brief communications NATURE CELL BIOLOGY VOL 3 NOVEMBER 2001 http://cellbio.nature.com 1014 Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo Sue C. Bodine*‡, Trevor N. Stitt*, Michael Gonzalez*, William O. Kline*, Gretchen L. Stover*, Roy Bauerlein*, Elizabeth Zlotchenko*, Angus Scrimgeour†, John C. Lawrence†, David J. Glass* and George D. Yancopoulos*‡ *Regeneron Pharmaceuticals, Inc. 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, USA † Departments of Pharmacology and Medicine, University of Virginia, Charlottesville, Virginia, 22908, USA ‡e-mail: sue.bodine@regeneron.com or george@regeneron.com Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms 1,2 . The roles of two signalling pathways implicated in muscle hypertro- phy on the basis of findings in vitro 3–6 , Akt/mTOR (mam- malian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atro- phy in vivo. The Akt/mTOR pathway was upregulated dur- ing hypertrophy and downregulated during muscle atro- phy. Furthermore, rapamycin, a selective blocker of mTOR 7 , blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the cal- cineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activa- tion of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genet- ic blockade of this pathway blocked hypertrophy in vivo. We conclude that the activation of the Akt/mTOR pathway and its downstream targets, p70 S6K and PHAS-1/4E-BP1, is requisitely involved in regulating skeletal muscle fibre size, and that activation of the Akt/mTOR pathway can oppose muscle atrophy induced by disuse. Initial studies in cardiac hypertrophy 3 , as well as early studies with skeletal muscle cells in vitro 4,5 , pointed towards a key role for the cyclosporin-inhibitable phosphatase known as calcineurin. We first examined the role of the calcineurin pathway in a model of com- pensatory muscle hypertrophy. When a fast-twitch skeletal muscle is subjected to a chronic workload increase by removing function- ally synergistic muscles, the muscle compensates by increasing fibre size and muscle weight, as well as by switching fibres to a slow- twitch phenotype 8,9 . The switch to a slow fibre phenotype has recently been suggested to be under the control of a calcineurin- dependent pathway 10 , although the Ras/MAPK (mitogen-activated protein kinase) pathway has also been implicated 11 . Functional overload of the rat plantaris muscle was induced by surgically removing the soleus and gastrocnemius muscles. Cyclosporin A (CsA) was given at a dosage (15 mg kg -1 , subcutaneously) sufficient to block completely the cardiac hypertrophy induced pharmaco- logically by the β 2 -adrenergic agonist clenbuterol (Fig. 1a) and which inhibited calcineurin activity in control skeletal muscle (Fig. 1b, first two columns). Treatment with CsA was unable to prevent compensatory hypertrophy of the plantaris at 7, 14 or 30 days after the surgical overload, as shown by the increases in muscle weight (Fig. 1d) and fibre size (Fig. 1c, e). Furthermore, treatment with CsA had no effect on the percentage of fibres expressing slow myosin heavy chain (MyHC) after compensatory hypertrophy (Fig. 1c, f) or the shifts in MyHC expression (data not shown). Consistent with the lack of a role for calcineurin induction during compensatory hypertrophy, calcineurin activity was not increased but rather decreased in hypertrophying muscle (Fig. 1b, third col- umn). Finally, treatment with FK506, a calcineurin inhibitor that functions through the binding of FK506-binding protein 12 (FKBP12), was unable to prevent the increase in muscle weight associated with compensatory hypertrophy (Fig. 1g). We have found 6 that hypertrophy of myotubes induced in vitro by insulin-like growth factor 1 (IGF-1) depended on a pathway ini- tiated by PtdIns-3-OH kinase (PI(3)K) and the PtdIns-regulated kinase Akt, which in turn led to activation of the rapamycin-sensi- tive kinase known as mTOR, whose downstream targets, p70 S6K and PHAS-1/4E-BP1, have been shown to promote protein synthesis through increases in translation initiation and elongation 12–16 . To obtain initial evidence for a role of the Akt pathway during muscle hypertrophy in vivo, we examined Akt phosphorylation in the model of compensatory hypertrophy of the plantaris described above. The amount of Akt, and more importantly the phosphory- lation state representing activated Akt, increased throughout the hypertrophy process. By 14 days, the total amount of Akt increased fourfold over control, whereas the level of phosphorylated/activat- ed form increased ninefold in the hypertrophying plantaris (Fig. 2a) as determined by densitometry. Akt seems to promote protein synthesis in several ways. For example, Akt phosphorylates glyco- gen synthase kinase 3β (GSK-3β), leading to its inhibition and the upregulation of protein synthesis 17,18 . More extensively character- ized is the ability of Akt to activate p70 S6K and PHAS-1/4E-BP1, presumably through mTOR, although this is controversial and has not been proved in vivo 7,16,19,20 . Phosphorylation of p70 S6K leads to its activation and to the promotion of protein synthetic pathways, whereas phosphorylation of PHAS-1/4E-BP1 releases it from with- in an inhibitory complex with the translation initiation factor eIF4E, thereby permitting the binding of eIF4E to eIF4G and pro- moting translation initiation. Consistent with a crucial role for the Akt pathway during muscle hypertrophy in vivo, the above Akt- inducible events were noted in hypertrophying plantaris. That is, in addition to Akt phosphorylation, GSK-3β phosphorylation and inhibition were noted (Fig. 2b, c). Furthermore, downstream tar- gets of mTOR were clearly involved, as p70 S6K was inducibly phos- phorylated and activated (Fig. 2b, d), whereas PHAS-1/4E-BP1 was released from eIF4E (Fig. 2e), allowing its binding to eIF4G (Fig. 2f). The above findings indicate that the Akt pathway and its down- stream targets are activated during muscle hypertrophy in vivo. To begin to determine the role of these activations, we used treatment in vivo with rapamycin, a quite specific inhibitor of one important Akt target, mTOR. Rapamycin binds to its intracellular receptor, © 2001 Macmillan Magazines Ltd