Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mtor complex 2 (mtorc2)

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1 Dietologi (212) 55: DOI 1.17/s ARTICLE myin toxiity in MIN6 ells nd rt nd humn islets is medited y the inhiition of mtor omplex 2 (mtorc2) A. D. Brlow & J. Xie & C. E. Moore & S. C. Cmpell & J. A. M. Shw & M. L. Niholson & T. P. Herert Reeived: 7 Septemer 211 / Aepted: 9 Jnury 212 / Pulished online: 8 Ferury 212 # The Author(s) 212. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis myin (sirolimus) is one of the primry immunosuppressnts for islet trnsplnttion. Yet there is evidene tht the long-term tretment of islet-trnsplnt ptients with rpmyin my e responsile for susequent loss of islet grft funtion nd viility. Therefore, the primry ojetive of this study ws to eluidte the moleulr mehnism of rpmyin toxiity in et ells. Methods Experiments were performed on isolted rt nd humn islets of Lngerhns nd MIN6 ells. The effets of rpmyin nd the roles of mmmlin trget of rpmyin omplex 2 (mtorc2)/protein kinse B () on et ell signlling, funtion nd viility were investigted using ell viility ssys, insulin ELISA ssys, kinse ssys, western lotting, phrmologil inhiitors, smll interfering (si)rna nd through the overprodution of onstitutively tive mutnt of. A. D. Brlow nd J. Xie ontriuted eqully to this study. Eletroni supplementry mteril The online version of this rtile (doi:1.17/s ) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. J. Xie : C. E. Moore : T. P. Herert () Deprtment of Cell Physiology nd Phrmology, University of Leiester, The Henry Wellome Building, University Rod, Leiester LE1 9HN, UK e-mil: tph4@le..uk A. D. Brlow : M. L. Niholson Trnsplnt Surgery Group, Deprtment of Infetion, Immunity nd Inflmmtion, University of Leiester, Leiester, UK S. C. Cmpell : J. A. M. Shw Institute of Cellulr Mediine, Newstle University, Newstle, UK Results myin tretment of MIN6 ells nd islets of Lngerhns resulted in loss of ell funtion nd viility. Although rpmyin utely inhiited mtor omplex 1 (mtorc1), the toxi effets of rpmyin were more losely orrelted to the dissoition nd intivtion of mtorc2 nd the inhiition of. Indeed, the overprodution of onstitutively tive proteted islets from rpmyin toxiity wheres the inhiition of led to loss of ell viility. Moreover, the seletive intivtion of mtorc2 using sirna direted towrds rpmyininsensitive ompnion of trget of rpmyin (), mimiked the toxi effets of hroni rpmyin tretment. Conlusions/interprettion This report provides evidene tht rpmyin toxiity is medited y the intivtion of mtorc2 nd the inhiition of nd thus revels the moleulr sis of rpmyin toxiity nd the essentil role of mtorc2 in mintining et ell funtion nd survivl. Keywords Apoptosis. Bet ell. Dietes mellitus. GSIS. Islet. Islet trnsplnttion. mtor. mtorc2.. myin. Arevitions AdC Adenovirus produing myristylted version (onstitutively tive) of CHAPS 3-[(3-Cholmidopropyl)dimethylmmonio]-1- propnesulfonte hydrte FOXO Forkhed ox O GSIS Gluose-stimulted insulin seretion GSK3 Glyogen synthetse kinse 3 mtor Mmmlin trget of rpmyin mtorc1 mtor omplex 1 mtorc2 mtor omplex 2 P- Phosphorylted PDK1 Phosphoinositide-dependent kinse 1

2 1356 Dietologi (212) 55: PKC βriko S6K sirna Introdution Protein kinse B Protein kinse C Regultory-ssoited protein of trget of rpmyin Bet ell-speifi Ritor knokout mie myin-insensitive ompnion of trget of rpmyin Riosoml protein S6 kinse Smll interfering RNA Sine the pulition of the lndmrk Edmonton study in 2 [1], use of rpmyin (sirolimus) hs een t the forefront of immunosuppression for islet trnsplnttion. The employment of rpmyin s the primry immunosuppressnt in the Edmonton protool llowed the voidne of gluoortioids nd minimistion of lineurin inhiitors, oth known to e profoundly dietogeni [1]. Although the initil results of the Edmonton study were very promising, enthusism ws tempered when the 5 yer results of the initil ohort of ptients were reported, with only pproximtely 1% of reipients mintining insulin independene [2]. Although the use of loss of grft funtion/ viility is poorly understood there is growing evidene tht it is, in prt, due to rpmyin toxiity [3 5]. myin hs een shown to hve detrimentl effets on the funtion nd survivl of murine pnreti et ell lines [4], ultured murine islets [3, 5] nd ultured humn islets [4] s well s of mouse syngenei islet-trnsplnt model [5]. In ddition, postmortem exmintion of ptient with filed islet grft trnsplnted under the Edmonton protool showed no evidene of utoimmune or lloimmune dmge to the trnsplnted islets, suggesting the filure ws due to non-immunologil uses, inluding drug toxiity [6]. myin exerts its phrmologil tions vi inhiition of the serine/threonine kinse mmmlin trget of rpmyin (mtor) [7]. mtor exists in two omplexes, mtor omplex 1 (mtorc1) nd mtor omplex 2 (mtorc2). Both mtor omplexes ontin mtor, mmmlin orthologue of lethl with se thirteen (mlst8), DEP (dishevelled, egl-1, plekstrin) domin-ontining mtor interting protein (DEPTOR), the newly disovered 58 kd gluose-regulted protein (GRP58), Tel2 interting protein 1 (TTI1), telomere mintenne 2 (TEL2) nd Rs (rt srom)-relted C3 otulinum toxin sustrte 1 (RAC1) [8]. In ddition, mtorc1 ontins regultoryssoited protein of trget of rpmyin () nd pro-rih Akt sustrte of 4 kd (PRAS4), while mtorc2 ontins rpmyin-insensitive ompnion of TOR (), mmmlin stress tivted protein kinse interting protein 1 (msin1) nd protein oserved with (PROTOR) [7, 8]. mtorc1 is highly sensitive to rpmyin wheres, in generl, mtorc2 is rpmyin insensitive [9]. However, it hs een shown in some ell types tht prolonged rpmyin tretment inhiits mtorc2 ssemly [1, 11]. mtorc1 is tivted y nutrients, growth ftors nd ellulr energy levels [7] nd plys key role in the regultion of et ell size nd prolifertion [8]. Indeed, et ell speifi tuerous slerosis omplex-2 (TSC2; n upstrem negtive regultor of mtorc1 [12]) knokout mie hve inresed et ell mss due to inresed ell size nd prolifertion [13, 14]. The effets of mtorc1 on ell size re likely to e medited y the tivtion of riosoml S6 protein kinse (S6K) 1 nd 2, downstrem trgets of mtorc1, s S6k1 (lso known s Rps6k1)-knokout mie [15] ndrps6-knokin mie, with non-phosphoryltle riosoml protein S6 () [16] hve omprtively smller et ells thn wild-type mie. mtorc2 ws originlly identified s meditor of tin ytoskeletl orgnistion, polristion nd ell migrtion [9], nd is responsile for the phosphoryltion nd tivtion of severl memers of the AGC kinse sufmily, inluding protein kinse B (, otherwise known s AKT), serum/ gluoortioid-indued kinse 1 (SGK1), onventionl protein kinse Cs (PKCs) nd PKCε [7]. Reently, it hs een reported tht et ell-speifi deletion of Ritor in mie (i.e. et ell-speifi Ritor knokout mie [βriko]) results in redution in et ell mss (due to impired prolifertion ut not hnges in ell size or ell deth) ompnied y moderte hyperglyemi nd gluose intolerne [17]. The initil ojetive of this study ws to eluidte the moleulr sis for rpmyin toxiity in islets. This led to the disovery tht rpmyin tretment of et ells not only inhiits mtorc1 ut lso inhiits mtorc2. More importntly, we provide evidene tht the moleulr sis of rpmyin toxiity is through the intivtion of mtorc2 nd its impt on tivity. These results revel hitherto unknown essentil role for mtorc2 in mintining et ell funtion nd viility. Methods Regents Unless otherwise stted, ll hemils nd regents were purhsed from Sigm-Aldrih (St Louis, MO, USA). FCS ws purhsed from Invitrogen (Crlsd, CA, USA). [γ 32 P]ATP ws purhsed from GE Helthre (Pistwy, NJ, USA). myin ws purhsed from Cliohem (Nottinghm, UK). Torin1 [18] wskindly provided y D. Stini (Whitehed Institute for Biomedil

3 Dietologi (212) 55: Reserh, Cmridge, MA, USA). Reominnt denovirus produing myristylted version (onstitutively tive) of (AdC) ws purhsed from Vetor Biols (Phildelphi, PA, USA). Cell ulture nd tretments MIN6 ells [19] wereused etween pssges 2 nd 45 t pproximtely 8% onfluene nd grown s previously desried [2]. Tretments were performed s desried in the figure legends. Islet isoltion, ulture nd tretment Pnreti islets were isolted from mle Sprgue Dwley rts, weighing 2 25 g, y ollgense digestion nd Histopque densitygrdient entrifugtion s previously desried [21]. Rt islets were ultured in RPMI 164 ontining 5.6 mmol/l gluose, 1 U/ml peniillin nd 1 μg/ml streptomyin. Humn islets were isolted from pnreses from herteting deesed humn donors following ethil pprovl nd informed onsent from the donors' reltives. Islets were isolted t the Sottish Ntionl Blood Trnsfusion Servie Islet Isoltion Fility, Edinurgh, UK [22], nd trnsported to Newstle University in CMRL 166 (Cellgro, Herndon, VA, USA), ontining.5% (wt/vol.) humn serum lumin nd 5, U heprin. Humn islets were ultured in CMRL-NCL1 (PAA Lortories, Yeovil, UK) ontining 1% humn serum lumin, 1 U/ml peniillin nd 1 μg/ml streptomyin, prior to experimenttion. Following tretment, rt nd humn islets were olleted y entrifugtion for 1 min t 2 g nd lysed in ie-old lysis uffer. SDS-PAGE nd western lotting SDS-PAGE nd western lotting were performed s desried previously [2]. AntimTOR, nti-, nti-, nti-, nti-leved spse 3, nti-, nti-s6k1, nti-phosphorylted (P)- Ser473, nti-p- Thr38, nti-p- Ser24/244, nti-p-s6k1 Thr389, nti-p-forkhed ox O (FOXO)1/ FOXO3 Thr24/Thr32, nti-p-glyogen synthetse kinse 3 (GSK3)α/β Ser21/9 nd nti-p-pkcα Thr638/641 ntiodies used for western lotting were purhsed from Cell Signlling Tehnologies (Beverly, MA, USA). Anti-glyerldehyde-3- phosphte dehydrogense (GAPDH) ws purhsed from Snt Cruz Biotehnology (Snt Cruz, CA, USA). Anti- PKCα ws purhsed from Trnsdution Lortories (Oxford, UK). Anti-mTOR nd nti- ntiodies used for immunopreipittion were purhsed from the Division of Signl Trnsdution Therpy, University of Dundee, UK. Infetion of ell lines with reominnt denoviruses Adenovirus-medited trnsdution of ell lines ws performed s previously desried [23]. Immunopreipittion of mtor nd For immunopreipittion, MIN6 ells were lysed in 3-[(3-holmidopropyl) dimethylmmonio]-1-propnesulfonte hydrte (CHAPS) lysis uffer nd the mtor omplexes were isolted essentilly s desried previously [24]. Gluose-stimulted insulin seretion ssy Following tretment, islets or MIN6 ells were inuted in KRB supplemented with 1 mmol/l gluose for 6 min t 37 C. The superntnt frtions were olleted nd the inution ontinued in KRB ontining 2 mmol/l gluose for further 6 min t 37 C. The superntnt frtions were gin olleted. For MIN6 ells, the ell pellets were lysed in ie-old id/ethnol solution (HCl 1.5% [vol./vol.], ethnol 75% [vol./vol.] nd H 2 O 23.5% [vol./vol.]) prior to mesurement of ellulr insulin ontent. Insulin onentrtion in the superntnt frtions or pellets were ssyed using n ntimouse (for MIN6 ells) or nti-rt (for rt islets) insulin ELISA kit (DRG Instruments, Mrurg, Germny) with mouse or rt insulin s stndrd in ordne with the mnufturer s instrutions. The sorne ws red t 45 nm on Novostr plte reder (BMG Lteh, Cry, NC, USA). kinse ssy MIN6 ells were infeted with reominnt denovirus produing onstitutively tive s desried ove. At 24 h post infetion, MIN6 ells were treted nd lysed s desried in the figure legends. ws immunopreipitted from the lystes using nti- ntiody (Millipore, Wtford, UK) s per the mnufturer s instrutions nd the tivity of determined using Crosstide s sustrte peptide (GRPRTSSFAEG; 3 mmol/l; Millipore) s previously desried [25]. Annexin V/propidium iodide stining Following tretment, the medi were removed nd kept. The ells were then inuted in 1 trypsin/edta (.5%) for 4 min t 37 C. DMEM ws dded nd the ells gently dispersed y pipetting, omined with the sved medi nd entrifuged t 2 g for 5 min t room temperture. The medi were disrded nd the ell pellets gently resuspended in DMEM nd equilirted y inution t 37 C for 3 min. The ells were pelleted y entrifugtion t 2 g for 1 min t room temperture nd the medi removed. Annexin V inding nd propidium iodide stining were performed using the Annexin-V-Fluos stining kit (Rohe, Burgess Hill, UK) s per the mnufturer s instrutions. Quntifition of stining ws performed using FACSn or FACSCliur flow ytometer, nd CellQuest softwre (BD Biosienes, Sn Jose, CA, USA). Cell deth detetion ssy Following islet ulture nd tretment, evlution of ell deth ws performed using the Cell Deth Detetion ELISA PLUS kit (Rohe, Burgess Hill, UK), s per the mnufturer s instrutions. Asorne ws

4 1358 Dietologi (212) 55: mesured t 45 nm ginst 2,2 -zino-is(3-ethylenzothizoline-6-sulphoni id) (ABTS) solution nd ABTS stop solution s lnk using Novostr plte reder (BMG Lteh, Aylesury, UK) nd the results expressed in ritrry units of oligonuleosome-ssoited histone. Smll interfering RNA trnsfetion of dispersed islets Islets were dispersed essentilly s desried y Jonkers et l. [26]. Smll interfering (si)rna trnsfetion ws performed using Lipofetmine 2 (Invitrogen, Crlsd, CA, USA) ording to the mnufturer s instrutions. For Ritor or tor (lso known s Rptor) knokdown, the ells were trnsfeted for 48 h with 2 nmol/l of on-trget plus SMARTpool smll interfering RNA (sirna) ginst Ritor (L ) or tor (L ), respetively. sigenome non-trgeting sirna (Dhrmon, Epsom, UK; srmled), 2 nmol/l, ws used s ontrol. Quntifition nd sttistil nlysis Immunolot-nd intensities were quntified using the ImgeJ (version 1.44) softwre. Sttistil nlyses were performed s indited in the figure legends using GrphPd Prism 5. (GrphPd Softwre, Sn Diego, CA, USA). Results myin hs deleterious effets on MIN6 ell viility nd funtion To investigte the effets of rpmyin on pnreti et ell viility nd funtion, the lonl pnreti et ell line MIN6 [19] ws inuted with 2 nmol/l rpmyin for up to 72 h (Fig. 1). Flow ytometry of nnexin V nd propidium iodide stined MIN6 ells demonstrted tht rpmyin used signifint loss of viility y 24 h through n inrese in poptosis rther thn nerosis (Fig. 1 f). In ddition, rpmyin used derese in et ell size (Fig. 1g, h) nd redution in oth sl nd gluose-stimulted insulin seretion (GSIS; Fig. 1i). However, there ws no signifint hnge in intrellulr insulin ontent, inditing tht the effets of rpmyin on GSIS re due to defet in insulin seretion, rther thn in insulin synthesis (Fig. 1j). myin inhiits oth mtorc1 nd mtorc2 in MIN6 ells nd rt- nd humn-isolted islets of Lngerhns In order to understnd the moleulr mehnism y whih rpmyin is using et ell toxiity, the effets of rpmyin on mtor signlling were investigted. MIN6 ells were treted with rpmyin for up to 72 h nd, s expeted, rpmyin utely (within 1 h) inhiited mtorc1 tivity s determined y derese in the phosphoryltion sttus of S6K on Thr389 nd on Ser24/244 (Fig. 2). Interestingly, y 24 h of rpmyin tretment, the phosphoryltion of t Ser473, downstrem trget of mtorc2 [27], ws lso signifintly inhiited, yet totl levels were unffeted (Fig. 2). Moreover, prolonged rpmyin tretment (48 72 h) lso inhiited the turn motif phosphoryltion of PKCα nd βii on Thr638/641, whih is lso medited y mtorc2 [28, 29]. Inution of MIN6 ells with s little s 1 nmol/l rpmyin for 24 h ws suffiient to inhiit oth mtorc1 nd mtorc2 tivity s determined y the phosphoryltion stte of on Ser24/244 nd on Ser473 (Fig. 2). This onentrtion is similr to tht seen in the portl venous system of islet-trnsplnt reipients [3]. These results lerly indite tht rpmyin inhiits oth mtorc1 nd mtorc2 in MIN6 ells. Given tht MIN6 ells re lonl et ell line, it ws importnt to onfirm these findings in primry ells. Therefore, isolted rt nd humn islets of Lngerhns were treted with 2 nmol/l rpmyin for 48 nd 72 h nd the tivities of mtorc1 nd mtorc2 were determined y ssessing the phosphoryltion sttes of on Ser24/244 nd on Ser473. myin tretment resulted in the inhiition of oth on Ser24/244 nd on Ser473, inditing tht rpmyin lso inhiits mtorc1 nd mtorc2 in rt (Fig. 2) nd humn islets of Lngerhns (Fig. 2d). myin, 2 nmol/l, ws used in these experiments nd susequent experiments to produe mximl effets. However, lower doses of rpmyin inhiit mtorc2 nd use ell deth in rt islets of Lngerhns (see eletroni supplementry mteril [ESM] Fig. 1). myin inhiits mtorc2 through the dissoition of the mtorc2 omplex The inhiition of mtorc2 y rpmyin ould e vi redution in the levels of mtorc2 omponents or the dissoition of the omplex. However, tretment of MIN6 ells with rpmyin for up to 72 h used no redution in the levels of either mtor or, the prinipl omponents of mtorc2 (Figs 2 nd 3). Isoltion of mtorc1 nd mtorc2 omplexes y immunopreipittion with nti-mtor ntiodies followed y nlysis reveled tht prolonged rpmyin tretment used the dissoition of oth nd from mtor, inditing tht rpmyin uses the dissoition of oth mtorc1 nd mtorc2 (Fig. 3). Moreover, isoltion of the mtorc2 y immunopreipittion with nti- ntiodies followed y nlysis of its omponents onfirmed tht prolonged rpmyin tretment used the dissoition of mtor from (Fig. 3). Therefore, rpmyin inhiits mtorc2 in et ells y using its dissoition. is essentil for ell survivl nd onstitutively tive protets MIN6 ells nd rt islets from the deleterious effets of rpmyin is n importnt pro-survivl ftor nd, s rpmyin uses derese in phosphoryltion nd deline in ell survivl, we further explored the

5 Dietologi (212) 55: FL1-H d Counts g i Insulin onentrtion (pmol/l) SuG FL2-H Control Control G/G1 G2/M FL2-H Counts FL1-H e Counts G/G1 SuG1 G2/M Control FSC-H FL2-H FL2-H Gluose (mmol) h 24 h 48 h h 24 h 24 h 48 h 72 h 48 h effets of rpmyin on the phosphoryltion nd tivtion of. Tretment of MIN6 ells with rpmyin for 24 h resulted in signifint derese in the phosphoryltion t Ser473 ut not t Thr38, s well s signifint derese in its tivity (Fig. 4). This derese in tivity ws oinident with n inrese in the leved form of spse-3, ellulr mrker of poptosis (Fig. 4). Moreover, the inhiition of tivity results in n inrese in poptosis nd derese in funtion in oth MIN6 ells nd rt islets (ESM Fig. 2). To determine whether the loss of tivity ws responsile for rpmyin toxiity, MIN6 ells were infeted with n denovirus produing myristylted version (onstitutively tive) of (AdC), whih direts to the plsm memrne nd thus onfers onstitutively tive phenotype [31]. In ells overproduing AdC, prolonged rpmyin tretment hd no detetle effet on kinse tivity or on the phosphoryltion of the sustrte GSK3 (Fig. 4). Moreover, produing AdC hd no effet on the ility of rpmyin to inhiit the phosphoryltion of on Ser24/ 244 nd hene mtorc1 (Fig. 4). Importntly, the prodution of AdC in MIN6 ells ws found to protet ells Apoptoti ells (%) f Per ent of ell popultion h Cell size (% of ontrol) j Insulin ontent (pmol/mg) , 4, 3, 2, 1, R Fig. 1 myin hs deleterious effets on MIN6 ell viility nd funtion. MIN6 ells were treted with 2 nmol/l rpmyin s indited., myin dereses viility. Following tretment, ells were dispersed, stined with nnexin V nd propidium iodide, nd then nlysed y flow ytometry: y-xis (FL1-H), propidium iodide; x- xis (FL2-H), nnexin V. Representtive histogrms of ontrol ells () nd ells treted with rpmyin () for 24 h re shown. Quntifition of perentge of poptoti ells ws performed y flow ytometry. p vlues were otined using one-wy ANOVA with Dunnett s test. d, e Representtive histogrms of ell yle distriution. MIN6 ells were treted with vehile (1% ethnol; d) or 2 nmol/l rpmyin (e) for 24 h, nd then nlysed y flow ytometry. f The perentge of ells in sug 1,G /G 1, S nd G 2 /M phses relted to the totl ell popultion were quntified. p vlues were otined using two-wy ANOVA with Bonferroni post test. Without rpmyin: white, sug 1, 1.3±.6; light grey, G /G 1, 66.5±1.3; drk grey, S, 8.5±.3; nd lk, G 2 /M, 14.9±1.3. With rpmyin: white, sug 1, 41.8±1.5; light grey, G /G 1, 44.4±2.1; drk grey, S, 8.5±.8; nd lk, G 2 /M, 5.3±.2. g myin used derese in ell size. The histogrm (FSC-H) for the G /G 1 popultion shows left-wrd shift in response to rpmyin tretment ompred with ontrol, demonstrting tht rpmyin uses derese in ell size. h Quntifition of dt from (g). Dt were expressed s perentge of ontrol. p vlues were otined y pired Student s t test. i myin inhiits GSIS. Following rpmyin tretment, ells were inuted in KRB ontining 1 mmol/l gluose for 1 h followed y KRB ontining 2 mmol/l gluose for further 1 h. Superntnt frtions were olleted nd ssyed for insulin onentrtion using ELISA. p vlues were otined using one-wy ANOVA followed y Bonferroni post-test. For simpliity, not ll sttistil signifines re shown. j Cells from (i) were lysed nd insulin ontent determined y ELISA. p vlues were otined using Dunnett s test with one-wy ANOVA. All dt re displyed s mens±se, n3. p.1.1, p<.1. Results shown re representtive of t lest three independent experiments., rpmyin from the negtive effets of prolonged rpmyin tretment on viility (Fig. 4) nd GSIS (Fig. 4d). To onfirm these findings in primry ells, rt islet of Lngerhns were infeted with AdC nd the effets of rpmyin on islet funtion nd viility were ompred with mok-infeted islets. myin tretment for 48 h resulted in inresed ell deth in oth infeted nd uninfeted islets. However, ell deth ws signifintly ttenuted in islets infeted with AdC ompred with mok-infeted islets (Fig. 4e). myin tretment for 72 h resulted in signifint redution in GSIS (Fig. 4f). Importntly, islets infeted with AdC were fully proteted ginst the effets of rpmyin on GSIS (Fig. 4f). Therefore, the deleterious effets of rpmyin on et ell viility nd funtion re likely to e used y the inhiition of medited y the intivtion of mtorc2. Evidene tht inhiition of mtorc2 is primrily responsile for rpmyin toxiity To provide supportive evidene tht mtorc2 plys ritil role in et ell survivl nd funtion, isolted rt islets of Lngerhns were treted for up to 24 h with rpmyin or Torin1, novel seletive mtor inhiitor tht, unlike rpmyin, rpidly inhiits oth mtorc1 nd mtorc2 [18] (Fig. 5). Torin1 hd inhiited oth mtorc1 nd mtorc2 y 8 h s determined y the

6 136 Dietologi (212) 55: mtorc2 mtorc1.5 1 (2 nmol/l) (nmol/l) h P-PKCα/βII Thr638/641 PKC P- Ser24/244 P-Ser6K1 Thr389 S6K1 mtor IP Lystes IP:mTOR IP: (h) mtor mtor Lystes Non-immune IgG mtor Non-immune IgG mtor Fig. 3 Prolonged rpmyin tretment inhiits mtorc2 tivity through dissoition of mtorc2. MIN6 ells were treted with 2 nmol/l rpmyin for 72 h. Cells were lysed in.3% CHAPS uffer nd immunopreipittion ws performed using nti-mtor nd nti- ntiodies. Non-immune IgG ws used s ontrol for the immunopreipittion. Immunopreipittes nd lystes were resolved on SDS-PAGE nd immunolotted using ntiser ginst mtor, nd. All results re representtive of three independent experiments. IP, immunopreipittes;, rpmyin IP mtorc2 mtorc1 Rt islets (2 nmol/l) C 48 h 72 h P- Ser24/244t GAPDH d P-PKCα/βII Thr638/641 P- Ser24/244 Humn islets 48 h 72 h (2 nmol/l) P- Ser24/244 GAPDH Fig. 2 myin inhiits downstrem trgets of oth mtorc1 nd mtorc2. MIN6 ells were treted with 2 nmol/l rpmyin for the time periods indited. MIN6 ells were treted with 2, 1, 2, 1 nd 2 nmol/l rpmyin for 24 h. Rt nd (d) humn islets of Lngerhns were treted with 2 nmol/l rpmyin for the times indited. Following ell lysis, proteins were resolved on SDS-PAGE nd immunolotted using ntiser ginst, P- Ser24/Ser244, P-S6K1 Thr389, P-PKCα/βII Thr638/Thr641, s well s totl levels of mtor,,,,, S6K1 nd GAPDH. All results re representtive of three or, in the se of humn islets, two independent experiments. GAPDH, glyerldehyde-3- phosphte dehydrogense;, rpmyin phosphoryltion stte of nd on Ser473, respetively (Fig. 5). Torin1 or rpmyin hd no effet on the phosphoryltion of t Thr38. Torin1 lso used rpid inrese in the rte of poptosis (Fig. 5), nd derese in GSIS (Fig. 5). Compred with islets treted with Torin1, the inhiitory effets of rpmyin on mtorc2 nd were delyed (Fig. 5). Moreover, the rte of ell deth ws elerted in Torin1-treted ells ompred with rpmyintreted ells (Fig. 5). Therefore, it is the inhiition of mtorc2, rther thn mtorc1, tht orreltes with deresed viility nd funtion. To provide further evidene to support these findings mtorc1 funtion ws inhiited in primry rt islets of Lngerhns y knoking down tor expression using sirna (Fig. 5d f). This hd no signifint effet on either islet viility (Fig. 5e) or GSIS(Fig. 5f), yet led to signifint derese in the phosphoryltion of S6K1 nd(fig.5d). Downregultion of Ritor expression uses loss of islet of Lngerhns viility As mtorc2 is required for full tivtion nd protets ells ginst rpmyin toxiity, we imed to determine whether there ws usl link etween the inhiition of mtorc2, the intivtion of nd the loss of islet viility. To investigte this, mtorc2 tivity ws downregulted in dispersed islets of Lngerhns y knoking down Ritor expression using sirna. sirnamedited knokdown of Ritor led to derese in the phosphoryltion of on Ser473, inditive of deresed mtorc2 tivity, without ffeting the phosphoryltion of, mrker of mtorc1 tivity (Fig. 6). This orrelted with n inrese in poptosis (Fig. 6) nd derese in GSIS (Fig. 6). Importntly, there ws no signifint hnge in the phosphoryltion of on Thr38 in islets trnsfeted with Ritor sirna (Fig. 6; see disussion). Tken together, these results indite tht mtorc2 is essentil for et ell viility. Disussion myin hs een used s the primry immunosuppressnt in mny islet-trnsplnt progrmmes over the lst dede [1]. This hoie hs een sed on the ssumption tht rpmyin is less toxi to pnreti et ells thn other immunosuppressnts, suh s ortiosteroids nd trolimus. However, rpmyin nd its nlogues n use

7 Dietologi (212) 55: CPM (% of ontrol) Apoptoti ells (%) e Oligonuleosome-ssoited histone (ritrry unit) P- Thr38 P- Ser24/244 l.sp 3 AdC P-GSK3α/β Ser21/9 P- Ser24/244 AdC AdC 2.5 AdC deleterious effets on et ell mss nd islet engrftment [3 5], primrily through indution of et ell poptosis. Prior to this study, the moleulr sis for this toxiity hd not een known. Although rpmyin is known to inhiit mtorc1 in et ells, we show tht prolonged rpmyin tretment is le to olish the tivity of mtorc2 in MIN6 ells, rt nd humn islets of Lngerhns s demonstrted y either derese in the phosphoryltion of its downstrem trget on Ser473 (Fig. 2) nd/or redution in kinse tivity (Fig. 4). Inhiition of using (Akt) inhiitor (AKTi) lso results in the loss of ell viility (ESM Fig. 2), wheres the overprodution of onstitutively tive resues MIN6 ells or islets from rpmyin-indued poptosis (Fig. 4). This is not speifilly medited y the reovery of Ser473 phosphoryltion ut through n inrese in overll tivity. Unlike rpmyin, Torin1 nd AKTi rpidly inhiit phosphoryltion t Ser473, nd lso led to more rpid loss in et ell viility ompred with rpmyin (Fig. 5 nd ESM Fig. 2). Moreover, the intivtion of mtorc2 y knokdown of Ritor expression in rt islets uses n CPM (% of ontrol) d Fold hnge in insulin seretion f Insulin onentrtion (pmol/l) mmol/l 2 mmol/l Gluose h 72 h h 72 h 1 mmol/l AdC 2 mmol/l Gluose R Fig. 4 is indispensle for et ell survivl nd the overprodution of onstitutively tive protets MIN6 ells from the deleterious effets of rpmyin., in vitro tivity ssy. MIN6 ells were inuted in DMEM supplemented with 15% (vol./vol.) FCS in the sene or presene of rpmyin (2 nmol/l) for 72 h. Endogenous ws immunopreipitted from the lystes nd sujeted to kinse ssy. Lystes previous to kinse ssy were olleted nd seprted y SDS-PAGE. Proteins were resolved on SDS-PAGE nd immunolotted using ntiser ginst, P- Thr38, P- Ser24/Ser244, P-GSK3α/β Ser21/Ser9, totl, nd leved spse 3. Following ell lysis, ws immunopreipitted using immoilised ntiody nd kinse tivity determined using [γp 32 ]ATP nd rosstide s the sustrte. p vlues were otined using one-wy ANOVA with Bonferroni post-test. d MIN6 ells were mok infeted or infeted with denovirus produing onstitutively tive (AdC) t multipliity of infetion of 5 for 24 h, followed y inution for 72 h ()or24h(, d) in the presene or sene of 2 nmol/l rpmyin. Following tretment, ells were dispersed, stined with nnexin V nd propidium iodide, nd then nlysed y flow ytometry. The perentge of poptoti ells ws quntified. p vlues were otined using one-wy ANOVA with Bonferroni post-test. d Following rpmyin tretment, ells were inuted in KRB ontining 1 mmol/l gluose for 1 h, followed y inution in KRB ontining 2 mmol/l gluose for further 1 h. Superntnt frtions were olleted nd ssyed for insulin onentrtion using ELISA. Results re expressed s fold hnge of stimulted insulin seretion over sl insulin seretion. p vlues were otined using one-wy ANOVA with Bonferroni post-test. All immunolots re representtive of three independent experiments. e, f Rt islets were infeted with denovirus produing onstitutively tive for 24 h, followed y further 48 h inution in the presene or sene of 2 nmol/l rpmyin. e To determine the rte of ell deth, internuleosoml DNA frgmenttion ws nlysed. p vlues were otined using one-wy ANOVA followed y Bonferroni post-test. f Following rpmyin tretment, islets were inuted in KRB ontining 1 mmol/l gluose for 1 h followed y further 1 h inution in KRB ontining 2 mmol/l gluose. Superntnt frtions were olleted nd ssyed for insulin onentrtion using ELISA. p vlues were otined using one-wy ANOVA followed y Tukey s multiple omprison test. For simpliity, not ll sttistil signifines re shown. All dt re shown s mens±se, n3. p.5.1, p.1.1 nd p<.1. All immunolots re representtive of three independent experiments. Cl. sp 3, leved spse 3;, rpmyin inrese in poptosis to similr extent s rpmyin (Fig. 6), wheres the intivtion of mtorc1 y knokdown of tor expression hd no signifint effet on ell viility (Fig. 5d,e). Therefore, we onlude tht the mintenne of mtorc2 tivity is ritil for et ell survivl nd tht the inhiition of mtorc2 y rpmyin is likely to e responsile for rpmyin toxiity to pnreti islets. Yet it ws reently reported tht βriko mie, with et ell-speifi ltion of Ritor, do not show n inrese in et ell deth, lthough there is redution in islet mss nd funtion [17]. One possile explntion for this pprent ontrdition is tht lthough phosphoryltion t Ser473 is ompromised in the islets isolted from βriko mie, the phosphoryltion of t Thr38, whih is medited y phosphoinositide-dependent kinse 1 (PDK1) [32], is enhned [17]. This is likely to hve ompenstory effet on the tivity of. However, the overll kinse tivity of in islets from βriko mie ws not

8 1362 Dietologi (212) 55: h 16 h 24 h Torin1 P- Ser24/244 P- Thr38 Oligonuleosome-ssoited histone (ritrry unit) Insulin onentrtion (pmol/l) Torin1 1 mmol/l 2 mmol/l Gluose 1. 8 h 16 h 24 h d sirna P-S6K1 Thr389 P- Ser24/244 e Oligonuleosome-ssoited histone (ritrry unit) sirna f Insulin onentrtion (pmol/l) sirna 1 mmol/l 2 mmol/l Gluose Fig. 5 Torin1 uses redution in the viility nd funtion of islets of Lngerhns. Rt islets were treted with rpmyin (2 nmol/l) or Torin1 (2 nmol/l) for the times indited. Rt islets were treted s in (), nd internuleosoml DNA frgmenttion ws determined s n inditor of ell poptosis using the ell deth detetion ELISA. p vlues were otined using two-wy ANOVA followed y Bonferroni post-test ompring rpmyin- nd Torin1-treted smples. Blk irles, ontrol; white squres, rpmyin; lk tringles, Torin1. Rt islets were treted with rpmyin (2 nmol/l) or Torin1 (2 nmol/l) for 4 h, nd then inuted in KRB ontining 1 mmol/l gluose for 1 h followed y further 1 h inution in KRB ontining 2 mmol/l gluose. Superntnt frtions were olleted nd ssyed for insulin onentrtion using ELISA. p vlues were otined using one-wy ANOVA followed y Bonferroni posttest. d Dispersed rt islets were trnsfeted with srmled or tor sirnas for 24 h nd then inuted for 48 h in the presene or sene of rpmyin (2 nmol/l). For () nd (d), fter ell lysis, proteins were seprted on SDS-PAGE nd western lotted using ntiser ginst,, P-S6K1 Thr389, P- Ser24/ Ser244, nd. e Dispersed rt islets were trnsfeted with srmled or tor sirnas for 24 h prior nd then further inuted for 48 h in the presene or sene of rpmyin (2 nmol/l). Apoptosis ws determined y mesuring using the ell deth detetion ELISA. p vlues were otined using two-wy ANOVA followed y Bonferroni post-test ompring eh olumn with the first olumn (); n3. f Rt islets were trnsfeted nd treted s in (e), nd then inuted in KRB ontining 1 mmol/l gluose for 1 h followed y further 1 h inution in KRB ontining 2 mmol/ l gluose. Superntnt frtions were olleted nd ssyed for insulin onentrtion using n ELISA. p vlues were otined using one-wy ANOVA followed y Tukey s multiple omprison test, n3. All dt re shown s mens±se. p.5.1, p.1.1 nd p<.1. All immunolots re representtive of three independent experiments., rpmyin; Sr, srmled reported y Gu et l. [17]. In ontrst, we hve een unle to detet ny signifint inrese in the phosphoryltion of t Thr38 in rt islets in whih Ritor expression ws utely knoked down (Fig. 6) or in islets in whih mtorc2 tivity ws inhiited y either rpmyin or Torin1 (Figs 5 nd 6). Therefore, it is plusile tht in βriko mie, signlling events downstrem of Thr38 phosphoryltion resue et ells from poptosis. integrtes upstrem survivl signls to mintin et ell viility. It protets et ells from streptozotoin-indued ell deth nd medites the nti-poptoti tions of insulin, IGF1 nd glugon-like peptide 1 (GLP-1; reviewed in Xie et l. nd Elghzi et l. [8, 33]). Of note, mie with knokout of Pkβ (lso known s Akt2), unlike those with knokout of Pkα (lso known s Akt1) [34], hve derese in et ell mss tht prllels n inrese in et ell poptosis [35]. Interestingly, it hs reently een reported tht the inhiition of phosphoryltion on Ser473 y rpmyin in primry rt nd humn pltelets orreltes with derese in the tivity of β rther thn α [36], Therefore, it is tempting to speulte tht rpmyin islet toxiity is used y the speifi impirment of β tivity vi the loss of Ser473 phosphoryltion used y the intivtion of mtorc2. Moreover, dereses in phosphoryltion in trnsgeni mie where omponents of the IRS PDK1 pthwy re lted (reviewed in Elghzi et l. [33]), or mie produing onstitutively tive S6K in whih IRS signlling is impired [37], orrelte with deresed viility. These nti-

9 Dietologi (212) 55: P- Thr38 P- Ser24/244 level (% of ontrol) P- Thr38 level (% of ontrol) P-rpS6 Ser24/244 level (% of ontrol) Oligonuleosome-ssoited histone (ritrry unit) Insulin onentrtion (pmol/l) mmol/l 2 mmol/l Gluose Fig. 6 sirna knokdown of Ritor expression uses loss of et ell viility. Dispersed rt islets were trnsfeted with srmled or Ritor sirnas for 24 h nd then inuted for 48 h in the presene or sene of rpmyin (2 nmol/l). Cells were then lysed, proteins were resolved on SDS-PAGE nd western lotted using ntiser ginst,,, P- Thr38, P- Ser24/Ser244, nd. Levels of, P- Thr38 nd P- Ser24/244 were quntified y densitometry nd expressed s perentge of ontrol (srmled sirna). p vlues were otined using one-wy ANOVA followed y Bonferroni post-test. Dispersed rt islets were trnsfeted with srmled sirna or Ritor sirna for 24 h nd then inuted for 48 h in the presene or sene of rpmyin (2 nmol/l). Apoptosis ws determined using the ell deth detetion ELISA (see Methods) following mnufturer s instrutions. p vlues were otined using two-wy ANOVA followed y Bonferroni post-test ompring eh olumn to the first olumn (); n4. Rt islets were trnsfeted nd treted s in (), nd then inuted in KRB ontining 1 mmol/l gluose for 1 h followed y further 1 h inution in KRB ontining 2 mmol/ l gluose. Superntnt frtions were olleted nd ssyed for insulin onentrtion using ELISA. p vlues were otined using one-wy ANOVA followed y Tukey s multiple omprison test, n3. All dt re shown s mens±se. p.5.1, p.1.1 nd p<.1. Results from () re from five independent experiments; immunolots from one representtive experiment re shown. Results from () nd () re from three independent experiments., rpmyin; RIC, ; Sr, srmled poptoti effets of my e medited y the nuler exlusion nd degrdtion of the FOXO fmily of proteins, whih is ontrolled y the phosphoryltion of on Ser473 [38]. However, rpmyin ws unle to inhiit the phosphoryltion of FOXO (ESM Fig. 3), nd mtorc2 ltion in et ells from βriko mie leds to n inrese in protein prodution of FOXO1 nd its nuler retention, yet it does not result in n inrese in poptosis [17]. Therefore, it is likely tht the positive role of in et ell viility is medited y other downstrem trgets implited in ell survivl, suh s B ell CLL/lymphom 2 (BCL-2) fmily memers, pro-spse-9 nd murine doule minute 2 (MDM2) (reviewed in Hers et l. [39]). We, in this report, nd others hve shown tht long-term rpmyin tretment inhiits GSIS [3 5]. However, GSIS is not ffeted y short-term rpmyin tretment [3, 4, 41]nd is unffeted y tor knokdown (Fig. 5f), inditing tht the deleterious effets of rpmyin on GSIS re not medited through the inhiition of mtorc1 ut used y the inhiition of mtorc2. Although mtorc1 regultes protein synthesis, rpmyin hs little effet on either insulin synthesis [42] or insulin ontent (Fig. 1j nd [43]) in vitro. Yet the hroni inhiition of mtorc1 in vivo my led to deresed insulin ontent whih, in turn, ould impt on GSIS. However, the inhiition of GSIS y rpmyin hs een reported to e used y redued mitohondril ATP prodution [43]. The findings of this study hve importnt implitions for linil islet trnsplnttion. First, it rings into question the use of rpmyin s primry immunosuppressnt in islet trnsplnttion. However, there re limited lterntives s

10 1364 Dietologi (212) 55: gluoortioids nd trolimus hve more signifint detrimentl effets on et ells. An lterntive is myophenolte mofetil (MMF); however, studies on humn islets hve shown tht MMF tretment results in signifint redution in GSIS [41]. The key role of mtorc2 nd in et ell funtion nd survivl (this report nd others [44 48]) is of importne to those involved in the development of novel immunosuppressive gents for islet trnsplnttion. Idelly, ny new gents should not ffet tivity. One potentil re for development is mtorc1-speifi inhiitors, whih should retin the immunosuppressive effets of rpmyin without ny mtorc2-medited toxiity. However, this mkes the ssumption tht the immunosuppressive effets of rpmyin re indeed medited solely vi mtorc1 rther thn mtorc2. In ddition, the in vivo tivtion of might improve the outome of islet trnsplnttion y improving the funtion nd survivl of trnsplnted et ells. In onlusion, we hve shown tht the moleulr sis of rpmyin-indued islet toxiity is through the dissoition nd inhiition of mtorc2 nd the susequent redution in phosphoryltion t Ser473 nd the suppression of its kinse tivity. As onsequene, this work hs reveled n importnt role for mtorc2 in et ell survivl. Aknowledgements We re very grteful to N. MGown, L. Frser nd G. Gle from the Sottish Ntionl Blood Trnsfusion Servie, Edinurgh, UK, for the isoltion nd provision of humn islets. We thnk R. Snowden from the MRC Toxiology Unit, Leiester, UK, for providing exellent tehnil dvie nd ssistne. We lso thnk E. Gomez from the University of Leiester, Leiester, UK, for ritil reding of the mnusript. TPH is ting s the gurntor for this rtile. Funding A.D. Brlow ws supported y Royl College of Surgeons of Englnd Reserh Fellowship (Fmily Rih hritle trust) nd Peel Medil Trust equipment grnt. J. Xie ws supported y CONACYT studentship (sholrship No. 2671) wrded y the Mexin government. C.E. Moore ws supported y Wellome Trust Projet Grnt (WT81268MA) wrded to T.P. Herert. Contriution sttement ADB nd JX nlysed nd interpreted the mjority of the dt nd drfted the mnusript. CEM, SCC nd JAMS nlysed nd interpreted some of the dt nd revised the mnusript. MLN helped oneive the study nd revised the mnusript ritilly. TPH ws responsile for the oneption of the study, the nlysis nd interprettion of the dt nd the drfting of the rtile. All uthors pproved the finl version. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution Liense whih permits ny use, distriution, nd reprodution in ny medium, provided the originl uthor(s) nd the soure re redited. Referenes 1. Shpiro AM, Lkey JR, Ryn EA et l (2) Islet trnsplnttion in seven ptients with type 1 dietes mellitus using gluoortioid-free immunosuppressive regimen. N Engl J Med 343: Ryn EA, Pty BW, Senior PA et l (25) Five-yer follow-up fter linil islet trnsplnttion. Dietes 54: Fin MC, Lkey JR, Rjotte RV, Knetemn NM (1993) The effiy nd toxiity of rpmyin in murine islet trnsplnttion. In vitro nd in vivo studies. Trnsplnttion 56: Bell E, Co X, Moii JA et l (23) myin hs deleterious effet on MIN-6 ells nd rt nd humn islets. Dietes 52: Zhng N, Su D, Qu S et l (26) Sirolimus is ssoited with redued islet engrftment nd impired et-ell funtion. Dietes 55: Smith RN, Kent SC, Ngle J et l (28) Pthology of n islet trnsplnt 2 yers fter trnsplnttion: evidene for nonimmunologil loss. Trnsplnttion 86: Zonu R, Efeyn A, Stini DM (211) mtor: from growth signl integrtion to ner, dietes nd geing. Nt Rev Mol Cell Biol 12: Xie J, Herert TP (211) The role of mmmlin trget of rpmyin (mtor) in the regultion of pnreti et-ell mss: implitions in the development of type-2 dietes. Cell Mol Life Si. doi:1.17/s Jinto E, Loewith R, Shmidt A et l (24) Mmmlin TOR omplex 2 ontrols the tin ytoskeleton nd is rpmyin insensitive. Nt Cell Biol 6: Srssov DD, Ali SM, Sengupt S et l (26) Prolonged rpmyin tretment inhiits mtorc2 ssemly nd Akt/. Mol Cell 22: Zeng Z, dos Srssov D, Smudio IJ et l (27) myin derivtives redue mtorc2 signling nd inhiit AKT tivtion in AML. Blood 19: Hung J, Mnning BD (28) The TSC1-TSC2 omplex: moleulr swithord ontrolling ell growth. Biohem J 412: Shigeym Y, Koyshi T, Kido Y et l (28) Biphsi response of pnreti et ell mss to ltion of TSC2 in mie. Mol Cell Biol 28: Rhdi L, Blzr N, Osorio-Duque F et l (28) Disruption of Ts2 in pnreti et ells indues et ell mss expnsion nd improved gluose tolerne in TORC1-dependent mnner. Pro Ntl Ad Si USA 15: Pende M, Kozm SC, Jquet M et l (2) Hypoinsulinemi, gluose intolerne nd diminished et-ell size in S6K1- defiient mie. Nture 48: Ruvinsky I, Shron N, Lerer T et l (25) Riosoml protein S6 phosphoryltion is determinnt of ell size nd gluose homeostsis. Genes Dev 19: Gu Y, Lindner J, Kumr A, Yun W, Mgnuson MA (211) Ritor/ mtorc2 is essentil for mintining lne etween β-ell prolifertion nd ell size. Dietes 6: Thoreen CC, Kng SA, Chng JW et l (29) An ATP-ompetitive mmmlin trget of rpmyin inhiitor revels rpmyin-resistnt funtions of mtorc1. J Biol Chem 284: Miyzki J, Arki K, Ymto E et l (199) Estlishment of pnreti et ell line tht retins gluose-induile insulin seretion: speil referene to expression of gluose trnsporter isoforms. Endorinology 127: Moore CE, Xie J, Gomez E, Herert TP (29) Identifition of AMP-dependent kinse s third in vivo riosoml protein S6 kinse in pnreti et-ells. J Mol Biol 389: Moore CE, Omikorede O, Gomez E, Willrs GB, Herert TP (211) PERK tivtion t low gluose onentrtion is medited

11 Dietologi (212) 55: y SERCA pump inhiition nd onfers preemptive ytoprotetion to pnreti et-ells. Mol Endorinol 25: Hung GC, Zho M, Jones P et l (24) The development of new density grdient medi for purifying humn islets nd islet-qulity ssessments. Trnsplnttion 77: Gomez E, Powell ML, Bevington A, Herert TP (28) A derese in ellulr energy sttus stimultes PERK-dependent eif2lph phosphoryltion nd regultes protein synthesis in pnreti et-ells. Biohem J 41: Xie J, Ponuwei GA, Moore CE, Willrs GB, Tee AR, Herert TP (211) AMP inhiits mmmlin trget of rpmyin omplex-1 nd 2 (mtorc1 nd 2) y promoting omplex dissoition nd inhiiting mtor kinse tivity. Cell Signl 23: Cross DA, Alessi DR, Cohen P, Andjelkovih M, Hemmings BA (1995) Inhiition of glyogen synthse kinse-3 y insulin medited y protein kinse B. Nture 378: Jonkers FC, Henquin JC (21) Mesurements of ytoplsmi C2 in islet ell lusters show tht gluose rpidly reruits et-ells nd grdully inreses the individul ell response. Dietes 5: Srssov DD, Guertin DA, Ali SM, Stini DM (25) Phosphoryltion nd regultion of Akt/ y the ritor-mtor omplex. Siene 37: Ikenoue T, Inoki K, Yng Q, Zhou X, Gun KL (28) Essentil funtion of TORC2 in PKC nd Akt turn motif phosphoryltion, mturtion nd signlling. EMBO J 27: Fhinetti V, Ouyng W, Wei H et l (28) The mmmlin trget of rpmyin omplex 2 ontrols folding nd stility of Akt nd protein kinse C. EMBO J 27: Desi NM, Goss JA, Deng S et l (23) Elevted portl vein drug levels of sirolimus nd trolimus in islet trnsplnt reipients: lol immunosuppression or islet toxiity? Trnsplnttion 76: Kohn AD, Tkeuhi F, Roth RA (1996) Akt, plekstrin homology domin ontining kinse, is tivted primrily y phosphoryltion. J Biol Chem 271: Alessi DR, Jmes SR, Downes CP et l (1997) Chrteriztion of 3-phosphoinositide-dependent protein kinse whih phosphoryltes nd tivtes protein kinse Blph. Curr Biol 7: Elghzi L, Bernl-Mizrhi E (29) Akt nd PTEN: et-ell mss nd pnres plstiity. Trends Endorinol Met 2: Cho H, Thorvldsen JL, Chu Q, Feng F, Birnum MJ (21) Akt1/lph is required for norml growth ut dispensle for mintenne of gluose homeostsis in mie. J Biol Chem 276: Groflo RS, Oren SJ, Rfidi K et l (23) Severe dietes, gedependent loss of dipose tissue, nd mild growth defiieny in mie lking Akt2/ et. J Clin Invest 112: Moore SF, Hunter RW, Hers I (211) mtorc2 Protein-medited protein kinse B (Akt) serine 473 phosphoryltion is not required for Akt1 tivity in humn pltelets. J Biol Chem 286: Elghzi L, Blzr N, Blndino-Rosno M et l (21) Deresed IRS signling impirs et-ell yle progression nd survivl in trnsgeni mie overexpressing S6K in et-ells. Dietes 59: Guertin DA, Stevens DM, Thoreen CC et l (26) Altion in mie of the mtorc omponents rptor, ritor, or mlst8 revels tht mtorc2 is required for signling to Akt-FOXO nd PKlph, ut not S6K1. Dev Cell 11: Hers I, Vinent EE, Tvre JM (211) Akt signlling in helth nd disese. Cell Signl 23: MDniel ML, Mrshll CA, Pppn KL, Kwon G (22) Metoli nd utorine regultion of the mmmlin trget of rpmyin y pnreti et-ells. Dietes 51: Johnson JD, Ao Z, Ao P et l (29) Different effets of FK56, rpmyin, nd myophenolte mofetil on gluose-stimulted insulin relese nd poptosis in humn islets. Cell Trnsplnt 18: Gomez E, Powell ML, Greenmn IC, Herert TP (24) Gluosestimulted protein synthesis in pnreti et-ells prllels n inrese in the vilility of the trnsltionl ternry omplex (eif2-gtp.met-trnai) nd the dephosphoryltion of eif2 lph. J Biol Chem 279: Shimodhir M, Fujimoto S, Muki E et l (21) myin impirs metolism-seretion oupling in rt pnreti islets y suppressing rohydrte metolism. J Endorinol 24: Contrers JL, Smyth CA, Bilo G, Young CJ, Thompson JA, De Ekhoff (22) Simvsttin indues tivtion of the serinethreonine protein kinse AKT nd inreses survivl of isolted humn pnreti islets. Trnsplnttion 74: Wng Q, Li L, Xu E, Wong V, Rhodes C, Bruker PL (24) Glugon-like peptide-1 regultes prolifertion nd poptosis vi tivtion of protein kinse B in pnreti INS-1 et ells. Dietologi 47: Li L, El-Kholy W, Rhodes CJ, Bruker PL (25) Glugon-like peptide-1 protets et ells from ytokine-indued poptosis nd nerosis: role of protein kinse B. Dietologi 48: Fvro E, Mieli I, Bussolti B et l (28) Hyperglyemi indues poptosis of humn pnreti islet endothelil ells: effets of prvsttin on the Akt survivl pthwy. Am J Pthol 173: Shui H, Zhng J, Zhng J et l (211) Erythropoietin protets pnreti et-ell line NIT-1 ells ginst ytokine-indued poptosis vi phosphtidylinositol 3-kinse/Akt signling. Endor Res 36:25 34