ARTICLE. I. Chopra & H. F. Li & H. Wang & K. A. Webster

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1 Dietologi (212) 55: DOI 1.17/s y ARTICLE Phosphoryltion of the insulin reeptor y AMP-tivted protein kinse (AMPK) promotes lignd-independent tivtion of the insulin signlling pthwy in rodent musle I. Chopr & H. F. Li & H. Wng & K. A. Wester Reeived: 1 Septemer 211 / Aepted: 1 Novemer 211 / Pulished online: 3 Deemer 211 # Springer-Verlg 211 Astrt Aims/hypothesis Musle my experiene hypoglyemi during ishemi or insulin infusion. During severe hypoglyemi energy prodution is loked, nd n inrese of AMP:ATP tivtes the energy sensor nd puttive insulinsensitiser AMP-tivted protein kinse (AMPK). AMPK promotes energy onservtion nd survivl y shutting down nolism nd tivting toli pthwys. We investigted the moleulr mehnism of unique gluose stress defene pthwy involving AMPK-dependent, insulin-independent tivtion of the insulin signlling pthwy. Methods Crdi or skeletl myoytes were sujeted to gluose nd insulin-free inution for inresing intervls up to 2 h. AMPK, nd omponents of the insulin signlling pthwy nd their trgets were quntified y western lot using phosphor-speifi ntiodies. Phosphomimetis were used to determine the funtion of Ser789 phosphoryltion nd in vitro [ 32 P]ATP kinse ssys were used to mesure the phosphoryltion of the purified insulin reeptor y AMPK. Results Gluose deprivtion inresed -Thr38 nd -Ser473 phosphoryltion y lmost tenfold. Phosphoryltion of glyogen synthse kinse 3 et inresed in prllel, ut phosphoryltion of riosoml 7S suunit-s6 protein kinse nd mmmlin trget of rpmyin deresed. AMPK Eletroni supplementry mteril The online version of this rtile (doi:1.17/s y) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. I. Chopr : H. F. Li : H. Wng : K. A. Wester () Deprtment of Moleulr nd Cellulr Phrmology, University of Mimi Miller Shool of Mediine, 16 NW 1th Ave, RMSB 638, Mimi, FL 33136, USA e-mil: kwester@med.mimi.edu inhiitors loked nd minoimidzole roxmide rionuleotide (AICAR) mimiked the effets of gluose strvtion. Gluose deprivtion inresed the phosphoryltion of on serine-789, ut phosphomimetis reveled tht this onferred negtive regultion. Gluose deprivtion enhned tyrosine phosphoryltion of nd the insulin reeptor, effets tht were loked y AMPK inhiition nd mimiked y AICAR. In vitro kinse ssys using purified proteins onfirmed tht the insulin reeptor is diret trget of AMPK. Conlusions/interprettion AMPK phosphoryltes nd tivtes the insulin reeptor, providing diret link etween AMPK nd the insulin signlling pthwy; this pthwy promotes energy onservtion nd survivl of musle exposed to severe gluose deprivtion. Keywords AICAR. AMPK. Gluose trnsport. Hypoglyemi. Insulin resistne. Insulin sensitivity. Insulin signlling.. Serine/threonine kinse. Tyrosine kinse Arevitions ACC Aetyl oenzyme A roxylse Ad Adenovirus AICAR Aminoimidzole roxmide rionuleotide AMPK AMP-tivted protein kinse CAP C1-ssoited protein CBL Csits B-linege lymphom dn Dominnt negtive dnampk Dominnt-negtive AMPK denovirus DOG 2-Deoxy-[ 3 H]gluose GFP Green fluoresent protein GSK3β Glyogen synthse kinse 3 et IR Insulin reeptor IRK Insulin reeptor kinse JNK1 -Jun N-terminl kinse 1

2 784 Dietologi (212) 55: mtor Mmmlin trget of rpmyin p7s6k Riosoml 7S suunit-s6 protein kinse PI3K Phosphtidylinositol 3-kinse PKC Protein kinse C S6K S6 protein kinse SIK2 Slt-induile kinse 2 TC1 Rs-like protein TC1 TORC1 Trget of rpmyin omplex 1 TSC2 Tuerous slerosis omplex-2 Introdution The insulin nd 5 -AMP-tivted protein kinse (AMPK) signlling pthwys regulte gluose nd ftty id metolism, nd numerous omponents of growth, differentition nd survivl. Both pthwys re uiquitously expressed in eukryoti tissues [1, 2]. Ativtion of the insulin signlling pthwy is n noli stimulus, while AMPK represses nolism nd tivtes toli pthwys to onserve ATP. The insulin signlling pthwy is tivted when insulin or IGF-1 ind the insulin reeptor (IR), tivte the insulin reeptor kinse (IRK) nd utophosphorylte the IR. The tivted IR inds nd tyrosine phosphoryltes IRS proteins, reting doking sites for SH2 domin-ontining proteins tht propgte the signl to the finl effetors [1, 3]. The mjor pthwys doking t the tivted IRSs re the phosphtidylinositol 3-kinse (PI3K) pthwy, the C1-ssoited protein (CAP) sits B-linege lymphom (CBL) rs-like protein TC1 (TC1) pthwy, nd the mitogen-tivted protein kinse nd the protein kinse C (PKC) pthwys [1, 4, 5]. PI3K, CAP CBL TC1 nd PKC re the mjor onduits for gluose (GLUT4) regultion. PI3K reruits the kinse, phosphoinositide-dependent kinse-1, nd is tivted y phosphoryltion of Thr38. As desried in reent reviews, phosphoryltes severl trgets tht regulte ell growth nd survivl, s well s metolism [6, 7]. The trgets inlude spse 9, the pro-poptoti B ell leukemi/lymphom 2 (BCL2) fmily memer, BCL2-ssoited gonist of ell deth (BAD), the forkhed trnsription ftors, glyogen synthse kinse 3 et (GSK3β) nd the riosoml 7S suunit-s6 protein kinse (p7s6k) [8 1]. The tivities of the IR nd IRS proteins re modulted y serine/threonine (Ser/Thr) phosphoryltion. Ser/Thr phosphoryltion typilly onfers negtive regultion nd insulin resistne, s desried y others [11 13]. This regultion mostly tkes ple t the level of /2. IR- Ser1275, -139, nd re sujet to utophosphoryltion y IRK, nd Ser117, -78, -82, -135 nd -137 re phosphorylted y PKC [14]. IRS proteins hve severl Ser/Thr sites, most of whih negtively regulte signl trnsdution nd onfer insulin resistne. As desried previously nd in reviews, IRS proteins ontin trgets for, S6 protein kinse (S6K), -Jun N-terminl kinse 1 (JNK-1), PKC, GSK3β, slt-induile kinse 2 (SIK2) nd AMPK [11 13]. -Ser37 is key regultory site tht, when phosphorylted y JNK-1, inhiitor of κb kinse et (IκK-β) or PKC, inhiits signl ondution. -Ser636 nd -639 phosphoryltion y S6K lso loks signl trnsdution, nd medites negtive feedk nd termintion of insulin signlling [15 17]. SIK2 nd AMPK phosphorylte t the sme Ser789 site [18 22]. Wheres positive regultion y Ser789 hs een suggested [2], suh positive regultion hs not een onfirmed, nd in light of more reent studies inluding the results desried here, it seems likely tht Ser789 exerts only negtive regultion of [18, 19, 21, 22]. AMPK is n energy sensor kinse tht responds to the ATP:ADP/AMP energy hrge of the ell. It is tivted llosterilly y AMP inding nd phosphoryltion y liver kinse 1 [23]. As reviewed y others, AMPK is tivted in musle y physiologil nd pthophysiologil stimuli tht inrese AMP levels, inluding ontrtion, hypoxi nd hypoglyemi [24, 25]. The tivity of AMPK is lso modulted y hormones nd ytokines tht ffet wholeody energy lne, nd y the insulin-sensitising drugs metformin nd the thizolidinediones, s desried in reent review [25]. AMPK tivity is inresed y gluose deprivtion of musle ells, whih orreltes with inresed gluose uptke nd glyolysis independently of insulin [26 28]. AMPK lso enhnes ftty id trnsport nd oxidtion, while swithing off ftty id, holesterol, glyogen nd protein synthesis pthwys. The ltter effets ontriute to the insulin-sensitising properties of AMPK [29]. AMPK loks ell growth oth indiretly y inhiiting the tivity nd prodution of iosyntheti enzymes, nd diretly y trgeting the tuerous slerosis omplex-2 (TSC2) nd trget of rpmyin omplex 1 (TORC1)-rptor, therey loking phosphoryltion of p7s6k. Severl pthwys hve een desried for AMPKenhned insulin sensitivity nd gluose trnsport; mjor moleulr omponent involves relief of the negtive feedk loop nd inhiition of S6K y phosphoryltion of TSC2 [25]. Here, we present evidene for nother link etween AMPK nd the insulin signlling pthwy vi diret phosphoryltion nd lignd-independent tivtion of the IR. Methods Regents Antiodies direted ginst, phosphor-- Thr38, phosphor--ser473, AMPK, phosphor-ampk- Thr172,, phosphor--ser789, GSK3β,

3 Dietologi (212) 55: phosphor-gsk3β-ser9, phosphor etyl oenzyme A roxylse (ACC)-Ser79, nti-green fluoresent protein (GFP) nd phosphor- (pntyr) (ELISA) were from Cell Signling Tehnology (Beverly, MA, USA). PI3K, tin, IR, phosphor- IR Tyr1158, Tyr1162 nd Tyr1163 ntiodies, nd AMPK (α1, β1, γ1) tive were from Millipore (Temeul, CA, USA). Anti-My ntiody ws from Sigm (St. Louis, MO, USA). Insulin ws from Sigm, nd LY2942 nd wortmnnin were from BioSoure (Cmrillo, CA, USA). Aminoimidzole roxmide rionuleotide (AICAR), ompound C nd inhiitor X were from EMD4 Biosienes (Sn Diego, CA, USA). 2-Deoxy-[ 3 H]gluose (DOG) ws from Amershm Biosienes (Pistwy, NJ, USA) nd [γ- 32 P]ATP ws from Perkin Elmer (Sn Jose, CA, USA). The ATP olorimetri ssy kit ws from BioVision (Mountin View, CA, USA) nd the QuikChnge Site-Direted Mutgenesis Kit ws from Strtgene (L Joll, CA, USA). The reominnt denovirl vetor expressing dominnt negtive (dn)ampk ws generous gift from M.J. Birnum (Institute of Oesity, Dietes nd Metolism, University of Pennsylvni, Phildelphi, PA, USA). Cell ulture Methods for primry ulture of neontl rt rdi myoytes hve een previously desried [3]. Before experiments, ells were trnsferred to defined serum nd gluose-free DMEM, supplemented with trnsferrin nd vitmin B12, with or without insulin s indited. For virl infetions, ultures were exposed to the indited mount of virus for 48 h efore exposure to experimentl onditions. Cultures were exposed to AMPK-modifying drugs, inluding Ar-A, ompound C or AICAR, 3 min efore olletion or s indited. Intt mouse myotues were isolted from dispersed hindlim musles y inution with ollgense nd dispse, nd plting on ollgen-oted dishes s desried [31]. Preprtions ontining intt myofires nd myotues were exposed to serum-free defined medium efore experimentl tretments. All experiments involving nimls were pproved y the University of Mimi Institutionl Animl Cre nd Use Committee. Western lot, immunopreipittion nd immunostining Detiled proedures for western lot nlyses nd immunostining hve een desried [3]. Immunopreipittion ssys followed the sme proedures s western lots, exept tht lered lystes were pre-inuted with ntiody overnight nd omplexes seprted using protein-g-grose efore gel eletrophoresis. Site-direted mutgenesis Methods desried y Luo et l. [32] were used to implement mutgenesis of -Ser794. Briefly, humn IRS1 DNA ws inserted into preeiver- Lv8 vetor nd mplified with PCR using kit (QuikChnge Site-Direted Mutgenesis; Strtgene). The primers were 5 - CACTGCCTCTGGTCGCCTTCTCTATG-3 (Ser-Al) or 5 - CACTGAATCTGGTCGCCT TCTCTATG-3 (Ser-Glu). Muttions were verified y sequening. Adenoviruses (Ad) enoding GFP- or mutnts were produed using RAPAd CMV Adenovirl Expression System (Cell Biols, Sn Diego, CA, USA). Myoytes were infeted with the viruses t multipliity of infetion of 5 for 48 h efore tretments. Gluose uptke mesurement Gluose ws mesured using DOG s desried y Chudry et l. [33]. Briefly, ells were wshed three times with KRB uffer t 37 C. Gluose uptke ws initited y dding.1 mmol/l DOG ontining 37 kbq/ml DOG, nd ells were inuted t 37 C for 5 to 15 min. At eh time point, ells were hilled on ie, wshed three times with ie-old KRB ontining 25 mmol/l gluose, lysed with NOH, neutrlised nd ounted y sintilltion. In vitro PI3K tivity ssy PI3K tivity ssoited with ws nlysed following the proedure desried elsewhere [34]. ws immunopreipitted from ell lystes nd retions inuted t room temperture using 2 μg/μl phosphtidylinositol nd [γ- 32 P]ATP (74 kbq). The PI3K phosphoryltion produt ws visulised y thin-lyer hromtogrphy nd utordiogrphy. In vitro AMPK ssy Equl liquots of IR immunopreipitted from serum-strved HepG2 ell lystes were mixed with kinse uffer,.5 mmol/l AMP, tive AMPK (3 ng/μl), 25 μmol/l ATP nd [γ- 32 P]ATP ( Bq), nd inuted t 3 C for 5 to 2 min. Retions were stopped y oiling in SDS-loding uffer. Proteins were seprted y PAGE nd lelled produts deteted y utordiogrphy. Sttistil nlysis Western lots were quntified using NIH Imge J softwre ( downloded June 211). Results re expressed s men±sem. Differenes etween mens were evluted y two-tiled Student s t test. Results Ativtion of in gluose- nd insulin-depleted myoytes Crdi myoytes were sujeted to gluose- nd insulin-free medium for progressive time periods up 24 h nd omponents of the insulin signlling pthwy mesured t intervls. As indited in Fig. 1, the phosphoryltion of on Thr38 nd Ser473 peked etween 4 nd 8 h t lmost tenfold ove the levels of prllel gluoseontining ultures nd remined elevted over 24 h. GSK3β phosphoryltion inresed in prllel, wheres the phosphoryltion of S6K nd mmmlin TORC1 Ser2448

4 786 Dietologi (212) 55: Time 2 h 4 h 8 h 16 h 24 h Gluose P-Ser473 -P-Thr38 GSK-3βP-Ser9 S6K-P-Thr389 S6K TOR-Ser2448 TOR Fold hnge Fold hnge d Fold hnge Gluose-free (h) Gluose-free (h) Gluose-free (h) Gluose-free (h) Fig. 1 Ativtion of the insulin signlling pthwy proteins y gluose deprivtion. Crdi myoytes were ultured in defined medium without insulin or gluose for 24 h nd pltes were removed t the indited times for western lot nlyses s desried. P, phosphoryltion. Western lots of phosphoryltion t Ser473 (grey rs) deresed in the gluose-free ondition. GSK3β is diret sustrte for, wheres S6K is phosphorylted y the mmmlin TORC1 [6, 1]. TORC1 is negtively regulted y TSC2, nd S6K phosphoryltion is normlly inresed trnsiently y insulin euse phosphoryltes nd intivtes TSC2 [35]. Ativted S6K then ontriutes to the negtive feedk regultion of the insulin signl y phosphorylting -Ser636 nd -639, therey inhiiting PI3K [15, 16, 36]. Downregultion of S6K simultneously with tivtion of -Thr38 nd -Ser473 suggests tht gluose strvtion mimis insulin stimultion upstrem ut not downstrem of. Gluose strvtion tivtes PI3K To investigte the effet of gluose strvtion on signlling omponents upstrem of, we mesured PI3K tivity nd the inding of the p85 suunit of PI3K to (Fig. 2, ). Gluose strvtion used signifint inrese of PI3K tivity t 4 nd 8 h, whih oinided with the pek of phosphoryltion nd with inresed inding of PI3K p85 to. PI3K tivity did not orrelte with phosphoryltion t lter time points, suggesting tht dditionl ftors ontriute to the nd Thr38 (lk rs) were quntified y densitometry in the liner exposure rnge using NIH Imge J s desried. Quntifition s ove () of GSK3β (grey rs) nd S6K (lk rs) phosphoryltion, nd (d) mtor phosphoryltion t Ser2448; n3; p<.5 y Student s t test regultion of when gluose strvtion is prolonged. To onfirm the role of PI3K, we pretreted rdi myoytes with the prtilly seletive inhiitors LY2942 nd wortmnnin, exposed ultures to gluose deprivtion for 4 h nd quntified phosphoryltion s desried in Fig. 1. As shown in Fig. 2, phosphoryltion t Thr38 ws fully loked (>95%) y oth inhiitors, wheres phosphoryltion t Ser473 ws unffeted y these inhiitors under norml gluose onditions nd prtilly (4±9%) inhiited y LY2942 in the gluose-deprived ondition. Gluose strvtion primes insulin signlling To determine whether enhned /PI3K tivity used y gluose depletion ugments insulin signlling, rdi myoytes were ultured in the presene or sene of gluose s desried (Fig. 1) nd stimulted with insulin. As shown in Fig. 2d, e, insulin phosphoryltion of -Thr38 nd GSK3β ws mrkedly enhned y gluose-free ulture. As expeted, nd GSK3β sl phosphoryltion were elevted in gluose-strved ultures prior to insulin stimultion. Gluose strvtion onferred out fourfold enhnement of insulin-stimulted -Thr38 nd twofold enhnement of

5 Dietologi (212) 55: Time 2 h 4 h 8 h 16 h 24 h Gluose IP: PI3KP PI3K-p85 d + Glu + Glu + LY + Glu + Wm Glu Glu + LY Glu + Wm + Gluose Gluose Insulin (nmol/l) P-T38 -P-S473 β-tuulin -P-T38 GSK3β-PS9 GSK3β P-ACC ACC 4 β-tuulin Fold inrese Time gluose-free (h) e Rtio Akr-P-T38 Glu:+ Glu Rtio GSK-P-S9 Glu:+ Glu Insulin (nmol/l) Insulin (nmol/l) Fig. 2 Gluose strvtion tivtes PI3K nd onfers enhned insulin sensitivity. Crdi myoytes were ultured s desried (Fig. 1). At eh time point, ell lystes were immunopreipitted (IP) with nti- ntiody nd PI3K ws ssyed s desried, or lystes were sujeted to western lot nlysis with ntiodies ginst the p85 suunit of PI3K or. PI3K-P, phosphorylted PI3K. Quntifition of PI3K-P nd p85 in lots () from three seprte experiments; p<.5. -Thr38 phosphoryltion (-P-T38) is sensitive to PI3K inhiitors. Crdi myoytes were ultured s desried (Fig. 1) nd inuted with (+) or without ( ) gluose (Glu) for 4 h in the presene nd sene of LY2942 (2 μmol/l) or wortmnnin (Wm, 1 μmol/l) s indited. Results re representtive of three experiments. -P-S473, -Ser473 phosphoryltion. d Gluose deprivtion enhnes insulin-indued phosphoryltion. Crdi myoytes were inuted for 2 h with (+) or without ( ) gluose, stimulted with 1 nmol/l insulin for the indited time periods nd equl ell lyste proteins nlysed y western lot with the indited proes. GSK3β-P-S9, phosphoryltion of GSK3β t Ser9; P-ACC, phosphorylted ACC. e -P-T38 ws quntified s the rtio of -P-T38: totl. Gluose strvtion signifintly inresed insulin-indued phosphoryltion of t 5 nd 15 min, p<.1; n4 GSK3β t oth insulin onentrtions. Therefore gluose strvtion primes insulin signlling nd is powerful stimulus of the pthwy with or without insulin. Gluose strvtion tivtes AMPK, n ute effet of whih is to phosphorylte TSC2 nd rptor, nd to lok the tivtion of mmmlin TORC1 nd S6K [35, 37]. This ntgonises the tion of insulin on TORC1 nd S6K. To egin investigting possile role of AMPK in the regultion of y gluose deprivtion, we quntified the phosphoryltion of its prinipl trget, ACC, during insulin stimultion in the presene nd sene of gluose. As shown in Fig. 2d, the tivity of AMPK s refleted y phosphorylted ACC- Ser79 ws mrkedly inresed in gluose-free ultures. To determine whether tretment with AICAR lso primed insulin signlling, we repeted the experiments desried in Fig. 2d, ut sustituted AICAR tretment for gluose deprivtion. As shown in eletroni supplementry mteril (ESM) Fig. 1, AICAR pretretment for 3 min inresed the phosphoryltion of AMPK nd ACC, nd enhned the phosphoryltion of -Thr38 in the presene nd in the sene of insulin. These results re onsistent with role for AMPK in the tivtion of the insulin signlling pthwy y gluose deprivtion. Stimultion of y gluose strvtion requires AMPK Figures 1 nd 2 show tht gluose strvtion differentilly regultes nd S6K, nd tivtes AMPK. To determine whether AMPK is involved in the reiprol regultion of nd S6K y gluose depletion, we gin monitored AMPK tivity through its mrkers phosphorylted ACC nd phosphorylted AMPK, nd determined the effets of AMPK inhiitors nd AICAR. These results re

6 788 Dietologi (212) 55: shown in Fig. 3 d. In Fig. 3 rdi myoytes were gin sujeted to gluose- nd insulin-free inution for 4 h, this time in the presene nd sene of dnampk Ad (dnampk) or Ad-GFP s ontrol. Phosphoryltion of on Thr38 nd Ser473 ws inresed y gluose strvtion nd this ws loked y dnampk. Similrly ACC phosphoryltion on Ser79 ws inresed y gluose strvtion nd loked y dnampk. Consistent with previous reports, we found tht -Ser789 phosphoryltion ws mrkedly inresed y gluose strvtion, gin in n AMPK-dependent mnner. Expression of the - My tg onfirmed high expression of the dnampk, nd the effet on ws prlleled y phosphoryltion of GSK3β- Ser9 (Fig. 3). To further investigte role for AMPK in these effets, rdi myoytes were strved of gluose for 4 h in the presene nd sene of the AMPK inhiitor Ar-A, or treted with AICAR for 3 min with norml gluose. As shown in Fig. 3, gluose strvtion inresed the phosphoryltion of ACC, AMPK,, GSK3β nd -Ser789, this effet eing loked in eh se y Ar-A nd mimiked y AICAR. To onfirm these effets over n extended time ourse, myoytes were sujeted to gluose strvtion for up to 16 h s desried (Fig. 1) nd the phosphoryltion of ACC- Ser79 nd of three key regultory sites ws quntified. As shown in Fig. 3, d, phosphoryltion of ACC-Ser79 ws inresed y out twofold t ll time points. -Ser789 phosphoryltion ws inresed y more thn fivefold, ut there ws no signifint hnge in the phosphoryltion of Gluose: Ad-GFP dnampk + -P-Thr38 -P-Ser473 ACC-P-Ser79 Time 2 h 4 h 8 h 16 h Gluose P-ACC-Ser79 AMPK -P-Ser636/639 -P-Ser789 -P-Ser37 -P-Ser789 My GSK3β-Ser9 β-atin d 4 Gluose + + AICAR Ar-A P-ACC-Ser79 AMPK-Thr172 AMPK -Ser473 -P-Thr38 GSK3β-Ser9 -Ser789 e Fold hnge Fold hnge Atin Gluose-free (h) Fig. 3 Ativtion of the insulin signlling sde y gluose strvtion requires AMPK nd is mimiked y AICAR. Crdi myoytes were infeted with dnampk or Ad-GFP for 48 h s desried, nd then inuted for 4 h in the presene or sene of gluose s in Fig. 1, fter whih equl lyste proteins were nlysed y western lot. Overprodution of dnampk in gluose-ontining ultures did not ffet sl levels of phosphorylted (-P), phosphoryltion (P) t Ser789 or ACC phosphoryltion (ACC-P) (dt not shown). Crdi myoytes were ultured s ove () nd sujeted to 4 h inution in the presene or sene of gluose, AICAR or Ar- A s indited. AICAR tretment ws t.5 μmol/l for 3 min, Ar-A ws t 5 mmol/l for 1 h efore ell olletion. Results re representtive of three seprte experiments. Crdi myoytes were ultured with or without gluose s desried (Fig. 1) nd equl proteins were nlysed y western lot with the indited proes. d Western lots of ACC-P were quntified s desried nd orreted for protein loding; plese note tht totl ACC protein levels were not signifintly ffeted y gluose strvtion (not shown). e Quntifition s ove (d) of phosphoryltion t Ser789 (grey rs), Ser636 nd Ser639 (lk rs), nd Ser37 (white rs). d, e n4; p<.5 nd p<.1; NS, phosphoryltion t Ser636 nd Ser639 nd Ser37

7 Dietologi (212) 55: Ser636, -639 or -37. The sene of n effet on IRS- 1-Ser636 nd -639 is onsistent with the suppression of S6K (Fig. 1). In ontrol experiments we found tht insulin tretment used the rpid phosphoryltion of -Ser37, -636 nd -639 s well s S6K, ut not of -Ser789 (ESM Fig. 2). Tken together, these results show tht tivtion y gluose strvtion requires AMPK tivtion. Negtive regultion of y -Ser789 To determine whether the inresed phosphoryltion of -Ser789 ontriutes to the tivtion of, muttions were introdued into humn GFP-tgged IRS1 DNA to reple Ser794 (humn -Ser794rt -Ser789) with lnine (-Ser794-A) or glutmine (-Ser794-E) to respetively lok or mimi phosphoryltion. wildtype nd mutnt DNAs were loned into denovirl vetors nd used to infet rdi myoytes prior to exposure to gluose strvtion s desried. The results of these experiments re shown in Fig. 4. Gluose-free ulture inresed the phosphoryltion of on Thr38 nd Ser473 s expeted, this eing mrkedly enhned in eh se y overundne of wild-type, inditing tht endogenous is limiting in the tivtion of. Infetion of ultures with -Ser794-A inresed nd GSK3β phosphoryltion to level omprle with wild-type IRS- 1, wheres infetion with -Ser794-E deresed phosphoryltion reltive to wild-type or uninfeted ells. Quntifition of these results is shown in Fig. 4. The phosphoryltion of -Thr38 ws inresed eightfold in gluose-strved myoytes infeted with wild-type or the Ser-Al muttion, wheres overexpression of the Ser- Glu mutnt used >5% loss of -Thr38 phosphoryltion. In dt not shown, we found the sme trends when AICAR tretments repled gluose strvtion. Phosphoryltion of on Ser473 displyed similr trends, ut ws less dependent on, finding tht is onsistent with the lterntive pthwy of tivtion [38]. Figure 4 shows tht infetion of rdi myoytes with the different denovirl vetors generted equivlent -GFP levels. The results support previous reports [18, 19, 21, 22] tht the - Ser789 (h794) site onfers negtive regultion nd nnot Fig. 4 Phosphomimeti nlyses of -Ser789/794 funtion. Crdi myoytes were infeted with wild-type (wt), or with muttions with glutmine (-S794 E) or lnine (-S794 A), eh with GFP tg. Infetion ws for 48 h s desried ove (Methods), followed y 4 h inution in gluose-free medi. Equl ell lyste proteins were nlysed y western lot for phosphoryltion (P), GSK3β P nd. The upper nd in the lnes indites levels of infeted -GFP. S, Ser; T, Thr. Quntifition of P undne y Ad infetion, reltive to ontrol (no Ad, i.e. wt); n4; p<.4 nd p<.1. Crdi myoytes were infeted with eh Ad s desried (Methods) nd visulised for GFP fluoresene, immunostined GFP ntiody nd DAPI. Results showing equivlent GFP undne with eh Ad re representtive of three seprte infetions. A, lnine; E, glutmine; S, Ser ount for enhned tivtion y gluose strvtion or AMPK. Gluose depletion inreses tyrosine phosphoryltion of Beuse gluose strvtion used inresed inding of to PI3K, we determined whether this ws ompnied S S No virus P- (rtio of : ontrol) A wt E + wt IRS1-S794-A wt -P-T38 -P-S473 Anti-GFP No virus + wt GFP + IRS1-S794-E -P-T38 -P-S473 GSK3β-P-S9 Tuulin -S794-A -S794-E -P-T38 -P-S473 -P-T38 -P-S473 DAPI

8 79 Dietologi (212) 55: y hnges in tyrosine phosphoryltion. Totl phosphotyrosine ws quntified y ELISA efore nd fter tretments with insulin or gluose-free inution s desried (Methods). As shown in Fig. 5, tyrosine phosphoryltion of ws signifintly inresed y insulin stimultion or gluose strvtion. residues Tyr612 nd Tyr632 re within the PI3K inding domin of nd essentil for this inding [39]. Therefore we lso exmined the effets of gluose strvtion nd insulin on the phosphoryltion of these residues. Figure 5 shows tht gluose strvtion or tretment with AICAR inresed the phosphoryltion of oth tyrosine residues y mounts tht were lose to tht used y insulin tretment. Figure 5 lso shows tht seletive inhiition of AMPK with ompound C eliminted the inresed phosphoryltion of Tyr632 simultneously with prllel inhiition of nd ACC phosphoryltion. Quntifition of these results (Fig. 5) onfirmed tht gluose strvtion medited signifint AMPK-dependent inrese in the phosphoryltion of IRS1 t Tyr632. Gluose depletion inreses tyrosine phosphoryltion of IR Ativted IRK is the only known pthwy for tyrosine phosphoryltion of. We therefore sked whether inresed tyrosine phosphoryltion of IR prlleled tht of. Previous studies hve shown tht IR tivtion loop tyrosine residues 1162 nd 1163 re essentil for tivtion of the IRK y insulin inding [4]. As shown in Fig. 6,, phosphoryltion of IR t Tyr1162 inresed signifintly during gluose strvtion. This ws prlleled y inresed nd GSK3β phosphoryltion, nd loked y dnampk. The lower moility nd representing AMPK- GFP (Fig. 6) onfirms overundne of dnampk. We found tht ompound C lso inhiited the enhned phosphoryltion of IR t Tyr1162 y gluose strvtion (not shown). Therefore gluose strvtion inreses tyrosine phosphoryltion of key residues of the IR nd in n AMPK-dependent mnner. As expeted, IR Tyr1162 phosphoryltion ws lso inresed y AICAR tretment (ESM Fig. 3). To determine whether these results extended to skeletl myoytes nd intt myotues, we mesured, -PY632 nd/or IR-PY1162 in C2C12 skeletl myoytes, nd in freshly isolted primry mouse myofilments nd myotues (see Methods) fter exposure to gluose-free inution. As shown in Fig. 6, d, -Thr38, - Y632 nd IR-Y1162 phosphoryltion were eh inresed y gluose strvtion or insulin tretment of C2C12 myoytes (Fig. 6) nd intt myotues (Fig. 6d), suggesting tht similr pthwys operte in skeletl musle. For myotues, we lso demonstrted tht GSK3β ws phosphorylted in prllel nd ll effets were mimiked y AICAR (Fig. 6d). Phosphoryltion of IR y AMPK Figures 5 nd 6 show tht gluose strvtion medites the AMPK-dependent inrese in tyrosine phosphoryltion of nd IR. Therefore we hypothesised tht AMPK diretly phosphoryltes the IR. To test for this, we mixed purified α-ampk nd immunopreipitted IR β-suunit in n in vitro kinse retion. As shown in Fig. 7, the IR β-suunit hs moleulr size of d Asorne units Ins +Ins Glu Gluose Compound C +Glu Glu AICAR Insulin PY632 -PY612 -P-T38 ACC-P-Ser79 -PY632 Tuulin Phosphor-IRS1-Y632:tuulin NS +Glu Glu Glu +CC Fig. 5 Gluose strvtion medites AMPK-dependent tyrosine phosphoryltion. Crdi myoytes were ultured s desried (Fig. 1) nd inuted with or without gluose for 4 h. Totl phosphor-tyrosine ws mesured on equl protein ell lystes using n ELISA kit. For the positive ontrol (with insulin, +Ins), ultures were stimulted with 1 nmol/l insulin for 7 min; n4; p<.3. Crdi myoytes were ultured s ove (), inuted with (+) or without ( ) 5 mmol/l gluose for 4 h, nd treted with.5 μmol/l AICAR for 3 min or with 1 nmol/l insulin for 7 min. Equl lyste proteins were nlysed y western lot using the indited proes. Cultures were dditionlly treted with or without ompound C (2 μmol/l) for 3 min efore olletion. d Phosphor- Y632 ws quntified s the rtio of phosphorylted Y632/totl ; n4; p<.2. P, phosphoryltion; Y, Tyr

9 Dietologi (212) 55: dnampk Insulin +Glu Glu Glu +Glu -P-T38 -P-S473 GSK3β-PS9 IR-PY1162 IR P-AMPK AMPK β-tuulin Phosphor-IR Y1162:IR +Glu Glu +Insulin Gluose -Thr38 -Ser473 IR-Y1162 IR Atin +Insulin Gluose Gluose +dnampk 1 kd 1 kd Superntnt frtion AMPK + AMPK + IR Antiody + IgG ontrol No ell extrt No ntiody + IR Antiody + IgG ontrol No ell extrt No ntiody + AMPK + CC Beds IR phosphoryltion ( 32 P inorportion) AMPK + AMPK + AMPK + CC Fig. 7 Phosphoryltion of the IR y AMPK. HepG2 ells ultured without serum or insulin for 24 h were olleted nd ell lystes used for immunopreipittion of the IR s desried (Methods). Proteins were frtionted y SDS-PAGE. Lnes show proteins remining in the superntnt frtion or tthed to the protein-a eds fter immunopreipittion. IR immunopreipittes ontining equl proteins were inuted with or without AMPK nd [ 32 P]ATP s desried (Methods) in the presene nd sene of 2 μmol/l ompound C (CC). Proteins were seprted y SDS-PAGE, followed y detetion of phosphorylted IR y utordiogrphy. Autordiogrms () were quntified y densitometry using NIH Imge J; n3; p<.1 d +G +G+Ly G G+Ly AICAR Ins -Thr38 GSK3β-PS9 GSK3β -Y632 IR-Y1162 β-tuulin Fig. 6 Gluose strvtion medites nd IR tyrosine phosphoryltion in rdi nd skeletl myoytes, nd isolted skeletl musle fires. Crdi myoytes were infeted or not with dnampk s desried (Methods), followed y ulture s desried (Fig. 1) nd inution with or without gluose for 4 h. Insulin tretment ws 1 nmol/l for 7 min. Equl ell lyste proteins were nlysed y western lot. P, phosphoryltion; S, Ser; T, Thr; Y, Tyr. Phosphor- IR Y1162 ws quntified s the rtio of phosphorylted Y1162:totl IR; n4; p<.5. C2C12 skeletl myoytes were serum-strved overnight nd inuted for 4 h without insulin nd with or without 5 mmol/l gluose. For insulin-treted lnes, ultures were treted with 1 nmol/l insulin for 1 min. Equl lyste proteins were nlysed y western lot s indited. d Skeletl myotues were isolted from mouse hindlims s desried (Methods) nd inuted in defined medium with or without gluose (G), LY2942 (Ly, 2 μmol/l), AICAR (.5 μmol/l for 3 min) or insulin (1 nmol/l for 1 min). Equl lyste proteins were nlysed y western lot. Results re representtive of t lest three experiments out 1 kd (Fig. 7). In the sene of AMPK, we oserved smll inorportion of [ 32 P]ATP into the 1 kd puttive IR nd tht my represent endogenous IR utophosphoryltion or ontminting kinses. There ws mrked inrese in the phosphoryltion of IR when AMPK ws inluded in the retion, nd this ws eliminted y inlusion of the AMPK inhiitor ompound C (Fig. 7), onfirming AMPK-seletive phosphoryltion of IR in vitro. Quntifition onfirmed signifint AMPK-dependent, ompound-c-sensitive inorportion of [ 32 P]ATP into the IR (Fig. 7). -dependent preservtion of intrellulr ATP The energy-onserving roles of AMPK re well doumented; however, hs lso een shown to preserve intrellulr ATP [41]. To see whether tivtion during gluose strvtion onserved energy, we mesured ATP in gluosestrved rdi myoytes in the presene nd sene of the PI3K inhiitor LY2942 nd the inhiitor -X. As shown in Fig. 8, intrellulr ATP levels delined signifintly t 4 nd 6 h of gluose depletion, ut reovered signifintly t 8 h in PI3K- nd -dependent mnner. AMPK- nd PI3K-dependent gluose uptke We predited tht simultneous tivtion of AMPK nd y

10 792 Dietologi (212) 55: ATP (mmol/l) Gluose Hypoglyemi AICAR AMPK 4 h 6 h 8 h Gluose Ser/Thr 8 h +Ly 8 h +X Y1161/1162 Y612/632 Ser473 PDK2 Gluose uptke (fold sl) IR PI3K Ser1345 AKT TSC1/2 Ri TORC2 PDK1 Thr38 Thr1462 TORC1 Rp Ser722/792 Ser636/639 S6K gluose strvtion would ugment gluose trnsport in n AMPK- nd PI3K-dependent mnner. To test for this, we mesured DOG uptke during the initil 5 min of reintrodution in the presene nd sene of PI3K nd AMPK inhiitors. As shown in Fig. 8, gluose uptke ws inresed y more thn threefold y gluose-free ulture, ompred with norml gluose ulture. The enhned gluose uptke ws signifintly inhiited y infetion with dnampk or y tretments with the PI3K inhiitors, LY2942 nd wortmnnin. Comined dnampk nd LY2942 ws no different from either tretment lone, onfirming tht omponent of gluose Control ( Glu) dnampk +Ly dnampk +Ly +Wm +Cyto-Cl B +Anti-insulin Fig. 8 Role of tivted in ATP homeostsis nd gluose uptke. Crdi myoytes were ultured s desried (Fig. 1) nd inuted with or without gluose for 4, 6 or 8 h. Where indited, ultures were treted with LY2942 (Ly, 2 μmol/l) or inhiitor X (X, 1 μmol/l) for the durtion of the experiment. The lystes were olleted nd ATP onentrtion mesured; n5; p<.1. Crdi myoytes were ultured s desried (Fig. 1) nd exposed to gluose- nd insulin-free onditions for 2 h, followed y gluose uptke quntifition. To inhiit AMPK, ultures were pre-infeted with 2 plque forming units (pfu)/ell of denovirl-dnampk for 48 h efore inutions, s desried (Fig. 3). LY2942 (2 μmol/l), wortmnnin (Wm, 1 μmol/l) or ytohlsin B (Cyto-Cl B, 1 mmol/l) were dded 3 min efore mesurement of gluose uptke; n5; p<.1 reltive to +gluose ondition: p<.5 reltive to ontrol. Proposed pthwy of gluose strvtion/ampk-medited sustined tivtion of. AMPK is tivted in musle ells y multiple physiologil nd pthophysiologil onditions, inluding gluose deprivtion nd ontrtion. We propose tht AMPK phosphoryltes IR on Ser/Thr residue(s) nd promotes llosteri tivtion of IRK with tyrosine phosphoryltion of IR nd, tivtion of PI3K nd phosphoinositide-dependent kinse 1 (PDK1), nd phosphoryltion of on Thr38. AMPK lso phosphoryltes TSC2 on Ser1235 nd TORC1-rptor (Rp) on Ser722 nd -792, oth of whih suppress the tivtion of S6K y. Suppression of S6K loks the negtive feedk termintion of insulin signlling nd llows sustined tivtion of y AMPK. TORC2/ritor (Ri) is not ffeted y AMPK nd PDK2 remins tive, llowing sustined phosphoryltion of -Ser473. AMPK-medited sustined tivtion of nd suppression of mmmlin TORC1 supports retention of the survivl funtions of, while suppressing the noli funtions tht would e detrimentl for ell survivl under ompromised energy sttes trnsport is ommon to oth inhiitors. To determine whether the PI3K-dependent omponent of gluose trnsport involved residul insulin, we lso exposed myoytes to sturting levels of n nti-insulin loking ntiody for 1 h efore mesuring gluose uptke. This tretment did not signifintly redue gluose uptke, onfirming insulin independene (Fig. 8). Cytohlsin B tretment onfirmed low non-speifi kground rdiotivity ssoited with the ssy. Disussion We hve shown tht gluose nd insulin strvtion of rdi nd skeletl myoytes tivtes AMPK nd stimultes the insulin-signlling pthwy with five- to tenfold sustined inreses in the phosphoryltion of nd GSK3β. During the sme time period, the phosphoryltion of mmmlin TORC1-Ser2448 nd S6K-Thr389 deresed. Experiments using seletive AMPK inhiitors or AICAR onfirmed tht AMPK ws essentil for gluose strvtion to tivte the insulin signlling pthwy. Importntly, the results with different hemil nd geneti inhiitors nd induers of AMPK were internlly onsistent in supporting these onlusions. The effets ould not e ounted for y AMPK-medited phosphoryltion of t Ser789 euse this ws shown to onfer negtive regultion (Fig. 4). To further investigte the mehnism, we mesured the phosphoryltion of key tyrosine residues of nd IR (Figs 5 nd 6). These

11 Dietologi (212) 55: results reveled signifintly inresed phosphoryltion of residues Y612 nd Y632, nd of IR residue Y1162 y gluose strvtion or AICAR. In vitro kinse ssys reveled AMPK-dependent IR phosphoryltion, whih ws fully inhiited y ompound C. We onlude tht AMPK n phosphorylte the humn IR. Wheres we hve not identified the site of phosphoryltion, the humn IR ontins numer of sequenes with homology to the AMPK onsensus [37]. We propose tht AMPK phosphoryltion of the IR uses llosteri tivtion of IRK independently of insulin. Previous work hs shown tht the IR is phosphorylted y PKC [42, 43] nd yli AMP-dependent kinse [44], nd tht the tivtion loop of the IR is llosterilly modified y ATP inding [45] Gluose strvtion onferred sustined tivtion of nd GSK3β, while depressing the phosphoryltion of mmmlin TORC1 nd S6K. These effets n e explined y the dul AMPK-medited phosphoryltion of IR nd TSC2. Figure 8 shows our proposed sheme for regultion of the insulin signlling pthwy y gluose strvtion. The pthwy is initited y AMPK phosphoryltion nd llosteri tivtion of IRK. Ativted IRK leds to lssil PI3K nd tivtion, ut the signl ends there euse S6K is suppressed due to AMPK-medited phosphoryltion of TSC2 nd/or rptor, nd repression of mmmlin TORC1. S6K normlly termintes the insulin signl y phosphorylting t the negtive regultory Ser636 nd Ser639 site. The negtive feedk regultion does not our when the pthwy is tivted y AMPK euse tivity is proteted. In dt not shown, we found tht when insulin stimultion ws superimposed on gluose-strved myoytes, the negtive feedk loop ws restored nd tivity returned to its sl unstimulted stte y 3 min fter stimultion. This my e due in prt to the ft tht phosphoryltion of TSC overrides tht of AMPK, nd in prt to inhiition of AMPK y [46, 47]. Mmmlin TORC2 ppers to e fully tivted y gluose strvtion or AICAR, s evidened y the sustined phosphoryltion of -Ser473 (Fig. 8). Our results onfirm previous work tht the phosphoryltion of t Ser789 onfers negtive regultion nd exludes this s mehnism for tivtion of the pthwy y AMPK [18, 19, 21, 22]. We provide diret evidene for negtive regultion of -Ser789. Our results, moreover, indite tht AMPK tivtes the pthwy despite, rther thn euse of -Ser789. Although positive regultory -Ser/Thr sites hve een reported [11, 17, 48, 49], none of these sites ontin sequenes with signifint homology to the AMPK onsensus. Consequently, we ttriute tivtion of the insulin signlling pthwy y gluose deprivtion of rdi nd skeletl myoytes to AMPKmedited phosphoryltion of the IR, llosteri tivtion of IRK nd trnsmission of the signl to, PI3K nd - GSK3β. Our results further suggest tht this improves ATP onservtion nd ugments gluose uptke. Our preliminry oservtions tht this pthwy is tivted y the sme stimuli in skeletl myofilments suggest tht it my trnslte into similr physiologil responses in more omplex tissues nd whole orgns. In summry, we provide the first evidene tht AMPK phosphoryltes nd tivtes the IR nd promotes tivtion of the insulin signlling pthwy in the sene of insulin or growth ftors in rdi nd skeletl myoytes, nd in primry skeletl myofilments. The pthwy onfers survivl signls for ells under energy stress with sustined tivtion of nd GSK3β, nd suppression of the noli pthwys regulted y mtor1 nd S6K. Funding This work ws supported y grnts HL44578 nd HL69812 (to K.A. Wester) from the Ntionl Institutes of Helth nd grnt from the Florid Hert Institute. H. Li nd I. Chopr re reipients of Amerin Hert Assoition, Florid Affilite predotorl fellowships. K.A. Wester holds the Wlter G. Ross Distinguished Chir in Vsulr Biology t the University of Mimi Miller Shool of Mediine. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. 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