. Islets were isolted s desried prior to tissue olletion [n = 5 9 per tretment, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (C) The testes of mie in eh tretment group were weighed [n = 9 per group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. Error rs represent stndrd error. pnreti islets from mie fter 8 weeks of rpmyin tretment nd performed n ex vivo gluose-stimulted insulin seretion ssy (Kimple et l., 213) (Fig. 2F H). Islets from mie treted dily with rpmyin sereted signifintly less insulin into the medi thn mie treted with vehile or intermittently treted with rpmyin (Fig. 2F), nd we lso oserved slight redution in insulin ontent in these islets (Fig. 2G). When we lulted the perentge of insulin sereted, we found tht islets from mie treted dily with rpmyin were signifintly less responsive to gluose stimultion thn islets from vehile-treted mie (Fig. 2H). Intermittent tretment with rpmyin hd little to no effet on sereted insulin, islet insulin ontent, or the perent insulin sereted in response to gluose stimultion, demonstrting tht et ell funtion is mintined in mie treted intermittently with rpmyin. Impt of intermittent rpmyin tretment on mtor signling nd testes weight We nlyzed mtor signling in severl tissues from the sme mie used for ex vivo islet nlysis. After 8 weeks of reeiving vehile, intermittent (19/5 dys) rpmyin, or dily rpmyin, mie were srified on dy 5 (D5) 5 dys following the lst dministrtion of rpmyin to the intermittent tretment group in order to determine whether mtor inhiition persists etween tretments. We oserved signifint derese in the phosphoryltion of S6 in the musle, liver, hert, nd pnreti islets of mie reeiving dily rpmyin, ut no hnge in S6 phosphoryltion in the D5 intermittent tretment group (Fig. 3A nd Fig. S2A C), lthough there ws ler trend towrd redued S6 phosphoryltion in pnreti islets isolted from the D5 intermittent tretment group (Fig. S2C). Similrly, we oserved deresed AKT S473 phosphoryltion in musle of the dily rpmyin tretment group, ut not the intermittent tretment group (Fig. 3A). Adipose tissue ws unique in tht we oserved sustined effet of intermittent rpmyin on S6 phosphoryltion even on dy 5 tht ws equivlent to tht oserved in dily rpmyin (Fig. 3B). Anlysis of mtor signling in hrvested tissue is informtive, ut idelly we would e le to mesure verge mtor tivity over time rther thn the preise tivity t single time point. A well-known side effet of rpmyin tretment in humns nd mie is testiulr degenertion (Wilkinson et l., 212), nd we oserved tht oth the intermittent rpmyin nd dily rpmyin tretment regimens signifintly deresed testes mss (Fig. 3C). Dily rpmyin deresed testes weight y pproximtely %, while the intermittent (19/5 dys) rpmyin regimen deresed testes weight y lmost 6%. ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

6 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. 33 % CD (A) (B) (C) # 2 1 FoxP3 + CD (D) FoxP3 + CD Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d (E) (F) (G) FoxP3 + CD + CD FoxP3 + CD CD FoxP3 CD + CD (H) FoxP3 CD CD Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Rp 1x/5 d Rp 1x/d Fig. 4 Intermittent rpmyin tretment hs redued ut signifint impt on the immune system. (A H) Flow ytometry nlysis (expressed s perent of totl live ells) of splenoytes from mle C57BL/6J mie treted with vehile or rpmyin (19/dy or 19/5 dys) for 8 weeks (n = 6 8 mie/group, # P <.52,P <.5, P <.5, Tukey Krmer test following one-wy ANOVA). Error rs represent stndrd error. Rpmyin delivered one every 5 dys hs redued impt on the immune system The immunosuppressive effets of rpmyin re potentil rrier to widespred use of rpmyin s therpy for ge-relted diseses (Lmming et l., 213), lthough the dosing strtegy utilized my e of mjor importne. The true impt of rpmyin on the immune system is still unler, nd rpmyin my not e generlly immunosuppressive indeed, rpmyin tretment improves survivl in mouse models of infetion (Hinojos et l., 212; Hsty et l., 214) nd improves the response to vines in oth nonhumn primtes (Turner et l., 211) nd elderly humns (Mnnik et l., 214). However, in ddition to reported inrese in virl nd fungl infetions in humns tking rpmyin (Mhe et l., ), even short-term low-dose rpmyin dereses defense ginst teril nd virl pthogens in mie (Golderg et l., 214). While not ttempting to resolve this importnt question, we deided to ompre the impt of dily rpmyin to our intermittent tretment regimen. We isolted splenoytes from the mie during the tissue nd islet hrvesting experiments desried ove nd nlyzed the splenoyte popultion using flow ytometry. Both the intermittent nd dily rpmyin tretment regimens hd signifint impt upon immune ell numers, with dily rpmyin tretment impting T-ell numers more thn the intermittent rpmyin tretment regimen (Fig. 4A). This effet ws oserved on oth CD3 + CD4 + ells (Fig. 4B) nd CD3 + CD8 + T ells (Fig. S3A). It hs previously een reported tht rpmyin tretment of mie results in derese in lood T regultory ells (Tregs), defined s CD3 + CD4 + CD + Foxp3 + (Mkki et l., 214), nd we oserved similr effet of dily rpmyin tretment upon Tregs isolted from the spleen (Fig. 4C). Interesting, while dily rpmyin indues n lmost 6% derese in Tregs, intermittent rpmyin tretment resulted in only % derese (Fig. 4C). Similrly, intermittent rpmyin hd redued impt on the frequeny of CD Tregs (Fig. 4D), whih my onstitute reservoir of ommitted regultory ells (Zeleny et l., ) nd hve een shown to hve similr iologil funtion to CD + Tregs (Fontenot et l., ). We oserved similr greter impt of dily rpmyin tretment on CD8 + ells expressing Foxp3 (Fig. S3B,C), whih lso funtion s regultory ells (Tng et l., ). mtorc2-defiient T ells hve deresed tivtion-indued inding to ICAM-1, key step in the immune response (Lee et l., 21). The intertion of T ells with ICAM-1 is medited y the integrin lymphoyte funtion-ssoited ntigen 1 (LFA1), whih onsists of two suunits, Cd11 nd Cd11. We therefore exmined the reltive impt of intermittent nd dily rpmyin on the expression of Cd11 y CD + Tregs (Fig. 4E), CD Tregs (Fig. 4F), nd ells lking Foxp3 expression (Fig. 4G-H). In oth CD + nd CD Tregs, nd lso in CD3 + CD4 + CD Foxp3 ells, dily rpmyin tretment redued the numer of Cd11 + T ells y 6 8%, with the intermittent rpmyin tretment regimen hving signifintly redued impt. ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

7 34 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. (A) Blood gluose (mg dl 1 ) 3 2, GTT, 1 Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Are under the urve Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy (B) Blood gluose (mg dl 1 ) 3 2 PTT 1 Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Are under the urve Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy, Blood gluose (mg dl 1 ) (C) Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy (E) (F) Testes weight (mg) # # Fsting Gluose stimulted 2 1, % CD Insulin (ng ml 1 ) Fsting Gluose stimulted (G) 2 1 (D) AKT p-s24/244 S6 S6 (H) FoxP3 + CD + Rpmyin 1.5 Musle Everolimus Temsirolimus Protein phosphoryltion (% of ) (I) FoxP3 + CD + CD p-s24/244 S6 Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy 1 5, Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Rpmyin 1x/dy Everolimus 1x/dy Temsirolimus 1x/dy Fig. 5 Rpmyin nlogs effiiently inhiit mtorc1 ut show redued impt on gluose homeostsis nd the immune system. (A) Gluose nd (B) pyruvte tolerne tests on mle C57BL/6J mie treted with vehile or with 2 mg/kg rpmyin (19/dy) or equimolr mounts of everolimus or temsirolimus for 2 or 3 weeks, respetively [n = 9 per tretment; for GTT/PTT, Tukey Krmer test following two-wy repeted-mesures ANOVA, = P <.5 vehile vs. ll groups, = P <.5 rpmyin vs. everolimus, = P <.5 rpmyin vs. temsirolimus. For AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following onewy ANOVA, P <.5)]. (C) Fsting nd gluose-stimulted insulin seretion (GSIS) were mesured y fsting mie treted for 5 weeks overnight, olleting serum, injeting 1 g/kg gluose, nd olleting serum 15 min fter injetion. [n = 9/group (gluose), 4/group (insulin), # P.8 vs. vehile, P.5 vs. vehile, Dunnett s test following one-wy ANOVA)]. (D) Musle lyste ws nlyzed y Western lotting nd the phosphoryltion of S6 24/244 nd AKT S473 reltive to their respetive totl protein ws quntified [n = 5 6 per tretment; mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (E) The testes of mie in eh tretment group were weighed [n = 9/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (F I) Flow ytometry nlysis (expressed s perent of totl live ells) on splenoytes isolted from eh tretment group [n = 3 8 mie/ group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. The experiments presented here were onduted in prllel with the experiment presented in Figs 2 4, nd the vehile nd dily (rpmyin 19/dy) dt re duplited here for ese of omprison. Error rs represent stndrd error. Dily everolimus or temsirolimus tretment hs redued impt on gluose nd pyruvte tolerne ompred to dily sirolimus Sine the initil disovery of rpmyin, severl rpmyin nlogs (rplogs) hve een developed to improve the phrmokinetis of rpmyin (sirolimus/rpmune). The most widely used re everolimus nd temsirolimus, whih re oth FDA-pproved for speifi types of ner nd re in numerous linil trils. While ompring side effets ross linil trils is diffiult, it hs een suggested tht the side effet profiles of rplogs in humns my differ (Snkhl et l., 29). We therefore deided to ompre the effet of dily dosing of 2 mg/kg rpmyin (sirolimus/rpmune) to dily dosing of equimolr quntities of everolimus nd temsirolimus (Figure 5). These experiments were ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

8 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. 35 onduted in prllel with the experiments onduted in Figures 2 4, nd the vehile nd dily (rpmyin 19/dy) dt re duplited for ese of omprison. We performed gluose (Fig. 5A) nd pyruvte (Fig. 5B) tolerne tests on everolimus- nd temsirolimus-treted mie nd ssessed fsting nd gluose-stimulted gluose nd insulin levels (Fig. 5C). Despite the very similr effets of dily rpmyin, everolimus, nd temsirolimus on fsting lood gluose (Fig. 5C), everolimus nd temsirolimus oth hd redued impt on gluose tolerne ompred to dily rpmyin (Fig. 5A). A similr effet ws oserved with respet to pyruvte tolerne, with everolimus hving 4% less impt thn rpmyin on AUC (Fig. 5B). With regrd to fsting nd gluose-stimulted gluose nd insulin levels, dily tretment with ll three rplogs showed similr effets (Fig. 5C). We likewise lulted HOMA2-IR nd found similr effets of ll three rplogs on HOMA2- IR, %S, nd %B (Fig. S4A C). We wondered whether the redued effet of the rpmyin nlogs on gluose nd pyruvte tolerne ws orrelted with redued inhiition of mtor signling, nd we nlyzed the impt of the three rplogs on S6 nd AKT S473 phosphoryltion. In musle, liver, dipose, nd hert tissue, we found tht ll three rplogs were eqully effiious in inhiiting S6 phosphoryltion (Fig. 5D nd S5A C). Interestingly, while dily rpmyin nd temsirolimus inhiited AKT S473 phosphoryltion in musle (Fig. 5D), everolimus hd no signifint effet. All three rplogs signifintly redued testes weight, lthough the testes of everolimus-treted mie were slightly hevier thn those from rpmyin-treted mie (Fig. 5E), nd were equivlent in weight to testes from mie treted 19/5 dys with rpmyin (Fig. 3C). Finlly, we nlyzed the reltive impt on immune ells of the three rplogs. All three rplogs showed signifint impt on the T-ell popultions nlyzed, inluding totl CD3 + ell (Fig. 5F), CD3 + CD4 + ells (Fig. 5G), Tregs (Fig. 5H), nd Tregs expressing CD11 (Fig. 5I). Similr effets were oserved on the CD8 + popultion, inluding those expressing Foxp3, nd on other CD3 + CD4 + ells popultions expressing Cd11 (Fig. S6). Everolimus showed slightly redued impt on Tregs expressing Cd11 reltive to oth sirolimus nd temsirolimus (Fig. 5I). Disussion The extensive side effet profile of rpmyin in humns my e signifint hllenge to the potentil use of rpmyin for ge-relted diseses. This is espeilly true if rpmyin needs to e tken ontinuously s prophylti mesure. If rpmyin ould e used intermittently, some of these effets might e voided. Severl experiments hve explored the use of intermittent rpmyin; while these experiments hve inresed mie lifespn, the dosing shedules used (e.g., 2 weeks on rpmyin followed y 2-week drug holidy) re, sed on previous work from mny lortories nd our present results, long enough to signifintly impir metolism nd immunity throughout the tretment intervl s well s during the drug holidy period (Anisimov et l., 21, 211). Here, we insted utilized n intermittent tretment regimen with single doses of rpmyin seprted y reltively short time intervl tht we hve experimentlly determined minimizes the effet of rpmyin on gluose tolerne. We found tht rpmyin remins in the lood t detetle levels for t lest 3 dys nd tht dosing mie with rpmyin one every 5 dys hs no signifint impt on gluose tolerne (Fig. 1). In ontrst to dily rpmyin dministrtion, whih used fsting hyperglyemi nd hyperinsulinemi nd impired gluose-stimulted insulin seretion (GSIS) from et ells, intermittent (19/5 dys) rpmyin tretment did not impt fsting gluose nd insulin levels, nd hd miniml impt on GSIS (Fig. 2). A redued ut still signifint impt of intermittent rpmyin on T ells ws lso oserved (Fig. 4). As mtorc2 in T ells my e extremely sensitive to rpmyin (Powell et l., 212), it is not ler whether this remining impt on the immune system is primrily the result of mtorc1 inhiition, or lso reflets residul inhiition of mtorc2. Surprisingly, while intermittent rpmyin dministrtion hd no impt on gluose tolerne (Fig. 2A), it hd signifint effet on pyruvte tolerne (Fig. 2B), inditing inresed hepti gluoneogenesis. The different outomes of these two tests were unexpeted, s we hve previously oserved tht hroni rpmyin tretment indues oth gluose nd pyruvte intolerne due to inresed hepti gluoneogenesis nd hepti insulin resistne (Lmming et l., 212, 213). Ritor is n essentil protein omponent of mtorc2, nd we showed tht the effets of rpmyin on hepti insulin resistne re medited y disruption of mtorc2 utilizing whole-ody tmoxifen-induile Ritor knokout mouse (Lmming et l., 212). As Ritor ws deleted throughout the ody of the mie in this study, the site (or sites) of rpmyin tion on mtorc2 tht medites these glyemi phenotypes is not ler. Although tissue-speifi deletion of Ritor in liver is suffiient to use hepti insulin resistne, this is lso feture of mie lking Ritor in dipose tissue, nd mie lking Ritor in skeletl musle or pnreti et ells lso hve impired gluose tolerne (Kumr et l., 28, 21; Gu et l., 211; Lmming et l., 212, 214). Inhiition of mtorc2 in multiple tissues my ontriute to the orgnisml phenotype of mie hronilly treted with rpmyin, nd the distint effets of hroni nd intermittent rpmyin on gluose tolerne my e due to hroni nd intermittent rpmyin impiring mtorc2 in distint sets of tissues. A full mehnisti explntion of this effet will likely require the development of dditionl mouse models. Consistent with its effets on gluose homeostsis, hroni rpmyin tretment n disrupt mtorc2 signling in liver, skeletl musle, nd dipose tissues s well s mny others (Lmming et l., 212; Shreier et l., 215). Interestingly, in the present study, we oserve deresed AKT S473 phosphoryltion in the skeletl musle of mie treted dily with rpmyin (Fig. 3A), ut not in dipose tissue (Fig. 3B) or liver (Fig. S2A). The effet of hroni rpmyin tretment on AKT S473 phosphoryltion n e diffiult to oserve nd is time sensitive (Shreier et l., 215), nd so we my hve only oserved this effet in skeletl musle due to the time point utilized. The impt of hroni rpmyin on AKT S473 phosphoryltion is lso influened y diet (Liu et l., 214). The urrent study utilized different how (LDiet 51) thn we utilized in our previous studies (ProL RMH 3), yet the phenotypes of rpmyin-treted mie were identil. One possile interprettion of our urrent results is tht the inhiition of mtorc2 in skeletl musle nd/or speifi other extr-hepti tissues is suffiient to inhiit hepti insulin sensitivity. Alterntively, the effet of hroni rpmyin tretment in the liver my e independent of AKT, nd medited y other hepti sustrtes of mtorc2 suh s SGK (Lmming et l., 214). Further studies will e required to distinguish etween these distint possiilities. As we ntiipted following our preliminry experiments, our intermittent rpmyin regimen does not ontinuously inhiit mtorc1 throughout the 5-dy period in mouse tissues (Fig. 3). Fsintingly, in dipose tissue (Fig. 3B), we oserved tht S6 phosphoryltion ws strongly inhiited even on dy 5 (D5). We theorize tht s rpmyin is highly lipophili, its residene in ft tissue my e prolonged. The ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

9 36 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. prolonged inhiition of mtorc1 signling in dipose tissue my hve signifint implitions for the tretment of oesity nd dietes, nd my prtilly explin reent report demonstrting enefiil effets of weekly rpmyin dministrtion to mie on high-ft diet (Leontiev et l., 214). We mesured testes weight s onvenient method of ssessing the time-integrted impt of rpmyin on mtor signling. Dily rpmyin redued testes weight y lmost 8%, while intermittent dosing of rpmyin redued testes weight y lmost 6% (Fig. 3C). While there is ler differene etween the effets of the two rpmyin dosing shedules on testes weight, these drmti results demonstrte the sustined iologil impt of intermittent rpmyin. Testes weight is losely orrelted with sperm prodution in mmmls (Moller, 1989), nd the reently demonstrted requirement for mtorc1 tivity in spermtogenesis (Bker et l., 214) leds us to hypothesize tht the effets of intermittent rpmyin tretment on testes weight my primrily result from mtorc1 inhiition. Severl different rpmyin nlogs (rplogs) hve een pproved y the FDA. These rplogs, whih inlude everolimus nd temsirolimus, were developed in prt to improve the phrmokinetis of sirolimus. For exmple, everolimus hs n pproximtely 5% shorter lood hlf-life thn sirolimus in humns (Formi et l., 24), while in vitro sirolimus hs slightly lower IC 5 ginst mtorc1 (Lmming et l., 213). While ompring side effets ross linil trils is diffiult, it hs een suggested tht the side effet profiles of rplogs in humns my differ (Snkhl et l., 29). We hypothesized tht the ltered phrmokinetis of everolimus nd temsirolimus might led to redued inhiition of mtorc2 nd therefore redution in undesirle side effets. Notly, we oserved signifintly deresed impt of everolimus nd temsirolimus on oth gluose nd pyruvte tolerne, despite the similr effets of ll three rplogs on lood gluose nd insulin levels (Fig. 5). Importntly, ll three ompounds were eqully effiious in inhiiting mtorc1 tivity in ll of the tissues exmined (Figs. 5D nd S5). Everolimus showed slightly deresed impt ompred to rpmyin with regrd to splenoyte Cd11 + Tregs nd testes weight. We onlude tht t lest in mie, everolimus nd temsirolimus effiiently inhiit mtorc1 while hving redued impt on gluose homeostsis reltive to rpmyin. The impt of this differene on the metoli effets of rplogs in humns remins to e determined. While the effiy of everolimus nd temsirolimus t extending lifespn nd helthspn hs not yet een tested, our predition from this dt is tht it will e t lest s effetive, nd possily more so, thn sirolimus. It is perhps fortuitous tht the first humn trils of rplogs for gerelted onditions hve utilized everolimus (Mnnik et l., 214). Our reserh here hs investigted the possiility tht the therpeuti window of rpmyin for ge-relted diseses ould e expnded through the use of either n intermittent rpmyin dosing shedule or FDA-pproved rpmyin nlogs. With regrd to the limited set of side effets exmined here notly the dverse effets on gluose homeostsis nd immune ell popultion we hve shown tht intermittent dministrtion of rpmyin or the dily dministrtion of rplogs n effiiently inhiit mtorc1 signling with redued negtive side effets ompred to dily dministrtion of rpmyin. While importnt unnswered questions remin, inluding the impt of these strtegies on other rpmyin-ssoited side effets nd the ility of these strtegies to dely ge-relted diseses, our results suggest tht refully designed dosing strtegy, possily using rplog suh s everolimus or temsirolimus, my enle the trnsltion of rpmyinsed therpies to the lini while minimizing side effets. Experimentl proedures Mterils For Western lotting, ntiodies to phospho-akt S473 (46), Akt (4691), phospho-s6 riosoml protein (2215), nd S6 riosoml protein (2217) were from Cell Signling Tehnology. For flow ytometery, ntiodies to CD4 (-41-U) nd CD8 (8-81-U) were from Tono, ntiodies to CD (47-1-8) nd Foxp3 ( ) were from ebiosiene, nd ntiodies to CD11 (56289) nd CD3 (562332) were from BD. Protese nd phosphtse inhiitor oktil tlets were from Fisher. Other hemils were purhsed from Sigm unless noted. Gluose mesurements were performed using Byer Contour lood gluose meter nd test strips. Mouse HA1 nd Mouse insulin ELISA kits were purhsed from Crystl Chem, Downers Grove, IL. Rpmyin (sirolimus), everolimus, nd temsirolimus were purhsed from LC Lortories. Monolonl insulin/proinsulin (1R-I136) nd iotin-onjugted (61R-I136BT) ntiodies for islet ELISAs were purhsed from Fitzgerld. Immunolotting Cells nd tissue smples were lysed in old RIPA uffer supplemented with phosphtse inhiitor nd protese inhiitor oktil tlets. Tissues were lysed in RIPA uffer s previously desried (Lmming et l., 212) using FstPrep 24 (M.P. Biomedils) with ed-eting tues nd ermi eds (Mo-Bio Lortories, Crlsd, CA), nd then entrifuged. Protein onentrtion ws determined y Brdford (Piere Biotehnology, Rokford, IL). Twenty mirogrm of protein ws seprted y sodium dodeylsulfte polyrylmide gel eletrophoresis (SDS-PAGE) on 1% resolving gels (Thermo Fisher Sientifi, Wlthm, MA). Imging ws performed using GE ImgeQunt LAS 4 imging sttion. Quntifition ws performed y densitometry using NIH ImgeJ softwre. Animls nd tretments Animl studies were pproved y the Institutionl Animl Cre nd Use Committee of the University of Wisonsin-Mdison nd the Willim S. Middleton Memoril Veterns Hospitl, Mdison WI. C57BL/6J mie were purhsed from The Jkson Lortory t 8 9 weeks of ge, nd rpmyin/rpmyin nlog tretment ws egun t 1 weeks of ge. Gluose, insulin, nd pyruvte tolerne tests were performed y fsting the mie overnight for 16 h nd then injeting either gluose (1 g/kg), insulin (. U/kg), or pyruvte (2 g/kg) intrperitonelly. Gluose mesurements were performed using Byer Contour lood gluose meter nd test strips. Rpmyin (2 mg/kg) nd equimolr quntities of rpmyin nlogs (2.1 mg/kg everolimus nd 2. mg/kg temsirolimus) were dissolved in ethnol nd diluted in vehile (5% Tween-8, 5% PEG-4) prior to intrperitonel injetion. Mie were typilly injeted etween 3 nd 5 pm, nd prior to tolerne tests, ny injetions were performed immeditely prior to ommenement of the overnight fst. Nomenlture for intermittent tretments with rpmyin: Dy 1 (D1) refers to mie nlyzed or srified whih were treted with rpmyin the previous fternoon, nd Dy 3 (D3), to mie nlyzed or srified 3 dys fter the most reent rpmyin tretment; similrly for Dy 5 (D5) nd Dy 7 (D7). All mie were nlyzed or srified etween 8 m nd 12 pm. ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

10 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. 37 Rpmyin quntifition, islet isoltion nd ex vivo gluosestimulted insulin seretion (GSIS) ssy, nd splenoyte preprtion See supplementl methods. Sttistis Sttistil nlysis ws onduted using PRISM 6 (GrphPd Softwre, Sn Diego, CA). Gluose, insulin, nd pyruvte tolerne tests were nlyzed with two-wy repeted-mesures ANOVA followed y Tukey Krmer post ho test. Are under the urves lulted from tolerne tests, nd ll other omprisons of three or more mens, ws nlyzed y one-wy ANOVA followed y Dunnett s or Tukey Krmer post ho test s pproprite. Aknowledgments We would like to thnk Dr. Christine Seroogy (UW-Mdison Deprtment of Peditris) for vlule disussion nd insight with regrd to the impt of rpmyin on Tregs nd knowledge of flow ytometry, s well s ritil reding of the mnusript. We would lso like to thnk Dgn Sheerr nd Kris Burmeister of the UWCCC Flow Cytometry Lortory for performing the stining nd flow ytometry of isolted splenoytes, nd for their dvie nd help with protools. We would like to thnk Mrtin Jvors of the Nthn Shok Center Sn Antonio Bionlytil Phrmology Core for performing the quntifition of rpmyin in lood nd tissue nd Rndy Strong for disussion. We would like to thnk ll the memers of the Lmming nd Kimple lortories for their ssistne nd insight, nd the Dvis nd Merrins lortory for their ontinul support, s well s Joseph Bur nd Den Cohen for dvie nd onsulttion. Funding The Lmming l is supported y K99/R Pthwy to Independene Awrd to D.W.L. from the Ntionl Institute of Helth/Ntionl Institute on Aging (AG41765), s well s strtup funds from the UW-Mdison Shool of Mediine nd Puli Helth nd the UW-Mdison Deprtment of Mediine. This work ws lso supported y grnts from the Amerin Dietes Assoition (1-14-BS-115) nd the NIH/NIDDK (R1 DK198) to M.E.K. D.W.L. is memer of the UW-Crone Cner Center (UWCCC), nd use of the UWCCC Flow Cytometry Lortory, Shred Servie of the UWCCC, ws supported in prt y the University of Wisonsin Crone Cner Center Support Grnt P3 CA1452. J.C.N. is supported in prt y trining grnt from the UW Institute on Aging (NIA T32 AG213). This work ws supported using filities nd resoures t the Willim S. Middleton Memoril Veterns Hospitl. This work does not represent the views of the Deprtment of Veterns Affirs or the United Sttes Government. Conflit of interest None delred. Referenes Anisimov VN, Zezhinski MA, Popovih IG, Piskunov TS, Semenhenko AV, Tyndyk ML, Yurov MN, Antoh MP, Blgosklonny MV (21) Rpmyin extends mximl lifespn in ner-prone mie. Am. J. Pthol. 176, Anisimov VN, Zezhinski MA, Popovih IG, Piskunov TS, Semenhenko AV, Tyndyk ML, Yurov MN, Rosenfeld SV, Blgosklonny MV (211) Rpmyin inreses lifespn nd inhiits spontneous tumorigenesis in inred femle mie. Cell Cyle 1, Bker MD, Ezzti M, Aloisio GM, Trnw ED, Cuevs I, Nkd Y, Cstrillon DH (214) The smll GTPse Rhe is required for spermtogenesis ut not oogenesis. Reprodution 147, Brlow AD, Niholson ML, Herert TP (213) Evidene for rpmyin toxiity in pnreti et-ells nd review of the underlying moleulr mehnisms. Dietes 62, Byles V, Covrruis AJ, Ben-Shr I, Lmming DW, Stini DM, Mnning BD, Horng T (213) The TSC-mTOR pthwy regultes mrophge polriztion. Nt. Commun. 4, Festui WT, Pouliot P, Bkn I, Stini DM, Lplnte M (214) Myeloid-speifi Ritor deletion indues M1 mrophge polriztion nd potentites in vivo proinflmmtory response to lipopolyshride. PLoS ONE 9, e Fontenot JD, Rsmussen JP, Willims LM, Dooley JL, Frr AG, Rudensky AY () Regultory T ell linege speifition y the forkhed trnsription ftor foxp3. Immunity 22, Formi RN Jr, Lorer KM, Friedmn AL, Bi MJ, Lkkis F, Smith JD, Lorer MI (24) The evolving experiene using everolimus in linil trnsplnttion. Trnsplnt. Pro. 36, 495S 499S. Golderg EL, Smithey MJ, Lutes LK, Uhrlu JL, Nikolih-Zugih J (214) Immune memory-oosting dose of rpmyin impirs mrophge vesile idifition nd urtils glyolysis in effetor CD8 ells, impiring defense ginst ute infetions. J. Immunol. 193, Gu Y, Lindner J, Kumr A, Yun W, Mgnuson MA (211) Ritor/mTORC2 is essentil for mintining lne etween et-ell prolifertion nd ell size. Dietes 6, Hsty P, Livi CB, Dodds SG, Jones D, Strong R, Jvors M, Fisher KE, Slone L, Murthy K, Hurd G, Sun L, Hurez V, Curiel TJ, Shrp ZD (214) erp restores norml life spn in FAP mouse model. Cner Prev. Res. (Phil.) 7, Hinojos CA, Mgemen V, Vn Roekel S, Austd SN, Miller RA, Bose S, Orihuel CJ (212) Enteri-delivered rpmyin enhnes resistne of ged mie to pneumool pneumoni throughredued ellulr senesene. Exp. 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Dietes 59, Lmming DW, Ye L, Ktjisto P, Gonlves MD, Sitoh M, Stevens DM, Dvis JG, Slmon AB, Rihrdson A, Ahim RS, Guertin DA, Stini DM, Bur JA (212) Rpmyin-indued insulin resistne is medited y mtorc2 loss nd unoupled from longevity. Siene 335, Lmming DW, Ye L, Astle CM, Bur JA, Stini DM, Hrrison DE (213) Young nd old genetilly heterogeneous HET3 mie on rpmyin diet re gluose intolernt ut insulin sensitive. Aging Cell 12, Lmming DW, Ye L, Stini DM, Bur JA (213) Rplogs nd mtor inhiitors s nti-ging therpeutis. J. Clin. Invest. 123, Lmming DW, Demirkn G, Boyln JM, Mihylov MM, Peng T, Ferreir J, Neretti N, Slomon A, Stini DM, Gruppuso PA (214) Hepti signling y the mehnisti trget of rpmyin omplex 2 (mtorc2). FASEB J. 28, Lmming DW, Mihylov MM, Ktjisto P, Br EL, Yilmz OH, Huthins A, Gultekin Y, Gither R, Stini DM (214) Depletion of Ritor, n essentil protein omponent of mtorc2, dereses mle lifespn. Aging Cell 13, Lee K, Gudpti P, Drgovi S, Spener C, Joye S, Killeen N, Mgnuson MA, Boothy M (21) Mmmlin trget of rpmyin protein omplex 2 regultes differentition of Th1 nd Th2 ell susets vi distint signling pthwys. Immunity 32, Leontiev OV, Pszkiewiz GM, Blgosklonny MV (214) Weekly dministrtion of rpmyin improves survivl nd iomrkers in oese mle mie on high-ft diet. Aging Cell 13, ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.

11 38 Intermittent rpmyin nd rplogs mitigte side effets, S. I. Arriol Apelo et l. Levy JC, Mtthews DR, Hermns MP (1998) Corret homeostsis model ssessment (HOMA) evlution uses the omputer progrm. Dietes Cre 21, Liu Y, Diz V, Fernndez E, Strong R, Ye L, Bur JA, Lmming DW, Rihrdson A, Slmon AB (214) Rpmyin-indued metoli defets re reversile in oth len nd oese mie. Aging (Alny NY) 6, Mhe E, Morelon E, Lehton S, Sng KH, Mnsouri R, Dusse MF, Mmzer- Bruneel MF, de Prost Y, Kreis H, Bodemer C () Cutneous dverse events in renl trnsplnt reipients reeiving sirolimus-sed therpy. Trnsplnttion 79, Mkki K, Tront S, Molendi-Coste O, Bouhert E, Neve B, Eury E, Loens S, Llette M, Duez H, Stels B, Domrowiz D, Froguel P, Wolowzuk I (214) Benefiil metoli effets of rpmyin re ssoited with enhned regultory ells in diet-indued oese mie. PLoS ONE 9, e Mnnik JB, Del Giudie G, Lttnzi M, Vlinte NM, Prestgrd J, Hung B, Lonetto MA, Meker HT, Kovrik J, Crson S, Glss DJ, Klikstein LB (214) mtor inhiition improves immune funtion in the elderly. Si. Trnsl. Med. 6, 268r179. Mther K (29) Surrogte mesures of insulin resistne: of rts, mie, nd men. Am. J. Physiol. Endorinol. Met. 296, E398 E399. Moller AP (1989) Ejulte qulity, testes size nd sperm prodution in mmmls. Funt. Eol. 3, Powell JD, Pollizzi KN, Heikmp EB, Horton MR (212) Regultion of immune responses y mtor. Annu. Rev. Immunol. 3, Snkhl K, Mit A, Kelly K, Mhlingm D, Giles F, Mit M (29) The emerging sfety profile of mtor inhiitors, novel lss of ntiner gents. Trget. Onol. 4, Srssov DD, Ali SM, Sengupt S, Sheen JH, Hsu PP, Bgley AF, Mrkhrd AL, Stini DM (26) Prolonged rpmyin tretment inhiits mtorc2 ssemly nd Akt/PKB. Mol. 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USA 12, Supporting Informtion Additionl Supporting Informtion my e found in the online version of this rtile t the pulisher s we-site. Figure S1 Impt of weekly rpmyin on gluose homeostsis nd mtorc1 tivity in the liver. A) Gluose tolerne test on mle C57BL/6J mie treted with vehile or with 2 mg/kg rpmyin (19/dy or 19/7 dys) for 5 weeks, tested 4 dys (D4) fter the most reent injetion of the Rpmyin 19/ 7 dys group [n = 1 vehile, n = 9 Rpmyin 19/dy, nd n = 11 Rpmyin 19/7 dys rpmyin; for GTT, P <.5, P <.1 vs. ll groups, Tukey-Krmer test following two-wy repeted-mesures ANOVA; for AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA,p <.5)]. B) HA1 ws ssyed from the whole lood of mie treted with vehile or with 2 mg/kg rpmyin (19/dy or 19/7 dys). C) Rpmyin onentrtion in liver from mle C57Bl/6J mie treted with 2 mg/kg rpmyin (19/dy or 19/7 dys) for 8 weeks; lood from 19/7 dys mie ws olleted 1 dy (D1), 3 dys (D3) or 7 dys (D7) fter the more reent rpmyin injetion (n = 3-6/group; = P <.5 vs. 19/7 dys D1; = P <.5 vs. 19/dy dily rpmyin mie; = P <.5 vs. 19/7 dys D3; two-tiled t-test). (D) Western lotting nlysis nd quntifition of phosphorylted S6 (Ser 24/244) in liver [n = 9 vehile, 7 19/dy rpmyin, 3 rpmyin 19/7dys D1, 6 rpmyin 19/ 7dys D3; mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. (E) Western lotting nlysis nd quntifition of phosphorylted S6 (Ser 24/ 244) in liver nd musle [n = 7 per group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. F) Insulin tolerne test on mie treted intermittently with either vehile or with 2 mg/kg rpmyin (19/3 or 5 dys) for 2 weeks [n = 1 mie/group, = P <.5, Tukey-Krmer test following two-wy repeted-mesures ANOVA; for AUC, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. Error rs represent stndrd error. Figure S2 Impt of intermittent rpmyin on liver nd hert. A) Liver lyste, B) Hert tissue lyste nd C) Pnreti islet lyste ws nlyzed y western lotting nd the phosphoryltion of S6 24/244 nd AKT S473 reltive to their respetive totl protein ws quntified. Tissues were olleted from mie treted with vehile or rpmyin (19/dy or 19/5 dys) for 8 weeks, with the tissue olletion sheduled suh tht the intermittent rpmyin tretment group ws srified 5 dys fter the previous rpmyin injetion. Mie were fsted overnight nd srified following stimultion with. U/ kg insulin for 15 min. Islets were isolted s desried prior to tissue olletion [n = 4 9/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. Error rs represent stndrd error. Figure S3 Intermittent rpmyin tretment hs redued ut signifint impt on the immune system. A-H) Flow ytometry nlysis (expressed s perent of totl live ells) of CD8 + splenoytes from mle C57BL/6J mie treted with vehile or rpmyin (19/dy or 19/5 dys) for 8 weeks (n = 6 8 mie/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5). Error rs represent stndrd error. Figure S4 Impt of rplogs on insulin resistne. A-C) HOMA2-IR nd HOMA2%B ws lulted using the fsting insulin dt in Figure 5C nd fsting gluose dt from the sme mie (n = 4/group, # = P.8 vs. vehile, =P.5 vs. vehile, Dunnett s test following one-wy ANOVA). The experiments presented here were onduted in prllel with the experiment presented in Figure 2, nd the vehile nd dily (Rpmyin 19/dy) dt is duplited here for ese of omprison. Error rs represent stndrd error. Figure S5 Impt of rpmyin nlogs on mtor signling in liver, dipose nd hert. A-C) Liver, dipose nd hert lyste ws nlyzed y western lotting nd the phosphoryltion of S6 24/244 nd AKT S473 reltive to their respetive totl protein ws quntified [n = 4 9/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. Error rs represent stndrd error. Figure S6 Impt of rpmyin nlogs on splenoyte popultions. Flow ytometry nlysis (expressed s perent of totl live ells) on splenoytes isolted from eh tretment group [n = 3 8 mie/group, mens with the sme letter re not signifintly different from eh other (Tukey Krmer test following one-wy ANOVA, P <.5)]. The experiments presented here were onduted in prllel with the experiment presented in Figures 4 nd S3, nd the vehile nd dily (Rpmyin 19/dy) dt is duplited here for ese of omprison. Error rs represent stndrd error. ª 215 The Authors. Aging Cell pulished y the Antomil Soiety nd John Wiley & Sons Ltd.